Reverse transcription recombinase polymerase amplification assay for the detection of middle East respiratory syndrome coronavirus.

Author(s):

Abd El Wahed, Ahmed; Patel, Pranav; Heidenreich, Doris; Hufert, Frank T; Weidmann, Manfred;

Journal:

PLoS Curr.

Year:

2013

Abstract:

The emergence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in the eastern Mediterranean and imported cases to Europe has alerted public health authorities. Currently, detection of MERS-CoV in patient samples is done by real-time RT-PCR. Samples collected from suspected cases are sent to highly-equipped centralized laboratories for screening. A rapid point-of-care test is needed to allow more widespread mobile detection of the virus directly from patient material. In this study, we describe the development of a reverse transcription isothermal Recombinase Polymerase Amplification (RT-RPA) assay for the identification of MERS-CoV. A partial nucleocapsid gene RNA molecular standard of MERS-coronavirus was used to determine the assay sensitivity. The isothermal (42°C) MERS-CoV RT-RPA was as sensitive as real-time RT-PCR (10 RNA molecules), rapid (3-7 minutes) and mobile (using tubescanner weighing 1kg). The MERS-CoV RT-RPA showed cross-detection neither of any of the RNAs of several coronaviruses and respiratory viruses affecting humans nor of the human genome. The developed isothermal real-time RT-RPA is ideal for rapid mobile molecular MERS-CoV monitoring in acute patients and may also facilitate the search for the animal reservoir of MERS-CoV.

PrimerBankID	Target Pathogen	Target Gene
RPB0039	MERS-CoV	NC gene

Recombinations in staphylococcal cassette chromosome mec elements compromise the molecular detection of methicillin resistance in Staphylococcus aureus

Author(s):

Grant A Hill-Cawthorne , Lyndsey O Hudson , Moataz Fouad Abd El Ghany , Olaf Piepenburg , Mridul Nair , Andrew Dodgson , Matthew S Forrest , Taane G Clark , Arnab Pain 

Journal:

PLOS ONE

Year:

2014

Abstract:

Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (∼4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates.

PrimerBankID	Target Pathogen	Target Gene
RPB0195	Staphylococcus aureus	orfX

Rapid Detection of Mycobacterium tuberculosis by Recombinase Polymerase Amplification

Author(s):

David S. Boyle, Ruth McNerney, Hwee Teng Low,  Brandon Troy Leader,  Ailyn C. Pérez-Osorio, Jessica C. Meyer, Denise M. O'Sullivan,  David G. Brooks,  Olaf Piepenburg, and Matthew S. Forrest

Journal:

PLOS ONE

Year:

2014

Abstract:

Improved access to effective tests for diagnosing tuberculosis (TB) has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA) is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC) DNA in <20 minutes at 39°C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110) and 20 fg (IS1081)were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9) and 86.1% (95%CI: 78.1, 94.1) respectively (n = 71). Specificities were 100% and 88.6% (95% CI: 80.8, 96.1) respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2) and 70.8% (95%CI: 62.9, 78.7) were obtained (n = 90). Specificities were 95.4 (95% CI: 92.3,98.1) and 88% (95% CI: 83.6, 92.4) respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB assays could be of use for integration into a point-of-care test for use in resource constrained settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0180	Mycobacterium tuberculosis	IS6110
RPB0181	Mycobacterium tuberculosis	IS1081

Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens

Author(s):

Sebastian Kersting,corresponding author, Valentina Rausch, Frank F. Bier, and Markus von Nickisch-Rosenegk

Journal:

Microchimica Acta

Year:

2014

Abstract:

We report on the development of an on-chip RPA (recombinase polymerase amplification) with simultaneous multiplex isothermal amplification and detection on a solid surface. The isothermal RPA was applied to amplify specific target sequences from the pathogens Neisseria gonorrhoeae, Salmonella enterica and methicillin-resistant Staphylococcus aureus (MRSA) using genomic DNA. Additionally, a positive plasmid control was established as an internal control. The four targets were amplified simultaneously in a quadruplex reaction. The amplicon is labeled during on-chip RPA by reverse oligonucleotide primers coupled to a fluorophore. Both amplification and spatially resolved signal generation take place on immobilized forward primers bount to expoxy-silanized glass surfaces in a pump-driven hybridization chamber. The combination of microarray technology and sensitive isothermal nucleic acid amplification at 38 °C allows for a multiparameter analysis on a rather small area. The on-chip RPA was characterized in terms of reaction time, sensitivity and inhibitory conditions. A successful enzymatic reaction is completed in <20 min and results in detection limits of 10 colony-forming units for methicillin-resistant Staphylococcus aureus and Salmonella enterica and 100 colony-forming units for Neisseria gonorrhoeae. The results show this method to be useful with respect to point-of-care testing and to enable simplified and miniaturized nucleic acid-based diagnostics.

PrimerBankID	Target Pathogen	Target Gene
RPB0194	Staphylococcus aureus	mecA

Influence of sequence mismatches on the specificity of recombinase polymerase amplification technology

Author(s):

Rana K Daher , Gale Stewart , Maurice Boissinot , Dominique K Boudreau, Michel G Bergeron

Journal:

Molecular and Cellular Probes

Year:

2015

Abstract:

Recombinase polymerase amplification (RPA) technology relies on three major proteins, recombinase proteins, single-strand binding proteins, and polymerases, to specifically amplify nucleic acid sequences in an isothermal format. The performance of RPA with respect to sequence mismatches of closely-related non-target molecules is not well documented and the influence of the number and distribution of mismatches in DNA sequences on RPA amplification reaction is not well understood. We investigated the specificity of RPA by testing closely-related species bearing naturally occurring mismatches for the tuf gene sequence of Pseudomonas aeruginosa and/or Mycobacterium tuberculosis and for the cfb gene sequence of Streptococcus agalactiae. In addition, the impact of the number and distribution of mismatches on RPA efficiency was assessed by synthetically generating 14 types of mismatched forward primers for detecting five bacterial species of high diagnostic relevance such as Clostridium difficile, Staphylococcus aureus, S. agalactiae, P. aeruginosa, and M. tuberculosis as well as Bacillus atropheus subsp. globigii for which we use the spores as internal control in diagnostic assays. A total of 87 mismatched primers were tested in this study. We observed that target specific RPA primers with mismatches (n > 1) at their 3'extrimity hampered RPA reaction. In addition, 3 mismatches covering both extremities and the center of the primer sequence negatively affected RPA yield. We demonstrated that the specificity of RPA was multifactorial. Therefore its application in clinical settings must be selected and validated a priori. We recommend that the selection of a target gene must consider the presence of closely-related non-target genes. It is advisable to choose target regions with a high number of mismatches (≥36%, relative to the size of amplicon) with respect to closely-related species and the best case scenario would be by choosing a unique target gene.

PrimerBankID	Target Pathogen	Target Gene
RPB0185	Mycobacterium tuberculosis	tuf
RPB0187	Pseudomonas aeruginosa	tuf
RPB0193	Staphylococcus aureus	cfb

Diagnostics-in-a-Suitcase: Development of a portable and rapid assay for the detection of the emerging avian influenza A (H7N9) virus

Author(s):

Ahmed Abd El Wahed,Manfred Weidmann,Frank T. Hufertd

Journal:

Journal of Clinical Virology

Year:

2015

Abstract:

Background In developing countries, equipment necessary for diagnosis is only available in few central laboratories, which are less accessible and of limited capacity to test large numbers of incoming samples. Moreover, the transport conditions of samples are inadequate, therefore leading to unreliable results. Objectives The development of a rapid, inexpensive, and simple test would allow mobile detection of viruses. Study design A suitcase laboratory “Diagnostics-in-a-Suitcase” (56 cm × 45.5 cm × 26.5 cm) containing all reagents and devices necessary for performing a reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed. As an example, two RT-RPA assays were established for the detection of hemagglutinin (H) and neuraminidase (N) genes of the novel avian influenza (H7N9) virus. Results The sensitivities of the H7 and the N9 RT-RPA assays were 10 and 100 RNA molecules, respectively. The assays were performed at a single temperature (42 °C). The results were obtained within 2–7 min. The H7N9 RT-RPA assays did not show a cross-detection either of any other respiratory viruses affecting humans and/or birds or of the human or chicken genomes. All reagents were used, stored, and transported at ambient temperature, that is, cold chain independent. In addition, the Diagnostics-in-a-Suitcase was operated by a solar-powered battery. Conclusions The developed assay protocol and mobile setup performed well. Moreover, it can be easily implemented to perform diagnoses at airports, quarantine stations, or farms for rapid on-site viral nucleic acid detection.

PrimerBankID	Target Pathogen	Target Gene
RPB0172	Influenza A virus (H7)	\
RPB0173	Influenza A virus (N9)	\

Development of reverse transcription recombinase polymerase amplification assay for avian influenza H5N1 HA gene detection

Author(s):

Nahed Yehiaa,Abdel-Satar Arafa, Ahmed Abd El Wahed, Ahmed A. El-Sanousi , Manfred Weidmann,Mohamed A. Shalaby

Journal:

Journal of Virological Methods

Year:

2015

Abstract:

The 2006 outbreaks of H5N1 avian influenza in Egypt interrupted poultry production and caused stagger_x005f_x0002_ing economic damage. In addition, H5N1 avian influenza viruses represent a significant threat to public health. Therefore, the rapid detection of H5 viruses is very important in order to control the disease. In this study, a qualitative reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of hemagglutinin gene of H5 subtype influenza viruses was developed. The results were compared to the real-time reverse transcription polymerase chain reaction (RT-PCR). An in vitro transcribed RNA standard of 970 nucleotides ofthe hemagglutinin gene was developed and used to determine the assay sensitivity. The developed H5 RT-RPA assay was able to detect one RNA molecule within 7 min,while in real-time RT-PCR, at least 90 min was required. H5 RT-RPA assay did not detect nucleic acid extracted from H5 negative samples or from other pathogens producing respiratory manifestation in poultry. The clinical performance of the H5 RT-RPA assay was tested in 30 samples collected between 2014 and 2015; the sensitivity of H5 RT-RPA and real-time RT-PCR was 100%. In conclusion, H5 RT-RPA was faster than real-time RT-PCR and easily operable in a portable device. Moreover, it had an equivalent sensitivity and specificity.

PrimerBankID	Target Pathogen	Target Gene
RPB0174	Influenza A virus (H5N1)	Influenza A virus (H5N1)

On-Chip Isothermal Nucleic Acid Amplification on Flow-Based Chemiluminescence Microarray Analysis Platform for the Detection of Viruses and Bacteria

Author(s):

A Kunze,M Dilcher,A Abd El Wahed,F Hufert,R Niessner,M Seidel

Journal:

Analytical Chemistry

Year:

2016

Abstract:

This work presents an on-chip isothermal nucleic acid amplification test (iNAAT) for the multiplex amplification and detection of viral and bacterial DNA by a flow-based chemiluminescence microarray. In a principle study, on-chip recombinase polymerase amplification (RPA) on defined spots of a DNA microarray was used to spatially separate the amplification reaction of DNA from two viruses (Human adenovirus 41, Phi X 174) and the bacterium Enterococcus faecalis, which are relevant for water hygiene. By establishing the developed assay on the microarray analysis platform MCR 3, the automation of isothermal multiplex-amplification (39 °C, 40 min) and subsequent detection by chemiluminescence imaging was realized. Within 48 min, the microbes could be identified by the spot position on the microarray while the generated chemiluminescence signal correlated with the amount of applied microbe DNA. The limit of detection (LOD) determined for HAdV 41, Phi X 174, and E. faecalis was 35 GU/μL, 1 GU/μL, and 5 × 10(3) GU/μL (genomic units), which is comparable to the sensitivity reported for qPCR analysis, respectively. Moreover the simultaneous amplification and detection of DNA from all three microbes was possible. The presented assay shows that complex enzymatic reactions like an isothermal amplification can be performed in an easy-to-use experimental setup. Furthermore, iNAATs can be potent candidates for multipathogen detection in clinical, food, or environmental samples in routine or field monitoring approaches.

PrimerBankID	Target Pathogen	Target Gene
RPB0170	HAdV	HAdV 41

Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform

Author(s):

Lars D Renner, Jindong Zan 1, Linda I Hu 1, Manuel Martinez , Pedro J Resto , Adam C Siegel , Clint Torres , Sara B Hall, Tom R Slezak , Tuan H Nguyen , Douglas B Weibel

Journal:

Applied and Environmental Microbiology

Year:

2017

Abstract:

An estimated 1.5 billion microbial infections occur globally each year and result in ∼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ∼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting.

PrimerBankID	Target Pathogen	Target Gene
RPB0168	Acinetobacter baumannii	Acinetobacter baumannii BJAB07104（429756-429939)
RPB0169	Acinetobacter baumannii	Acinetobacter baumannii BJAB07104（599665-599840)
RPB0175	Klebsiella pneumoniae	Klebsiella pneumoniae CG43（2388833-2389025)
RPB0176	Klebsiella pneumoniae	Klebsiella pneumoniae CG43（2398713-2398848)
RPB0188	Pseudomonas aeruginosa	\
RPB0189	Pseudomonas aeruginosa	\
RPB0190	Staphylococcus aureus	\
RPB0191	Staphylococcus aureus	\

Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of the Mycobacterium avium subsp. paratuberculosis

Author(s):

Sören Hansen, Jenny Schäfer, Kim Fechner, Claus-Peter Czerny, and Ahmed Abd El Wahed

Journal:

PLoS One

Year:

2016

Abstract:

Background The detection of Mycobacterium avium subsp. paratuberculosis (MAP) infections in ruminants is crucial to control spread among animals and to humans. Cultivation of MAP is seen as the gold standard for detection, although it is very time consuming and labour intensive. In addition, several PCR assays have been developed to detect MAP in around 90 minutes, but these assays required highly sophisticated equipment as well as lengthy and complicated procedure. Methodology/Principal Findings In this study, we have developed a rapid assay for the detection of MAP based on the recombinase polymerase amplification (RPA) assay targeting a MAP specific region, the IS900 gene. The detection limit was 16 DNA molecules in 15 minutes as determined by the probit analysis on eight runs of the plasmid standard. Cross reactivity with other mycobacterial and environmentally associated bacterial strains was not observed. The clinical performance of the MAP RPA assay was tested using 48 MAP-positive and 20 MAP-negative blood, sperm, faecal and tissue samples. All results were compared with reads of a highly sensitive real-time PCR assay. The specificity of the MAP RPA assay was 100%, while the sensitivity was 89.5%. Conclusions/Significance The RPA assay is quicker and much easier to handle than real-time PCR. All RPA reagents were cold-chain independent. Moreover, combining RPA assay with a simple extraction protocol will maximize its use at point of need for rapid detection of MAP.

PrimerBankID	Target Pathogen	Target Gene
RPB0184	Mycobacterium avium subsp. paratuberculosis （ATCC 19698）	IS900 gene

Self-powered integrated microfluidic point-of-care low-cost enabling (SIMPLE) chip

Author(s):

Erh-Chia Yeh,Chi-Cheng Fu, Lucy Hu, Rohan Thakur, Jeffrey Feng, and Luke P. Lee

Journal:

Science Advances

Year:

2017

Abstract:

Portable, low-cost, and quantitative nucleic acid detection is desirable for point-of-care diagnostics; however, current polymerase chain reaction testing often requires time-consuming multiple steps and costly equipment. We report an integrated microfluidic diagnostic device capable of on-site quantitative nucleic acid detection directly from the blood without separate sample preparation steps. First, we prepatterned the amplification initiator [magnesium acetate (MgOAc)] on the chip to enable digital nucleic acid amplification. Second, a simplified sample preparation step is demonstrated, where the plasma is separated autonomously into 224 microwells (100 nl per well) without any hemolysis. Furthermore, self-powered microfluidic pumping without any external pumps, controllers, or power sources is accomplished by an integrated vacuum battery on the chip. This simple chip allows rapid quantitative digital nucleic acid detection directly from human blood samples (10 to 105 copies of methicillin-resistant Staphylococcus aureus DNA per microliter, ~30 min, via isothermal recombinase polymerase amplification). These autonomous, portable, lab-on-chip technologies provide promising foundations for future low-cost molecular diagnostic assays.

PrimerBankID	Target Pathogen	Target Gene
RPB0192	Staphylococcus aureus	\

Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Common Bacterial Urinary Tract Infection Pathogens

Author(s):

Balakrishnan Raja,Heather J. Goux, Archana Marapadaga, Sri Rajagopalan, Katerina Kourentzi, and Richard C. Willson

Journal:

Journal of Applied Microbiology

Year:

2017

Abstract:

Aims To develop and evaluate the performance of a panel of isothermal real-time recombinase polymerase amplification (RPA) assays for detection of common bacterial urinary tract infection (UTI) pathogens. Methods and Results The panel included RPAs for Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, and Enterococcus faecalis. All five RPAs required reaction times of under 12 minutes to reach their lower limit of detection of 100 genomes per reaction or less, and did not cross-react with high concentrations of non-target bacterial genomic DNA. In a 50-sample retrospective clinical study, the five-RPA assay panel was found to have a specificity of 100% (95% CI, 78%-100%) and a sensitivity of 89% (95% CI, 75%-96%) for UTI detection. Conclusions The analytical and clinical validity of RPA for the rapid and sensitive detection of common UTI pathogens was established. Significance and Impact Rapid identification of the causative pathogens of urinary tract infections (UTIs) can be valuable in preventing serious complications by helping avoid the empirical treatment necessitated by traditional urine culture’s 48–72 hour turnaround time. The routine and widespread use of RPA to supplement or replace culture-based methods could profoundly impact UTI management and the emergence of multidrug-resistant pathogens.

PrimerBankID	Target Pathogen	Target Gene
RPB0177	Klebsiella pneumoniae	khe
RPB0186	Pseudomonas aeruginosa	lasB

Rapid and visual detection of Mycobacterium tuberculosis complex using recombinase polymerase amplification combined with lateral flow strips

Author(s):

Qinglin Ma , Houming Liu , Feidi Ye , Guangxin Xiang , Wanshui Shan , Wanli Xing

Journal:

Molecular and Cellular Probes

Year:

2017

Abstract:

To definitively diagnose active pulmonary Tuberculosis (TB), Mycobacterium tuberculosis complex (MTBC) bacilli must be identified within clinical specimens from patients. In this study, we introduced a rapid and visual detection method of MTBC using recombinase polymerase amplification (RPA) combined with lateral flow (LF) strips. The LF-RPA assay, read results with naked eyes, could detect as few as 5 genome copies of M. tuberculosis H37Rv (ATCC 27294) per reaction and had no cross-reactions with other control bacteria even using excessive amount of template DNA. The system could work well at a broad range of temperature 25-45 °C and reach detectable level even within 5 min. When testing a total of 137 clinical specimens, the sensitivity and specificity of the LF-RPA assay were 100% (95% CI: 95.94%-100%) and 97.92% (95% CI: 88.93%-99.95%), respectively, compared to culture identification method. Therefore, the LF-RPA system we have demonstrated is a rapid, simple, robust method for MTBC detection which, subject to the availability of a suitable sample extraction method, has the potentiality to diagnose TB at the point-of-care testing.

PrimerBankID	Target Pathogen	Target Gene
RPB0183	Mycobacterium tuberculosis	IS1081

Quantification of viable and non-viable Legionella spp. by heterogeneous asymmetric recombinase polymerase amplification (haRPA) on a flow-based chemiluminescence microarray

Author(s):

Catharina Kober , Reinhard Niessner, Michael Seidel 2

Journal:

Biosensors & bioelectronics

Year:

2018

Abstract:

Increasing numbers of legionellosis outbreaks within the last years have shown that Legionella are a growing challenge for public health. Molecular biological detection methods capable of rapidly identifying viable Legionella are important for the control of engineered water systems. The current gold standard based on culture methods takes up to 10 days to show positive results. For this reason, a flow-based chemiluminescence (CL) DNA microarray was developed that is able to quantify viable and non-viable Legionella spp. as well as Legionella pneumophila in one hour. An isothermal heterogeneous asymmetric recombinase polymerase amplification (haRPA) was carried out on flow-based CL DNA microarrays. Detection limits of 87 genomic units (GU) µL-1 and 26GUµL-1 for Legionella spp. and Legionella pneumophila, respectively, were achieved. In this work, it was shown for the first time that the combination of a propidium monoazide (PMA) treatment with haRPA, the so-called viability haRPA, is able to identify viable Legionella on DNA microarrays. Different proportions of viable and non-viable Legionella, shown with the example of L. pneumophila, ranging in a total concentration between 101 to 105GUµL-1 were analyzed on the microarray analysis platform MCR 3. Recovery values for viable Legionella spp. were found between 81% and 133%. With the combination of these two methods, there is a chance to replace culture-based methods in the future for the monitoring of engineered water systems like condensation recooling plants.

PrimerBankID	Target Pathogen	Target Gene
RPB0178	Legionella pneumophila	16S rRNA

Handheld isothermal amplification and electrochemical detection of DNA in resource-limited settings

Author(s):

Maria-Nefeli Tsaloglou , Alex Nemiroski , Gulden Camci-Unal , Dionysios C Christodouleas , Lara P Murray , John T Connelly , George M Whitesides

Journal:

Analytical Biochemistry

Year:

2018

Abstract:

This paper demonstrates a new method for electrochemical detection of specific sequences of DNA present in trace amounts in serum or blood. This method is designed for use at the point-of-care (particularly in resource-limited settings). By combining recombinase polymerase amplification (RPA)- an isothermal alternative to the polymerase chain reaction - with an electroactive mediator, this electrochemical methodology enables accurate detection of DNA in the field using a low-cost, portable electrochemical analyzer (specifically designed for this type of analysis). This handheld device has four attributes: (1) It uses disposable, paper-based strips that incorporate screen-printed carbon electrodes; (2) It accomplishes thermoregulation with ±0.1 °C temperature accuracy; (3) It enables electrochemical detection using a variety of pulse sequences, including square-wave and cyclic voltammetry, and coulometry; (4) It is operationally simple to use. Detection of genomic DNA from Mycobacterium smegmatis (a surrogate for M. tuberculosis-the main cause of tuberculosis), and from M. tuberculosis itself down to ∼0.040 ng/μL provides a proof-of-concept for the applicability of this method of screening for disease using molecular diagnostics. With minor modifications to the reagents, this method will also enable field monitoring of food and water quality.

PrimerBankID	Target Pathogen	Target Gene
RPB0182	Mycobacterium tuberculosis	16S rRNA

Rapid visual detection of Mycobacterium avium subsp. paratuberculosis by recombinase polymerase amplification combined with a lateral flow dipstick

Author(s):

Guimin Zhao,Hongmei Wang,Peili Hou,Chengqiang He,Hongbin He

Journal:

Journal of veterinary science

Year:

2018

Abstract:

Paratuberculosis (Johne's disease) is a chronic debilitating disease of domestic and wild ruminants. However, widespread point-of-care testing is infrequent due to the lack of a robust method. The isothermal recombinase polymerase amplification (RPA) technique has applied for rapid diagnosis. Herein, RPA combined with a lateral flow dipstick (LFD) assay was developed to estimate DNA from Mycobacterium avium subsp. paratuberculosis. First, analytical specificity and sensitivity of the RPA-nfo primer and probe sets were assessed. The assay successfully detected M. paratuberculosis DNA in 30 min at 39°C with a detection limit of up to eight copies per reaction, which was equivalent to that of the real-time quantitative polymerase chain reaction (qPCR) assay. The assay was specific, as it did not amplify genomes from five other Mycobacterium spp. or five pathogenic enteric bacteria. Six hundred-twelve clinical samples (320 fecal and 292 serum) were assessed by RPA-LFD, qPCR, and enzyme-linked immunosorbent assay, respectively. The RPA-LFD assay yielded 100% sensitivity, 97.63% specificity, and 98.44% concordance rate with the qPCR results. This is the first report utilizing an RPA-LFD assay to visualize and rapidly detect M. paratuberculosis. Our results show this assay should be a useful method for the diagnosis of paratuberculosis in resource-constrained settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0123	Mycobacterium avium subsp. Paratuberculosis	IS900

Automated real-time detection of drug-resistant Mycobacterium tuberculosis on a lab-on-a-disc by Recombinase Polymerase Amplification

Author(s):

I L G Law,J F C Loo,H C Kwok,H Y Yeung,C C H Leung,M Hui,S Y Wu,H S Chan,Y W Kwan,H P Ho,S K Kong

Journal:

Analytical biochemistry

Year:

2018

Abstract:

With the emergence of multi- and extensive-drug (MDR/XDR) resistant Mycobacterium tuberculosis (M. tb), tuberculosis (TB) persists as one of the world's leading causes of death. Recently, isothermal DNA amplification methods received much attention due to their ease of translation onto portable point-of-care (POC) devices for TB diagnosis. In this study, we aimed to devise a simple yet robust detection method for M. tb. Amongst the numerous up-and-coming isothermal techniques, Recombinase Polymerase Amplification (RPA) was chosen for a real-time detection of TB with or without MDR. In our platform, real-time RPA (RT-RPA) was integrated on a lab-on-a-disc (LOAD) with on-board power to maintain temperature for DNA amplification. Sputa collected from healthy volunteers were spiked with respective target M. tb samples for testing. A limit of detection of 102 colony-forming unit per millilitre in 15 min was achieved, making early detection and differentiation of M. tb strains highly feasible in extreme POC settings. Our RT-RPA LOAD platform has also been successfully applied in the differentiation of MDR-TB from H37Ra, an attenuated TB strain. In summary, a quantitative RT-RPA on LOAD assay with a high level of sensitivity was developed as a foundation for further developments in medical bedside and POC diagnostics.

PrimerBankID	Target Pathogen	Target Gene
RPB0121	Mycobacterium tuberculosis	IS6110
RPB0122	Mycobacterium tuberculosis	katG

Development and evaluation of a rapid recombinase polymerase amplification assay for the detection of human enterovirus 71

Author(s):

Dan Yin,Yanan Zhu,Kaifeng Wang,Jing Wang,Xiru Zhang,Min Han,Yaqing He,Qing Chen,Guifang Hu

Journal:

Archives of virology

Year:

2018

Abstract:

Enterovirus 71 (EV71) is one of the most common pathogens of hand, foot, and mouth disease (HFMD). A rapid reverse transcription recombinase polymerase amplification (RT-RPA) assay was established to detect EV71 subgenotype C4 (EV71-C4). The 95% detection limit of the RT-RPA was 3.767 log10 genomic copies (LGC)/reaction. The specificity was 100%. In a clinical sample evaluation, this approach demonstrated sufficient clinical performance when compared with a commercial RT-qPCR diagnostic kit. Thus, the RT-RPA assay may be a promising alternative for the detection of EV71-C4.

PrimerBankID	Target Pathogen	Target Gene
RPB0087	EV-A71	VP1 gene

Rapid and Visual Detection of Coxiella burnetii Using Recombinase Polymerase Amplification Combined with Lateral Flow Strips

Author(s):

Yong Qi,Qiong Yin,Yinxiu Shao,Suqin Li,Hongxia Chen,Wanpeng Shen,Jixian Rao,Jiameng Li,Xiaoling Li,Yu Sun,Yu Lin,Yi Deng,Wenwen Zeng,Shulong Zheng,Suyun Liu,Yuexi Li

Journal:

BioMed research international

Year:

2018

Abstract:

Coxiella burnetii, a global-distributed biological warfare agent, is the causative agent of Q fever. Correct diagnosis of Q fever is challenging and developing a fast, simple, and reliable detection method is necessary. In this study, recombinase polymerase amplification (RPA) assay combined with lateral flow (LF) test was developed targeting 23S rRNA gene of C. burnetii Xinqiao strain. Primers and probe were designed and synthesized, with one set with high amplification efficiency screened for establishment of the method. Reaction conditions were optimized. Sensitivity, specificity, and accuracy were evaluated. The established RPA-LF reaction could be completed in 30 minutes by combining RPA at 37°C with LF at room temperature, with visually judged results. The method showed good specificity without recognizing other bacteria evaluated. It detected positive plasmid and genomic DNA at levels of 10 copies/reaction and 7 copies/reaction, respectively, levels comparable to that of real-time quantitative PCR (RT-qPCR) targeting 23S rRNA gene established previously. Both RPA-LF and RT-qPCR were used to detect C. burnetii-infected mouse samples and the results were fully consistent. The method showed superior detection performance and will provide technical support against C. burnetii in resources-limited areas.

PrimerBankID	Target Pathogen	Target Gene
RPB0086	Coxiella burnetii	23S rRNA gene

Naked eye detection of the Mycobacterium tuberculosis complex by recombinase polymerase amplification-SYBR green I assays

Author(s):

Nuntita Singpanomchai,Yukihiro Akeda,Kazunori Tomono,Aki Tamaru,Pitak Santanirand,Panan Ratthawongjirakul

Journal:

Journal of clinical laboratory analysis

Year:

2019

Abstract:

Background: Rapid diagnosis of Mycobacterium tuberculosis (Mtb) is key to controlling the spread of tuberculosis, which is a global health concern. In this study, isothermal recombinase polymerase amplification (RPA) was developed to detect specific targets of Mtb, IS6110 and IS1081. Additionally, SYBR Green I was used for endpoint detection of the RPA products by the naked eye. Method: A total of 146 genomic Mtb DNA samples and 24 genomic nontuberculous mycobacteria (NTM) DNA samples were amplified at IS6110 and IS1081 by RPA. After a complete amplification, the RPA amplicons were examined by agarose gel electrophoresis (RPA-AGE) and SYBR Green I (RPA-S) assays. The performance of the RPA assays was evaluated by comparing them to a conventional PCR. Results: The RPA assay demonstrated to have a good capability to differentiate Mtb from NTM with a very short turnaround time at a constant temperature. Compared to conventional PCR, the sensitivities and specificities of RPA-AGE for IS6110 and IS1081 were 100%. The specificity of RPA-S was 100% for both targets; however, its sensitivities for IS6110 and IS1081 were 97.95% and 99.32%, respectively. The limits of detection of IS6110 RPA-AGE and RPA-S were 0.05 and 0.5 ng, respectively, while the LODs of IS1081 RPA-AGE and RPA-S were 0.00005 and 0.05 ng, respectively. Both RPA assays showed a satisfying diagnostic specificity, with no cross-reaction with other bacteria. Conclusion: A rapid, sensitive, naked eye RPA assay can be integrated into point-of-care diagnosis for Mtb detection, especially in remote areas where laboratory instrument resources are limited.

PrimerBankID	Target Pathogen	Target Gene
RPB0119	Mycobacterium tuberculosis	IS6110
RPB0120	Mycobacterium tuberculosis	IS1081

Development of propidium monoazide-recombinase polymerase amplification (PMA-RPA) assay for rapid detection of Streptococcus pyogenes and Streptococcus agalactiae

Author(s):

Jing Chen,Yuanyang Wang,Xiaoqing Liu,Guopei Chen,Xuejian Chen,Jiaping Chen,Zhongdong Liu,Jingwen Gong,Guowu Yang,Quanxue Lan

Journal:

Molecular and cellular probes

Year:

2018

Abstract:

Streptococcus pyogenes (Group A Streptococcus, GAS) and Streptococcus agalactiae (Group B Streptococcus, GBS) are common pathogens that threaten public health. In this study, a double recombinase polymerase (RPA) amplification assay was developed to rapidly detect these pathogens. Specificity tests revealed that the GAS and GBS strains were positive for speB and SIP genes, respectively. In clinical samples, the double assay performed similarly to the traditional biochemical method. The limits of detection were both ≤100 copies per reaction. In tests for simulant-contaminated samples, bacterial-culture media containing 103 CFU/mL original concentrations of S. pyogenes and S. agalactiae were positive in RPA assays after incubating for 4 h. Results can be obtained at 37 °C in 20 min. To determine whether propidium monoazide (PMA) can eliminate the influence of DNA extracted from dead cells, a bacterial suspension was treated with PMA before DNA extraction. Findings of RPA assay showed that DNA extracted from dead cells had no fluorescence signal. Therefore, the PMA-RPA assay is a promising technology for field tests and rapid point-of-care diagnosis.

PrimerBankID	Target Pathogen	Target Gene
RPB0166	Streptococcus pyogenes	speB
RPB0167	Streptococcus pyogenes	SIP
RPB0512	Group A Streptococcus	speB
RPB0513	Group B Streptococcus	SIP

Rapid Detection of Avian Influenza A Virus (H7N9) by Lateral Flow Dipstick Recombinase Polymerase Amplification

Author(s):

Shiwei Ma,Xue Li,Bo Peng,Weihua Wu,Xin Wang,Hui Liu,Lihong Yuan,Shisong Fang,Jiahai Lu

Journal:

Biological & pharmaceutical bulletin

Year:

2018

Abstract:

Avian influenza A (H7N9) virus has caused several epidemics and infection in both human and poultry. With mutation, the H7N9 virus gained its fifth endemic in China. Early diagnosis is crucial for the control of viral spread in poultry and prognosis of infected patients. In this study, we developed and evaluated a lateral flow dipstick recombinase polymerase amplification (LFD-RPA) assay for rapid detection of both hemagglutinin and neuraminidase gene of H7N9. Our H7-LFD-RPA and N9-LFD-RPA assay were able to detect 32 fg H7N9 nucleic acid which is more convenient and rapid than previous methods. Through detecting 50 influenza positive samples, cross-reaction was not found with other subtypes of influenza virus. The 100% analytical specificity and sufficient analytical sensitivity results agreed the real time RT-PCR assay. The results data demonstrated that our method performed well and could be applied to the detection of H7N9 virus. This LFD-RPA assay provides a candidate method for rapid point-of-care diagnosis of H7N9.

PrimerBankID	Target Pathogen	Target Gene
RPB0101	Influenza A virus (H7N9)	HA gene
RPB0102	Influenza A virus (H7N9)	NA gene

Reverse transcription recombinase polymerase amplification with lateral flow dipsticks for detection of influenza A virus and subtyping of H1 and H3

Author(s):

Ning Sun,Weiping Wang,Jie Wang,Xinyue Yao,Fangfang Chen,Xiaojun Li,Yi Yinglei,Bo Chen

Journal:

Molecular and cellular probes

Year:

2018

Abstract:

Three reverse transcription recombinase polymerase amplification assays with lateral flow dipsticks (RT-RPA-LFD) were developed for identification of the matrix and hemagglutinin (HA) genes to detect influenza A virus and distinguish subtypes H1 and H3. Assessment of the assays' specificity showed that there was no cross-reactivity with other targets. Their limits of detection were 123.6 copies per reaction for the matrix gene, 677.1 copies per reaction for the H1 HA gene, and 112.2 copies/reaction for the H3 HA gene. Of 111 samples tested by RT-RPA-LFD assays, 27 were positive for influenza A virus, 14 were positive for H1, and 10 were positive for H3. Compared to the results obtained from real-time RT-PCR assays, the sensitivity of RT-RPA-LFD assays was 75%, 93.33% and 71.43% for the matrix, H1, and H3, with 100% specificity. The sensitivity of RT-RPA-LFD assays is lower than that of real-time RT-PCR, comparable or better than that of conventional RT-PCR, and much better than that of RIDTs. In conclusion, these assays offer an efficient and reliable tool for identification and subtyping of influenza A virus (subtype H1 and H3) in the resource-limited setting.

PrimerBankID	Target Pathogen	Target Gene
RPB0098	Influenza A virus	matrix gene
RPB0099	Influenza A virus	H1
RPB0100	Influenza A virus	H3

Rapid detection of Mycobacterium ulcerans with isothermal recombinase polymerase amplification assay

Author(s):

Michael Frimpong,Hubert Senanu Ahor,Ahmed Abd El Wahed,Bernadette Agbavor,Francisca Naana Sarpong,Kenneth Laing,Mark Wansbrough-Jones,Richard Odame Phillips

Journal:

PLoS neglected tropical diseases

Year:

2019

Abstract:

Background: Access to an accurate diagnostic test for Buruli ulcer (BU) is a research priority according to the World Health Organization. Nucleic acid amplification of insertion sequence IS2404 by polymerase chain reaction (PCR) is the most sensitive and specific method to detect Mycobacterium ulcerans (M. ulcerans), the causative agent of BU. However, PCR is not always available in endemic communities in Africa due to its cost and technological sophistication. Isothermal DNA amplification systems such as the recombinase polymerase amplification (RPA) have emerged as a molecular diagnostic tool with similar accuracy to PCR but having the advantage of amplifying a template DNA at a constant lower temperature in a shorter time. The aim of this study was to develop RPA for the detection of M. ulcerans and evaluate its use in Buruli ulcer disease. Methodology and principal findings: A specific fragment of IS2404 of M. ulcerans was amplified within 15 minutes at a constant 42°C using RPA method. The detection limit was 45 copies of IS2404 molecular DNA standard per reaction. The assay was highly specific as all 7 strains of M. ulcerans tested were detected, and no cross reactivity was observed to other mycobacteria or clinically relevant bacteria species. The clinical performance of the M. ulcerans (Mu-RPA) assay was evaluated using DNA extracted from fine needle aspirates or swabs taken from 67 patients in whom BU was suspected and 12 patients with clinically confirmed non-BU lesions. All results were compared to a highly sensitive real-time PCR. The clinical specificity of the Mu-RPA assay was 100% (95% CI, 84-100), whiles the sensitivity was 88% (95% CI, 77-95). Conclusion: The Mu-RPA assay represents an alternative to PCR, especially in areas with limited infrastructure.

PrimerBankID	Target Pathogen	Target Gene
RPB0118	Mycobacterium ulcerans	IS2404

Development of a reverse transcription recombinase polymerase amplification assay for rapid detection of human respiratory syncytial virus.

Author(s):

Xi, Yun; Xu, Chang-Zhi; Xie, Zhi-Zhi; Zhu, Dong-Lin; Dong, Jie-Ming; Xiao, Gang;

Journal:

MOL CELL PROBE

Year:

2019

Abstract:

Respiratory syncytial virus (RSV) is one of the most important causative agents that causing respiratory tract infection in children and associated with high morbidity and mortality. A diagnostic method would be a robust tool for identification of RSV infection, especially in the resource-limited settings. Recombinase polymerase amplification (RPA) is a novel isothermal amplification technique which has been widely employed to detect human/animal pathogens. In present study, a probe-based reverse transcription RPA (RT-RPA) assay was established for the detection of RSV. The primers and probe were designed based on the sequences of the conserved nucleocapsid (N) gene. The minimal detection limit of the RT-RPA assay for the detection of RSV B was 19 copies of RNA molecules at 95% probability, whereas the detection limit for RSV A was 104 copies molecule. The assay was RSV-specific since it had no non-specific reactions with other common human pathogens. The clinical performance of the RT-RPA assay was validated using 188 nasopharyngeal aspirates (NPAs). The nucleic acid extraction of the samples was performed by use of the magnetic bead-based kit which didn't require the heavy and expensive centrifuge. The coincidence rates between RT-RPA and qRT-PCR for the clinical samples was 96%, indicating the RT-RPA assay had good diagnostic performance on clinical samples. The real-time RT-RPA assay combined with the manual genome extraction method make it potential to detect clinical samples in field, providing a possible solution for RSV diagnosis in remote rural areas in developing countries.

PrimerBankID	Target Pathogen	Target Gene
RPB0050	RSV A/B	N gene
RPB0129	RSV	(N) gene

Utilization of recombinase polymerase amplification method combined with lateral flow dipstick for visual detection of respiratory syncytial virus.

Author(s):

Xu, Yu-Zhong; Fang, Du-Zhi; Chen, Fang-Fang; Zhao, Qin-Fei; Cai, Chao-Ming; Cheng, Ming-Gang;

Journal:

MOL CELL PROBE

Year:

2020

Abstract:

Respiratory syncytial virus (RSV) is a major causative agent of respiratory tract infection necessitating hospitalization in children. A rapid diagnostic method would facilitate early detection of RSV infection and timely implementation of special treatment. Here, a reverse transcription recombinase polymerase amplification (RT-RPA) assay combined with lateral flow dipstick (LFD) was evaluated for rapid visual detection of RSV. The primers were designed to target the conserved L gene. The RT-RPA-LFD assay could simultaneously detect RSV subtype A and B with the same detection limit of 10 copies of a given RNA molecule. Moreover, the assay showed no cross-reactivity with other common human pathogens. The performance of the RT-RPA-LFD assay was evaluated by testing 136 nasopharyngeal aspirates (NPAs). The agreement of the detection results between RT-RPA-LFD and qRT-PCR was 100% (34 positive and 102 negative cases). In summary, the developed RT-RPA-LFD assay had good performance in detecting RSV in clinical specimens, thus providing a novel alternative solution for the detection of RSV under field conditions.

PrimerBankID	Target Pathogen	Target Gene
RPB0049	RSV A/B	UTR gene
RPB0128	RSV	conserved L gene

Rapid Detection of SARS-CoV-2 by Low Volume Real-Time Single Tube Reverse Transcription Recombinase Polymerase Amplification Using an Exo Probe with an Internally Linked Quencher (Exo-IQ).

Author(s):

Behrmann, Ole; Bachmann, Iris; Spiegel, Martin; Schramm, Marina; Abd El Wahed, Ahmed; Dobler, Gerhard; Dame, Gregory; Hufert, Frank T;

Journal:

CLIN CHEM

Year:

2020

Abstract:

BACKGROUND:The current outbreak of SARS-CoV-2 has spread to almost every country with more than 5 million confirmed cases and over 300,000 deaths as of May 26, 2020. Rapid first-line testing protocols are needed for outbreak control and surveillance. METHODS:We used computational and manual designs to generate a suitable set of reverse transcription recombinase polymerase amplification (RT-RPA) primer and exonuclease probe, internally quenched (exo-IQ), sequences targeting the SARS-CoV-2 N gene. RT-RPA sensitivity was determined by amplification of in vitro transcribed RNA standards. Assay selectivity was demonstrated with a selectivity panel of 32 nucleic acid samples derived from common respiratory viruses. To validate the assay against full-length SARS-CoV-2 RNA, total viral RNA derived from cell culture supernatant and 19 nasopharyngeal swab samples (8 positive and 11 negative for SARS-CoV-2) were screened. All results were compared to established RT-qPCR assays. RESULTS:The 95% detection probability of the RT-RPA assay was determined to be 7.74 (95% CI: 2.87-27.39) RNA copies per reaction. The assay showed no cross-reactivity to any other screened coronaviruses or respiratory viruses of clinical significance. The developed RT-RPA assay produced 100% diagnostic sensitivity and specificity when compared to RT-qPCR (n = 20). CONCLUSIONS:With a run time of 15 to 20 minutes and first results being available in under 7 minutes for high RNA concentrations, the reported assay constitutes one of the fastest nucleic acid based detection methods for SARS-CoV-2 to date and may provide a simple-to-use alternative to RT-qPCR for first-line screening at the point of need.

PrimerBankID	Target Pathogen	Target Gene
RPB0079	SARS-CoV-2	N gene

An Isothermal Method for Sensitive Detection of Mycobacterium tuberculosis Complex Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a Cis and Trans Cleavage

Author(s):

Haipo Xu,Xiaolong Zhang,Zhixiong Cai,Xiuqing Dong,Geng Chen,Zhenli Li,Liman Qiu,Lei He,Bin Liang,Xiaolong Liu,Jingfeng Liu

Journal:

The Journal of molecular diagnostics : JMD

Year:

2020

Abstract:

Tuberculosis is one of the most serious infectious diseases, resulting in death worldwide. Traditional detection methods are not enough to meet the clinical requirements of rapid diagnosis, high specificity, and high sensitivity. Fast, sensitive, and accurate detection of Mycobacterium tuberculosis (MTB) is urgently needed to treat and control tuberculosis disease. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas12a) exhibit strong nonspecific degradation ability of exogenous single-strand nucleic acids (trans cleavage) after specific recognition of target sequence. We purified Cas12a protein and selected a proper guide RNA based on conserved sequences of MTB from designed guide RNA library. Then, we proposed a novel detection method based on recombinase polymerase amplification and CRISPR/Cas12a nuclease system for specific and sensitive detection of MTB DNA. The assay, based on fluorescence detection, showed 4.48 fmol/L of limit of detection and good linear correlation of concentration with fluorescence value (R2 = 0.9775). It also showed good performance in distinguishing other bacteria. Furthermore, its clinical performance was evaluated by 193 samples and showed sensitivity of 99.29% (139/140) and specificity of 100% (53/53) at 99% CI, compared with culture method. Taken together, the CRISPR/Cas12a system showed good specificity, excellent sensitivity, and excellent accuracy for MTB detection, and it meets requirements of MTB detection in clinical samples and has great potential for clinical translation.

PrimerBankID	Target Pathogen	Target Gene
RPB0117	Mycobacterium	IS1081

Recombinase polymerase amplification with polymer flocculation sedimentation for rapid detection of Staphylococcus aureus in food samples

Author(s):

Jinqiang Hu,Yi Wang,Huimin Ding,Chunpeng Jiang,Yao Geng,Xincheng Sun,Jianzhou Jing,Hui Gao,Zhangcun Wang,Caiwen Dong

Journal:

International journal of food microbiology

Year:

2020

Abstract:

Currently, rapid, sensitive, and convenient visual detection methods for Staphylococcus aureus (S. aureus) are scarce. In this study, a novel detection method based on recombinase polymerase amplification (RPA) and polymer flocculation sedimentation (PFS) was developed. Twelve effective primer combinations derived from four forward primers F1, F2, F3, F4, and three reverse primers R1, R2, R3 targeting the nuc gene of S. aureus were designed and screened by a polymerase chain reaction and RPA methods. RPA reaction conditions, including temperature, time, and volume as well as PEG8000 and NaCl concentrations range, were optimized. Moreover, the specificity and sensitivity of the RPA-PFS assay were further analyzed. Finally, the potential use of the RPA-PFS assay was evaluated using artificially S. aureus contaminated food samples, including pork, beef, shrimp, fish, cheese, cabbage, leftover rice, egg, milk, and orange juice. Results showed that the SA5 (F2/R2) combination was the optimal primer candidate. The optimal temperature range, the shortest time and the minimal volume of RPA reaction were 40-42 °C, 10 min and 10 μL, respectively and the optimal PEG8000/NaCl concentrations were 0.2 g/mL and 2.5 M, respectively, for the adsorption between magnetic beads and RPA products. The RPA-PFS method could detect as little as 13 fg genomic DNA of S. aureus and was also specific for five target S. aureus as well as twenty-seven non-target foodborne bacteria. The limit of detection of RPA-PFS for S. aureus in artificially contaminated food samples was 38 CFU/mL (g). Besides, RPA-PFS has directly been judged by the naked eye and has totally taken less than 20 min. In short, the assay RPA-PFS developed in this study is a rapid, sensitive, and specific visual detection method for S. aureus.

PrimerBankID	Target Pathogen	Target Gene
RPB0159	Staphylococcus aureus	nuc gene

Ultra-sensitive and high-throughput CRISPR-p owered COVID-19 diagnosis.

Author(s):

Huang, Zhen; Tian, Di; Liu, Yang; Lin, Zhen; Lyon, Christopher J; Lai, Weihua; Fusco, Dahlene; Drouin, Arnaud; Yin, Xiaoming; Hu, Tony; Ning, Bo;

Journal:

BIOSENS BIOELECTRON

Year:

2020

Abstract:

Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to detect target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ~50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved quantitative RT-PCR (RT-qPCR) assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the RT-qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts.

PrimerBankID	Target Pathogen	Target Gene
RPB0077	SARS-CoV-2	ORF1ab and N gene
RPB0078	SARS-CoV-2	N gene

3D Printed Monolithic Microreactors for Real-Time Detection of Klebsiella pneumoniae and the Resistance Gene blaNDM-1 by Recombinase Polymerase Amplification

Author(s):

Ole Behrmann,Matthias Hügle,Franz Eckardt,Iris Bachmann,Cecilia Heller,Marina Schramm,Carrie Turner,Frank T Hufert,Gregory Dame

Journal:

Micromachines

Year:

2020

Abstract:

We investigate the compatibility of three 3D printing materials towards real-time recombinase polymerase amplification (rtRPA). Both the general ability of the rtRPA reaction to occur while in contact with the cured 3D printing materials as well as the residual autofluorescence and fluorescence drift in dependence on post curing of the materials is characterized. We 3D printed monolithic rtRPA microreactors and subjected the devices to different post curing protocols. Residual autofluorescence and drift, as well as rtRPA kinetics, were then measured in a custom-made mobile temperature-controlled fluorescence reader (mTFR). Furthermore, we investigated the effects of storage on the devices over a 30-day period. Finally, we present the single- and duplex rtRPA detection of both the organism-specific Klebsiella haemolysin (khe) gene and the New Delhi metallo-β-lactamase 1 (blaNDM-1) gene from Klebsiella pneumoniae. Results: No combination of 3D printing resin and post curing protocol completely inhibited the rtRPA reaction. The autofluorescence and fluorescence drift measured were found to be highly dependent on printing material and wavelength. Storage had the effect of decreasing the autofluorescence of the investigated materials. Both khe and blaNDM-1 were successfully detected by single- and duplex-rtRPA inside monolithic rtRPA microreactors printed from NextDent Ortho Clear (NXOC). The reaction kinetics were found to be close to those observed for rtRPA performed in a microcentrifuge tube without the need for mixing during amplification. Singleplex assays for both khe and blaNDM-1 achieved a limit of detection of 2.5 × 101 DNA copies while the duplex assay achieved 2.5 × 101 DNA copies for khe and 2.5 × 102 DNA copies for blaNDM-1. Impact: We expand on the state of the art by demonstrating a technology that can manufacture monolithic microfluidic devices that are readily suitable for rtRPA. The devices exhibit very low autofluorescence and fluorescence drift and are compatible with RPA chemistry without the need for any surface pre-treatment such as blocking with, e.g., BSA or PEG.

PrimerBankID	Target Pathogen	Target Gene
RPB0104	Klebsiella pneumoniae	khe
RPB0105	Klebsiella pneumoniae	blaNDM-1

Rapid detection of Mycobacterium tuberculosis based on antigen 85B via real-time recombinase polymerase amplification

Author(s):

Y Xu,P Wu,H Zhang,J Li

Journal:

Letters in applied microbiology

Year:

2021

Abstract:

Tuberculosis (TB), as a common infectious disease, still remains a severe challenge to public health. Due to the unsatisfied clinical needs of currently available diagnostic vehicles, it is desired to establish a new approach for universally detecting Mycobacterium tuberculosis. Herein, we designed a real-time recombinase polymerase amplification (RPA) technology for identifying M. tuberculosis within 20 min at 39°C via custom-designed oligonucleotide primers and probe, which could specifically target antigen 85B (Ag85B). Particularly, the primers F4-R4 produced the fastest fluorescence signal with the probe among four pairs of designed primers in the RPA assays. The optimal primers/probe combination could effectively identify M. tuberculosis with the detection limit of 4·0 copies per μl, as it could not show a positive signal for the genomic DNA from other mycobacteria or pathogens. The Ag85B-based RPA could determine the genomic DNA extracted from M. tuberculosis with high reliability (100%, 22/22). More importantly, when testing clinical sputum samples, the real-time RPA displayed an admirable sensitivity (90%, 95% CI: 80·0-96·0%) and specificity (98%, 95% CI: 89·0-100·0%) compared to traditional smear microscopy, which was similar to the assay of Xpert MTB/RIF. This real-time RPA based Ag85B provides a promising strategy for the rapid and universal diagnosis of TB.

PrimerBankID	Target Pathogen	Target Gene
RPB0116	Mycobacterium	Ag85B

Single-strand RPA for rapid and sensitive detection of SARS-CoV-2 RNA.

Author(s):

Kim, Youngeun; Yaseen, Adam B; Kishi, Jocelyn Y; Hong, Fan; Saka, Sinem K; Sheng, Kuanwei; Gopalkrishnan, Nikhil; Schaus, Thomas E; Yin, Peng;

Journal:

medRxiv.

Year:

2020

Abstract:

We report the single-strand Recombinase Polymerase Amplification (ssRPA) method, which merges the fast, isothermal amplification of RPA with subsequent rapid conversion of the double-strand DNA amplicon to single strands, and hence enables facile hybridization-based, high-specificity readout. We demonstrate the utility of ssRPA for sensitive and rapid (4 copies per 50 渭L reaction within 10 min, or 8 copies within 8 min) visual detection of SARS-CoV-2 RNA spiked samples, as well as clinical saliva and nasopharyngeal swabs in VTM or water, on lateral flow devices. The ssRPA method promises rapid, sensitive, and accessible RNA detection to facilitate mass testing in the COVID-19 pandemic.

PrimerBankID	Target Pathogen	Target Gene
RPB0076	SARS-CoV-2	\

Rapid and accurate detection of carbapenem-resistance gene by isothermal amplification in Acinetobacter baumannii

Author(s):

Shuang Liu,Guangtao Huang,Yali Gong,Xiaojun Jin,Yudan Meng,Yizhi Peng,Junning Zhao,Xiaolu Li,Qin Li

Journal:

Burns & trauma

Year:

2020

Abstract:

Background:Acinetobacter baumannii (A. baumannii) is one of the pivotal pathogens responsible for nosocomial infections, especially in patients with low immune response, and infection with carbapenem-resistant A. baumannii has been increasing in recent years. Rapid and accurate detection of carbapenem-resistance genes in A. baumannii could be of immense help to clinical staff. Methods: In this study, a 15-μL reaction system for recombinase polymerase amplification (RPA) was developed and tested. We collected 30 clinical isolates of A. baumannii from the Burn Institute of Southwest Hospital of Third Military Medical University (Army Medical University) for 6 months and tested antibiotic susceptibility using the VITEK 2 system. A. baumannii was detected based on the bla OXA-51 gene by PCR, qPCR and 15 μL-RPA, respectively. Sensitivity and specificity were evaluated. In addition, PCR and 15 μL-RPA data for detecting the carbapenem-resistance gene bla OXA-23 were comparatively assessed. Results: The detection limit of the bla OXA-51 gene by 15 μL RPA was 2.86 CFU/ml, with sensitivity comparable to PCR and qPCR. No positive amplification signals were detected in non-Acinetobacter isolates, indicating high specificity. However, only 18 minutes were needed for the 15 μL RPA assay. Furthermore, an antibiotic susceptibility test showed that up to 90% of A. baumannii strains were resistant to meropenem and imipenem; 15 μL RPA data for detecting bla OXA-23 showed that only 10% (n = 3) of A. baumannii isolates did not show positive amplification signals, and the other 90% of (n = 27) isolates were positive, corroborating PCR results. Conclusion: We demonstrated that the new 15 μL RPA assay for detecting bla OXA-23 in A. baumannii is faster and simpler than qPCR and PCR. It is a promising alternative molecular diagnostic tool for rapid and effective detection of A. baumannii and drug-resistance genes in the field and point-of-care testing.

PrimerBankID	Target Pathogen	Target Gene
RPB0082	Acinetobacter baumannii	blaOXA-51
RPB0083	Acinetobacter baumannii	bla OXA-23

An enhanced isothermal amplification assay for viral detection.

Author(s):

Qian, Jason; Boswell, Sarah A; Chidley, Christopher; Lu, Zhi-Xiang; Pettit, Mary E; Gaudio, Benjamin L; Fajnzylber, Jesse M; Ingram, Ryan T; Ward, Rebecca H; Li, Jonathan Z; Springer, Michael;

Journal:

Nat Commun

Year:

2020

Abstract:

Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. Here we report the development of a molecular diagnostic test for SARS-CoV-2 based on an enhanced recombinase polymerase amplification (eRPA) reaction. eRPA has a detection limit on patient samples down to 5 viral copies, requires minimal instrumentation, and is highly scalable and inexpensive. eRPA does not cross-react with other common coronaviruses, does not require RNA purification, and takes ~45鈥塵in from sample collection to results. eRPA represents a first step toward at-home SARS-CoV-2 detection and can be adapted to future viruses within days of genomic sequence availability.

PrimerBankID	Target Pathogen	Target Gene
RPB0074	SARS-CoV-2	S gene
RPB0075	SARS-CoV-2	N gene

Simultaneous Dual-Gene Diagnosis of SARS-CoV-2 Based on CRISPR/Cas9-Mediated Lateral Flow Assay

Author(s):

Xiong, Erhu; Jiang, Ling; Tian, Tian; Hu, Menglu; Yue, Huahua; Huang, Mengqi; Lin, Wei; Jiang, Yongzhong; Zhu, Debin; Zhou, Xiaoming;

Journal:

ANGEW CHEM INT EDIT

Year:

2020

Abstract:

Few methods for the detection of SARS-CoV-2 currently have the capability to simultaneously detect two genes in a single test, which is a key measure to improve detection accuracy, as adopted by the gold standard RT-qPCR method. Developed here is a CRISPR/Cas9-mediated triple-line lateral flow assay (TL-LFA) combined with multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and simultaneous dual-gene detection of SARS-CoV-2 in a single strip test. This assay is characterized by the detection of envelope (E) and open reading frame 1ab (Orf1ab) genes from cell-cultured SARS-CoV-2 and SARS-CoV-2 viral RNA standards, showing a sensitivity of 100 RNA copies per reaction (25 μL). Furthermore, dual-gene analysis of 64 nasopharyngeal swab samples showed 100 % negative predictive agreement and 97.14 % positive predictive agreement. This platform will provide a more accurate and convenient pathway for diagnosis of COVID-19 or other infectious diseases in low-resource regions

PrimerBankID	Target Pathogen	Target Gene
RPB0059	SARS-CoV-2	E gene
RPB0060	SARS-CoV-2	Orf1ab gene

Rapid detection of SARS-CoV-2 with CRISPR-Cas12a.

Author(s):

Xiong, Dan; Dai, Wenjun; Gong, Jiaojiao; Li, Guande; Liu, Nansong; Wu, Wei; Pan, Jiaqiang; Chen, Chen; Jiao, Yingzhen; Deng, Huina; Ye, Junwei; Zhang, Xuanxuan; Huang, Huiling; Li, Qianyun; Xue, Liang; Zhang, Xiuming; Tang, Guanghui; 

Journal:

PLOS BIOL.

Year:

2020

Abstract:

The recent outbreak of betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is responsible for the Coronavirus Disease 2019 (COVID-19) global pandemic, has created great challenges in viral diagnosis. The existing methods for nucleic acid detection are of high sensitivity and specificity, but the need for complex sample manipulation and expensive machinery slow down the disease detection. Thus, there is an urgent demand to develop a rapid, inexpensive, and sensitive diagnostic test to aid point-of-care viral detection for disease monitoring. In this study, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) 12a-based diagnostic method that allows the results to be visualized by the naked eye. We also introduced a rapid sample processing method, and when combined with recombinase polymerase amplification (RPA), the sample to result can be achieved in 50 minutes with high sensitivity (1-10 copies per reaction). This accurate and portable detection method holds a great potential for COVID-19 control, especially in areas where specialized equipment is not available.

PrimerBankID	Target Pathogen	Target Gene
RPB0072	SARS-CoV-2	ORF1ab
RPB0073	SARS-CoV-2	N gene

Development of a reverse transcription recombinase polymerase amplification assay for rapid and direct visual detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

Author(s):

Lau, Yee Ling; Ismail, Ilyiana Binti; Mustapa, Nur Izati Binti; Lai, Meng Yee; Tuan Soh, Tuan Suhaila; Haji Hassan, Afifah; Peariasamy, Kalaiarasu M; Lee, Yee Leng; Abdul Kahar, Maria Kahar Bador; Chong, Jennifer; Goh, Pik Pin;

Journal:

PLoS One

Year:

2021

Abstract:

Rapid diagnosis is an important intervention in managing the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak. Real time reverse transcription polymerase chain reaction (RT-qPCR) remains the primary means for diagnosing the new virus strain but it is time consuming and costly. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that does not require a PCR machine. It is an affordable, rapid, and simple assay. In this study, we developed and optimized a sensitive reverse transcription (RT)-RPA assay for the rapid detection of SARS-CoV-2 using SYBR Green I and/or lateral flow (LF) strip. The analytical sensitivity and specificity of the RT-RPA assay were tested by using 10-fold serial diluted synthetic RNA and genomic RNA of similar viruses, respectively. Clinical sensitivity and specificity of the RT-RPA assay were carried out using 78 positive and 35 negative nasopharyngeal samples. The detection limit of both RPA and RT-qPCR assays was 7.659 and 5 copies/渭L RNA, respectively with no cross reactivity with other viruses. The clinical sensitivity and specificity of RT-RPA were 98% and 100%, respectively. Our study showed that RT-RPA represents a viable alternative to RT-qPCR for the detection of SARS-CoV-2, especially in areas with limited infrastructure.

PrimerBankID	Target Pathogen	Target Gene
RPB0071	SARS-CoV-2	N gene

Suitcase Lab for Rapid Detection of SARS-CoV-2 Based on Recombinase Polymerase Amplification Assay.

Author(s):

El Wahed, Ahmed Abd; Patel, Pranav; Maier, Melanie; Pietsch, Corinna; Rüster, Dana; Böhlken-Fascher, Susanne; Kissenkötter, Jonas; Behrmann, Ole; Frimpong, Michael; Diagne, Moussa Moïse; Faye, Martin; Dia, Ndongo; Shalaby, Mohamed A; Amer, Haitham; Elgamal, Mahmoud; Zaki, Ali; Ismail, Ghada; Kaiser, Marco; Corman, Victor M; Niedrig, Matthias; Landt, Olfert; Faye, Ousmane; Sall, Amadou A; Hufert, Frank T; Truyen, Uwe; Liebert, Uwe G; Weidmann, Manfred; 

Journal:

ANAL CHEM

Year:

2021

Abstract:

In March 2020, the SARS-CoV-2 virus outbreak was declared as a world pandemic by the World Health Organization (WHO). The only measures for controlling the outbreak are testing and isolation of infected cases. Molecular real-time polymerase chain reaction (PCR) assays are very sensitive but require highly equipped laboratories and well-trained personnel. In this study, a rapid point-of-need detection method was developed to detect the RNA-dependent RNA polymerase (RdRP), envelope protein (E), and nucleocapsid protein (N) genes of SARS-CoV-2 based on the reverse transcription recombinase polymerase amplification (RT-RPA) assay. RdRP, E, and N RT-RPA assays required approximately 15 min to amplify 2, 15, and 15 RNA molecules of molecular standard/reaction, respectively. RdRP and E RT-RPA assays detected SARS-CoV-1 and 2 genomic RNA, whereas the N RT-RPA assay identified only SARS-CoV-2 RNA. All established assays did not cross-react with nucleic acids of other respiratory pathogens. The RT-RPA assay's clinical sensitivity and specificity in comparison to real-time RT-PCR (n = 36) were 94 and 100% for RdRP; 65 and 77% for E; and 83 and 94% for the N RT-RPA assay. The assays were deployed to the field, where the RdRP RT-RPA assays confirmed to produce the most accurate results in three different laboratories in Africa (n = 89). The RPA assays were run in a mobile suitcase laboratory to facilitate the deployment at point of need. The assays can contribute to speed up the control measures as well as assist in the detection of COVID-19 cases in low-resource settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0063	SARS-CoV-2	RdRp gene
RPB0064	SARS-CoV-2	E gene
RPB0065	SARS-CoV-2	N gene

Reverse Transcription Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a for Facile and Highly Sensitive Colorimetric SARS-CoV-2 Detection.

Author(s):

Zhang, Wei S; Pan, Jianbin; Li, Feng; Zhu, Min; Xu, Mengting; Zhu, Hongyan; Yu, Yanyan; Su, Gaoxing;

Journal:

ANAL CHEM

Year:

2021

Abstract:

The outbreak of the pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for an urgent unmet need for developing a facial and cost-effective detection method. The requirement of well-trained personnel and sophisticated instrument of current primary mean (reverse transcription polymerase chain reaction, RT-PCR) may hinder the practical application worldwide. In this regard, a reverse transcription recombinase polymerase amplification (RT-RPA) coupled with CRISPR-Cas12a colorimetric assay is proposed for the SARS-CoV-2 detection. The methodology we have described herein utilizes DNA-modified gold nanoparticles (AuNPs) as a universal colorimetric readout and can specifically target ORF1ab and N regions of the SARS-CoV-2 genome. After the virus genome is amplified through RT-RPA, the resulting abundant dsDNA will bind and activate Cas12a. Under trans-cleavage degradation, the capped DNA substrate will be hydrolyzed gradually from AuNPs, demonstrating a change in the surface plasmon resonance (SPR), which can be facially monitored by UV-vis absorbance spectroscopy and naked eye observation. The high amplification efficiency from RT-RPA and Cas12a trans-cleavage process bring the sensitivity of our method to 1 copy of viral genome sequence per test. Notably, under the dual variations inspecting from the isothermal amplification and Cas12a activation process, the false positive events from other beta coronavirus members can be effectively avoided and thus significantly improve the specificity. Furthermore, the reliability of this colorimetric assay is validated by standard clinical samples from the hospital laboratory department. Through integration of the inherently high sensitivity and specificity from an RPA-coupled Cas12a system with the intrinsic simplicity of AuNP-based colorimetric assay, our method increases the practical testing availability of SARS-CoV-2.

PrimerBankID	Target Pathogen	Target Gene
RPB0066	SARS-CoV-2	ORF1ab gene
RPB0067	SARS-CoV-2	N gene

One-tube SARS-CoV-2 detection platform based on RT-RPA and CRISPR/Cas12a.

Author(s):

Sun, Yangyang; Yu, Lei; Liu, Chengxi; Ye, Shanting; Chen, Wei; Li, Dechang; Huang, Weiren;

Journal:

19 J Transl Med

Year:

2021

Abstract:

BACKGROUND:COVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component.METHODS:We tried to develop a one-tube detection platform based on RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, termed OR-DETECTR, to detect SARS-CoV-2. We designed RT-RPA primers of the RdRp and N genes following the SARS-CoV-2 gene sequence. We optimized reaction components so that the detection process could be carried out in one tube. Specificity was demonstrated by detecting nucleic acid samples from pseudoviruses from seven human coronaviruses and Influenza A (H1N1). Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards and pseudoviruses diluted by different gradients were used to demonstrate the detection limit. Additionally, we have developed a lateral flow assay based on OR-DETECTR for detecting COVID-19.RESULTS:The OR-DETECTR detection process can be completed in one tube, which takes approximately 50min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/ul input (RNA standard) and 1 copy/ul input (pseudovirus). Results of six samples from SARS-CoV-2 patients, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. The lateral flow assay based on OR-DETECTR can be used for the detection of COVID-19, and the detection limit is 2.5 copies/ul input.CONCLUSIONS:The OR-DETECTR platform for the detection of COVID-19 is rapid, accurate, tube closed, easy-to-operate, and free of large instruments.

PrimerBankID	Target Pathogen	Target Gene
RPB0061	SARS-CoV-2	RdRp
RPB0062	SARS-CoV-2	RdRp and Ngene

One-Step Reverse-Transcription Recombinase Polymerase Amplification Using Lateral Flow Strips for the Detection of Coxsackievirus A6

Author(s):

Jia Xie,Xiaohan Yang,Lei Duan,Keyi Chen,Pan Liu,Wenli Zhan,Changbin Zhang,Hongyu Zhao,Mengru Wei,Yuan Tang,Mingyong Luo

Journal:

Frontiers in microbiology

Year:

2021

Abstract:

Hand, foot, and mouth disease (HFMD) is a common infectious disease affecting mainly children under 5 years of age. Coxsackievirus A6 (CVA-6), a major causative pathogen of HFMD, has caused outbreaks in recent years. Currently, no effective vaccine or antiviral treatments are available. In this study, one-step reverse-transcription recombinase polymerase amplification (RT-RPA), combined with a disposable lateral flow strip (LFS) assay, was developed to detect CVA-6. This assay can be performed in less than 35 min at 37°C without expensive instruments, and the result can be observed directly with the naked eye. The sensitivity of the RT-RPA-LFS was 10 copies per reaction, which was comparable to that of the conventional real-time quantitative polymerase chain reaction (qPCR) assays. Moreover, the assay specificity was 100%. The clinical performance of the RT-RPA-LFS assay was evaluated using 142 clinical samples, and the coincidence rate between RT-RPA-LFS and qPCR was 100%. Therefore, our RT-RPA-LFS assay provides a simple and rapid approach for point-of-care CVA-6 diagnosis.

PrimerBankID	Target Pathogen	Target Gene
RPB0085	Coxsackievirus A6	VP1 gene

Nucleic acid visualization assay for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) by targeting the UpE and N gene.

Author(s):

Huang, Pei; Jin, Hongli; Zhao, Yongkun; Li, Entao; Yan, Feihu; Chi, Hang; Wang, Qi; Han, Qiuxue; Mo, Ruo; Song, Yumeng; Bi, Jinhao; Jiao, Cuicui; Li, Wujian; He, Hongbin; Wang, Hongmei; Ma, Aimin; Feng, Na; Wang, Jianzhong; Wang, Tiecheng; Yang, Songtao; Gao, Yuwei; Xia, Xianzhu; Wang, Hualei;

Journal:

PLoS Negl Trop Dis

Year:

2021

Abstract:

Since its first emergence in 2012, cases of infection with Middle East respiratory syndrome coronavirus (MERS-CoV) have continued to occur. At the end of January 2020, 2519 laboratory confirmed cases with a case-fatality rate of 34.3% have been reported. Approximately 84% of human cases have been reported in the tropical region of Saudi Arabia. The emergence of MERS-CoV has highlighted need for a rapid and accurate assay to triage patients with a suspected infection in a timely manner because of the lack of an approved vaccine or an effective treatment for MERS-CoV to prevent and control potential outbreaks. In this study, we present two rapid and visual nucleic acid assays that target the MERS-CoV UpE and N genes as a panel that combines reverse transcription recombinase polymerase amplification with a closed vertical flow visualization strip (RT-RPA-VF). This test panel was designed to improve the diagnostic accuracy through dual-target screening after referencing laboratory testing guidance for MERS-CoV. The limit of detection was 1.2脳101 copies/渭l viral RNA for the UpE assay and 1.2 copies/渭l viral RNA for the N assay, with almost consistent with the sensitivity of the RT-qPCR assays. The two assays exhibited no cross-reactivity with multiple CoVs, including the bat severe acute respiratory syndrome related coronavirus (SARSr-CoV), the bat coronavirus HKU4, and the human coronaviruses 229E, OC43, HKU1 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furthermore, the panel does not require sophisticated equipment and provides rapid detection within 30 min. This panel displays good sensitivity and specificity and may be useful to rapidly detect MERS-CoV early during an outbreak and for disease surveillance.

PrimerBankID	Target Pathogen	Target Gene
RPB0047	MERS-CoV	MERS-CoV UpE gene
RPB0048	MERS-CoV	MERS-CoV N gene
RPB0108	MERS-CoV	UpE
RPB0109	MERS-CoV	N gene

Sensitive electrochemical biosensor combined with isothermal amplification for point-of-care COVID-19 tests

Author(s):

Hyo Eun Kim, Ariadna Schuck, See Hi Lee, Yunjong Lee, Minhee Kang, and Yong-Sang Kim

Journal:

Biosensors and Bioelectronics

Year:

2021

Abstract:

We report an electrochemical biosensor combined with recombinase polymerase amplification (RPA) for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2. The electrochemical biosensor based on a multi-microelectrode array allows the detection of multiple target genes by differential pulse voltammetry. The RPA reaction involves hybridization of the RPA amplicon with thiol-modified primers immobilized on the working electrodes, which leads to a reduction of current density as amplicons accumulate. The assay results in shorter “sample-to-answer” times than conventional PCR without expensive thermo-cycling equipment. The limits of detection are about 0.972 fg/μL (RdRP gene) and 3.925 fg/μL (N gene), which are slightly lower than or comparable to that of RPA assay results obtained by gel electrophoresis without post-amplification purification. The combination of electrochemical biosensors and the RPA assay is a rapid, sensitive, and convenient platform that can be potentially used as a point-of-care test for the diagnosis of COVID-19.

PrimerBankID	Target Pathogen	Target Gene
RPB0217	SARS-CoV-2	N gene
RPB0218	SARS-CoV-2	RdRP gene

Combined recombinase polymerase amplification/rkDNA-graphene oxide probing system for detection of SARS-CoV-2.

Author(s):

Choi, Moon Hyeok; Lee, Jaehyeon; Seo, Young Jun;

Journal:

ANAL CHIM ACTA

Year:

2021

Abstract:

The development of rapid, highly sensitive, and selective methods for the diagnosis of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) should help to prevent the spread of this pandemic virus. In this study, we combined recombinase polymerase amplification (RPA), as a means of isothermal DNA amplification, with an rkDNA-graphene oxide (GO) probe system to allow the rapid detection of SARS-CoV-2 with high sensitivity and selectivity. We used in situ enzymatic synthesis to prepare an rkDNA probe that was complementary to an RPA-amplified sequence of the target N-gene of SARS-CoV-2. The fluorescence of this rkDNA was perfectly quenched in the presence of GO. When the quenched rkDNA-GO system was added to the RPA-amplified sequence of the target SARS-CoV-2, the fluorescence recovered dramatically. The combined RPA/rkDNA-GO system exhibited extremely high selectivity (discrimination factor: 17.2) and sensitivity (LOD = 6.0 aM) for the detection of SARS-CoV-2. The total processing time was only 1.6 h. This combined RPA/rkDNA-GO system appears to be a very efficient and simple method for the point-of-care detection of SARS-CoV-2.

PrimerBankID	Target Pathogen	Target Gene
RPB0058	SARS-CoV-2	N gene

A microfluidic-integrated lateral flow recombinase polymerase amplification (MI-IF-RPA) assay for rapid COVID-19 detection.

Author(s):

Liu, Dan; Shen, Haicong; Zhang, Yuqian; Shen, Danyu; Zhu, Mingyang; Song, Yanling; Zhu, Zhi; Yang, Chaoyong;

Journal:

LAB CHIP

Year:

2019

Abstract:

The COVID-19 pandemic, caused by SARS-CoV-2, currently poses an urgent global medical crisis for which there remains a lack of affordable point-of-care testing (POCT). In particular, resource-limited areas need simple and easily disseminated testing solutions to manage the outbreak. In this work, a microfluidic-integrated lateral flow recombinase polymerase amplification (MI-IF-RPA) assay was developed for rapid and sensitive detection of SARS-CoV-2, which integrates the reverse transcription recombinase polymerase amplification (RT-RPA) and a universal lateral flow (LF) dipstick detection system into a single microfluidic chip. The single-chamber RT-RPA reaction components are mixed with running buffer, and then delivered to the LF detection strips for biotin- and FAM-labelled amplified analyte sequences, which can provide easily interpreted positive or negative results. Testing requires only a simple nucleic acid extraction and loading, then incubation to obtain results, approximately 30 minutes in total. SARS-CoV-2 armored RNA particles were used to validate the MI-IF-RPA system, which showed a limit of detection of 1 copy per 渭L, or 30 copies per sample. Chip performance was further evaluated using clinically diagnosed cases of COVID-19 and revealed a sensitivity of 97% and specificity of 100%, highly comparable to current reverse transcription-polymerase chain reaction (RT-PCR)-based diagnostic assays. This MI-IF-RPA assay is portable and comprises affordable materials, enabling mass production and decreased risk of contamination. Without the need for specialized instrumentation and training, MI-IF-RPA assay can be used as a complement to RT-PCR for low-cost COVID-19 screening in resource-limited areas.

PrimerBankID	Target Pathogen	Target Gene
RPB0057	SARS-CoV-2	N gene

Harnessing recombinase polymerase amplification for rapid multi-gene detection of SARS-CoV-2 in resource-limited settings.

Author(s):

Cherkaoui, Dounia; Huang, Da; Miller, Benjamin S; Turbé, Valérian; McKendry, Rachel A;

Journal:

BIOSENS BIOELECTRON

Year:

2021

Abstract:

The COVID-19 pandemic is challenging diagnostic testing capacity worldwide. The mass testing needed to limit the spread of the virus requires new molecular diagnostic tests to dramatically widen access at the point-of-care in resource-limited settings. Isothermal molecular assays have emerged as a promising technology, given the faster turn-around time and minimal equipment compared to gold standard laboratory PCR methods. However, unlike PCR, they do not typically target multiple SARS-CoV-2 genes, risking sensitivity and specificity. Moreover, they often require multiple steps thus adding complexity and delays. Here we develop a multiplexed, 1-2 step, fast (20-30 min) SARS-CoV-2 molecular test using reverse transcription recombinase polymerase amplification to simultaneously detect two conserved targets - the E and RdRP genes. The agile multi-gene platform offers two complementary detection methods: real-time fluorescence or dipstick. The analytical sensitivity of the fluorescence test was 9.5 (95% CI: 7.0-18) RNA copies per reaction for the E gene and 17 (95% CI: 11-93) RNA copies per reaction for the RdRP gene. The analytical sensitivity for the dipstick method was 130 (95% CI: 82-500) RNA copies per reaction. High specificity was found against common seasonal coronaviruses, SARS-CoV and MERS-CoV model samples. The dipstick readout demonstrated potential for point-of-care testing in decentralised settings, with minimal or equipment-free incubation methods and a user-friendly prototype smartphone application. This rapid, simple, ultrasensitive and multiplexed molecular test offers valuable advantages over gold standard tests and in future could be configurated to detect emerging variants of concern.

PrimerBankID	Target Pathogen	Target Gene
RPB0042	MERS-CoV	E gene
RPB0043	MERS-CoV	RdRP gene

Autonomous Lab-on-Paper for Multiplexed, CRISPR-based Diagnostics of SARS-CoV-2

Author(s):

Kun Yin , Xiong Ding , Ziyue Li , Maroun M Sfeir , Enrique Ballesteros , Changchun Liu

Journal:

Lab on a Chip

Year:

2021

Abstract:

The COVID-19 pandemic, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has become a public health emergency and widely spread around the world. Rapid, accurate and early diagnosis of COVID-19 infection plays a crucial role in breaking this pandemic. However, the detection accuracy is limited for current single-gene diagnosis of SARS-CoV-2. Herein, we develop an autonomous lab-on-paper platform for multiplex gene diagnosis of SARS-CoV-2 by combining reverse transcription recombinase polymerase amplification (RT-RPA) and CRISPR-Cas12a detection. The autonomous lab-on-paper is capable of simultaneously detecting nucleoprotein (N) gene and spike (S) gene of SARS-CoV-2 virus as well as human housekeeping RNAse P gene (an internal control) in a single clinical sample. With the developed platform, 102 copies viral RNA per test can be detected within one hour. Also, the lab-on-paper platform has been used to detect 21 swab clinical samples and obtains a comparable performance to the conventional RT-PCR method. Thus, the developed lab-on-paper platform holds great potential for rapid, sensitive, reliable, multiple molecular diagnostics of COVID-19 and other infectious diseases in resource-limited settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0215	SARS-CoV-2	N gene
RPB0216	SARS-CoV-2	S gene

A Ligation/Recombinase Polymerase Amplification Assay for Rapid Detection of SARS-CoV-2.

Author(s):

Wang, Pei; Ma, Chao; Zhang, Xue; Chen, Lizhan; Yi, Longyu; Liu, Xin; Lu, Qunwei; Cao, Yang; Gao, Song;

Journal:

Front Cell Infect Microbiol

Year:

2021

Abstract:

The pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to more than 117 million reported cases and 2.6 million deaths. Accurate diagnosis technologies are vital for controlling this pandemic. Reverse transcription (RT)-based nucleic acid detection assays have been developed, but the strict sample processing requirement of RT has posed obstacles on wider applications. This study established a ligation and recombinase polymerase amplification (L/RPA) combined assay for rapid detection of SARS-CoV-2 on genes N and ORF1ab targeting the specific biomarkers recommended by the China CDC. Ligase-based strategies usually have a low-efficiency problem on RNA templates. This study has addressed this problem by using a high concentration of the T4 DNA ligase and exploiting the high sensitivity of RPA. Through selection of the ligation probes and optimization of the RPA primers, the assay achieved a satisfactory sensitivity of 101 viral RNA copies per reaction, which was comparable to RT-quantitative polymerase chain reaction (RT-qPCR) and other nucleic acid detection assays for SARS-CoV-2. The assay could be finished in less than 30 min with a simple procedure, in which the requirement for sophisticated thermocycling equipment had been avoided. In addition, it avoided the RT procedure and could potentially ease the requirement for sample processing. Once validated with clinical samples, the L/RPA assay would increase the practical testing availability of SARS-CoV-2. Moreover, the principle of L/RPA has an application potential to the identification of concerned mutations of the virus.

PrimerBankID	Target Pathogen	Target Gene
RPB0068	SARS-CoV-2	N gene
RPB0069	SARS-CoV-2	ORF1ab gene

Optimization of reaction condition of recombinase polymerase amplification to detect SARS-CoV-2 DNA and RNA using a statistical method

Author(s):

Kevin Maafu Juma, Teisuke Takita, Kenji Ito, Masaya Yamagata,Shihomi Akagi, Emi Arikawa, Kenji Kojima, Manish Biyani,Shinsuke Fujiwara, Yukiko Nakura, Itaru Yanagihara, and Kiyoshi Yasukawa

Journal:

Biochemical and Biophysical Research Communications

Year:

2021

Abstract:

Recombinase polymerase amplification (RPA) is an isothermal reaction that amplifies a target DNA sequence with a recombinase, a single-stranded DNA-binding protein (SSB), and a strand-displacing DNA polymerase. In this study, we optimized the reaction conditions of RPA to detect SARS-CoV-2 DNA and RNA using a statistical method to enhance the sensitivity. In vitro synthesized SARS-CoV-2 DNA and RNA were used as targets. After evaluating the concentration of each component, the uvsY, gp32, and ATP concentrations appeared to be rate-determining factors. In particular, the balance between the binding and dissociation of uvsX and DNA primer was precisely adjusted. Under the optimized condition, 60 copies of the target DNA were specifically detected. Detection of 60 copies of RNA was also achieved. Our results prove the fabrication flexibility of RPA reagents, leading to an expansion of the use of RPA in various fields.

PrimerBankID	Target Pathogen	Target Gene
RPB0213	SARS-CoV-2	nucleocapsid phosphoprotein gene
RPB0214	SARS-CoV-2	ORF8 protein gene