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