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