Isothermal Recombinase Polymerase Amplification Assay Applied to the Detection of Group B Streptococci in Vaginal/Anal Samples

Abstract BACKGROUND Group B streptococcal infections are the leading cause of sepsis and meningitis in newborns. A rapid and reliable method for the detection of this pathogen at the time of delivery is needed for the early treatment of neonates. Isothermal amplification techniques such as recombinase polymerase amplification have advantages relative to PCR in terms of the speed of reaction and simplicity. METHODS We studied the clinical performance of recombinase polymerase amplification for the screening of group B streptococci in vaginal/anal samples from 50 pregnant women. We also compared the limit of detection and the analytical specificity of this isothermal assay to real-time PCR (RT-PCR). RESULTS Compared to RT-PCR, the recombinase polymerase amplification assay showed a clinical sensitivity of 96% and a clinical specificity of 100%. The limit of detection was 98 genome copies and the analytical specificity was 100% for a panel of 15 bacterial and/or fungal strains naturally found in the vaginal/anal flora. Time-to-result for the recombinase polymerase amplification assay was <20 min compared to 45 min for the RT-PCR assay; a positive sample could be detected as early as 8 min. CONCLUSIONS We demonstrate the potential of isothermal recombinase polymerase amplification assay as a clinically useful molecular diagnostic tool that is simple and faster than PCR/RT-PCR. Recombinase polymerase amplification offers great potential for nucleic acid–based diagnostics at the point of care.

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Comparison of commercial RT-PCR diagnostic kits for COVID-19

10.1101/2020.04.22.056747 ◽

2020 ◽

Cited By ~ 3

Author(s):

Puck B. van Kasteren ◽

Bas van der Veer ◽

Sharon van den Brink ◽

Lisa Wijsman ◽

Jørgen de Jonge ◽

...

Keyword(s):

Limit Of Detection ◽

Cross Reactivity ◽

Molecular Diagnostic ◽

Clinical Samples ◽

Specific Method ◽

Preliminary Evaluation ◽

Rt Pcr ◽

Accurate Identification ◽

Clinical Sensitivity ◽

Pcr Efficiency

ABSTRACTThe final months of 2019 witnessed the emergence of a novel coronavirus in the human population. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since spread across the globe and is posing a major burden on society. Measures taken to reduce its spread critically depend on timely and accurate identification of virus-infected individuals by the most sensitive and specific method available, i.e. real-time reverse transcriptase PCR (RT-PCR). Many commercial kits have recently become available, but their performance has not yet been independently assessed.The aim of this study was to compare basic analytical and clinical performance of selected RT-PCR kits from seven different manufacturers (Altona Diagnostics, BGI, CerTest Biotec, KH Medical, PrimerDesign, R-Biopharm AG, and Seegene).We used serial dilutions of viral RNA to establish PCR efficiency and estimate the 95% limit of detection (LOD95%). Furthermore, we ran a panel of SARS-CoV-2-positive clinical samples (n=16) for a preliminary evaluation of clinical sensitivity. Finally, we used clinical samples positive for non-coronavirus respiratory viral infections (n=6) and a panel of RNA from related human coronaviruses to evaluate assay specificity.PCR efficiency was ≥96% for all assays and the estimated LOD95% varied within a 6-fold range. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona)viruses, except as expected for the SARS-CoV-1 E-gene.We conclude that all RT-PCR kits assessed in this study may be used for routine diagnostics of COVID-19 in patients by experienced molecular diagnostic laboratories.

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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)

PLoS ONE ◽

10.1371/journal.pone.0245164 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0245164

Author(s):

Yee Ling Lau ◽

Ilyiana binti Ismail ◽

Nur Izati binti Mustapa ◽

Meng Yee Lai ◽

Tuan Suhaila Tuan Soh ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Sensitivity And Specificity ◽

Reverse Transcription ◽

Cross Reactivity ◽

Recombinase Polymerase Amplification ◽

Analytical Sensitivity ◽

Clinical Sensitivity ◽

Primary Means ◽

Amplification Assay ◽

Recombinase Polymerase Amplification Assay

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/μL 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.

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Development and performance evaluation of a recombinase polymerase amplification assay for the rapid detection of group B streptococcus

BMC Microbiology ◽

10.1186/s12866-016-0836-y ◽

2016 ◽

Vol 16 (1) ◽

Cited By ~ 8

Author(s):

Christina Clarke ◽

Louise O’Connor ◽

Heather Carré-Skinner ◽

Olaf Piepenburg ◽

Terry J. Smith

Keyword(s):

Performance Evaluation ◽

Rapid Detection ◽

Group B Streptococcus ◽

Recombinase Polymerase Amplification ◽

Amplification Assay ◽

And Performance ◽

Group B ◽

Recombinase Polymerase Amplification Assay

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Rapid molecular detection of Zika virus in urine using the recombinase polymerase amplification assay

10.1101/078501 ◽

2016 ◽

Author(s):

Ahmed Abd El Wahed ◽

Sabri S. Sanabani ◽

Oumar Faye ◽

Rodrigo Pessôa ◽

João Veras Patriota ◽

...

Keyword(s):

Real Time ◽

Zika Virus ◽

Recombinase Polymerase Amplification ◽

Control Measures ◽

Mosquito Vector ◽

Rt Pcr ◽

Low Resource Settings ◽

Low Resource ◽

Amplification Assay ◽

Recombinase Polymerase Amplification Assay

AbstractBackgroundCurrently the detection of Zika virus (ZIKV) in patient samples is done by real-time RT-PCR. Samples collected from rural area are sent to highly equipped laboratories for screening. A rapid point-of-care test is needed to detect the virus, especially at low resource settings.Methodology/Principal FindingsIn this report, we describe the development of a reverse transcription isothermal recombinase polymerase amplification (RT-RPA) assay for the identification of ZIKV. RT-RPA assay was portable, sensitive (21 RNA molecules), and rapid (3-15 minutes). No cross-reactivity was detected to other flaviviruses, alphaviruses and arboviruses. Compared to real-time RT-PCR, the diagnostic sensitivity was 92%, while the specificity was 100%.Conclusions/SignificanceThe developed assay is a promising platform for rapid point of need detection of ZIKV in low resource settings and elsewhere (e.g. during mass gathering).Author SummaryCurrently, Dengue (DENV), Zika (ZIKV) and Chikungunya (CHIKV) viruses represent a global threat. The clinical picture of the acute febrile diseases caused by DENV, ZIKV and CHIKV is very similar, in addition, the same mosquito vector is involved in the transmission cycle. The differentiation between them is of great importance as supportive treatment differs and the identification of any of the three viruses prompts implementation of control measures to avoid spreading of an outbreak. We have developed an assay for the detection of ZIKV genome. The assay based on isothermal “recombinase polymerase amplification” assay, which was performed at one temperature (42°C). The result was obtained in maximum of 15 minutes. Moreover, the assay is easy to be implemented at low resource settings.

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