scholarly journals Development of a recombinase-aided amplification assay for rapid detection of human norovirus GII.4

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Zhiwei Qin ◽  
Liang Xue ◽  
Weicheng Cai ◽  
Junshan Gao ◽  
Yueting Jiang ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Enzyme Reaction ◽  
Detection Sensitivity ◽  
Health Concern ◽  
Clinical Samples ◽  
Nucleic Acid Detection ◽  
Human Norovirus ◽  
Amplification Assay ◽  
Norovirus Gii

Abstract Background Human noroviruses are one of the main causes of foodborne illnesses and represent a serious public health concern. Rapid and sensitive assays for human norovirus detection are undoubtedly necessary for clinical diagnosis, especially in regions without more sophisticated equipment. Method The rapid reverse transcription recombinase-aided amplification (RT-RAA) is a fast, robust and isothermal nucleic acid detection method based on enzyme reaction. This method can complete the sample detection at 39 °C in 30 min. In this study, we successfully established a rapid reverse transcription recombinase-aided amplification (RT-RAA) assay for the detection of human norovirus GII.4 and applied this assay to clinical samples, as well as comparison with commercial reverse transcription real-time fluorescence quantitative PCR (RT-qPCR). Results At 95% probability, the detection sensitivity of RT-RAA was 3.425 log10 genomic copies (LGC)/reaction. Moreover, no cross-reaction was observed with other norovirus genogroups and other common foodborne viruses. Stool samples were examined by RT-RAA and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Compared of RT-qPCR, kappa values for human norovirus detection with RT-RAA were 0.894 (p < 0.001), indicating that both assays were in agreement. Conclusion This RT-RAA assay provides a rapid, specific, and sensitive assay for human norovirus detection and is suitable for clinical testing.

scholarly journals Development of a Recombinase-Aided Amplification Assay for Rapid Detection of Human Norovirus GII.4

2020 ◽  
Author(s):  
Zhiwei Qin ◽  
Liang Xue ◽  
Weicheng Cai ◽  
Junshan Gao ◽  
Yueting Jiang ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Enzyme Reaction ◽  
Health Concern ◽  
Clinical Samples ◽  
Nucleic Acid Detection ◽  
Analytical Sensitivity ◽  
Human Norovirus ◽  
Amplification Assay ◽  
Stool Samples ◽  
Norovirus Gii

Abstract Background: Human noroviruses are one of the main causes of foodborne illnesses and represent a serious public health concern. Rapid and sensitive assays for human norovirus detection are undoubtedly necessary for clinical diagnosis, especially in regions without more sophisticated equipment.Method: The recombinase-aided amplification (RT-RAA) is a fast, robust and isothermal nucleic acid detection method based on enzyme reaction. This method can complete the sample detection at 39°C in 30 minutes. In this study, we successfully established a rapid reverse transcription recombinase-aided amplification (RT-RAA) assay for the detection of human norovirus GII.4 and applied this assay to clinical samples, as well as comparison with commercial RT-qPCR.Results: An analytical sensitivity detection of the RT-RAA at a 95% probability of 3.425 log10 genomic copies (LGC)/reaction. Moreover, no cross-reaction was observed with other norovirus genogroups and other common foodborne viruses. Stool samples were examined by RT-RAA and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Compared with RT-qPCR, kappa values for human norovirus detection with RT-RAA were 0.894 (p < 0.001), indicating that both assays were in agreement.Conclusion: This RT-RAA assay provides a rapid, specific, and sensitive assay for human norovirus detection and is suitable for clinical testing.

scholarly journals Schnellnachweis von SARS-CoV-2 mit recombinase polymerase amplification

BIOspektrum ◽  
2020 ◽  
Vol 26 (6) ◽  
pp. 624-627
Author(s):  
Ole Behrmann ◽  
Iris Bachmann ◽  
Frank Hufert ◽  
Gregory Dame
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Dna Polymerase ◽  
Fluorescent Probes ◽  
Reverse Transcription ◽  
Nucleic Acid Detection ◽  
Detection Scheme ◽  
Recombination Proteins ◽  
Target Dna ◽  
Rapid Amplification

Abstract The COVID-19 pandemic highlights the need for fast and simple assays for nucleic acid detection. As an isothermal alternative to RT-qPCR, we outline the development of a detection scheme for SARS-CoV-2 RNA based on reverse transcription recombinase polymerase amplification (RT-RPA) technology. RPA uses recombination proteins in combination with a DNA polymerase for rapid amplification of target DNA at a constant temperature (39–42 °C) within 10 to 20 minutes and can be monitored in real-time with fluorescent probes.

scholarly journals Multiplex Real-Time Reverse-Transcription Polymerase Chain Reaction Assays for Diagnostic Testing of Severe Acute Respiratory Syndrome Coronavirus 2 and Seasonal Influenza Viruses: A Challenge of the Phase 3 Pandemic Setting

2020 ◽  
Author(s):  
Fabiola Mancini ◽  
Fabrizio Barbanti ◽  
Maria Scaturro ◽  
Stefano Fontana ◽  
Angela Di Martino ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Reverse Transcription ◽  
Seasonal Influenza ◽  
Influenza Viruses ◽  
Activity Period ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract Background Pandemic coronavirus disease 2019 (COVID-19) disease represents a challenge for healthcare structures. The molecular confirmation of samples from infected individuals is crucial and therefore guides public health decision making. Clusters and possibly increased diffuse transmission could occur in the context of the next influenza season. For this reason, a diagnostic test able to discriminate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from influenza viruses is urgently needed. Methods A multiplex real-time reverse-transcription polymerase chain reaction (PCR) assay was assessed using 1 laboratory protocol with different real-time PCR instruments. Overall, 1000 clinical samples (600 from samples SARS-CoV-2–infected patients, 200 samples from influenza-infected patients, and 200 negative samples) were analyzed. Results The assay developed was able to detect and discriminate each virus target and to intercept coinfections. The limit of quantification of each assay ranged between 5 and 10 genomic copy numbers, with a cutoff value of 37.7 and 37.8 for influenza and SARS-CoV-2 viruses, respectively. Only 2 influenza coinfections were detected in COVID-19 samples. Conclusions This study suggests that multiplex assay is a rapid, valid, and accurate method for the detection of SARS-CoV-2 and influenza viruses in clinical samples. The test may be an important diagnostic tool for both diagnostic and surveillance purposes during the seasonal influenza activity period.

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