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 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 Reverse-transcription recombinase-aided amplification assay for H5 subtype avian influenza virus

2020 ◽  
Author(s):  
Suchun Wang ◽  
Yang Li ◽  
Nan Jiang ◽  
Fuyou Zhang ◽  
Qingye Zhuang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Reverse Transcription ◽  
Economic Losses ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Single Temperature ◽  
Amplification Assay ◽  
H5 Subtype

AbstractThe H5 subtype Avian Influenza Virus has caused huge economic losses to the poultry industry and is a threat to human health. A rapid and simple test is needed to confirm infection in suspected cases during disease outbreaks. In this study, we developed a reverse-transcription recombinase-aided amplification assay for the detection of H5 subtype avian influenza virus. Assays were performed at a single temperature (39°C), and the results were obtained within 20 min. The assay showed no cross-detection with Newcastle disease virus or infectious bronchitis virus. The analytical sensitivity was 103 RNA copies per reaction at a 95% confidence interval according to probit regression analysis, with 100% specificity. Compared with published reverse-transcription quantitative real-time polymerase chain reaction assays, the κ value of the reverse transcription recombinase-aided amplification assay in 365 avian clinical samples was 0.970 (p < 0.001). The sensitivity for avian clinical sample detection was 94.44% (95%CI, 70.63% - 99.71%), and the specificity was 100% (95%CI, 98.64% - 100%). These results indicated that our reverse-transcription recombinase-aided amplification assay may be a valuable tool for detecting H5 subtype avian influenza virus.

scholarly journals Development and application of a method to detect 27 respiratory pathogens using multiplex RT-PCR combined with MassARRAY technology

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Huan Zhao ◽  
Yichao Yang ◽  
Jiangfeng Lyu ◽  
Xuyi Ren ◽  
Wei Cheng
Keyword(s):  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Early Recognition ◽  
Detection System ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Respiratory Pathogens ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Tract Infections

Abstract Background Respiratory tract infections are the most common infections that lead to morbidity and mortality worldwide. Early recognition and precise diagnosis of microbial etiology is important to treat LRTIs promptly, specifically and effectively. Objectives To establish a method based on multiplex reverse transcription (MRT)-PCR and MassARRAY technology for the simultaneous detection of 27 respiratory pathogens and explore its clinical application value. Methods Analytical sensitivity and specificity of the MRT-PCR-MassARRAY system were validated using inactivated bacterial and viral strains. Also we analyzed samples from 207 patients by MassARRAY methods and compared the results with consensus PCR/reverse transcription (RT)-PCR. Results The minimum detection limit of our MRT-PCR-MassARRAY method for pathogens was 10–100 copies/μl, with high specificity. Comparison test with consensus PCR/RT-PCR on 207 clinical samples, the positive, negative, and total correlation rates were 100, 98.68, and 99.03%, respectively. There was a high degree of agreement between the test results of the two methods (P < 0.01 by McNemar’s test). Conclusion Our detection system of 27 respiratory pathogens based on MassARRAY technology has high sensitivity and specificity, high throughput, and is simple to operate. It provides diagnostic value for the clinical diagnosis of respiratory pathogens and is of great significance in the screening of respiratory pathogens.

scholarly journals 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 ◽  
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.

scholarly journals Evaluation of a New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate Surfaces

2015 ◽  
Vol 81 (17) ◽  
pp. 5987-5992 ◽  
Author(s):  
Geun Woo Park ◽  
David Lee ◽  
Aimee Treffiletti ◽  
Mario Hrsak ◽  
Jill Shugart ◽  
...  
Keyword(s):  
Stainless Steel ◽  
Clinical Samples ◽  
Environmental Sampling ◽  
Viral Gastroenteritis ◽  
Human Norovirus ◽  
Drying Time ◽  
Viral Loads ◽  
Toilet Seat ◽  
Outbreak Investigations ◽  
Norovirus Gii

ABSTRACTInanimate surfaces are regarded as key vehicles for the spread of human norovirus during outbreaks. ISO method 15216 involves the use of cotton swabs for environmental sampling from food surfaces and fomites for the detection of norovirus genogroup I (GI) and GII. We evaluated the effects of the virus drying time (1, 8, 24, or 48 h), swab material (cotton, polyester, rayon, macrofoam, or an antistatic wipe), surface (stainless steel or a toilet seat), and area of the swabbed surface (25.8 cm2to 645.0 cm2) on the recovery of human norovirus. Macrofoam swabs produced the highest rate of recovery of norovirus from surfaces as large as 645 cm2. The rates of recovery ranged from 2.2 to 36.0% for virus seeded on stainless-steel coupons (645.0 cm2) to 1.2 to 33.6% for toilet seat surfaces (700 cm2), with detection limits of 3.5 log10and 4.0 log10RNA copies. We used macrofoam swabs to collect environmental samples from several case cabins and common areas of a cruise ship where passengers had reported viral gastroenteritis symptoms. Seventeen (18.5%) of 92 samples tested positive for norovirus GII, and 4 samples could be sequenced and had identical GII.1 sequences. The viral loads of the swab samples from the cabins of the sick passengers ranged from 80 to 31,217 RNA copies, compared with 16 to 113 RNA copies for swab samples from public spaces. In conclusion, our swab protocol for norovirus may be a useful tool for outbreak investigations when no clinical samples are available to confirm the etiology.

scholarly journals Development and Application of a Method to Detect 27 Respiratory Pathogens Using Multiplex RT-PCR Combined with MassARRAY Technology

2021 ◽  
Author(s):  
Huan Zhao ◽  
Yichao Yang ◽  
Jiangfeng Lyu ◽  
Xuyi Ren ◽  
Wei Cheng
Keyword(s):  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Early Recognition ◽  
Detection System ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Respiratory Pathogens ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Tract Infections

Abstract Background: Respiratory tract infections are the most common infections that lead to morbidity and mortality worldwide. Early recognition and precise diagnosis of microbial etiology is important to treat LRTIs promptly, specifically and effectively.Objectives: To establish a method based on multiplex reverse transcription (MRT)-PCR and MassARRAY technology for the simultaneous detection of 27 respiratory pathogens and explore its clinical application value.Methods: Analytical sensitivity and specificity of the MRT-PCR-MassARRAY system were validated using inactivated bacterial and viral strains. Also we analyzed samples from 207 patients by MassARRAY methods and compared the results with consensus PCR/reverse transcription (RT)-PCR.Results: The minimum detection limit of our MRT-PCR-MassARRAY method for pathogens was 10–100 copies/µl, with high specificity. Comparison test with consensus PCR/RT-PCR on 207 clinical samples, the positive, negative, and total correlation rates were 100%, 98.68%, and 99.03%, respectively. There was a high degree of agreement between the test results of the two methods (P < 0.01 by McNemar’s test).Conclusion: Our detection system of 27 respiratory pathogens based on MassARRAY technology has high sensitivity and specificity, high throughput, and is simple to operate. It provides diagnostic value for the clinical diagnosis of respiratory pathogens and is of great significance in the screening of respiratory pathogens.

scholarly journals Reverse Transcription Recombinase-aided Amplification Assay for H5 Subtype avian Influenza Virus

2021 ◽  
Author(s):  
Suchun Wang ◽  
Yang Li ◽  
fuyou zhang ◽  
Nan Jiang ◽  
Qingye Zhuang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Reverse Transcription ◽  
Clinical Sample ◽  
Economic Losses ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Single Temperature ◽  
H5 Subtype

Abstract Background: The H5 subtype avian influenza virus (AIV) has caused huge economic losses to the poultry industry and is a threat to human health. A rapid and simple test is needed to confirm infection in suspected cases during disease outbreaks. Methods: In this study, we developed a reverse transcription recombinase-aided amplification (RT-RAA) assay for the detection of H5 subtype AIV. Assays were performed at a single temperature (39°C), and the results were obtained within 20 min. Results: The assay showed no cross-detection with Newcastle disease virus or infectious bronchitis virus. The analytical sensitivity was 103 RNA copies/μL at a 95% confidence interval according to probit regression analysis, with 100% specificity. Compared with published reverse transcription quantitative real-time polymerase chain reaction assays, the κ value of the RT-RAA assay in 420 avian clinical samples was 0.983 (p < 0.001). The sensitivity for avian clinical sample detection was 97.26% (95% CI, 89.56–99.52%), and the specificity was 100% (95% CI, 98.64–100%). Conclusions: These results indicated that our RT-RAA assay may be a valuable tool for detecting H5 subtype AIV.

scholarly journals Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of Avian Influenza Virus H9N2 HA Gene

2021 ◽  
Vol 8 (7) ◽  
pp. 134
Author(s):  
Nahed Yehia ◽  
Fatma Eldemery ◽  
Abdel-Satar Arafa ◽  
Ahmed Abd El Wahed ◽  
Ahmed El Sanousi ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Reverse Transcription ◽  
Rapid Detection ◽  
Influenza A ◽  
Recombinase Polymerase Amplification ◽  
Economic Losses ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Ha Gene

The H9N2 subtype of avian influenza A virus (aIAV) is circulating among birds worldwide, leading to severe economic losses. H9N2 cocirculation with other highly pathogenic aIAVs has the potential to contribute to the rise of new strains with pandemic potential. Therefore, rapid detection of H9 aIAVs infection is crucial to control virus spread. A qualitative reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of aIAV subtype H9N2 was developed. All results were compared to the gold standard (real-time reverse transcription polymerase chain reaction (RT-PCR)). The RT-RPA assay was designed to detect the hemagglutinin (HA) gene of H9N2 by testing three pairs of primers and a probe. A serial concentration between 106 and 100 EID50 (50% embryo infective dose)/mL was applied to calculate the analytical sensitivity. The H9 RT-RPA assay was highly sensitive as the lowest concentration point of a standard range at one EID50/mL was detected after 5 to 8 min. The H9N2 RT-RPA assay was highly specific as nucleic acid extracted from H9 negative samples and from other avian pathogens were not cross detected. The diagnostic sensitivity when testing clinical samples was 100% for RT-RPA and RT-PCR. In conclusion, H9N2 RT-RPA is a rapid sensitive and specific assay that easily operable in a portable device for field diagnosis of aIAV H9N2.

scholarly journals Reverse transcription loop-mediated isothermal amplification combined with nanoparticles-based biosensor for diagnosis of COVID-19

2020 ◽  
Author(s):  
Xiong Zhu ◽  
Xiaoxia Wang ◽  
Limei Han ◽  
Ting Chen ◽  
Licheng Wang ◽  
...  
Keyword(s):  
Public Health ◽  
Reverse Transcription ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Clinical Samples ◽  
Public Health Issue ◽  
Analytical Sensitivity ◽  
Sample Collection ◽  
Loop Mediated Isothermal Amplification ◽  
One Step

ABSTRACTGiven the scale and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, known as 2019-nCov) infection (COVID-19), the ongoing global SARS-CoV-2 outbreak has become a huge public health issue. Rapid and precise diagnostic methods are thus immediately needed for diagnosing COVID-19, providing timely treatment and facilitating infection control. A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) coupled with nanoparticles-based biosensor (NBS) assay (RT-LAMP-NBS) was successfully established for rapidly and accurately diagnosing COVID-19. A simple equipment (such as heating block) was required for maintaining a constant temperature (63°C) for only 40 min. Using two designed LAMP primer sets, F1ab (opening reading frame 1a/b) and np (nucleoprotein) genes of SARS-CoV-2 were simultaneously amplified and detected in a ‘one-step’ and ‘single-tube’ reaction, and the detection results were easily interpreted by NBS. The sensitivity of SARS-CoV-2 RT-LAMP-NBS was 12 copies (each of detection target) per reaction, and no cross-reactivity was generated from non-SARS-CoV-2 templates. Among clinically diagnosed COVID-19 patients, the analytical sensitivity of SARS-CoV-2 was 100% (33/33) in the oropharynx swab samples, and the assay’s specificity was also 100% (96/96) when analyzed the clinical samples collected from non-COVID-19 patients. The total diagnosis test from sample collection to result interpretation only takes approximately 1 h. In sum, the RT-LAMP-NBS is a promising tool for diagnosing the current SARS-CoV-2 infection in first line field, public health and clinical laboratories, especially for resource-challenged regions.

scholarly journals Highly sensitive and specific diagnosis of COVID-19 by reverse transcription multiple cross-displacement amplification-labelled nanoparticles biosensor

2020 ◽  
Vol 56 (6) ◽  
pp. 2002060 ◽  
Author(s):  
Shijun Li ◽  
Weijia Jiang ◽  
Junfei Huang ◽  
Ying Liu ◽  
Lijuan Ren ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Low Cost ◽  
Test Procedure ◽  
Diagnostic Techniques ◽  
Health Concern ◽  
Clinical Samples ◽  
Specific Diagnosis ◽  
Nucleoprotein Gene ◽  
Multiple Cross ◽  
Positive Results

BackgroundThe ongoing outbreak of the novel human coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (also known as 2019-nCoV) has become a global health concern. Rapid and easy-to-use diagnostic techniques are urgently needed to diagnose SARS-CoV-2 infection.MethodsWe devised a reverse transcription multiple cross-displacement amplification (RT-MCDA) coupled with a nanoparticle-based biosensor assay (RT-MCDA-BS) for rapid, sensitive and specific diagnosis of coronavirus disease 2019 (COVID-19). Two primer sets were designed to target the open reading frame 1a/b and nucleoprotein gene of SARS-CoV-2. A total of 183 clinical samples, including 65 patients with COVID-19 infection and 118 patients with other pathogen infections were used to testify the assay's feasibility. Diagnosis results were reported visually using the biosensor.FindingsThe assay designed was performed using a simple instrument which could maintain the reaction in a constant temperature at 64°C for only 35 min. The total COVID-19 RT-MCDA-BS test procedure could be finished within 1 h. The COVID-19 RT-MCDA-BS could detect down to five copies of target sequences. Among 65 clinical samples from the COVID-19 patients, 22 (33.8%) positive results were obtained from faeces, nasal, pharyngeal and anal swabs via COVID-19 RT-MCDA-BS assay, while real-time reverse transcription-PCR assay only detected 20 (30.7%) positive results in these samples. No positive results were obtained from clinical samples with non-COVID-19 infections.InterpretationCOVID-19 RT-MCDA-BS was a rapid, reliable, low-cost and easy-to-use assay, which could provide an attractive laboratory tool to diagnose COVID-19 in multiple clinical specimens, especially for field, clinic laboratories and primary care facilities in resource-poor settings.

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