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 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-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 Rapid Identification of Nine Microorganisms Causing Acute Respiratory Tract Infections by Single-Tube Multiplex Reverse Transcription-PCR: Feasibility Study

1999 ◽  
Vol 37 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Britta Gröndahl ◽  
Wolfram Puppe ◽  
Andrea Hoppe ◽  
Inka Kühne ◽  
Josef A. I. Weigl ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Reverse Transcription ◽  
Influenza A ◽  
Respiratory Tract Infections ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Tract Infections ◽  
Type 3

Acute respiratory tract infections (ARIs) are leading causes of morbidity and, in developing countries, mortality in children. A multiplex reverse transcription-PCR (RT-PCR) assay was developed to allow in one test the detection of nine different microorganisms (enterovirus, influenza A and B viruses, respiratory syncytial virus [RSV], parainfluenzaviruses type 1 and type 3, adenovirus,Mycoplasma pneumoniae, and Chlamydia pneumoniae) that do not usually colonize the respiratory tracts of humans but, if present, must be assumed to be the cause of respiratory disease. Clinical samples from 1,118 children admitted to the Department of Pediatrics because of an ARI between November 1995 and April 1998 were used for a first clinical evaluation. Detection of one of the microorganisms included in the assay was achieved for 395 of 1,118 (35%) clinical samples, of which 37.5% were RSV, 20% were influenza A virus, 12.9% were adenovirus, 10.6% were enterovirus, 8.1% were M. pneumoniae, 4.3% were parainfluenzavirus type 3, 3.5% were parainfluenzavirus type 1, 2.8% were influenza B virus, and 0.2% were C. pneumoniae. Seasonal variations in the rates of detection of the different organisms were observed, as was expected from the literature. The levels of concordance with the data obtained by commercially available enzyme immunoassays were 95% for RSV and 98% for influenza A. The results show that the multiplex RT-PCR–enzyme-linked immunosorbent assay is a useful and rapid diagnostic tool for the management of children with ARI. Studies of the overall benefit of this method with regard to the use of antibiotics, the use of diagnostic procedures including additional microbiological tests, and hospitalization rate and duration are warranted.

scholarly journals Comparison of a New Gold Immunochromatographic Assay for the Rapid Diagnosis of the Novel Influenza A (H7N9) Virus with Cell Culture and a Real-Time Reverse-Transcription PCR Assay

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Changzhong Jin ◽  
Nanping Wu ◽  
Xiaorong Peng ◽  
Hangping Yao ◽  
Xiangyun Lu ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Influenza A ◽  
High Specificity ◽  
High Viral Load ◽  
Immunochromatographic Assay ◽  
Clinical Samples ◽  
H7n9 Virus ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Viral Culture

We assessed a colloidal gold immunochromatographic assay (GICA) for rapid detection of influenza A (H7N9) and compared it with reverse-transcription-polymerase chain reaction (RT-PCR) and viral culture. Samples from 35 H7N9 infected patients were collected, including 45 throat swab samples, 56 sputum samples, and 39 feces samples. All samples were tested by GICA, viral culture, and RT-PCR. GICA specifically reacted with recombinant HA proteins, virus lysates, and clinical samples from H7 subtype viruses. Compared with RT-PCR, GICA demonstrated low sensitivity (33.33%) but high specificity (97.56%). The positive rate of GICA tests for samples collected in the period from 8 to 21 days after contact with poultry was much higher than those for samples collected before or after this period. Compared with viral culture, GICA showed sensitivity of 91.67% and specificity of 82.03%. Sputum specimens were more likely to test positive for H7N9 virus than samples from throat swabs and feces. The GICA-based H7 test is a reliable, rapid, and convenient method for the screening and diagnosis of influenza A (H7N9) disease, especially for the sputum specimens with high viral load. It may be helpful in managing H7N9 epidemics and preliminary diagnosis in early stages in resource-limited settings.

scholarly journals Recombinase polymerase amplification assay combined with a dipstick-readout for rapid detection of Mycoplasma ovipneumoniae infections

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0246573
Author(s):  
Sandeep K. Gupta ◽  
Qing Deng ◽  
Tanushree B. Gupta ◽  
Paul Maclean ◽  
Joerg Jores ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Recombinase Polymerase Amplification ◽  
Economic Losses ◽  
Clinical Samples ◽  
The Real ◽  
Positive Rate ◽  
Mycoplasma Ovipneumoniae

Mycoplasma ovipneumoniae infects both sheep and goats causing pneumonia resulting in considerable economic losses worldwide. Current diagnosis methods such as bacteriological culture, serology, and PCR are time consuming and require sophisticated laboratory setups. Here we report the development of two rapid, specific and sensitive assays; an isothermal DNA amplification using recombinase polymerase amplification (RPA) and a real-time PCR for the detection of M. ovipneumoniae. The target for both assays is a specific region of gene WP_069098309.1, which encodes a hypothetical protein and is conserved in the genome sequences of ten publicly available M. ovipneumoniae strains. The RPA assay performed well at 39°C for 20 min and was combined with a lateral flow dipstick (RPA-LFD) for easy visualization of the amplicons. The detection limit of the RPA-LFD assay was nine genome copies of M. ovipneumoniae per reaction and was comparable to sensitivity of the real-time PCR assay. Both assays showed no cross-reaction with 38 other ovine and caprine pathogenic microorganisms and two parasites of ruminants, demonstrating a high degree of specificity. The assays were validated using bronchoalveolar lavage fluid and nasal swab samples collected from sheep. The positive rate of RPA-LFD (97.4%) was higher than the real-time PCR (95.8%) with DNA as a template purified from the clinical samples. The RPA assay was significantly better at detecting M. ovipneumoniae in clinical samples compared to the real-time PCR when DNA extraction was omitted (50% and 34.4% positive rate for RPA-LFD and real-time PCR respectively). The RPA-LFD developed here allows easy and rapid detection of M. ovipneumoniae infection without DNA extraction, suggesting its potential as a point-of-care test for field settings.

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.

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