scholarly journals Specific Detection of Influenza A and B Viruses by CRISPR-Cas12a-Based Assay

Biosensors ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 88
Author(s):  
Bum Ju Park ◽  
Man Seong Park ◽  
Jae Myun Lee ◽  
Yoon Jae Song
Keyword(s):  
Diagnostic Test ◽  
Reverse Transcription ◽  
Influenza A ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Influenza Viruses ◽  
Nucleic Acid Amplification ◽  
Lateral Flow Strip ◽  
Influenza Pandemics

A rapid and accurate on-site diagnostic test for pathogens including influenza viruses is critical for preventing the spread of infectious diseases. Two types of influenza virus, A and B cause seasonal flu epidemics, whereas type A can cause influenza pandemics. To specifically detect influenza A (IAV) and B (IBV) viruses, we developed a clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system-based assay. By coupling reverse transcription recombinase polymerase amplification (RT-RPA) and reverse transcription loop-mediated isothermal amplification (RT-LAMP), a CRISPR-Cas12a DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) detected IAV and IBV titers as low as 1 × 100 plaque forming units (PFUs) per reaction without exhibiting cross-reactivity. Only 75 to 85 min were required to detect IAV and IBV, depending on isothermal nucleic acid amplification methods, and results were verified using a lateral flow strip assay that does not require additional analytic equipment. Taken together, our findings establish RT-RPA and RT-LAMP-coupled DETECTR-based diagnostic tests for rapid, specific and high-sensitivity detection of IAV and IBV using fluorescence and lateral flow assays. The diagnostic test developed in this study can be used to distinguish IAV and IBV infections, a capability that is necessary for monitoring and preventing the spread of influenza epidemics and pandemics.

scholarly journals Applicability of duplex real time and lateral flow strip reverse-transcription recombinase aided amplification assays for the detection of Enterovirus 71 and Coxsackievirus A16

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Xin-na Li ◽  
Xin-xin Shen ◽  
Ming-hui Li ◽  
Ju-ju Qi ◽  
Rui-huan Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Enterovirus 71 ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Coxsackievirus A16 ◽  
Fluorescence Detector ◽  
Lateral Flow Strip ◽  
Clinical Diagnostic

Abstract Background Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two main etiological agents of Hand, Foot and Mouth Disease (HFMD). Simple and rapid detection of EV71 and CA16 is critical in resource-limited settings. Methods Duplex real time reverse-transcription recombinase aided amplification (RT-RAA) assays incorporating competitive internal amplification controls (IAC) and visible RT-RAA assays combined with lateral flow strip (LFS) for detection of EV71 and CA16 were developed respectively. Duplex real time RT-RAA assays were performed at 42 °C within 30 min using a portable real-time fluorescence detector, while LFS RT-RAA assays were performed at 42 °C within 30 min in an incubator. Recombinant plasmids containing conserved VP1 genes were used to analyze the sensitivities of these two methods. A total of 445 clinical specimens from patients who were suspected of being infected with HFMD were used to evaluate the performance of the assays. Results The limit of detection (LoD) of the duplex real time RT-RAA for EV71 and CA16 was 47 copies and 38 copies per reaction, respectively. The LoD of the LFS RT-RAA for EV71 and CA16 were both 91 copies per reaction. There was no cross reactivity with other enteroviruses. Compared to reverse transcription-quantitative PCR (RT-qPCR), the clinical diagnostic sensitivities of the duplex real time RT-RAA assay were 92.3% for EV71 and 99.0% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. The clinical diagnostic sensitivities of the LFS RT-RAA assay were 90.1% for EV71 and 94.9% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. Conclusions The developed duplex real time RT-RAA and LFS RT-RAA assays for detection of EV71 and CA16 are potentially suitable in primary clinical settings.

scholarly journals Nucleic Acid-Based Sensing Techniques for Diagnostics and Surveillance of Influenza

Biosensors ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 47 ◽  
Author(s):  
Samantha J. Courtney ◽  
Zachary R. Stromberg ◽  
Jessica Z. Kubicek-Sutherland
Keyword(s):  
Influenza Virus ◽  
Nucleic Acid ◽  
Diagnostic Test ◽  
Influenza A ◽  
Direct Detection ◽  
Point Of Care ◽  
Influenza Viruses ◽  
Nucleic Acid Amplification ◽  
High Genetic Diversity ◽  
Mild Disease

Influenza virus poses a threat to global health by causing seasonal outbreaks as well as three pandemics in the 20th century. In humans, disease is primarily caused by influenza A and B viruses, while influenza C virus causes mild disease mostly in children. Influenza D is an emerging virus found in cattle and pigs. To mitigate the morbidity and mortality associated with influenza, rapid and accurate diagnostic tests need to be deployed. However, the high genetic diversity displayed by influenza viruses presents a challenge to the development of a robust diagnostic test. Nucleic acid-based tests are more accurate than rapid antigen tests for influenza and are therefore better candidates to be used in both diagnostic and surveillance applications. Here, we review various nucleic acid-based techniques that have been applied towards the detection of influenza viruses in order to evaluate their utility as both diagnostic and surveillance tools. We discuss both traditional as well as novel methods to detect influenza viruses by covering techniques that require nucleic acid amplification or direct detection of viral RNA as well as comparing advantages and limitations for each method. There has been substantial progress in the development of nucleic acid-based sensing techniques for the detection of influenza virus. However, there is still an urgent need for a rapid and reliable influenza diagnostic test that can be used at point-of-care in order to enhance responsiveness to both seasonal and pandemic influenza outbreaks.

scholarly journals Rapid Detection of blaKPC, blaNDM, blaOXA-48-like and blaIMP Carbapenemases in Enterobacterales Using Recombinase Polymerase Amplification Combined With Lateral Flow Strip

2021 ◽  
Vol 11 ◽  
Author(s):  
Fang Wang ◽  
Lei Wang ◽  
Huimin Chen ◽  
Na Li ◽  
Yan Wang ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Global Public Health ◽  
Special Equipment ◽  
Lateral Flow Strip ◽  
Carbapenemase Gene ◽  
Primer Sets

The emergence of carbapenemase-producing Enterobacterales (CPE) infections is a major global public health threat. Rapid and accurate detection of pathogenic bacteria is essential to optimize treatment and timely avoid further transmission of these bacteria. Here, we aimed to develop a rapid on site visualization detection method for CPE using improved recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) method, based on four most popular carbapenemase genes: blaKPC, blaNDM, blaOXA-48-like, and blaIMP. All available allelic variants of the above carbapenemases were downloaded from the β-lactamase database, and the conserved regions were used as targets for RPA assay. Five primer sets were designed targeting to each carbapenemase gene and the RPA amplification products were analyzed by agarose gel electrophoresis. FITC-labeled specific probes were selected, combined with the best performance primer set (Biotin-labeled on the reverse primer), and detected by RPA-LFS. Mismatches were made to exclude the false positive signals interference. This assay was evaluated in 207 clinically validated carbapenem-resistant Enterobacterales (CRE) isolates and made a comparison with conventional PCR. Results showed that the established RPA-LFS assay for CPE could be realized within 30 min at a constant temperature of 37°C and visually detected amplification products without the need for special equipment. This assay could specifically differentiate the four classes of carbapenemases without cross-reactivity and shared a minimum detection limit of 100 fg/reaction (for blaKPC, blaNDM, and blaOXA-48-like) or 1000 fg/reaction (for blaIMP), which is ten times more sensitive than PCR. Furthermore, the detection of 207 pre-validated clinically CRE strains using the RPA-LFS method resulted in 134 blaKPC, 69 blaNDM, 3 blaOXA-48-like, and 1 blaIMP. The results of the RPA-LFS assay were in consistent with PCR, indicating that this method shared high sensitivity and specificity. Therefore, the RPA-LFS method for CPE may be a simple, specific, and sensitive method for the rapid diagnosis of carbapenemase Enterobacterales.

scholarly journals Detection of severe acute respiratory syndrome coronavirus 2 and influenza viruses based on CRISPR-Cas12a

2020 ◽  
pp. 153537022096379
Author(s):  
Oraphan Mayuramart ◽  
Pattaraporn Nimsamer ◽  
Somruthai Rattanaburi ◽  
Naphat Chantaravisoot ◽  
Kritsada Khongnomnan ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Severe Acute Respiratory Syndrome ◽  
Influenza A ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Influenza Viruses ◽  
Influenza B Virus ◽  
Influenza B ◽  
Influenza Virus A ◽  
B Virus

Due to the common symptoms of COVID-19, patients are similar to influenza-like illness. Therefore, the detection method would be crucial to discriminate between SARS-CoV-2 and influenza virus-infected patients. In this study, CRISPR-Cas12a-based detection was applied for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus which would be a practical and attractive application for screening of patients with COVID-19 and influenza in areas with limited resources. The limit of detection for SARS-CoV-2, influenza A, and influenza B detection was 10, 103, and 103 copies/reaction, respectively. Moreover, the assays yielded no cross-reactivity against other respiratory viruses. The results revealed that the detection of influenza virus and SARS-CoV-2 by using RT-RPA and CRISPR-Cas12a technology reaches 96.23% sensitivity and 100% specificity for SARS-CoV-2 detection. The sensitivity for influenza virus A and B detections was 85.07% and 94.87%, respectively. In addition, the specificity for influenza virus A and B detections was approximately 96%. In conclusion, the RT-RPA with CRISPR-Cas12a assay was an effective method for the screening of influenza viruses and SARS-CoV-2 which could be applied to detect other infectious diseases in the future.

HIGHLY SENSITIVE H7N9 AVIAN INFLUENZA VIRUS DETECTION USING REVERSE TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION AND OLIGONUCLEOTIDE MICROARRAY

2015 ◽  
Vol 41 (04) ◽  
pp. 251-255
Author(s):  
Lih-Chiann Wang ◽  
Dean Huang
Keyword(s):  
Avian Influenza ◽  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
High Sensitivity ◽  
Oligonucleotide Microarray ◽  
Influenza Viruses ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
Loop Mediated Isothermal Amplification ◽  
Viral Copy Number

H7N9 avian influenza viruses have circulated in the human population and poultry flocks in China since 2013. H7N9 virus monitoring is imperative in Taiwan due to the frequent contact between China and Taiwan. Traditional viral molecular detection methods using RT-PCR and sequencing are time- and labor-intensive, thus a simpler and cheaper method with high sensitivity and specificity is worth developing. We successfully detected human and wild bird H7N9 viruses in this study using reverse transcription loop-mediated isothermal amplification (RT-LAMP) and oligonucleotide microarray. The detection limit was as low as one viral copy number. The specific matching reaction between the templates and microarray probes during hybridization ensured the high detection effectiveness. The excellent sensitivity and specificity of the RT-LAMP-microarray makes it a powerful H7N9 surveillance approach in Taiwan.

scholarly journals From influenza infection to anti-ADAMTS13 autoantibodies via cross-reactivity

2018 ◽  
Vol 7 (4) ◽  
pp. 113-120
Author(s):  
Darja Kanduc
Keyword(s):  
Immune Responses ◽  
Thrombotic Thrombocytopenic Purpura ◽  
Von Willebrand Factor ◽  
Influenza A ◽  
Influenza Infection ◽  
Cross Reactivity ◽  
Influenza Viruses ◽  
Thrombocytopenic Purpura ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Autoantibodies (AAbs) against von Willebrand factor (vWF)-cleaving protease ADAMTS13 causally relate to thrombotic thrombocytopenic purpura (TTP). How anti-ADAMTS13 AAbs are generated is unknown. Starting from reports according to which influenza infection can trigger TTP by the production of ADAMTS13 AAbs, this study explores influenza viruses and ADAMTS13 protein for common peptide sequences that might underlie anti-influenza immune responses able to cross-react with ADAMTS13. Results document that numerous peptides are shared between influenza A and B viruses and ADAMTS13, thus supporting the hypothesis of cross-reactivity as a mechanism driving the generation of anti-ADAMTS13 AAbs.

scholarly journals One-Step Reverse-Transcription Recombinase Polymerase Amplification Using Lateral Flow Strips for the Detection of Coxsackievirus A6

2021 ◽  
Vol 12 ◽  
Author(s):  
Jia Xie ◽  
Xiaohan Yang ◽  
Lei Duan ◽  
Keyi Chen ◽  
Pan Liu ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Clinical Performance ◽  
Quantitative Polymerase Chain Reaction ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Clinical Samples ◽  
Effective Vaccine ◽  
Lateral Flow Strip ◽  
Coxsackievirus A6 ◽  
One Step

Hand, foot, and mouth disease (HFMD) is a common infectious disease affecting mainly children under 5 years of age. Coxsackievirus A6 (CVA-6), a major causative pathogen of HFMD, has caused outbreaks in recent years. Currently, no effective vaccine or antiviral treatments are available. In this study, one-step reverse-transcription recombinase polymerase amplification (RT-RPA), combined with a disposable lateral flow strip (LFS) assay, was developed to detect CVA-6. This assay can be performed in less than 35 min at 37°C without expensive instruments, and the result can be observed directly with the naked eye. The sensitivity of the RT-RPA-LFS was 10 copies per reaction, which was comparable to that of the conventional real-time quantitative polymerase chain reaction (qPCR) assays. Moreover, the assay specificity was 100%. The clinical performance of the RT-RPA-LFS assay was evaluated using 142 clinical samples, and the coincidence rate between RT-RPA-LFS and qPCR was 100%. Therefore, our RT-RPA-LFS assay provides a simple and rapid approach for point-of-care CVA-6 diagnosis.

High-quality RNA improves sensitivity of SARS-CoV-2 detection by colorimetric RT-LAMP

2021 ◽  
pp. 153537022110547
Author(s):  
Marta Puigmulé ◽  
Mònica Coll ◽  
Alexandra Pérez-Serra ◽  
Laura López ◽  
Ferran Picó ◽  
...  
Keyword(s):  
Diagnostic Test ◽  
Quantitative Pcr ◽  
Standard Method ◽  
Reverse Transcription ◽  
Gold Standard ◽  
High Sensitivity ◽  
High Quality ◽  
Gold Standard Method ◽  
Increased Sensitivity ◽  
User Friendly

The global SARS-CoV-2 pandemic requires a rapid, reliable, and user-friendly diagnostic test to help control the spread of the virus. Reverse transcription and quantitative PCR (RT-qPCR) is currently the gold standard method for SARS-CoV-2 detection. Here, we develop a protocol based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) and demonstrate increased sensitivity of this technique using fresh RNA extracts compared to RNA samples subjected to freezing/thawing cycles. We further compare RT-LAMP to RT-qPCR and demonstrate that the RT-LAMP approach has high sensitivity in fresh RNA extracts and can detect positive samples with Ct values between 8 and 35.

scholarly journals Effect of Priming with H1N1 Influenza Viruses of Variable Antigenic Distances on Challenge with 2009 Pandemic H1N1 Virus

2012 ◽  
Vol 86 (16) ◽  
pp. 8625-8633 ◽  
Author(s):  
Christopher D. O'Donnell ◽  
Amber Wright ◽  
Leatrice N. Vogel ◽  
Chih-Jen Wei ◽  
Gary J. Nabel ◽  
...  
Keyword(s):  
Influenza A ◽  
New Caledonia ◽  
Protective Efficacy ◽  
Negative Control ◽  
Cross Reactivity ◽  
Glycosylation Site ◽  
Influenza Viruses ◽  
Influenza B Virus ◽  
Influenza B ◽  
Pandemic H1n1

Compared to seasonal influenza viruses, the 2009 pandemic H1N1 (pH1N1) virus caused greater morbidity and mortality in children and young adults. People over 60 years of age showed a higher prevalence of cross-reactive pH1N1 antibodies, suggesting that they were previously exposed to an influenza virus or vaccine that was antigenically related to the pH1N1 virus. To define the basis for this cross-reactivity, ferrets were infected with H1N1 viruses of variable antigenic distance that circulated during different decades from the 1930s (Alaska/35), 1940s (Fort Monmouth/47), 1950s (Fort Warren/50), and 1990s (New Caledonia/99) and challenged with 2009 pH1N1 virus 6 weeks later. Ferrets primed with the homologous CA/09 or New Jersey/76 (NJ/76) virus served as a positive control, while the negative control was an influenza B virus that should not cross-protect against influenza A virus infection. Significant protection against challenge virus replication in the respiratory tract was observed in ferrets primed with AK/35, FM/47, and NJ/76; FW/50-primed ferrets showed reduced protection, and NC/99-primed ferrets were not protected. The hemagglutinins (HAs) of AK/35, FM/47, and FW/50 differ in the presence of glycosylation sites. We found that the loss of protective efficacy observed with FW/50 was associated with the presence of a specific glycosylation site. Our results suggest that changes in the HA occurred between 1947 and 1950, such that prior infection could no longer protect against 2009 pH1N1 infection. This provides a mechanistic understanding of the nature of serological cross-protection observed in people over 60 years of age during the 2009 H1N1 pandemic.

Sign in / Sign up

Export Citation Format

Share Document