August 20, 2020
Quick assessment of influenza a virus infectivity with a long-range reverse-transcription quantitative polymerase chain reaction assay
Background: polymerase chain reaction (PCR) which is used to detect viral pathogens because of the high sensitivity and specificity. However, the conventional PCR method can not determine the virus infectivity. Virus infectivity is conventionally checked by methods such as plaque test, although the test like this takes a few days. Long-Range quantitative reverse-transcription PCR (RT-qPCR) has previously been suggested for a rapid assessment of RNA virus infectivity which the loss of infectivity due to the fragmentation of the genome.
Methods: IAV is irradiated with ultraviolet rays of 253.7 nm (UV) to induce genomic strand breaks were confirmed by the full-length RT-PCR assay. The IAV is then subjected to the test plaque, conventional RT-qPCR and RT-qPCR long term to examine the relationship between infectious titer and number of copies. A simple linear regression analysis was conducted to test the correlation between the test results.
Results: A remote RT-qPCR assay was developed and validated for influenza A virus (IAV). Although only a few minutes of UV radiation required to completely disable IAV, genomic RNA remains detectable by conventional RT-qPCR and full-length RT-PCR for the following NS inactivation of the viral genome. A long-distance RT-qPCR assay was then designed using RT-priming at the 3 ‘termini of each segment and subsequent genomic qPCR of the 5’ region. UV-mediated inactivation IAV successfully analyzed by RT-qPCR assay distance especially when targeting the PA of the viral genome. It is also supported by a regression analysis that within RT-qPCR is highly correlated with plaque test (adjusted R2 = 0.931, P = 0.000066).
Conclusion: This study shows that IAV infectivity can be predicted without testing infectivity. Rapid detection of pathogenic IAV has, therefore, been achieved with this sensing technology.
The one-step immunoassay without washing steps for the detection of influenza A virus using isfet
A one-step immunoassay for the detection of influenza A virus were developed using two different microbeads and bottle filter-inserted. Two types of beads with a diameter of 15 (capture beads) and 3 (detection beads) um prepared specifically detect the influenza virus A. Anti-influenza A virus antibody coated on both types of beads, while urease was immobilized only on the detection beads.
Samples of influenza A-positive can form a sandwich complex with the capture and detection of beads; This complex will not pass through the filter, which has a controlled pore size. As the beads used at a concentration detection limit, it will be prevented from crossing the filter; thus, the more it will react with the substrate urea and consequently increase the pH. Influenza A-negative samples will fail to form a sandwich complex in the presence of the capture and detection beads.
Thus, the detection beads will pass through the filter into urea buffer and increasing the pH. Changes in pH in the reaction of urease can be quantitatively measured by indicators such as phenol red or using ion-selective field-effect transistors (isfet). one-step immunoassay is used to detect influenza A viruses in real samples.
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Receiver operating characteristics (ROC) plot analysis shows the value of area under the curve (AUC) of 0.931; the sensitivity and specificity of the test was 80% and 90%, respectively, with a cutoff value of 0.9986. These results indicate that one-step immunoassay can increase the sensitivity of detection of influenza A viruses in real samples.