Abstract
Avian influenza virus (AIV) outbreaks occur frequently worldwide, causing a potential public health risk and great economic losses to poultry industries. Considering the high mutation rate and frequent genetic reassortment between segments in the genome of AIVs, emerging new strains are a real threat that may infect and spread through the human population, causing a pandemic. Therefore, rapid AIV diagnostic tests are essential tools for surveillance and assessing virus spreading. Real-time reverse transcription PCR (rRT-PCR), targeting the matrix gene, is the main official standard test for AIV detection, but the method requires well-equipped laboratories. Reverse transcription Loop-Mediated Isothermal Amplification (RT-LAMP) has been reported as a rapid method and an alternative to PCR in pathogen detection. The high mutation rate in the AIV genome increases the risk of false negative in nucleic acid amplification methods for detection, such as PCR and LAMP, due to possible mismatched priming. In this study, we analyzed 800 matrix gene sequences of newly isolated AIV in the EU and designed a highly efficient LAMP primer set that covers all AIV subtypes. The designed LAMP primer set was optimized in real-time RT-LAMP (rRT-LAMP) assay. The rRT-LAMP assay detected AIV samples belonging to nine various subtypes with the specificity and sensitivity comparable to the official standard rRT-PCR assay. Further, a two-color visual detection RT-LAMP assay protocol was adapted with the aim to develop on-site diagnostic tests. The on-site testing successfully detected spiked AIV in birds oropharyngeal and cloacal swabs samples at a concentration as low as 100.8 EID50 per reaction within 30 minutes including sample preparation. The results revealed a potential of this newly developed rRT-LAMP assay to detect AIV in complex samples using a simple heat treatment step without the need for RNA extraction.
Highlights
Influenza viruses have a negative-sense single-stranded RNA genome, and are classified in four genera, Alphainfluenzavirus, Betainfluenzavirus, Deltainfluenzavirus, and Gammainfluenzavirus under the Orthomyxoviridae family
Ten reference inactivated Avian influenza virus (AIV) strains and three non-Influenza avian virus strains (Table 1) as well as 14 AIV samples isolated from wild birds in Denmark in 2018 were used to evaluate the newly developed Reverse transcription Loop-Mediated Isothermal Amplification (RT-Loop-Mediated Isothermal Amplification (LAMP)) method
The whole Matrix gene sequences of 800 AIV strains acquired from Global Initiative on Sharing All Influenza Data (GISAID) were aligned with the UGENE program to find the sequence of relatively conserved regions on recently circulating AIVs
Summary
Influenza viruses have a negative-sense single-stranded RNA genome, and are classified in four genera, Alphainfluenzavirus, Betainfluenzavirus, Deltainfluenzavirus, and Gammainfluenzavirus under the Orthomyxoviridae family. AIV surveillance including early and accurate detection in wild birds and poultry farms is a critical measure to control the spreading of the virus and to reduce the risk of emerging new strains Different methods such as cultivating in embryonated chicken eggs, serological based assays, as well as nucleic acid amplification techniques have been developed for AIV detection. LAMP has been reported to be more resistant to inhibitors that ease the sample preparation steps (Francois et al, 2011) Having several advantages such as being simple to operate, rapid, resistant to inhibitors, cost efficient, detectable results, and high sensitivity and specificity, LAMP is an ideal nucleic acid amplification technique to be adapted for on-site, rapid, diagnostic assays. The newly designed primer set was adapted into a commercial LAMP test for on-site testing applicable to perform a rapid and accurate AIV detection
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