Establishment of two assays based on reverse transcription recombinase-aided amplification technology for rapid detection of H5 subtype avian influenza virus

Author:

Li Yang1ORCID,Shang Jiajing12ORCID,Wang Yixin3,Luo Juan12,Jiang Wenming1,Yin Xin1,Zhang Fuyou1,Deng Chunran12,Yu Xiaohui1,Liu HuaLei1ORCID

Affiliation:

1. China Animal Health and Epidemiology Center , Qingdao, China

2. School of Life Science and Food Engineering, Hebei University of Engineering , Hebei, China

3. College of Veterinary Medicine, Shandong Agricultural University , Shandong, China

Abstract

ABSTRACT The avian influenza virus (AIV), a member of the type A influenza virus group, is harbored by avian hosts. The highly pathogenic H5 subtype of AIV has inflicted significant financial damage on the poultry industry and also presents a possible pandemic hazard to the worldwide human population. There is an urgent need for a detection method that is both rapid and accurate in identifying H5-AIV. In this study, two assays based on reverse transcription recombinase-aided amplification technology (RT-RAA) have been developed to detect H5-AIV, namely, real-time fluorescence and reverse transcription recombinase-aided amplification (RF-RT-RAA) and reverse transcription recombinase-aided amplification combined lateral flow dipstick (RT-RAA-LFD). The completion of the two methods can be achieved in 30 min, and there was no cross-reaction with the nucleic acid of other avian pathogens. The results indicated that the lowest detectable limit of RF-RT-RAA and RT-RAA-LFD for H5-AIV detection was 1 copy/μL, which was 100 times higher than that of RT-PCR (10 2 copies/μL). A total of 376 samples, consisting of 350 clinical and 26 experimental samples, underwent testing utilizing virus isolation, RF-RT-RAA and RT-RAA-LFD methods, respectively. The results demonstrated a high degree of consistency between those assays. In summary, both the RF-RT-RAA and RT-RAA-LFD methods demonstrated excellent specificity and sensitivity, making them ideal for use in both laboratory and field settings. IMPORTANCE Avian influenza virus (AIV) subtype H5 is a highly contagious zoonotic disease and a serious threat to the farming industry and public health. Traditional detection methods, including virus isolation and real-time PCR, require tertiary biological laboratories and are time-consuming and complex to perform, making it difficult to rapidly diagnose H5 subtype avian influenza viruses. In this study, we successfully developed two methods, namely, RF-RT-RAA and RT-RAA-LFD, for rapid detection of H5-AIV. The assays are characterized by their high specificity, sensitivity, and user-friendliness. Moreover, the results of the reaction can be visually assessed, which are suitable for both laboratory testing and grassroots farm screening for H5-AIV.

Funder

National key Research and Program

Publisher

American Society for Microbiology

Subject

Infectious Diseases,Cell Biology,Microbiology (medical),Genetics,General Immunology and Microbiology,Ecology,Physiology

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