H Gu1, X Qi, X Li, H Jiang, Y Wang, F Liu, S Lu, Y Yang, F Liu. 1. Institute of Virology, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu, China.
Abstract
AIM: The main objective of our study is to develop a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP)-based system for rapid and specific detection of H3 swine influenza virus (SIV). METHODS AND RESULTS: The system, H3 RT-LAMP, contained a set of six novel primers that targeted eight distinct regions of the viral haemagglutinin (HA) gene that are highly conserved among H3 influenza A viruses but not between H3 and other subtypes. H3 RT-LAMP accurately and specifically detected H3 SIV of different isolates from culture and from swine lung samples. The system is at least 10-fold more sensitive than the conventional RT-PCR assay and even comparable to the real-time RT-PCR method, with the detection limit of about one plaque-forming unit per reaction. Of 27 swine lung samples tested, 11 samples were positive in reactions with the RT-LAMP and real-time RT-PCR methods, while only 7 were positive with the conventional RT-PCR assay. Importantly, the assay can be completed within 45 min and is faster than the conventional RT-PCR and real-time RT-PCR approaches. CONCLUSIONS: Our results provide the first direct evidence that RT-LAMP is highly specific and sensitive for detecting H3 SIV. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that LAMP offers a promising alternative tool for rapid, inexpensive and specific diagnosis of influenza virus infection of swine and other animals in frontline settings.
AIM: The main objective of our study is to develop a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP)-based system for rapid and specific detection of H3 swine influenza virus (SIV). METHODS AND RESULTS: The system, H3 RT-LAMP, contained a set of six novel primers that targeted eight distinct regions of the viral haemagglutinin (HA) gene that are highly conserved among H3 influenza A viruses but not between H3 and other subtypes. H3 RT-LAMP accurately and specifically detected H3 SIV of different isolates from culture and from swine lung samples. The system is at least 10-fold more sensitive than the conventional RT-PCR assay and even comparable to the real-time RT-PCR method, with the detection limit of about one plaque-forming unit per reaction. Of 27 swine lung samples tested, 11 samples were positive in reactions with the RT-LAMP and real-time RT-PCR methods, while only 7 were positive with the conventional RT-PCR assay. Importantly, the assay can be completed within 45 min and is faster than the conventional RT-PCR and real-time RT-PCR approaches. CONCLUSIONS: Our results provide the first direct evidence that RT-LAMP is highly specific and sensitive for detecting H3 SIV. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that LAMP offers a promising alternative tool for rapid, inexpensive and specific diagnosis of influenza virus infection of swine and other animals in frontline settings.
Authors: Abhijeet A Bakre; Les P Jones; Hailey K Bennett; Davis E Bobbitt; Ralph A Tripp Journal: J Virol Methods Date: 2020-11-30 Impact factor: 2.014