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Rapid and specific detection of H3 swine influenza virus using reverse transcription loop‐mediated isothermal amplification method
Author(s) -
Gu H.,
Qi X.,
Li X.,
Jiang H.,
Wang Y.,
Liu F.,
Lu S.,
Yang Y.,
Liu F.
Publication year - 2010
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2009.04520.x
Subject(s) - loop mediated isothermal amplification , reverse transcription loop mediated isothermal amplification , virology , biology , reverse transcriptase , virus , reverse transcription polymerase chain reaction , real time polymerase chain reaction , detection limit , influenza a virus , microbiology and biotechnology , polymerase chain reaction , gene , chemistry , messenger rna , chromatography , dna , genetics
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.