Reverse-transcription, loop-mediated isothermal amplification assay for the sensitive and rapid detection of H10 subtype
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RESEARCH
Open Access
Reverse-transcription, loop-mediated isothermal amplification assay for the sensitive and rapid detection of H10 subtype avian influenza viruses Sisi Luo1, Zhixun Xie1*, Liji Xie1, Jiabo Liu1, Zhiqin Xie1, Xianwen Deng1, Li Huang1, Jiaoling Huang1, Tingting Zeng1 and Mazhar I. Khan2
Abstract Background: The H10 subtype avian influenza viruses (H10N4, H10N5 and H10N7) have been reported to cause disease in mammals, and the first human case of H10N8 subtype avian influenza virus was reported in 2013. Recently, H10 subtype avian influenza viruses (AIVs) have been followed more closely, but routine diagnostic tests are tedious, less sensitive and time consuming, rapid molecular detection assays for H10 AIVs are not available. Methods: Based on conserved sequences within the HA gene of the H10 subtype AIVs, specific primer sets of H10 subtype of AIVs were designed and assay reaction conditions were optimized. A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was established for the rapid detection of H10 subtype AIVs. The specificity was validated using multiple subtypes of AIVs and other avian respiratory pathogens, and the limit of detection (LOD) was tested using concentration gradient of in vitro-transcribed RNA. Results: The established assay was performed in a water bath at 63 °C for 40 min, and the amplification result was visualized directly as well as under daylight reflections. The H10-RT-LAMP assay can specifically amplify H10 subtype AIVs and has no cross-reactivity with other subtypes AIVs or avian pathogens. The LOD of the H10-RT-LAMP assay was 10 copies per μL of in vitro-transcribed RNA. Conclusions: The RT-LAMP method reported here is demonstrated to be a potentially valuable means for the detection of H10 subtype AIV and rapid clinical diagnosis, being fast, simple, and low in cost. Consequently, it will be a very useful screening assay for the surveillance of H10 subtype AIVs in underequipped laboratories as well as in field conditions.
Introduction The influenza A viruses belong to the family Orthomyxoviridae and are classified into 18 hemagglutinin (HA) and 11 neuraminidase (NA) subtypes based on their antigenic properties [1, 2]. Avian influenza viruses (AIVs) can be divided into two distinct groups based on their virulence: highly pathogenic avian influenza viruses (HPAIVs) and low pathogenic avian influenza viruses (LPAIVs). HPAIVs generally cause high morbidity and mortality in poultry flock, even directly infect human or cause death such as * Correspondence: [email protected] 1 Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, Guangxi 530001, P.R. China Full list of author information is available at the end of the article
H5N1 in Hong Kong in 1997 [3], but the H7N9 of HPAIVs broke out and infected human was low pathogenic in poultry in 2013 [4]; LPAIVs such as H9N2 cause mild or no symptoms in poultry, wild birds and human, and most AIV subtypes are LPAIVs [5, 6]. Most H10 subtype
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