Reverse-transcription, loop-mediated isothermal amplification assay for the sensitive and rapid detection of H10 subtype avian influenza viruses.

Sisi Luo,Tingting Zeng,Mazhar I Khan,Zhiqin Xie,Jiabo Liu,Zhixun Xie,Li Huang,Jiaoling Huang,Liji Xie,Xianwen Deng

doi:10.1186/s12985-015-0378-1

Sisi Luo, Tingting Zeng + Show 8 more

Open Access

https://doi.org/10.1186/s12985-015-0378-1

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Abstract

BackgroundThe 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.MethodsBased 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.ResultsThe 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.ConclusionsThe 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.

Highlights

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]
We developed an effective reverse-transcription loop-mediated isothermal amplification (RT-loop-mediated isothermal amplification (LAMP)) assay with calcein and MnCl2 [25], to visual detect the H10 subtype avian influenza viruses (AIVs), which might be suitable in the surveillance of the H10 subtype of AIVs for the rapid detection of H10 subtype AIVs in poultry and wild birds
Specificity and sensitivity of the H10-RT-LAMP assay As expected, the turbidity value of the H10 subtype AIVs gradually increased at 20 min

Summary

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]. 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]. H10N8 was reported to cause human disease for the first time in December 2013 following the emergence of the H7N9 and H5N1 subtype AIVs that cause serious disease in humans and have become a threat to public health [7, 8]. 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. 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

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