H5亚型禽流感病毒现场实时荧光RT-LAMP检测方法点的建立与优化

Establish and Optimization of Real-Time Fluorescent Reverse Transcription Loop-Mediated Isothermal Amplification for Detection of Avian Influenza H5 Hemagglutinin Gene

H5亚型禽流感是重要的人兽共患病,建立现场快速分子学检测方法是其防控关键。本实验首次借助热裂解法抽提病毒RNA,建立H5亚型禽流感病毒(H5subtype avian influenza Virus,AIV-H5)实时荧光反转录环介导等温扩增(Fluorescent quantitative real-time reversetranscription-LAMP,qRT-LAMP)检测法,并对该法进行特异性、敏感性、可重复性及可靠性分析;同时对比钙黄绿素(Calcein)、SYBR Green I、羟基萘酚蓝(Hydroxynaphthol blue,HNB)、SYTO 81四种荧光染色剂在颜色判定中的工作情况,筛选最佳染料。试验结果显示本实验所建AIV-H5qRT-LAMP法可有效鉴别AIV-H5,其敏感性较国家标准实时荧光RT-PCR检测法高100倍,最低检测限可达1个基因拷贝;该法与IBV、NDV等常见禽呼吸道传染病病原无交叉反应,特异性强;同时该法具有良好重复性。本实验筛选出最佳染色剂Calcein。小规模田间试验显示该法用于AIV-H5的普查监测结果可靠。可见本实验建立AIV-H5qRT-LAMP检测法可满足AIV-H5现场快速分子检测需求,具广阔应用空间。

H5 subtype avian influenza （AIV-HS） is a major causative agent of animalloimia a rapid and sen- sitive molecular biological diagnosis is crucial to the control program of AIV-HS. AIV-H5 real-time fluo- rescent reverse transcription loop-mediated isothermal amplification （qRT-LAMP） was established by means of heat treatment of the samples. The sensitivity, specificity and repeatability of this method were assessed and the performance of Calcein, SYBR Green I, H NB, SYTO 81 in colorimetric detection was com- paratively analyzed to screen the optimum dye. The results showed the sensitivity of this method was 100 times higher than that of standard real-time fluorescent RT-PCR, and the detection limit was one copy of the gene per reaction. This method had no cross-reactivity with other common avian respiratory tract infec tious disease-related pathogens such as IBV and NDV. The present study suggested Calcein was the opti- mum dye. Small-scale tests suggested this method was reliable for survey monitoring of AIV-H5 on the spot, indicating its potential applications in field investigation.