病毒样颗粒为阳性对照的H5N1实时荧光定量PCR检测方法的建立

Establishment of H5N1 Real-time Fluorescence Quantitative PCR Detection Method Using Virus-like Particles as the Positive Control

合适的标准品对实时荧光定量PCR(qPCR)检测高致病性H5N1禽流感病毒(avain influenza virus,AIV)十分重要.本研究将H5N1AIV HA基因的部分序列插入到能够表达MS2噬菌体病毒样颗粒(virus-like particle,VLP)DNA序列的表达载体上,诱导表达后得到了包裹有H5N1AIV HA基因RNA片段的VLP.该VLP能够耐受核酶的消化,形态与MS2噬菌体病毒颗粒形态相同.利用表达的VLP作为阳性标准品及设计的特异性荧光探针、淬灭链,使用优化的qPCR反应体系,得到qPCR检测H5N1亚型AIV的阳性对照标准曲线.研究结果为高致病性H5N1亚型AIV的准确定量检测提供了基础.

Appropriate standards are very important in detecting highly pathogenic H5N1 subtype avain influenza virus （AIV） by real time fluorescent quantitative PCR （qPCR）.In this study,part of HA gene sequence of the H5N1 AIV was inserted into the expression vector with the expression of MS2 bacteriophage virus like particles （VLP）.After that,the derivative HA VLP products were obtained,containing the RNA of parts of HA gene sequences by isopropyl β-D-1-thiogalactopyranoside induced expression.The HAVLP was able to tolerate nuclear enzyme digestion,of which morphological structure was the same as VLP.Using the expressed HAVLP as a positive control,the specific fluorescent probes and quenching were designed,and the positive control standard curve of qPCR detection of H5N1 subtype AIV was described through the optimum qPCR reaction system.The results laid a basic means for the quantitative detection of the highly pathogenic H5N1 subtype AIV.