鹅星状病毒Ⅰ、Ⅱ型和鹅细小病毒多重荧光定量PCR检测方法的建立与应用

Establishment and application of Real-time PCR for the detection of GAstV type I、II and GPV

关键词:  鹅星状病毒 Ⅰ 型  鹅星状病毒 Ⅱ 型  鹅细小病毒  多重荧光定量PCR

Key words:  GAstV Ⅰ, GAstV Ⅱ, GPV, Multiple Realtime PCR

摘要:

为建立一种可快速、特异检测鹅星状病毒Ⅰ型(GAstVⅠ)、Ⅱ型(GAstVⅡ)及鹅细小病毒(GPV)多重荧光定量PCR方法，根据GAstVⅠ型、GAstVⅡ型及GPV保守基因序列设计合成了特异性引物及三种荧光基团TaqMan探针，分别构建了带有三种病原靶基因的重组质粒标准品。通过筛选引物，优化反应条件，分析了该方法的特异性、敏感性及重复性，并进行临床样品检测评价。结果显示：该方法特异性强，能够特异检测GAstVⅠ型、GAstVⅡ型与GPV，且与其他常见鹅病病原无交叉反应；敏感性高，对GAstVⅠ型、GAstVⅡ型及GPV对相应的标准质粒最低检出限均为10拷贝/μL；重复性好，重复性试验批内及批间变异系数均小于3%；该方法对90份临床样品检测结果与已发表的三种病毒单重荧光定量PCR方法的检测结果进行比较，其中GAstVⅠ型符合率为98.89%，GAstVⅡ型符合率为96.67%，GPV符合率为96.67%。研究建立的多重荧光定量PCR检测方法能够快速、特异、灵敏的检测GAstVⅠ型、GAstVⅡ型及GPV，实现一管检多病，实用性强，效率更高，为GAstVⅠ、Ⅱ型和GPV的监测和防控提供了高效技术手段。

Abstract:

To establish a rapid and specific multiplex real-time PCR method for the detection of Goose Astrovirus TypeⅠ(GAstVⅠ),TypeⅡ(GAstVⅡ), and Goose Parvovirus (GPV), this study designed and synthesized specific primers and TaqMan probes with three different fluorescent labels based on the conserved gene sequences of GAstVⅠ,GAstVⅡ, and GPV. Recombinant plasmid standards containing the target genes of the three viruses were constructed. By screening primers, optimizing reaction conditions, and analyzing the specificity, sensitivity, and repeatability of the method, as well as the effect by detecting clinical samples, a multiplex real-time PCR detection method for GAstVⅠ,GAstVⅡ, and GPV was established. The results showed that this method has strong specificity, capable of specifically detecting GAstVⅠ,GAstVⅡ, and GPV without cross-reactivity with other common goose pathogens; high sensitivity, with a minimum detection limit of 10 copies/μL for the standard plasmids of GAstVⅠ, GAstVⅡ, and GPV; and good repeatability, with intra- and inter-assay coefficients of variation both less than 3%. When the detection results of 90 clinical samples using this method were compared with those obtained by previously published single real-time PCR methods for the three viruses. Among them, the coincidence rate of GAstVⅠ, GAstVⅡ and GPV was 98.89%、96.67% and 96.67%, respectively. The multiplex real-time PCR detection method established in this study can rapidly, specifically, and sensitively detect GAstVⅠ, GAstV Ⅱ, and GPV, enabling the detection of multiple pathogens in a single tube. It is highly practical and efficient, providing an effective technique for the monitoring and control of GAstVⅠ,GAstV Ⅱ, and GPV.