猪星状病毒5型TaqMan荧光定量PCR检测方法的建立

Establishment of a method for TaqMan real-time PCR detection of procine astrovirus type 5

摘要

猪星状病毒5型（PAstV5）是引起仔猪病毒性腹泻的重要病原，常与其他猪肠道病毒混合感染，给全球养猪业造成严重经济损失。为建立特异、快速的PAstV5检测方法，本研究选取实验室分离的PDS2021毒株，通过与NCBI PAstV5全基因组序列比对，针对ORF1a保守区设计引物及TaqMan探针，通过反应条件和反应体系的不断优化，成功建立了针对TaqMan荧光定量PCR（qRT-PCR）检测方法。结果表明：该方法灵敏度达10 copies/μL，较普通PCR高100倍，与猪δ冠状病毒（PDCoV）、猪传染性胃肠炎病毒（TGEV）、猪流行性腹泻病毒（PEDV）等其他常见猪病毒性腹泻病原无交叉反应，特异性良好；变异系数小于2%，重复性良好。对60份临床猪腹泻粪便样品进行检测，其阳性率为25%。综上，本研究建立的qRT-PCR方法为PAstV5感染的流行病学研究和防控提供了高效检测工具。

Abstract

Porcine astrovirus type 5 (PAstV5) is a major causative agent of viral diarrhea in piglets, frequently co-circulating with other porcine enteric viruses and causing substantial economic losses to the global swine industry. To establish a specific and rapid method for detection of PAstV5, the PDS2021 strain isolated in our laboratory was aligned with NCBI PAstV5 reference genomes to design primers and a TaqMan probe targeting the conserved ORF1a region. Through systematic optimization of reaction conditions and components, we successfully developed a TaqMan real-time PCR assay. The validation results showed that the assay achieved a sensitivity of 10 copies/μL, representing a 100-fold improvement over conventional PCR. The method possessed excellent specificity with no cross-reactivity to other common porcine diarrheal pathogens, including porcine delta coronavirus (PDCoV), transmissible gastroenteritis virus (TGEV), and porcine epidemic diarrhea virus (PEDV). Both the intra- and inter-assay coefficients of variation were below 2%, indicating superior reproducibility of the assay. Clinical evaluation of 60 porcine diarrheal fecal samples revealed a 25% positivity rate. This reliable and sensitive detection method may serve as an efficient tool for epidemiological surveillance and control of PAstV5 infections.

关键词

猪星状病毒5型 / TaqMan探针 / 实时荧光定量PCR

Key words

porcine astrovirus type 5 / TaqMan probes / real-time quantitative PCR

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基金资助

河南省重点研发专项(231111113100)