中国兽医杂志2025，Vol.61Issue(5)：58-64,7.DOI:10.20157/j.cnki.zgsyzz.2025.05.007
FHV-1和FCV双重TaqMan实时荧光定量PCR方法的建立和应用
Establishment and Application of a Duplex TaqMan Real-Time Fluorescence Quantitative PCR Assay for FHV-1 and FCV
摘要
Abstract
To develop a rapid,convenient,and accurate diagnostic method for detecting feline herpesvirus-1(FHV-1)and feline calicivirus(FCV),a duplex TaqMan real-time quantitative PCR assay was established.Primers and probes were designed based on the glycoprotein C(GC)gene of FHV-1(GenBank accession number OR504662),the capsid viral protein 1(VP1)gene of FCV(GenBank accession number OP904215),and an exogenous feline internal control sequence.Recombinant plasmid standards(pMD19-T-G,pMD19-T-P,and pMD19-T-C)were constructed and used to generate standard curves to evaluate the assay's specificity,sensitivity,and reproducibility.The method was validated using 33 clinical samples and applied to 292 samples of ocular,oral,and nasal secretions collected from stray cats in Taizhou.The results demonstrated excellent linearity,no cross-reactivity with other pathogens,and a limit of detection of 1×101 copies/µL for both FHV-1 and FCV.The intra-and inter-assay coefficients of variation were all below 2％.Clinical validation showed a 100％positive agreement for FHV-1 and FCV,with total agreement rates of 93.9％for FHV-1 and 90.9％for FCV.Among the 292 stray cat samples,the positive rates were 20.9％for FHV-1,46.2％for FCV,and 12.0％for co-infection.In conclusion,the established duplex TaqMan qPCR assay provides a highly specific,sensitive,and reproducible tool for the differential diagnosis of feline respiratory pathogens and supports pet disease surveillance and clinical diagnostics.关键词
猫疱疹病毒1型(FHV-1)/猫杯状病毒(FCV)/实时荧光定量PCRKey words
feline herpesvirus-1(FHV-1)/feline calicivirus(FCV)/real-time fluorescence quantitative PCR分类
畜牧业引用本文复制引用
静争,金红岩,王春艳,王文杰,沈佳宇,范志坚,侯绍华..FHV-1和FCV双重TaqMan实时荧光定量PCR方法的建立和应用[J].中国兽医杂志,2025,61(5):58-64,7.基金项目
江苏农牧科技职业学院校级自然科学基金储备项目(NSF2023CB14,NSF2024CB06) （NSF2023CB14,NSF2024CB06）
