研究旨在建立实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,qPCR)检测猫疱疹病毒Ⅰ型(Feline herpesvirus type Ⅰ,FHV-1)的方法,用于检测临床上猫的上呼吸道传染病样本.据GenBank已发表的猫疱疹病毒的gE基因序列的保守区域设计并合成一对特异性引物及探针,建立FHV-1的实时荧光定量PCR检测方法,对此方法进行特异性、灵敏度及重复性的验证,并对广东佛山某流浪猫基地猫疱疹病毒的流行情况进行调查,即对该基地的94份临床样品进行检测.结果表明:研究成功建立了 FHV-1实时荧光定量聚合酶链式反应检测方法,此方法特异性强,与大多流行的犬猫病毒均未出现交叉反应;敏感性高,最低检出限为10 copies·μL-1;重复性好,批内变异系数为0.29％～0.71％,批间变异系数为0.88％～1.38％.应用此方法在94份临床样品中检出28份FHV-1核酸阳性样品.综上所述,研究建立的FHV-1荧光定量聚合酶链式反应方法具有较好的特异性、敏感性和重复性,为临床上FHV-1的快速检测提供了可行的解决方案.
Establishment of a fluorescence quantitative polymerase chain reaction detection method for Feline Herpesvirus-1
The aim of this study was to establish a real-time fluorescence quantitative polymerase chain reaction(qPCR)method for the detection of Fe-line herpesvirus type Ⅰ(FHV-1)in cats with upper respiratory tract infectious diseases.A set of specific primers and probe were designed and synthesized according to the conserved region of the gE gene sequence of feline herpesvirus published by GenBank,and a real-time fluorescence quantitative PCR method of FHV-1 was established.The specificity,sensitivity and repeatability of the method were verified,and the epidemic situation of feline herpesvirus in a stray cat base in Foshan,Guangdong Province was investigated.94 clinical samples of cats from the base were tested using the method above.The re-sults showed that the real-time fluorescence quantitative PCR method for FHV-1 detection with high specificity was successfully established in this study.There was no cross-reaction with most popular canine and cat viruses.The minimum detection limit was 10 copies·μ L-1.The coefficient of variation in batch was 0.29％～0.71％,which was 0.88％～1.38％between batches.28 samples were detected positive for FHV-1 within 94 feline clinical samples us-ing the method established in this study.In conclusion,the real-time fluorescence quantitative PCR method for FHV-1 established in this study shows ro-bust specificity,sensitivity and repeatability,which provides a possible solution for the rapid detection of FHV-1 in clinical.
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