5种常见犬腹泻病毒基因芯片检测方法的建立

为建立一种简便、快速、高效的可同时区分犬腺病毒1型（canine adenovirus type 1，CAV-1）、犬腺病毒2型（canine adenovirus type 2，CAV-2）、犬冠状病毒（canine coronavirus，CCV）、犬瘟热病毒（canine distemper virus，CDV）、犬细小病毒（canine parvovirus，CPV） 5种常见犬腹泻病毒的基因芯片诊断方法，本研究以CAV-1、CAV-2、CCV、CDV、CPV 5种犬腹泻病毒为靶病毒，根据NCBI上收录的病毒基因序列在其保守区域内设计引物，在此基础上根据变异区域设计针对每种病毒的探针2~3条，优化检测体系的各反应条件，确定该检测方法的特异性与敏感性，建立可同时区分5种病毒的基因芯片检测方法。结果显示，建立的基因芯片检测方法可同时检测以上述5种犬腹泻病毒，其中PCR的退火温度为55℃、延伸时间为1 min 15 s；探针与PCR产物的杂交温度40℃、杂交时间2.5 h时，该方法对CAV-1、CAV-2、CCV、CDV和CPV 5种病毒标准品的检测限分别为0.2 fg/μL、2 fg/μL、2 fg/μL、20 pg/μL和0.02 fg/μL，具有较高的灵敏性；同时对犬副流感病毒进行特异性试验，发现无阳性信号出现，具有较强的特异性；对14份临床腹泻样品检测结果显示，基因芯片方法对CAV-1、CAV-2、CCV、CDV和CPV 5种病毒的阳性检出率分别为28.57%、50.00%、64.28%、14.28%和85.71%，并且基因芯片检测方法的敏感性较PCR要高10~100倍。以上结果表明，本研究建立的基因芯片检测方法具有特异、敏感等特点，对临床中犬类混合感染病毒检测具有一定的诊断意义。

Establishment of Gene Chip Detection Method for 5 Common Canine Diarrhea Viruses

This study was aimed to establish a simple, rapid and efficient method for the diagnosis of canine adenovirus type 1 (CAV-1), canine adenovirus type 2 (CAV-2), canine coronavirus (CCV), canine distemper virus (CDV) and canine parvovirus (CPV).5 canine diarrhea viruses CAV-1, CAV-2, CCV, CDV and CPV were used as target viruses.The primers were designed in the conserved region according to the viral gene sequences included in NCBI.Then 2 to 3 probes for each virus were designed according to the variation region.After optimizing the reaction conditions of the detection system and detecting the specificity and sensitivity of the detection method, the gene chip detection method which could simultaneously distinguish 5 viruses was established.The results showed that the chip was able to simultaneously detect the above five kinds of canine diarrhea viruses.The optimized reaction system in this study was that the annealing temperature of PCR was 55℃, the extension time was 1 min 15 s, the hybridization temperature was 40℃, and the hybridization time was 2.5 h.The detection limit of 5 virus standard were as follows:CAV-1 0.2 fg/μL, CAV-2 2 fg/μL, CCV 2 fg/μL, CDV 20 pg/μL and CPV 0.02 fg/μL.The specificity results showed that no positive signals were found for the CPIV.The detection of 14 clinical diarrhea samples showed that the positive detection rates of CAV-1, CAV-2, CCV, CDV and CPV by the gene chip method were 28.57%, 50.00%, 64.28%, 14.28% and 85.71%, respectively, and the sensitivity of the microarray assay was 10 to 100 times higher than that of PCR.These results indicated that the developed method in this study was specific and sensitive, and had diagnostic meaning for the clinical mixed infection of canine viruses.