鹅细小病毒PCR-DHPLC检测方法的建立

Development and Preliminary Application of PCR-DHPLC for Goose Parvovirus Detection

为建立一种敏感、自动化,高通量的检测病料中鹅细小病毒(GPV)的PCR-DHPLC检测方法,本研究首先根据GenBank上GPV标准毒株VP3基因序列设计一对引物,建立检测GPV的PCR方法,然后将PCR检测方法与DHPLC法进行有机结合,建立了GPV PCR-DHPLC检测方法。以GPV、鹅副粘病毒、鸭瘟病毒及正常番鸭胚尿囊液进行特异性试验,结果无交叉反应,表明该检测方法具有较高的特异性。对GPV阳性样品检测结果表明该方法的敏感性较高,是常规PCR电泳法的100倍,且具有较好的重复性。对临床上采集的100份疑似病料,分别应用PCR电泳法和本试验建立的PCR-DHPLC方法进行检测,结果阳性符合率为100%。表明该方法具有特异、敏感、快速、重复性好、可高通量检测等优点,可用于临床样品的大批量检测。

In order to establish a sensitive, automated, high-throughput PCR-DHPLC detection method for GPV of pathological tissue, a pair of primers, based on VP3 gene sequence of GPV standard strains in GenBank, was first designed in this study to establish the PCR method. Then, this PCR was combined with DHPLC to establish a new detection method （PCR-DHPLC） for the detection of GPV, after which, specificity tests were conducted on GPV, Goose paramyxovirus, DPV and normal duck embryo allantoic fluid. No cross-reactivity was observed, suggesting that this method had high specificity. Another test on the positive samples of GPV showed that this method not only had a very high sensitivity, 100 times more sensitive than the conventional PCR electrophoresis method, but also had a very good reproducibility. 100 suspected diseased parts of clinical materials were detected by applying electrophoresis PCR and then the established PCR-DHPLC test method. The results of both methods were positive, displaying 100 % coinci- dence rate. All the results mentioned above showed that this established PCR-DHPLC method is quite spe- cific, sensitive, rapid, reproducible and high-throughput in detecting GPV. It is appropriate for high-vol- ume clinical detection.