Ⅰ型鸭肝炎病毒和鸭瘟病毒一步法二重RT-PCR检测方法的建立

Establishment of one-step duplex RT-PCR for the simultaneous detection of duck hepatitis type Ⅰ and duck plague virus

为了建立一种能同时检测I型鸭肝炎病毒（duck hepatitis virustype I，DHV—I）和鸭瘟病毒（duckplague virus，DPV）的检测方法，根据GrenBank中DHV-I和DPV的基因保守序列．设计合成两对特异性引物，扩增得到DHV—I和DPV片段，大小分别为399bp和232bp。据此建立了双重PCR检测方法，并将反应条件进行了优化。结果显示，该方法特异性强，能同时扩增出DHV—I和DPV目的条带，而对其他常见的水禽病病原的扩增结果均为阴性。该方法灵敏度高，最低能检出5．3PgI型鸭肝炎病毒和2．7pg鸭瘟病毒的RNA或DNA。采用该方法对在江苏省徐州地区所采集的166份鸭肛拭子进行检测，DHV—I阳性率为1．2％：DPV阳性率为26％。同时。利用该方法对建立的DHV—I和DPV雏鸭感染模型的肝组织和肝组织接种的鸭胚尿囊液进行检测，结果均为阳性．与常规PCR和RT—PCR检测结果一致。上述结果表明。本研究建立的一步法二重RT—PCR方法可以用于DHV—I和DPV快速准确的诊断。

In order to establish a method for simultaneous detection of duck hepatitis virus type I （DHV-I）and duck plague virus（DPV）,the one-step duplex PCR was established with 2 pairs of specific primers which were designed based on the conserved sequences of DHV- I and DPV, of which one was used for specifically amplification of a 399 bp fragment of DHV-I,and the other for amplification of a 232 bp fragment of DPV. The reaction conditions of the annealing temperature and the primer concentration were optimized. The one-step duplex PCR assay is specific and was able to simultaneously detect DHV- I and DPV and there were not specifically amplified for other common pathogenic for waterfowl diseases. Tem- plates of DHV- I and DPV were detected 5.3pg and 2. Tpg at a minimum which were proved to have high sensi- tivity. Furthermore,the established assay was used to detect the 166 clinical samples of duck anus swabs from Xuzhou,Jiangsu province. The results showed that the positive rates of DHV- I and DPV were 1.2% and 26%. Meanwhile, duck embryo allantoic fluid and liver tissues of DHV- I and DPV in duckling infection models were detected by this method and the results were positive in accord with that of the conventional PCR and RT-PCR results. The above-mentioned results indicated that the one-step duplex RT-PCR assay could be used for rapid and accurate diagnosis of DHV- I and DPV,simultaneously.