犬瘟热病毒反转录-聚合酶链反应诊断方法的建立和初步应用

ESTABLISHMENT AND APPLICATION OF REVERSE- TRANSCRIPTION POLYMERASE CHAIN REACTION FOR DIAGNOSIS OF CANINE DISTEMPER VIRUS

根据Ｂａｒｅｔ报道的犬瘟热病毒Ｏｎｄｅｒｓｔｅｐｏｏｒｔ弱毒株的融合蛋白基因序列，设计合成了一对能扩增３２４ｂｐ基因片段的引物。将异硫氰酸胍－酚－仿一步法提取得到的细胞总ＲＮＡ进行反转录，以此产物为模板进行ＰＣＲ扩增，并对ＰＣＲ扩增条件进行了优化。经鉴定，以此对引物进行的ＰＣＲ扩增，得到了与设计片段大小和酶切位点相同的产物，且不扩增犬细小病毒、犬腺病毒和狂犬病病毒犬的三种病原的核酸，这表明此方法为特异性的。对２７条（只）临床病例和３２份细胞培养物进行检查，并与电子显微镜负染检查结果进行了比较。初步应用研究的结果表明，此方法可用于犬瘟热病毒的临床诊断和实验研究。

One pair of primers which may amplify 324 bp fragment had been designed and synthesized based on the fusion(F) protein gene of the Onderstepoort strain of canine distemper virus(CDV). The total RNA were extracted from CDV infected Vero cells or the tissues of infected animals by single step method of acid guanidinium thiocyanate-phenol-chloroform extraction and was used for reverse transcription. About 320 bp cDNA fragment had been amplified from product of RT-PCR. The conditions for PCR had been optimized. The length of cDNA fragment and restriction enzyme sites were examined as same as that of the CDV Onderstepoort strain. Not any gene fragments had been amplified from the cells that were infected by canine parvovirus, canine adenovirus or rabies virus respectively. 27 animals and 32 cells cultures had been tested by PCR. The results were compared with electron microscopy and they were greatly coincident. It showed that this method may be used in clinical diagnosis and laboratory research.