逆转录-聚合酶链反应(RT-PCR)检测轮状病毒

Detection of Rotavirus by Reverse Transcription polymerase Chain Reaction

为了给轮状病毒感染的诊断和流行病学研究提供更为敏感和可靠的手段，选取Ａ组轮状病毒ＶＰ７基因上的２段高度保守序列作为引物，在优化逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）条件的基础上，建立了检测轮状病毒的ＲＴ－ＰＣＲ方法。通过对比试验，确定了ＰＣＲ的最优模式：９４℃变性１ｍｉｎ→５５℃退火１ｍｉｎ→７２℃延伸２ｍｉｎ，３０个循环后再在７２℃下延伸１０ｍｉｎ。用此模式进行了ＲＴ－ＰＣＲ的特异性和敏感性试验。检测的６株轮状病毒分离株（牛ＨＮ－７、ＢＲＶ００７、ＢＲＶ０１４、ＢＲＶ６５５５、猪Ｌｉ９９、Ｎａｎ８６）及２株参考株（牛ＮＣＤＶ、猴ＳＡ１１），都能扩增出唯一的３４２ｂｐ的目的条带；对猪流行性腹泻病毒（ＰＥＤＶ）及传染性胃肠炎病毒（ＴＧＥＶ）感染猪的粪样、正常ＭＡ１０４细胞检测结果均呈阴性；检测的敏感度可达１ｐｇ水平。对４０份猪、牛、兔的腹泻粪样检测，３０份呈阳性，而用作平行对照的夹心ＥＬＩＳＡ法检测，有２５份呈阳性，两者符合率为８７．５％。两法检测不符的５份粪样的ＰＣＲ扩增产物，用地高辛标记探针进行了斑点杂交，结果均呈阳性，表明ＲＴ－ＰＣＲ法比ＥＬＩＳＡ法敏感性高。

Two oligonucleotide primers appropriate for PCR amplication were selected based on the two conserved regions of VP7 genome of group A rotavirus. A reserve transcription polymerase chain reaction (RT PCR) assay was developed for detection of the rotavirus after the reaction conditions being optimized. The best model of PCR was obtained as follows: PCR was conducted for 30 cycles (94℃ 1 min denature, 55℃ 1 min annealing, 72℃ 2 min extension) and 72℃ 10 min final extension. Eight rotavirus strains including six isolates of bovine HN 7, BRV007, BRV014, BRV6555, porcine Li99 and Nan86 as well as two reference strains (bovine NCDV and simian SA11) were subjected to RT PCR. A single band of the PCR product of the expected size (342 bp) from each strain was visible on 2% agarose gel stained with ethidium bromide. Using the same pairs, no PCR product was detected from normal MA104 cell culture and from the faecal samples of pigs which were infected with transimissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV). The PCR could detect 1 pg of rotavirus RNA. The sandwich ELISA was also evaluated on the same faecal samples. Of 40 samples, 30 were positive detected by RT PCR, meanwhile only 25 were positive detected by sandwich ELISA with the corresponding rate of the two methods being 87.5%. Furthermore, 5 samples which were positive for detection of RT PCR, but negative of sandwich ELISA. The result showed that the RT PCR products of the 5 samples were positive for detection by dot hybridization with Dig labelled probe suggesting that the RT PCR is more sensitive than sandwich ELISA and quite useful for the detection of rotavirus and the epidemiological survey.