摘要
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.
出处
《中国兽医学报》
CAS
CSCD
北大核心
1998年第5期437-440,共4页
Chinese Journal of Veterinary Science
关键词
轮状病毒
夹心ELISA
PT-PCR
检测
rotavirus
reverse transcription polymerase chain reaction
sandwich ELISA