五种常用轮状病毒检测方法的评价及应用策略

Evaluation on performance of five routine rotavirus detection assays and application strategies

目的比较胶体金免疫层析法（GICA）、酶联免疫吸附试验（ELISA）、反转录一聚合酶链反应（RT—PCR）、实时荧光定量一反转录聚合酶链反应（real—timeRT—PCR）和聚丙烯酰胺凝胶电泳（polyacrylamide gelelectrophoresis，PAGE）5种常用的轮状病毒检测方法的准确性。方法取A组轮状病毒阳性标本，绥：1×10’～1×10。10倍系列稀释，采用上述5种方法平行检测，比较检测限；选择分别含有以下9种病原体且轮状病毒证实为阴性的粪便标本作为阴性对照：脊髓灰质炎病毒疫苗株I～Ⅲ型、肠道病毒EV71、柯萨奇病毒A16、ECHO病毒6、诺如病毒I型、诺如病毒Ⅱ型和星状病毒，采用上述5种方法平行检测，比较特异性；采用上述方法，分别对184例婴幼儿腹泻粪便标本进行检测，以PAGE检测结果作为参考标准，比较其余4种方法的灵敏度和特异性。结果上述5种方法检测A组轮状病毒的检测限相差5个数量级，分别为：real—timeRT—PCR法10^-6稀释度，胶体金法10。稀释度，ELISA和PAGE方法均为1012稀释度，RT．PCR法10。稀释度；5种方法对9种对照病原体均无交叉反应；以PAGE方法检测结果作为标准，real—timeRT—PCR、ELISA、胶体金和RT—PCR灵敏度分别为95．45％、96．97％、93．94％和62．12％，特异性分别为82．2％、86．44％、82．20％和94．07％。结论PAGE作为检测轮状病毒的经典方法，具有高特异性，可用于确认实验；real—timeRT—PCR方法检测A组轮状病毒的灵敏度优于其他4种方法，可用于轮状病毒的高通量检测；ELISA和胶体金方法具有良好的灵敏度与特异性，且胶体金方法成本低，可用于初筛检测；行业标准推荐的RT-PCR方法特异性较高，但灵敏度较低，有待改进。

Objective To evaluate the performance of five routine rotavirus detection assays-gold Immunochromatography assay （GICA）, enzyme-linked immuno sorbent assay （ELISA）, reverse transcriptase polymerase chain reaction （ RT-PCR）, real-time RT PCR and polyacrylamide gel electrophoresis （PAGE）. Methods First, one rotavirus group A positive sample was 10-fold serially diluted from 1 x 101 fold to 1 x 108 fold and tested by above mentioned assays for lower limit of detection （LLD） evaluation. Second, nine sanlples, which were confirmed to be rotavirus negative, of poliovirus vaccine strain type I to 111, enterovirus 71, coxsackie virus A16, ECHO virus 6, norovirus type I , norovirus type 11 and astrovirus were used to evaluate the specificities of the five assays. Third, a total of 184 infantile diarrhea stool samples were collected and tested with the five assays for sensitivity and specificity evaluation. Results The LLDs of the five assays were different from each other by five orders of magnitude, i.e. 10 -6 dilution for real-time RT PCR, 10 -3 dilution for GICA, 10 -2 dilution for ELISA and PAGE and 10 -1 dilution for RT- PCR. The five assays had no cross reactions to the negative control samples. Based on the PAGE detection results of the clinical samples, the sensitivity was 95.45% for real-time RT-PCR, 93.94% for GICA, 96. 97% for ELISA and 62. 12% for RT-PCR, and the specificity was 82. 20% for real-time RT-PCR,86. 44% for GICA,82. 20% for ELISA, 94. 07% for RT-PCR. Conclusion PAGE, the classic assay for rotavirus detection, had the highest specificity and would be the best choice for the laboratory confirmation. Real time RT-PCR showed higher sensitivity and specificity than the other assays and could be used for the high throughout detection of rotavirus group A. GICA and ELISA assay are good screening assays because of their low cost. RT-PCR was proved to be with high specificity but low sensitivity and need improvement.