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首页> 外文期刊>Journal of Clinical Microbiology >Broadly Reactive Nested Reverse Transcription-PCR Using an Internal RNA Standard Control for Detection of Noroviruses in Stool Samples
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Broadly Reactive Nested Reverse Transcription-PCR Using an Internal RNA Standard Control for Detection of Noroviruses in Stool Samples

机译：使用内部RNA标准对照检测粪便样品中诺如病毒的广泛反应性巢式逆转录PCR

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We developed a nested reverse transcription-PCR (nRT-PCR) for the detection of noroviruses in stools, using random primers for RT, the JV12/JV13 primer pair in the first round of nPCR, and a set of nine inner primers for the second, comprising the reverse sequences of primers SR46, SR48, SR50, and SR52, and five novel oligonucleotide sequences (113-1, 113-2, 115-1, 115-2, and 115-3). The specificity of the nRT-PCR was confirmed by testing 61 stools containing enteric viruses other than noroviruses. In comparative assays on either stools or RNA dilutions from two genogroup I and three genogroup II (GII) norovirus-positive samples, nRT-PCR was always at least as sensitive as RT-PCR and Southern hybridization. With some of the samples tested, the increase in sensitivity was 10-fold or higher. For GII viruses, the detectable range of nRT-PCR was estimated to be 8.4 × 104 to 2 RNA viral particles. When used on 85 stools from pediatric patients with acute gastroenteritis negative for viruses by electron microscopy and cell culture, the nRT-PCR detected norovirus in 19 samples (22.3%), while it failed to detect one reference RT-PCR-positive sample containing a Desert Shield strain. Sixteen of the 19 nRT-PCR-positive samples gave concordant results with reference RT-PCR and Southern hybridization, and all with sequence analysis. Partial sequencing of the polymerase region revealed that from January to April 2000 all GII strains except two (Rotterdam- and Leeds-like viruses) formed a tight cluster related to Hawaii virus. The nRT-PCR described could prove suitable for large epidemiological studies and for specialized clinical laboratories performing routine molecular testing.
机译：我们开发了巢式逆转录PCR（nRT-PCR），用于检测粪便中的诺如病毒，使用RT随机引物，在nPCR的第一轮使用JV12 / JV13引物对，第二套使用9个内部引物包括引物SR46，SR48，SR50和SR52的反向序列以及五个新的寡核苷酸序列（113-1、113-2、115-1、115-2和115-3）。通过检测61份含有诺如病毒以外的肠病毒的粪便，证实了nRT-PCR的特异性。在对粪便或来自两个基因组I和三个基因组II（GII）诺如病毒阳性样品的RNA稀释液进行的比较分析中，nRT-PCR始终至少与RT-PCR和Southern杂交一样敏感。对于某些测试样品，灵敏度的提高是10倍或更高。对于GII病毒，nRT-PCR对2个RNA病毒颗粒的可检测范围估计为8.4×10 4 。当通过电子显微镜和细胞培养在85例小儿急性胃肠炎病毒阴性的小儿粪便中使用时，nRT-PCR在19个样品（22.3％）中检测到诺如病毒，而未能检测到一个包含沙漠盾牌应变。在19个nRT-PCR阳性样品中，有16个与参考RT-PCR和Southern杂交以及序列分析均给出了一致的结果。对聚合酶区域的部分测序表明，从2000年1月至2000年4月，除两种（鹿特丹和利兹样病毒）外，所有GII菌株均形成了与夏威夷病毒有关的紧密簇。描述的nRT-PCR可能适合大型流行病学研究以及进行常规分子检测的专业临床实验室。

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