Simultaneous Detection and Genotyping of “Norwalk-Like Viruses” by Oligonucleotide Array in a Reverse Line Blot Hybridization Format
Simultaneous Detection and Genotyping of “Norwalk-Like Viruses” by Oligonucleotide Array in a Reverse Line Blot Hybridization Format
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通过寡核苷酸阵列以反向线印迹杂交形式同时检测“诺瓦克样病毒”并进行基因分型
DOI：
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发表时间：
2000
影响因子：
9.4
通讯作者：
M. Koopmans
中科院分区：
文献类型：
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作者：
J. Vinjé;M. Koopmans
“诺瓦克样病毒”（NLVs）是世界范围内非细菌性胃肠炎爆发的最常见原因。迄今为止，最广泛用于NLV菌株分型的方法是测序和随后的反转录(RT)-PCR产物的系统发育分析，这已经揭示了稳定的不同谱系（基因型）的存在。这种分型方法相当昂贵，不常用于临床实验室，也不太适合分析大量样本。因此，我们开发了一种快速简便的nlv基因分型方法。该方法被称为逆行杂交，是基于RNA聚合酶基因区域的核苷酸差异，可用于将nlv分类为基因型。通过RT-PCR扩增NLV RNA，并与18种不同的膜结合寡核苷酸杂交，这些寡核苷酸能够区分13种NLV基因型。系统发育分析证实，将该方法应用于6年（1994 - 1999年）期间收集的34例暴发和20例散发性胃肠炎病例的132份阳性粪便样本，成功地对124份样本（94%）进行了基因分型。来自三次暴发的其余8株（6%）的核苷酸序列未与已知的NLV基因型聚集。对这些菌株的完整和部分开放阅读框2（衣壳基因）序列进行系统发育分析，发现存在一个新的基因型（Alphatron）和一个潜在的新基因型（Amsterdam）。这种新方法可以同时检测nlv并对其进行基因分型，可用于从暴发中获得的nlv的诊断和分型以及大规模流行病学研究。
ABSTRACT “Norwalk-like viruses” (NLVs) are the most common cause of outbreaks of nonbacterial gastroenteritis worldwide. To date, the method most widely used for typing of NLV strains is sequencing and subsequent phylogenetic analysis of reverse transcription (RT)-PCR products, which has revealed the existence of stable distinct lineages (genotypes). This typing method is rather costly, not routinely used in clinical laboratories, and not very suitable for the analysis of large numbers of samples. Therefore, we have developed a rapid and simple method for genotyping of NLVs. The method, designated reverse line blot hybridization, is based on the nucleotide divergence of a region of the gene for RNA polymerase which can be used to classify NLVs into genotypes. NLV RNA was amplified by RT-PCR and then hybridized to 18 different membrane-bound oligonucleotides that were able to discriminate among 13 NLV genotypes. Application of the method to a panel of 132 positive stool samples from 34 outbreaks and 20 sporadic cases of gastroenteritis collected in a 6-year period (1994 to 1999) resulted in successful genotyping of 124 samples (94%), as confirmed by phylogenetic analysis. The nucleotide sequences of the remaning eight strains (6%) from three outbreaks did not cluster with the known NLV genotypes. Phylogenetic analysis of the complete and partial open reading frame 2 (capsid gene) sequences of these strains revealed the existence of one novel genotype (Alphatron) and one potentially novel genotype (Amsterdam). This novel method, which allows simultaneous detection and genotyping of NLVs, is useful in the diagnosis and typing of NLVs obtained from outbreaks and in large-scale epidemiological studies.
影响因子：
3.7
作者：
JIANG, X;WANG, M;ESTES, MK
通讯作者：
ESTES, MK