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首页> 外文期刊>Journal of Clinical Microbiology >Molecular Identification and Typing of Enteroviruses Isolated from Clinical Specimens
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Molecular Identification and Typing of Enteroviruses Isolated from Clinical Specimens

机译：从临床标本中分离出肠病毒的分子鉴定和分型

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Enterovirus characterization and typing require an integrated technological approach, using both immunological and molecular methods. The seventy-nine enteroviruses included in this study were isolated from cell cultures and classified as enteroviruses on the basis of an indirect immunofluorescence assay (IFA) against common enterovirus antigens and a neutralization test based on the Lim Benyesh-Melnick (LBM) pool. The final identification was carried out using a number of different molecular approaches, including reverse transcription (RT)-PCR, restriction fragment length polymorphism (RFLP) analysis, and nucleotide sequence analysis of amplicons from various regions of the genome. Twenty-seven poliovirus strains (set A) were identified using LBM pool analysis, RFLP analysis, and IFA. Use of the LBM pool method showed that 35 out of 79 strains were nonpoliovirus (set B), while 17 specimens tested negative (set C). Sets B and C were further investigated. Twenty-five specimens from set B and 8 from set C were identified by IFA. Six specimens from set B and five from set C were identified by RFLP analysis. Specimens in sets B and C were treated using RT-PCR; the resulting amplicons were subjected to nucleotide sequence analysis. The VP1 region was analyzed using two sets of deoxyinosine degenerate primers. Where the VP1 test gave no signal, the VP4-VP2 region was analyzed. Where both tests were negative, a 5′ noncoding region analysis was performed. Interestingly, analysis of the VP1 region showed that two specimens from set C were strains of enterovirus 71, whose presence was unexpected in Italy. As in other European epidemiological studies, the strain found most frequently was echovirus 30.
机译：肠病毒的鉴定和分型需要采用免疫和分子方法的综合技术方法。从细胞培养物中分离出本研究中包括的79种肠病毒，并根据针对普通肠病毒抗原的间接免疫荧光测定（IFA）和基于Lim Benyesh-Melnick（LBM）库的中和测试将其分类为肠病毒。使用多种不同的分子方法进行最终鉴定，包括逆转录（RT）-PCR，限制性片段长度多态性（RFLP）分析和来自基因组各个区域的扩增子的核苷酸序列分析。使用LBM池分析，RFLP分析和IFA鉴定了27株脊髓灰质炎病毒株（A组）。使用LBM池方法显示，在79株中，有35株是非脊髓灰质炎病毒（B组），而有17份标本呈阴性（C组）。对集合B和C进行了进一步研究。 IFA鉴定了B组的25个标本和C组的8个标本。通过RFLP分析鉴定出B组的六个标本和C组的五个标本。 B组和C组的样品使用RT-PCR处理;对得到的扩增子进行核苷酸序列分析。使用两组脱氧肌苷简并引物分析VP1区。在VP1测试未给出信号的地方，分析了VP4-VP2区域。当两个测试均为阴性时，进行5'非编码区分析。有趣的是，对VP1区域的分析表明，来自C组的两个标本是肠道病毒71株，在意大利出乎意料。与其他欧洲流行病学研究一样，最常发现的病毒株是回声病毒30。

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