应用 RT-nested PCR 与 RFLP 联合检测及基因分型汉坦病毒的临床应用：在中国山东省的一项前瞻性研究。

Clinical application of RT-nested PCR integrated with RFLP in Hantavirus detection and genotyping: a prospective study in Shandong Province, PR China.

机构信息

Department of Nosocomial Infection Management and Disease Control, Chinese People's Liberation Army General Hospital, 28 Fuxing Road, Haidian, Beijing, 100853, People's Republic of China,

出版信息

Cell Biochem Biophys. 2013;67(3):1521-7. doi: 10.1007/s12013-013-9655-z.

DOI:10.1007/s12013-013-9655-z

PMID:23723002

Abstract

The aim of the present study was to evaluate the clinical usefulness of applying RT-nested PCR along with RFLP as a method for diagnosis and genotypic differentiation of Hantavirus in the acute-stage sera of HFRS patients as compared to the ELISA technique. A prospective study of patients with suspected HFRS patients was carried out. Sera were collected for serological evaluation by ELISA and RT-nested PCR testing. Primers were selected from the published sequence of the S segment of HTNV strain 76-118 and SEOV strain SR-11, which made it possible to obtain an amplicon of 403 bp by RT-nested PCR. The genotypic differentiations of the RT-nested PCR amplicons were carried out by RFLP. Sequence analyses of the amplicons were used to confirm the accuracy of the results obtained by RFLP. Of the 48 acute-stage sera from suspected HFRS patients, 35 were ELISA-positive while 41 were positive by RT-nested PCR. With Hind III and Hinf I, RFLP profiles of the RT-nested PCR amplicons of the 41 positive sera exhibited two patterns. 33 had RFLP profiles similar to the reference strain R22, and thus belonged to the SEOV type. The other 8 samples which were collected during October-December had RFLP profiles similar to the reference strain 76-118, and thus belonged to the HTNV type. Sequence phylogenetic analysis of RT-nested PCR amplicons revealed sdp1, sdp2 YXL-2008, and sdp3 as close relatives of HTNV strain 76-118, while sdp22 and sdp37 as close relatives of SEOV strain Z37 and strain R22 located in two separate clusters in the phylogenetic tree. These results were identical to those acquired by RFLP. RT-nested PCR integrated with RFLP was a rapid, simple, accurate method for detecting and differentiating the genotypes of Hantavirus in the acute-stage sera of suspected HFRS patients. In Shandong province, the main genotypes of Hantavirus belonged to the SEOV types, while the HTNV types were observed during the autumn-winter season.

摘要

本研究的目的是评估 RT-巢式 PCR 与 RFLP 联合应用于 HFRS 患者急性期血清中汉坦病毒的诊断和基因分型的临床实用性，与 ELISA 技术相比。对疑似 HFRS 患者进行了前瞻性研究。采集血清进行 ELISA 和 RT-巢式 PCR 检测。根据 HTNV 株 76-118 和 SEOV 株 SR-11 的 S 片段发表序列选择引物，通过 RT-巢式 PCR 可获得 403bp 的扩增子。通过 RFLP 对 RT-巢式 PCR 扩增子进行基因分型。对扩增子的序列分析用于确认 RFLP 结果的准确性。在 48 份疑似 HFRS 患者的急性期血清中，35 份 ELISA 阳性，41 份 RT-巢式 PCR 阳性。用 Hind III 和 Hinf I 酶切，41 份阳性血清的 RT-巢式 PCR 扩增子的 RFLP 图谱显示两种模式。33 种具有与参考株 R22 相似的 RFLP 图谱，因此属于 SEOV 型。另外 8 份样本于 10-12 月采集，其 RFLP 图谱与参考株 76-118 相似，因此属于 HTNV 型。RT-巢式 PCR 扩增子的序列系统发育分析表明，sdp1、sdp2 YXL-2008 和 sdp3 与 HTNV 株 76-118 密切相关，而 sdp22 和 sdp37 与 SEOV 株 Z37 和 R22 密切相关，位于系统发育树的两个不同分支中。这些结果与 RFLP 获得的结果一致。RT-巢式 PCR 与 RFLP 联合应用是一种快速、简单、准确的方法，可用于检测和区分疑似 HFRS 患者急性期血清中的汉坦病毒基因型。在山东省，汉坦病毒的主要基因型属于 SEOV 型，而 HTNV 型则在秋冬季节出现。