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目的探讨山东肾综合征出血热重疫区病人血清中HV分子生物学特征,同时寻找准确、简便、迅速的HV检测与分型方法,从而为制定防治决策提供科学根据。方法从山东肾综合征出血热重疫区临沂市费县收集病人早期血清,应用巢式RT-PCR对病人血清中的HV进行基因扩增,采用RFLP、SSCP分型,并进行序列测定与分析。结果48份临床和ELISA检测确诊的HFRS病人血清经巢式RT-PCR扩增后,41份阳性,阳性率85.42%。41份阳性标本巢式RT-PCR产物经HindIII、HinfI酶切后,呈现2种不同的RFLP图谱:33份显示与R22株相似的酶切图谱,应属SEOV型;另外8份显示出与HTN76-118株相似的图谱,属HTNV型,这8份HTNV型标本均为10-12月间收集的病人血清。41份阳性标本巢式RT-PCR产物经SSCP分析,亦呈现2种不同的图谱:33份具有与R22株相似的SSCP图谱,应属SEOV型;而另8份则与HTN76-118株具有相似的SSCP图谱,属HTNV型。对部分扩增产物序列采用系统进化树分析也得出类似的结果:sdp1、sdp2、sdp3与HTN型76-118株亲缘关系相近,属同一簇,而sdp22、sdp37与SEOV型Z37、R22株亲缘关系相近,属另外一簇,这与RFLP和SSCP获得的结果相一致。结论山东肾综合征出血热重疫区病人基因型以SEOV型为主,但在秋冬季节也存在HTNV型。巢式RT-PCR结合RFLP、SSCP法可对HV准确分型,而且简便、迅速,适合于大规模流行病学调查。
Objective To investigate the molecular characteristics of HV in sera of patients with severe hemorrhagic fever with HSH and to find accurate, simple and rapid HV detection and typing methods in order to provide a scientific basis for making prevention and control decisions. Methods The early serum of patients was collected from Feixian County, Linyi City, Shandong Province, China. The HV in patients’ sera was amplified by nested RT-PCR and sequenced and analyzed by RFLP and SSCP . RESULTS: Forty-eight HFRS patients diagnosed by clinical and ELISA tests were positive for serum by nested RT-PCR and the positive rate was 85.42%. Nested RT-PCR products of 41 positive samples were digested with HindIII and HinfI and showed two different RFLP patterns: 33 showed similar digestion pattern to R22 strain, which should belong to SEOV type; -118 strains of similar profiles, is HTNV type, the eight HTNV specimens were collected from October to December patient serum. SSCP analysis of 41 positive samples of nested RT-PCR showed two different patterns: 33 SSCP profiles similar to R22 strains should be SEOV type; and the other 8 were similar to HTN76-118 strains SSCP map, is HTNV type. Phylogenetic tree analysis of partial amplification products also yielded similar results: sdp1, sdp2 and sdp3 were closely related to HTN type 76-118 strains, belonging to the same cluster, whereas sdp22, sdp37 and SEOV type Z37 and R22 strains The relationship is similar and belongs to another cluster, which is consistent with the results obtained by RFLP and SSCP. Conclusion The genotypes of patients with severe HFD in Shandong province are SEOV type, but HTNV type is also found in autumn and winter. Nested RT-PCR combined with RFLP, SSCP method can accurately type HV, and simple, rapid, suitable for large-scale epidemiological investigations.