PCR/SSCP在恙虫病东方体基因分型中的应用

刘运喜; 赵仲堂; 高媛; 张景兰; 杨占清; 步秀萍; 苏静静

PCR/SSCP在恙虫病东方体基因分型中的应用

Improvement on conditions of PCR/SSCP and its application in rapid genotyping of orientia tsutsugamushi

摘要

摘要:
目的采用优化聚合酶链反应/单链构象多态性分析(PCR/SSCP)方法分析恙虫病东方体(Orientia tsutsugamushi, Ot)基因型的各项条件。
方法对恙虫病东方体山东分离株、小盾纤恙螨匀浆及恙虫病人全血中提取的Ot-Sta56基因进行分析。采用PCR/SSCP检测, 并与Nested PCR基因分型的结果进行比较。
结果山东地区35份标本提取的Ot-DNA群引物扩增产物呈现出2种不同的单链构象图谱, 其中33份与国际参考株Gilliam, Karp, Kato均不相同, 应属这3型以外的一种基因型, 经Nested PCR分型证实为Kawasaki型; 2份与Karp株相同。表明山东地区恙虫病东方体至少存在2种型别。检测结果与Nested-PCR基因分型结果相符。条件优化以8%的非变性聚丙烯酰胺凝胶电泳, 常温下, 200V电泳3h获得的电泳结果较为理想, 并易于操作。
结论 PCR/SSCP条件优化后可用于快速分析恙虫病东方体基因型, 且操作简便, 结果准确。

Abstract:
Objective In order to rapidly analyze the gene type of orientia tsutsugamushi(Ot)from Shandong province by PCR/SSCP.
Methods Comparing the SSCP maps of the amplification products by group primers of Ot-DNA acquired from 23 strains of Ot isolated, 2 pools of homogenized Leptotrombidium(L.)scutellare, and 10 blood of scrub typhus with the reference strains Gilliam、Karp、Kato, and also compared with the results acquired by Nested PCR.
Results Ot-Sta56 gene amplification fragments by group primers of Ot-DNA acquired from 23 strains of Ot isolated, 2 pools of homogenized L.scutellare, and 10 blood of scrub typhus appeared two different maps of SSCP, 33 had the same gene types different from that of the reference strain Gilliam、Karp、Kato, 2 had the same gene type with the reference strain Karp.This results were identical to that acquired by Nested PCR.
Conclusion PCR/SSCP was a rapid, simple, accurate method for analyzing the gene type of Ot.

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