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

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 (Orientiatsut sugamushi,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-Sta5 6基因进行分析。采用PCR/SSCP检测 ,并与NestedPCR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NestedPCR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 ,2 0 0V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。

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.