随机扩增的多态性DNA分析在Hp菌株鉴定中的应用

Distinguishing Different H. pylori by PCR Based RAPD Fingerprinting

目的:建立一种快速准确鉴定不同Hp菌株的RAPD分析方法,并评价其在区分Hp复发和再感染中的应用价值。方法:用苯酚—氯仿方法提取Hp DNA,以一个10nt的寡核苷酸为引物,建立Hp PCR扩增体系,所得PCR产物以2％琼脂糖凝胶电泳分析(RAPD分析),以50株临床分离的Hp菌株检测该方法的重复性。对4例十二指肠溃疡伴Hp感染者在三联治疗前后及根除后一年内再现的Hp菌株进行RAPD分析,鉴别复发和再感染。结果:(1)同-Hp菌株于不同时间进行的2次RAPD分析,所得的PCR产物完全一致,说明该方法具有良好的可重复性。(2)50株不同的Hp菌株各产生不同的RAPD图谱,可按此图谱的不同将它们区分开来。(3)对4例十二指肠溃疡伴Hp感染者在治疗前及治疗后1年再现的菌株的RAPD分析,发现3例患者的菌株具有相同的RAPD图谱,证实为原菌株的复发,而1例患者的菌株则产生不同的RAPD图谱,推测为新菌株的再感染。4例患者随访1年均为十二指肠溃疡复发。结论:RAPD分析只需单一的随机引物,方法简便、快速、经济、重复性好,可用于区分不同来源的Hp菌株,特别对治疗前后再现的菌株复发和再感染的鉴别更具临床应用价值。

To establish a rapid and sensitive polymerase chain reaction (PCR) basedrandom amplified polymorphic DNA (RAPD) fingerprinting method to distinguish different H. pylori isolates and evaluate its use in distinguishing recrudescence and reinfection of H. pylori after eradication. Methods: H. pylori DNA was extracted and used for PCR based RAPD fingerprinting with a 10 nucleotide arbitrory primer. The reproducibility and sensitivity of RAPD patterns was tested using 50 different H. pylori strains. Strains which isolated from 4 duodenal ulcer patients with H. pylori infection before treated with triple therapy and one year after eradication were tested by this method to confirm recrudescence and reinfection. Results: RAPD fingerprinting yielded 10 to 20 main bands on the agarose gels. The profiles of the PCR products from the same isolates at two different times were indistinguishable. 50 different clinical strains each yielded a distinct RAPD profiles, different from all others. By comparing 4 pairs of isolates before and after treatment, we confirm recrudescence accured in 3 patients for the same RAPD profiles each and reinfection accured in 1 patient for the different RAPD profiles. Conclusions: PCR based RAPD fingerprinting which only needs a single primer is a rapid, simple and reproducible method in distinguishing different H. pylori strains, especially in distinguishing recrudescene and reinfection of H. pylori.