套式聚合酶链反应扩增尿素酶A基因检测幽门螺杆菌

Detection of Helicobacter pylori in gastric biopsies by using nested polymerase chain reaction

应用互补于幽门螺杆菌(HP)尿素酶A基因片段的两对引物进行套式聚合酶链反应(N-PCR),外引物的扩增片段为552bp,内引物扩增片段为310bp,用该方法检测1株HP标准菌株(N-CTC14126)和20个HP临床分离株均阳性,而其他12种肠道菌均为阴性,特异性100％,敏感性可检测到0.1fg细菌DNA的水平,优于目前国外的报道。取胃粘膜活检标本30例,用细菌培养、尿素酶试验、组织学染色作为参照方法检测,20例为阳性,10例阴性,N-PCR检测结果与之完全一致。

A nested polymerase chain reaction (N-PCR) for the specific detection of Helicobacter pylori (H. pylori) was developed with two primer pairs (nested primers) derived from the Urease A gene of H. pylori. The N-PCR could detect 21 different samples of H. pylori including 20 clinical isolates and 1 reference strain NCTC14126, but could not detect other bacterial species, showing the N-PCR assay to be 100% specific. Tenfold serial dilution experiments revealed the detection of as little as 0. 1fg of H. pylori DNA by N-PCR. To evaluate the PCR assay for clinical samples, gastric biopsies were tested by N-PCR, and the results were compared with those of culture, urease test and histologic examination (reference standard, RS). In 30 biopsy specimens, H. pylori sequences were detected by PCR in 20 of 20(100%) positive tissues and none of 10 negative tissues. PCR is a specific and sensitive method that can detect the presence of H. pylori without the need for culture and that would have significant diagnostc and epidemiologic importance.