Molnar Bela, Szoke Dominika, Ruzsovics Agnes, Tulassay Zsolt
Clinical Research Unit, Hungarian Academy of Sciences, Budapest, Hungary.
Eur J Gastroenterol Hepatol. 2008 Apr;20(4):305-13. doi: 10.1097/MEG.0b013e3282f2fda4.
Determination of the local densities of Helicobacter pylori and its genotypic variations in gastric biopsy specimens by using novel real-time PCR-based methods could support the precise diagnosis and understanding of H. pylori infections.
Serial dilutions of H. pylori (0.016-16 microg/microl), control, bacterial, and human DNA samples were prepared. Fresh-frozen gastric biopsy specimens were taken from 103 patients, and the DNA was isolated. Quantitative determination of the ureaseA gene using hybridization probes with parallel evaluation of an internal human control gene (beta-globin) was performed by real-time PCR. CagA and VacA s1 genotypic characterizations were also performed. The data were compared with urea breath test (UBT), histology, and serological testing.
The presence of H. pylori could be detected by ureaseA-fluorescence energy transfer (53%), UBT (51%), serological testing (48%), and histology (52%) when compared with the gold standard (54%). A significant correlation was found between the quantitative real-time ureaseA/beta-globin ratio-based H. pylori frequency and the UBT results (P<0.01). Significantly increased bacterial density was found in the erosions when compared with the healthy part of the antrum and corpus (P<0.01). Real-time PCR VacA s1 results were in significant correlation (P<0.01) with those of serological tests, but CagA results were not. The genomic profiles (VAC/GAC) were different in 13.7% of the cases, which involved three different locations in the stomach.
Real-time PCR was the most reliable method for H. pylori diagnosis. Furthermore, quantification and genotyping could also be performed using this technique. The density of H. pylori was significantly increased in macroscopic erosions.
运用基于新型实时聚合酶链反应（PCR）的方法测定胃活检标本中幽门螺杆菌的局部密度及其基因分型变异，有助于对幽门螺杆菌感染进行精确诊断和深入了解。
制备幽门螺杆菌（0.016 - 16微克/微升）、对照、细菌及人类DNA样本的系列稀释液。采集103例患者的新鲜冷冻胃活检标本，提取DNA。通过实时PCR，利用杂交探针并行评估内部人类对照基因（β - 珠蛋白）来定量测定脲酶A基因。同时进行细胞毒素相关基因A（CagA）和空泡毒素A s1（VacA s1）基因分型鉴定。将数据与尿素呼气试验（UBT）、组织学检查及血清学检测结果进行比较。
与金标准（54%）相比，脲酶A - 荧光能量转移法可检测出53%的幽门螺杆菌，UBT为51%，血清学检测为48%，组织学检查为52%。基于实时定量脲酶A/β - 珠蛋白比值的幽门螺杆菌感染率与UBT结果之间存在显著相关性（P < 0.01）。与胃窦和胃体的健康部位相比，糜烂部位的细菌密度显著增加（P < 0.01）。实时PCR VacA s1结果与血清学检测结果显著相关（P < 0.01），但CagA结果则不然。13.7%的病例基因组图谱（VAC/GAC）不同，涉及胃内三个不同部位。
实时PCR是诊断幽门螺杆菌最可靠的方法。此外，该技术还可进行定量和基因分型。在宏观糜烂部位，幽门螺杆菌密度显著增加。
