交叉等温扩增技术快速检测幽门螺杆菌方法的建立

Establishment of a Rapid Detection Method for Helicobacter Pylori by Cross Isothermal Amplification

目的建立一种新型的交叉引物等温扩增技术(cross-primer amplification,CPA)检测组织中的幽门螺杆菌(Helicobacter pylori,H.Pylori),为临床诊断幽门螺杆菌感染提供快速、灵敏、有效的检测方法。方法针对幽门螺杆菌尿霉素B基因(UreB)为靶序列设计3套交叉引物等温扩增方法的特异性引物,随后对反应体系的扩增温度、Bst DNA聚合酶浓度和扩增时间进行优化;选取2株幽门螺杆菌与其他7株致病菌进行特异性和敏感性试验;以尿素呼气法(UBT)为参考标准,检验CPA法对75例临床胃黏膜组织样本中幽门螺旋菌的检测灵敏度。结果幽门螺杆菌反应最佳引物组合为HCPA-3,最优反应温度为62℃、Bst DNA聚合酶浓度为6 U、反应时间为60 min;建立的方法具有较好的特异性,且检测灵敏度为1.26×102 cfu/mL;75例临床胃黏膜组织样本进行检测结果显示,CPA方法共检测出阳性标本26例,检出率为34.57%,准确率为96.30%(26/27)。结论 CPA法是一种快速、简便且高灵敏性和特异性的诊断方法,为临床上快速诊断幽门螺杆菌奠定了基础。

Objective To establish a new cross-primer isothermal amplification(CPA)technique for detection of Helicobacter pylori in tissues,which could provide a rapid,sensitive and effective method for clinical diagnosis of H.pylori infection.Methods Three sets of specific primers for isothermal amplification of UreB gene of H.pylori were designed,subsequently,the amplification temperature,concentration of Bst DNA polymerase and amplification time of the reaction system were optimized.Two strains of H.pylori and seven other pathogenic bacteria were selected for specificity and sensitivity tests.The sensitivity of CPA method for detecting H.pylori in 75 clinical gastric mucosa samples,which was tested by using UBT as a control standard.Results The results showed that HCPA-3 was the best primer group for H.pylori reaction,and the optimum reaction temperature was 62℃,the concentration of Bst DNA polymerase was 6 U,and the reaction time was 60 min.Meanwhile,the CPA method had good specificity,and the detection sensitivity was 1.26×102 cfu/mL.The results of 75 clinical samples of gastric mucosa showed that 26 positive samples were detected by CPA,the detection rate was 34.57%,and the accuracy rate was 96.30%(26/27).Conclusion The CPA method established in this study is a rapid,simple,highly sensitive and specific diagnostic method,which lays a foundation for rapid clinical diagnosis of H.pylori.