焦磷酸测序法在幽门螺杆菌克拉霉素耐药基因检测及药物疗效评价中的应用

Application of pyrophosphate sequencing in detection of clarithromycin-resistance gene of Helicobacter pylori and evaluating drug effectiveness

目的本研究建立了幽门螺杆菌(Hp)克拉霉素耐药基因检测的焦磷酸测序方法,在获知耐药情况的同时根据半定量结果初步评估治疗效果。方法通过考察最合适的扩增引物、体系、退火温度建立最优的PCR扩增体系。分别对方法的灵敏度,特异性及一致性进行了评价。通过对44例临床标本的检测,比较本方法与快速尿素酶试验和13 C呼气试验的检出率。结果最优的PCR扩增体系为HiFi体系,最佳退火温度为59.8℃。本方法特异性良好,灵敏度达到100 copies/μL,与Sanger测序结果100.0%一致。36例标本快速尿素酶和焦测序法的检出率分别为48.7%和81.1%,耐药率为10.81%。8例标本13 C呼气试验和焦测序法的检出率分别为75.0%和87.5%,阳性标本检出一致率为100.0%。结论焦测序检测方法具备灵敏度高、快速、半定量的特点,为临床Hp克拉霉素耐药检测及初步疗效评价提供了一种新的方法。

Objective To establish a method of pyrosequencing for the detection of clarithromycin-resistance gene in Helicobacter pylori(Hp),and to evaluate the therapeutic effect based on semi-quantitative results.Methods To establish the optimal PCR amplification system by examining the optimum amplification primer,system and annealing temperature.The sensitivity,specificity and consistency of the methods were evaluated respectively.44 clinical specimens were examined to compare the detection rate of this method with the rapid urease test and the 13 C breath test.Results The best PCR amplification system was HiFi system,and the optimum annealing temperature was 59.8℃.This method was specific and has a sensitivity of 100 copies/μL,which is 100.0%consistent with the results of the Sanger sequencing.For the 36 specimens,the detection rates of rapid urease test and pyrosequencing method was 48.7%and 81.1%respectively.The resistance rate was 10.81%.In 8 specimens,The detection rates of 13 C breath test and pyrosequencing method was 75.0%and 87.5%,respectively,and the consistent rate of positive specimen was 100.0%.Conclusion Pyrosequencing has the characteristics of high sensitivity,rapid and semi-quantitative,which provides a new method for the detection of clarithromycin-resistance and preliminary therapeutic evaluation.