Detection Of Drug Resistance Gene Mutation Of Helicobacter Pylori By Multiplex Fluorescent PCR

Background:Helicobacter pylori is a gram-negative bacterium with flagella,which has a certain correlation with the occurrence of peptic ulcer and gastric cancer.Studies have shown that more than 80%of gastric ulcers are caused by Helicobacter pylori infection.WHO has listed Helicobacter pylori as the first risk factor for gastric cancer.At this stage,the current clinical treatment for Helicobacter pylori infection mainly includes"triple therapy"and"quadruple therapy",which can only rely on the experience of doctors and the preference of antibiotics in the hospital.Therefore,there is an urgent need to carry out drug resistance tests on the strains infected in patients to provide guidance for the selection of antibiotics and implement precise treatment.The traditional drug sensitivity test and Sanger sequencing method for detecting HP drug resistance are cumbersome and have a high failure rate,which is not conducive to clinical promotion and application.This study intends to establish a method for rapid detection of HP drug resistant genes based on multiple real-time fluorescent PCR,and achieve the goal of high accuracy,convenient operation and short detection time through multi angle optimization,so as to provide effective auxiliary guidance for clinical treatment of Helicobacter pylori.Objective:This study designed a specific fluorescent PCR method for rapid detection of HP based on the Ure A urease gene of HP,and designed a multiple fluorescent PCR method to detect the drug resistance gene of HP.Through comparison and analysis with the traditional drug sensitivity test and Sanger sequencing method,the accuracy and efficiency were discussed to establish a new method system for rapid and accurate detection of HP drug resistance gene sites.Methods:1.Design primer probes for HP Ure A urease gene,explore the optimal primer concentration,reaction volume and annealing temperature,and establish a qualitative detection system for HP Ure A urease gene.2.Using immunohistochemical reagents and the newly established HP Ure A urease gene qualitative detection system to detect 232 paraffin embedded gastric mucosal tissue samples with positive Helicobacter pylori from Fujian Provincial Hospital respectively.After comparing and analyzing the results,PCR amplification system and positive reference value range were established.3.Sequence the drug resistant samples of 85 clinical patients with Helicobacter pylori infection randomly enrolled from Beijing area,analyze the sequencing results,and establish a multiplex fluorescent PCR detection system for HP drug resistant mutation sites according to the sequencing sites and sequences.4.Evaluate the specificity and sensitivity of multiplex fluorescent PCR for detecting HP drug resistance mutation site system by comparing with traditional drug sensitivity test and Sanger sequencing method.5.285 clinical samples randomly enrolled from Beijing area were detected by multiplex fluorescent PCR-HP drug resistance mutation site system,and compared with Sanger sequencing method and drug sensitivity test method simultaneously to analyze the consistency of the three methods.Results:1.Established a nucleic acid detection system for Helicobacter pylori Ure A urease gene,with a sensitivity of 1.0×10~2copies/m L.2.Using immunohistochemistry to detect 112 known HP-positive samples,the results were that the staining intensity was+in 27 cases,the staining intensity was++in 38 cases,and the staining intensity was+++in 47 cases.The newly established HP Ure A urease gene nucleic acid detection system was used to detect 112 known HP positive samples.The results showed that there were 27 cases with 32≤Ct<38,38 cases with 24<Ct<32,and 47 cases with Ct≤24,which were related to the results of immunohistochemical intensity.Ct value<38 was determined as the positive reference value range of this PCR detection method.3.The nucleic acid detection system of HP Ure A urease gene was verified for 120 samples.The Kappa value was 0.962,which was highly correlated with the immunohistochemical results.It can be determined that the detection result of fluorescent PCR is consistent with the staining method,and the range of Ct value detected by fluorescent PCR is consistent with the clinical infection of Helicobacter pylori in different degrees.4.85 drug resistant samples were sequenced.The results showed that there were 11mutations in 23S r RNA,PBP1,Gyr A and 6S r RNA genes.According to the proportion of mutations and multiple fluorescent channel restrictions,10 of the mutations were selected as targets to establish the next drug resistance PCR detection system.5.A single-tube detection was established for 4 sites of clarithromycin,2 sites of amoxicillin,3 sites of levofloxacin,and three consecutive bases of tetracycline.In the system,different sites are distinguished by different fluorescent markers of probes,and the optimal primer dosage,the optimal reaction volume of 25μL,and the optimal annealing temperature of60℃are found,and the sensitivity can reach 1.0×10~2copies/m L.6.Performance verification of the HP resistance mutation site system of multiple fluorescent PCR:the specificity was 100%,the sensitivity is up to 1.0×10~2copies/m L,and the repeatability CV value is less than 5%.7.285 clinical samples were tested,and it was found that the Kappa value of multiplex fluorescent PCR and Sanger sequencing was 0.998,which was highly correlated;The Kappa value of this method is 0.935,which is also highly correlated with the drug sensitivity test method.It can be determined that the reliability of the multiplex fluorescent PCR method is consistent with that of the traditional drug sensitivity test.It only takes 4 hours for the multiplex fluorescent PCR method to complete the detection from obtaining samples,while the whole process of commonly used drug sensitivity test takes 5 to 7 days.The detection cycle of Sanger sequencing method is 5 days.The PCR method is simpler and faster.Conclusions:1.In this study,a fluorescent PCR method for detecting HP urease gene was established,which has the same detection effect as the immunohistochemical method,and can be used together with the multiple fluorescent PCR method for HP drug resistance gene mutation to monitor HP drug resistance;2.The multiplex fluorescent PCR method for drug resistance gene mutation of Helicobacter pylori established in this study has higher detection sensitivity and accuracy,which is consistent with traditional drug sensitivity detection methods,but it is simpler and faster to operate and can bring convenience for clinical application.