| Helicobacter pylori(H.pylori)is a kind of microaerobic Gram-negative bacteria.Studies have shown that H.pylori is the main pathogenic factor of chronic gastritis and peptic ulcer,and its virulence factor cytotoxin-associated gene A(cagA)protein can induce canceration of gastric epithelial cells,which is closely related to the occurrence and development of gastric cancer.According to whether cagA gene is contained in the genome of H.pylori,it can be divided into type I H.pylori(cagA+,high toxic type)and type II H.pylori(cagA-,low toxic type).Type I H.pylori infection needs drug treatment,and type II H.pylori infection does not need treatment if there are no clinical symptoms.Many detection methods have been developed for H.pylori diagnosis.At present,endoscopy and urea breath test are the most used in hospitals.However,the above methods can only detect H.pylori infection in the stomach,but cannot judge whether the detected H.pylori is high or low toxic type,and provide accurate information for patients.Considering the harm of highly toxic H.pylori to health,it is urgent to establish a method for rapid detection of H.pylori and rapid classification of H.pylori,so as to improve the detection efficiency and shorten the detection time.In our previous work,our research group developed Accelerated strand exchange amplification(ASEA),and we further studied and optimized the detection method on this basis.Development of a novel Accelerated cycling Polymerase Chain Reaction(AC-PCR)method for nucleic acid amplification and simultaneous detection of multiple target genes by changing the fluorescent groups of the probe.The method,called Multiplex accelerated cycling PCR(MAC-PCR),can complete the detection and typing of H.pylori within 30 min.The establishment of this method can provide guidance for the formulation of early treatment plan of patients.The first part is the optimization of nucleic acid rapid extraction reagent of pathogenic microorganism.In this part,three targets were selected to optimize the formulation and extraction steps of Salmonella enterica(Gram-negative bacteria),Staphylococcus aureus(Gram-positive bacteria)and SARS-Co V-2 pseudovirus.Firstly,for the optimization of the lysis system,under the condition of controlling the only variable,each reagent is set with four concentration gradients.The results show that the optimal concentrations of each reagent are Na OH: 50 m M,EDTA: 0.5 m M,Y:0.5 g/L and chelex-100: 5%.The Ct value of the nucleic acid extracted is 1 to 3 less than that of other extraction reagents.Secondly,in order to optimize the extraction steps,the extraction time was shortened by avoiding heating and centrifugation steps and shortening the time of standing and shaking.The results showed that the extraction reagent could complete the extraction of sample nucleic acid within 3 min.Therefore,this method was applied to nucleic acid extraction in subsequent clinical samples of gastric mucosa in this study.Then the rapid nucleic acid detection and typing identification system of H.pylori was established.This part first studied the feasibility,specificity,anti-interference ability and sensitivity of AC-PCR detection of 16 S r DNA and cagA genes of H.pylori.Secondly,the specificity and anti-interference ability of MAC-PCR were evaluated.The results showed that MAC-PCR could specifically detect helicobacter pylori and classify it under the interference of multiple pathogens and host nucleic acids,and the limit of detection(LOD)was as low as 20copies/reaction,which can meet the requirements of clinical testing.The last part is the application of H.pylori clinical samples.In this part,100 clinical samples of gastric mucosa were tested and compared with the results of hospital histological examination.The results showed that 27 positive samples and 73 negative samples were detected by hospital histological examination.This method detected 28 positive samples and 72 negative samples.The results showed that one sample was inconsistent with the results of hospital examination.To further identify the suspected sample,we conducted sequencing analysis on the sample,and the results showed that the sample was H.Pylori positive sample.In addition,all samples with cagA+ genotype H.pylori were sequenced and analyzed in this part,and the results were consistent with the MAC-PCR method.The H.pylori detection system established in this study can realize the processing of gastric mucosal samples to the reading of detection results within 30 min,and MAC-PCR method can realize the detection and typing of H.pylori in one step.This method has good specificity and high sensitivity.The positive and negative detection rates of clinical samples of H.pylori are both 100%,which can meet the requirements of clinical detection,and is of great significance for the prevention and treatment of gastritis,gastric cancer,and other diseases. |
PDF Full Text Request