Detection Of Helicobacter Pylori By Loop-Mediated Isothermal Amplification And Typing

Helicobacter pylori infection is a common infectious bacterium in the Department of Gastroenterology,and the infection rate has been high all over the world,which can cause gastritis,stomach ulcers,stomach cancer and other diseases.Studies have found that Hp I is more toxic and more harmful to the gastric mucosa,while Hp II has less damage to the gastric mucosa.Accurate and efficient detection and genotyping have great significance for the diagnosis of Hp.The carbon-14 breath test is usually used to diagnose Hp,but it requires expensive equipment and is difficult to carry out in primary medical units.Genotyping detection is more complex and not widespread.Although fluorescent quantitative PCR is widely used as a common nucleic acid detection technology in large and medium-sized hospitals at home and abroad,but the fluorescent quantitative PCR reaction instrument is relatively expensive,except that it requires relatively high requirements for the operator's ability and quality,and is also strict in environmental requirements,which is not suitable for popularization in primary hospitals and in the field.In recent years,the loop-mediated isothermal amplification method has simple operation,strong specificity,high sensitivity and stable results.The results of the experiment can be distinguished by the naked eye by adding a color indicator such as calcein or hydroxynaphthol blue.Loop-mediated isothermal amplification(LAMP)as a fast and efficient method is suitable for detection in primary hospitals and in areas with poor medical conditions.In this study,primers were designed for Hp conserved regions,and LAMP technology was established to detect and genotype Hp.Part ?.Detection of Helicobacter pylori by loop-mediated isothermal amplification Objective:Establish of a fast,simple and efficient LAMP technology for detection of Hp.Methods: The entire sequence of Hp 16 s r RNA was downloaded from the NCBI website,multiple sequence alignments were performed using Clusaw X software,conserved sections were selected,and LAMP primers were designed by the LAMP primer software Primer Eplorer V5.There were 152 cases of saliva and fecal specimens collected from patients with clinical symptoms of upper abdominal discomfort,nausea,acid reflux,vomiting,etc,including 76 saliva specimens and 76 fecal specimens.There were 24 specimens of saliva and feces that were determined to be healthy after the test,including 12 specimens of gastric juice and 12 specimens of fecal specimens.Salivary and fecal specimens were collected from the same patient.There were 76 patients with gastric diseases and 12 healthy people.All patients with gastric diseases underwent carbon-14 breath test.The saliva was extracted according to a one-tube universal sample DNA extraction kit method,and the fecal specimen was extracted with the fecal genomic DNA extraction kit.The bacteria were quantified by fluorescence quantitative PCR.LAMP reaction conditions were optimizated by changing some factors,verification of specificity by amplification of non-specific templates.The sensitivity was verified by amplification ratio of diluted samples.At the same time,the experimental results were electrophoresed,and the results of the electrophoresis were compared with those of the color indicator.Results: After many experiments,the suitable reaction conditions of LAMP were explored.LAMP was used for the progressive amplification of non-specific templates,and no positive results were found.The reaction product was observed with the naked eye by adding the color indicator calcein or hydroxynaphthol blue.The results were the same as those obtained by gel electrophoresis.After adding the color indicator,the result was read directly with the naked eye without opening the cover,and aerosol pollution was avoided.In the 76 cases of fecal specimens detected by LAMP,53 cases were positive,and the positive rate was 69.7%.A total of 76 saliva samples were detected by LAMP,and 43 positive samples were detected.The positive rate was 56.6%.The carbon-14 breath test was underwent by 76 patients,and 51 positive cases were detected,with a positive rate of 67.1%.Fluorescence quantitative PCR was used to detect 76 fecal samples,and 47 of them were positive,with a positive rate of 61.8%.Fluorescence quantitative PCR was performed on 76 saliva samples,and positive results were obtained in 37 cases,with a positive rate of 48.7%.LAMP was used to detect 24 negative specimens.There was no specific amplification and the specificity was good.The sensitivity of LAMP was 10 times higher than that of fluorescent quantitative PCR by amplifying the diluted template.Conclusion: In this experiment,the special primers were designed for the 16 S r RNA region gene of Hp.By optimizing the experimental conditions,the specificity and sensitivity were ensured,and the reaction efficiency was improved.A visual LAMP technology for Hp detection was established,which showed good specificity and sensitivity,and was suitable for promotion in primary hospitals and areas with insufficient equipment.Part ?.Study of Hp typing by loop-mediated isothermal amplification methodObjective: Establish a visual LAMP technology for genotyping Hp I and Hp II genotypes.Methods: We collected 76 cases of stool samples from patients with clinical symptoms of upper abdominal discomfort,nausea and vomiting,and 12 specimens of fecal samples from healthy people after tested.The entire sequence of Cag A gene and Vac A gene were downloaded from the NCBI website,Clusaw X software was used to compare multiple sequences and select conservative segments.LAMP primer software Primer Eplorer V5 was used to design LAMP primers,and LAMP primers were designed using the LAMP primer software Primer Eplorer V5.The specificity of the primers was verified by amplification of non-specific templates.The experimental results were analyzed by electrophoresis,and the experimental products added with the color indicator were compared to see if the results were consistent.Fecal specimens were also typed by PCR.The SPSS21.0 software was used to compare whether the two methods of LAMP and PCR were statistically different and the consistency was good.Results: The LAMP primer had good specificity,no amplification to the non-specific template,and it specifically identifies of Cag A and Vac A genes genotyping-related gene polymorphisms.The electrophoresis analysis of the experimental results showed no difference between the results obtained and the experimental results observed by the naked eye after the addition of the color indicator.In the first part,LAMP detected 53 positive cases in 76 stool specimens.Detection of 47 positive cases by fluorescence quantitative PCR.Choosing 47 cases of common positive samples,and LAMP and PCR were used for typing.LAMP detected 35 cases of Hp I type,the positive rate was 74.5%,7 cases of Hp II type were detected,the positive rate was 14.9%,and the intermediate type was 5 cases,the positive rate was 10.6%.In the PCR reaction,31 cases of Hp I type were detected,the positive rate was 66.0%,9 cases of Hp II type were detected,the positive rate was 19.1%,and 7 cases were detected by intermediate type,the positive rate was 14.9%.Using SPSS21.0 software to compare the two detection methods of LAMP and PCR,it could be concluded that the difference between the two detection methods was not statistically significant.Conclusion: Specific primers were designed for genotyping polymorphisms of Caga gene and Vaca gene of Hp.We had established LAMP technology for Hp typing.Through a simple method,this study provided a basis for the treatment of patients with Hp infection.