| In recent years,with the rise of precision medicine,a variety of molecular diagnostic technology has developed rapidly.The medical technology,represented by gene sequencing,has been concerned by all parties,starting to usher in the day of a comprehensively explosive growth.Since Sanger sequencing technology emerges in early period,accuracy has been regarded as the gold standard of molecular diagnosis industry.Single nucleotide polymorphism(SNP),that is,SNP plays an important role in drug resistance and typing of viruses,susceptibility genes,drug sensitivity,tumor targeting drugs,human evolution and population genetics.With the evolution of virus microbes,changes in the structure of human genes caused by environmental and dietary factors have increased the amount,difficulty and complexity of Sanger sequencing data.More importantly,the sensitivity of Sanger sequencing has been criticized all the time.In order to obtain useful clinical information,researchers must use bioinformatics tools to analyze and process the data.Although these tools hide complex computing process from users,but there are limitations in the operational use,the management and processing of biological data is usually carried out in the linux environment,and its specialized operation interface is not well known to clinicians.Infectious disease is a major disease threatening human health,rapid and accurate diagnosis is an important prerequisite to effective treatment,disease monitoring and control of the spread of disease.With the development and ever-increasing improvement of molecular detection technology,molecular detection has become an important tool in the diagnosis of infectious diseases and the evaluation of curative effect.In December 1st 2017,the National Health and Family Planning Commission issued the guidelines for individualized Medical Molecular Detection of Infectious Diseases,pointing out that DNA sequencing technology is the gold standard for infectious disease detection at this stage.However,gene mutations with low abundance are often not recognized in clinical therapy.For example,the peripheral blood samples of patients with early drug resistance studies of chronic hepatitis B often contain the healthy peripheral blood of patients with early stage viruses,suspected viral components.Therefore,a simple,user-friendly and powerful analysis tool can better solve the sensitivity problem in early diagnosis.The main contents of this paper can be divided into three parts.The first part briefly introduces the background of sensitivity research of Sanger sequencing method.The current challenges of clinical drug resistance in infectious diseases are the current situation of Sanger data analysis and the limitations and shortcomings of current software in processing low-frequency sequencing peak.The necessity and significance of ab1 peak reporter analysis tool for quantitative analysis of low level mutation in clinical drug resistance treatment of infectious diseases were put forward.In the second part,the feasibility of using human p53 gene mutants as standard test tool is introduced.Ab1 peak reporter,an online analysis tool of Thermo Fisher Scientific website,and sequencing data of human p53 gene test samples with known mutation sensitivity are used as internal reference.Eight different proportions of wild and mutant strains were constructed.Two groups of parallel experiments used ab1 peak reporter analysis and seqscanner to output their respective electrophoretic patterns to realize the quantitative analysis of mutation,and to analyze the mutation sites by searching for conservative gene sequences.Correlation coefficient was used to verify the correlation and accuracy of ab1 peak reporter with known mutants in different mixing proportions.In the third part of this paper,the most representative chronic hepatitis B drug resistant disease and rifampicin resistant disease of Mycobacterium tuberculosis were selected as the research objects,and some clinical data were selected for experimental verification.The results show that ab1 peak reporter can assist in the early diagnosis and low level mutation study of two kinds of disease resistance,and is an effective tool for Sanger sequencing data processing and analysis.The main contents and methods are as follows:A total of 27 clinical blood samples of chronic hepatitis B resistance in Shenzhen Hospital were selected to extract hepatitis B DNA nucleic acid(namely 1*10~3)by centrifugal-column method,which accord with the lowest detection limit nucleic acid of Sanger sequencing method.Then referring to the mutation detection kit of hepatitis B virus resistance gene produced by Daan Gene of Sun Yat-sen University(PCR-sequencing)PCR amplification,gel electrophoresis and purification of P region of hepatitis B virus gene together with capillary electrophoresis of 3500DX gene analyzer,those were used to analyze27 cases of ab1 data uploaded to ab1 peak reporter.Results compared with the Seqscanner of gene analyzer,the ab1 peak reporter tool can realize the quantitative analysis of mutation abundance,help to find 11 cases of low level mutation,improve the detection rate and reduce the probability of error.Among them,5 patients with chronic hepatitis B received low level drug resistance mutations in advance,and changed drug treatment protocols for the benefit of patients and clinical practice.24 cases of rifampicin resistance of Mycobacterium tuberculosis in the first affiliated Hospital of Guangzhou Medical University were selected.The DNA of Mycobacterium tuberculosis was extracted from sputum,then the rifampicin mutation test kit of Sun Yat-sen University Daan gene(PCR-Sanger sequencing)was used to detect the rifampicin resistance of Mycobacterium tuberculosis and amplify The rpoB gene of rifampin resistance associated gene of Mycobacterium tuberculosis with electrophoretic and purified practice.The ab1 data of 24 patients with rifampicin resistance associated gene were uploaded to ab1 peak reporter and analyzed by using 3500DX gene analyzer.Results compared with the software of gene analyzer,Seqscanner,the ab1 peak reporter tool was used to find 9 cases of low abundance resistance mutation in clinic.Two of the patients could change the drug treatment plan early according to the low abundance resistance mutation,so as to improve the therapeutic effect and save the treatment cost.It has good application value. |
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