Rapid And Specific Method To Detect Mycobacterium Tuberculosis In Sputum Specimens

Tuberculosis(TB)is one of the oldest infectious disease,which is caused by Mycobacterium tuberculosis(MTB)infection.TB is one of the seriously public healthy problems,and China belongs to top 30 countries with high TB incidence.Yunnan Province locates in Southwest of China,which is economic backwardness and imbalance of medical resource.Thus,the TB patients,especially drug-resistant TB patients,are more in Yunnan than in other provinces.Early diagnosis will provide the basis for the following treatment.Until now,the diagnosis methods of TB are based on phenotype and genotype experiment,including smear microscopy,Lowenstein-Jensen culturing,BATCEC MGIT 960 culturing,and Gene Xpert MTB/RIF.Due to the shortcoming of these diagnosis methods,the purpose of this study is to establish a rapid,special,and sensitive method to detect MTB.Firstly,we analyzed the clinical information of 392 TB patients.The results showed that number of males was more than that of females(male:female is 2.35 :1).51.3% of TB patients(N= 201)is farmers.97% TB patients(N= 384)existed complication.337 patients were secondary pulmonary TB or secondary pulmonary TB with complication,and 55 patients were extrapulmonary TB.The primary symptom of pulmonary TB patients included cough,hemoptysis,dyspnea,heating,chest tightness,and chest pain.The extrapulmonary TB patients expressed the corresponding clinical symptoms.The incidence of TB is negative associated with economic condition.In poor regions,misdiagnosis is common,which could lead to more TB patients and serious TB disease.The clinical analysis will provide reference for TB diagnosis and treatment,and be benefit for protection of TB disease.We firstly collected clinic samples of TB patients,which mainly included sputum together with fester,cerebrospinal fluid,ascites and so forth.After optimizing the method of genomic DNA from MTB,DNA from each sample was obtained.Through bioinformatic analysis of genome,the TB18.5 gene was selected as the specific gene for MTB detection.Primers were designed for nest PCR or nest real-time PCR.Correct band could be obtained when 10 copy templets existed in the PCR system by using the constructed plasmid with TB18.5.the specificity of primers was identified after amplifying 6 common clinical pathogenic bacteria and 3 non-MTB.We compared the positive ratio of three MTB detecting method(BATCEC MGIT 960 <BD-960>,Gene Xpert,and TB18.5-PCR in 230 TB patients and 10 controls.The results showed that positive ratio of BD-960,Gene Xpert,and TB18.5-PCR were 62.69%(163/230),82.61%(190/230),and 85.65%(197/230),respectively.The positive ratio of TB18.5-PCR was significantly higher than that of BD-960(P< 0.001).In order to rapidly detect mutations in the drug-resistant genes from drug-resistant MTB,we designed primers to amply the rpo B,kat G,emb B,pnc Aand rps L gene.The results showed that mutant ratio reached 97.83% in the rpo B gene of rifampin-resistant MTB;mutant ratio was 92.31% in the kat G gene of isoniazide-resistant MTB;mutations were found in the rps L gene of 84.21% streptomycin-resistant MTB;and mutant ratio was 66.67%in the emb B gene of ethambutol-resistant MTB.In this study,we established a rapid,sensitive,and specific method to detect MTB in clinical samples.The TB18.5 gene was predicted as the specific gene for MTB strains by using bioinformation analysis.Then the TB18.5-PCR method was optimized and compared with BD-960 and Gene Xpert.The high MTB positive ratio was identified in our TB18.5-PCR method.Our study will provide a new detecting method in clinical,and it might help to diagnosis and provide reference for further treatment.