Rapid Detection Of Mycobacterium Tuberculosis Based On Novel Cross Primer Amplification Method

Tuberculosis(TB)is one of the top 10 diseases that threaten global health.High prevalence of TB mainly happens in low-or middle-income and source-limited areas,and most high TB burden countries are developing countries,where detection methods which are simple,good accuracy and low-cost are urgently needed.In addition,sputum is usually taken for TB clinical diagnosis.But for someone who is the hard to take sputum,timely discovery and treatment of TB may be delayed.To address these issues,we proposed a study on the rapid and accurate detection of Mycobacterium tuberculosis(MTB)and on the feasibility of replament of sputum samples with oral swab samples for TB patients.In chapter one,the biological characteristics,pathogenicity and spread of MTB were introduced first.The epidemiology,control strategies and challenge of TB in the whole world and China were summarized.Then,the common in vitro diagnostic methods of tuberculosis bacterium were reviewed.Next,loop-mediated isothermal amplification(LAMP)and cross primer amplification(CPA),which are the two common isothermal amplification technologies,were introduced.Finally,the purpose,content,meaning of this thesis were proposed.In chapter two,CPA and specific fluorescent probe were combined to establish a dual real-time detection system based on IS6110 target gene and internal control gene for MTB detection for the first time.The system was proved to have excellent specificity for MTB and the detection limit was 10 copies/?L.This method was used to test 165 clinical sputum samples and the test results were compared with results obtained by IS6110-PCR control system,positive coincidence rate was 96.7%(87/90),negative coincidence rate was 97.3%(73/75),the total coincidence rate was 97%(160/165).This method was used to test 46 sputum samples which were taken from TB patients with a rough extraction.The positive rate was 95.7%(44/46),which was higher than 84.8%(37/46)of IS6110-PCR control system.In chapter three,the CPA and PCR were combined to establish the CPA-PCR method which is highly sensitive for MTB detection.This method was used to evaluate the feasibility of replacement sputum samples with oral swab samples for TB detection.This method has good specificity for MTB and the sensitivity was 1 copy/?L.Then,this method was used to test oral swabs samples obtained from TB patients,whose sputum smear was tested to be positive.24 oral samples from 12 TB patients,each of which includes a cheek swab sample and a tongue swab sample,were tested respectively with this method and IS6110-PCR control method.The results showed that positive rate of the tongue swabs samples was significantly higher than that of the cheek swabs.Further,two methods were used to test 20 tongue swabs samples of TB patients,positive rate of CPA-PCR system and IS6110-PCR control system were 70%(14/20)and 60%(12/20),respectively.In addition,this method was used to test 40 tongue samples obtained from 20 healthy people and 20 patients who did not have tuberculosis but have other lung diseases.The results showed this method is highly specific and reliable.