Establishment And Clinical Application Of Multiplex PCR And LAMP Detection Methods With M. Tuber Culosis-Complex

Tuberculosis(TB) is an ancient chronic infectious diseases, It’s first discovered the famous biologist Koch in 1882. TB was once the disease caused largest number of deaths in human and animal. Although TB had been controlled in history, but since the 19th century, due to the rapid development of world economy, large numbers of people gathered in the city of residence, and the development of animal husbandry, resulting pandemic of tuberculosis and had multiple drug resistance to humans, the development of social and livestock face tremendous threat, The cross-infection of Mycobacterium tuberculosis and Mycobacterium bovis bring great difficulty for prevention and treatment of TB, but it was lack of an effective, rapid, inexpensive detection of TB in the present.This study established a effective multiple PCR and LAMP detection method for epidemiological investigation and control of tuberculosis, the following is the mainly studies:The first part, the method of multiple PCR for detection and clinical application. In this study, four pairs of primers were designed for the four specific genes of Mycobacterium tuberculos:16sRNA, IS6110, RV3873, RV2652, and four pairs of primers have the same annealing temperature, the molecular weight of gradient PCR products ordered (were 824bp、621bp、254bp and 418bp), and therefore agarose gel electrophoresis when the formation of four bands of different sizes, can specifically detect Mycobacterium tuberculosis and Mycobacterium bovis, and is able to distinguish them, the detection sensitivity came up to 50 CFU/template.Part II, LAMP detection technique and clinical application. Loop mediated isothermal amplification (LAMP) is a high specifici and sensitive technology, simply and fastly to operate, and it is a low-cost detection methods. In this study, specific gene sequence RV3877 primers designed to detect Mycobacterium tuberculosis and Mycobacterium bovis, and it had high specificity, sensitivity reached 16个菌/template.The last part,234 clinical samples from PPD-positive blood, the detection rate was 4.7% with multiplex PCR,; 42 isolates from clinical lung samples, the detection rate was 30.9% with multiplex PCR; 86 Clinical nez de boeuf samples from PPD-positive swab, the detection rate was 8.3% with multiplex PCR, while the detection rate was 24.4% with multiplex LAMP; 56 clinical isolated samples from PPD-positive milk, detection rate of 5.4% with multiplex PCR, while the detection rate was 16.1% with LAMP; 74 sputum samples of patients with suspected tuberculosis, the detection rate was 48.6% with multiplex PCR, while the detection rate was 66.2% with LAMP.LAMP technology has high sensitivity in the clinical detection, and multiple PCR is able to distinguish Mycobacterium tuberculosis and Mycobacterium bovis. LAMP detection technology is suit to the detection of tuberculosis and the large-scale epidemiological investigation and monitoring, and multiple PCR technique for Mycobacterium tuberculosis and Mycobacterium bovis cross-infection and epidemiological investigations. The establishment of these two methods provides an effective way for the tuberculosis detection and monitoring in human and animals.