Development Of Real-Time Fluorescence Quantitative PCR Method For Mycobacterium Tuberculosis In Milk And Dairy Products

Mycobacterium tuberculosis is the key zoonotic pathogen in inducing Bovine tuberculosis, which has hindered the development of dairy cow industry on a global scale,endangering the wellbeing of human beings and other animals, as well as incurring severe financial losses to the societies. The World Organisation for Animal Health(OIE) has listed it as a Type B zoonosis. It is listed as a Class B zoonosis in China. Every year a significant amount of resources, in terms of manpower, materials and funding, is invested across the world in order to prevent and control the occurrence of this disease. The testing method designated by the OIE is the Mantoux test. However, this test can be influenced by physical,chemical and biological factors, resulting in poor specificity and sensitivity. The classic method of cultivating bacteria has long testing cycles due to the slow development of the Mycobacterium. However, the length of testing cycle is at the essence of tests conducted for Entry-Exit Inspection and Quarantine of milk and dairy products. Therefore, it is of special significance for the Entry-Exit Inspection and Quarantine to develop a testing method that is fast, sensitive and specific for milk and dairy products.This study establishes a real-time fluorescent quantitative polymerase chain reaction(PCR) testing method based on the specific primer in the conserved sequence of the mycobacterium tuberculosis. This study exam the stability of outcomes, sensitivity and specificity of this testing method. The result shows that the standard curve of real-time PCR method is of a linear relationship. The standard curve equation is, and the correlation coefficient was 0.9980. The sensitivity is high, which is up to 1×102 copies/?L or 10CFU/5m L. Anti-interference ability is strong and there is only the mycobacterium tuberculosis specific amplification curve. There is also high stability of outcomes.Comparative studies in terms intra group and inter group parallel testings are conducted with standard plasmid of various density. The result shows the inter and intra assaycoefficients was less than 1%.Tested by animal disease prevention and control center of Dalian and Entry exit inspection and Quarantine Bureau of Jilin, Henan, Shandong,Heilongjiang, this real-time fluorescent quantitative PCR method can quickly and accurately detect mycobacterium tuberculosis in milk and dairy products. At the same time,this test method had become a inspection and quarantine industry standard, the method of Detection of Mycobacterium tuberculosis in the export of milk and dairy products-fluorescent quantitative PCR method will be implemented in January 1, 2016.