Real-time fluorescent quantitative PCR method used for detecting Mycobacterium tuberculosis, and primer, probe and kit thereof

Abstract

The invention discloses a real-time fluorescent quantitative PCR method used for detecting Mycobacterium tuberculosis. The method comprises the following steps: 1, collecting and processing a patient sputum sample; 2, extracting the DNA of the sample; 3, carrying out a real-time fluorescent quantitative PCR reaction of the DNA of the sample through treating the DNA of a standard strain as a positive control and disinfected water as a negative control; and 4, judging the detection result of the sample according to a Ct value obtained through the reaction, and calculating to obtain the copy number of the Mycobacterium tuberculosis in the sample. The invention also discloses a primer and a probe used in the detection method, and a kit containing the primer and the probe. The detection method has the advantages of simplicity, rapidness and high specificity, and can realize the rapid and quantitative detection of the Mycobacterium tuberculosis in clinic sputum samples, so the detection method is helpful for the prevention and the timely prevention of diseases.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a fluorescent quantitative PCR method for detecting mycobacterium tuberculosis. The invention also relates to primers and probes for use in the method, and kits comprising the primers and probes. Background technique [0002] Tuberculosis is one of the major public health problems facing the world, and it is the main source of infection causing adult deaths. Especially in recent years, due to large-scale population movement and the emergence of drug resistance of bacteria itself, the incidence of tuberculosis has a tendency to rise. [0003] Mycobacterium tuberculosis (MTB) is the causative agent of tuberculosis. Currently, the diagnosis of MTB mainly adopts the combination of laboratory diagnosis and clinical signs. Laboratory diagnosis mainly includes smear staining, bacterial culture, antigen antibody detection, PCR and other methods. Among them, the bacterial culture of patient t...

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Problems solved by technology

At the same time, using the application of gene chips in bacterial drug resistance, many methods for detecting MTB and its drug resistance genes combining fluorescent quantitative PCR technology and gene chips have appeared in recent years. The Chinese invention patent (Application No. 200810105172.X) of the method, kit and application for extracting bacterial nucleic acid from bacteria in China uses gene chip technology to design primers and probes for MTB by comparing the differences of 16S rRNA genes in different strains. detection, but because the 16S gene is short (1500bp), the detection specificity of this method is low; another Chinese invention patent application titled "a fluorescent quantitative RT-PCR detection method for Mycobacterium tuberculosis complex" ( Application No. 201010522800.1), when designing primers and probes, the method of comparing 16S rRNA genes in different bacterial strains was also adopted. Therefore, the above-mentioned problems also existed. In addition, this method uses human genome DNA as the most negative control for detection. The specificity of the test results needs to be improved

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