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

A real-time fluorescence quantitative technology for Mycobacterium tuberculosis, which is applied in the field of biomedicine, can solve the problems that the specificity of detection results needs to be improved, the 16S gene is short, and the detection specificity is low, and achieves the effect of good application prospect and good detection specificity.

Active Publication Date: 2013-03-06
SHANGHAI BIOCHIP +2
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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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  • Real-time fluorescent quantitative PCR method used for detecting Mycobacterium tuberculosis, and primer, probe and kit thereof
  • Real-time fluorescent quantitative PCR method used for detecting Mycobacterium tuberculosis, and primer, probe and kit thereof
  • Real-time fluorescent quantitative PCR method used for detecting Mycobacterium tuberculosis, and primer, probe and kit thereof

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Embodiment Construction

[0028] The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments.

[0029] The following examples are only used to illustrate the present invention, but not to limit the scope of the present invention. The experimental method that does not indicate specific condition in the embodiment, generally according to conventional conditions, such as people such as Sambrook, molecular cloning: the condition described in the laboratory handbook (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturer's suggested conditions.

[0030] 1. Sample collection and processing, and sample DNA extraction

[0031] Sputum samples were collected from patients in accordance with clinical sample collection requirements, and the collected samples were stored at -20°C.

[0032] Thaw the sample on ice, take 2 mL of the sample, add 4 mL of 4% NaOH solution to the sample, mix well, and treat at room temperature ...

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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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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06C12N15/11G01N21/64
Inventor 张春秀朱建民陈金丝周颖周佳菁李先茜肖华胜
Owner SHANGHAI BIOCHIP
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