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Kit and method for detecting mycobacterium tuberculosis and non-mycobacterium tuberculosis

A technology for Mycobacterium tuberculosis and Mycobacterium tuberculosis, which is applied in the field of biomedical detection, can solve the problems of difficult to achieve rapid detection, time-consuming, cumbersome and the like, and achieves the effects of short time, long time to solve, accurate and reliable classification

Inactive Publication Date: 2017-08-18
HEBEI CHEST HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are relatively cumbersome and time-consuming, and it is difficult to achieve rapid detection

Method used

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  • Kit and method for detecting mycobacterium tuberculosis and non-mycobacterium tuberculosis
  • Kit and method for detecting mycobacterium tuberculosis and non-mycobacterium tuberculosis

Examples

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Embodiment 1

[0039] The kit for detecting Mycobacterium tuberculosis and non-tuberculous mycobacteria includes nucleic acid extraction reagents, fluorescent PCR reaction system, negative reference, positive reference and instructions. All reagents in the kit are dry powder, and their specifications are shown in the table below.

[0040] Table 3 Nucleic acid detection kits for mycobacterium tuberculosis and non-tuberculosis mycobacteria

[0041] name Specification Nucleic acid extraction reagent 48 tubes / box Fluorescence PCR reaction system 48 tubes / box negative reference 1 tube / box positive reference 1 tube / box manual 1 copy / box

[0042] The above fluorescent PCR reaction system contains buffer, 1.5mM Mg2+, 1U Taq DNA polymerase, 20UUNG, 0.2mM dUTP, 0.2mM dNTP, 0.2mM primer pair 1, 0.2mM probe 1, 0.2mM primer pair 2, 0.2 mM Probe 2 et al.

[0043] Above-mentioned primer pair 1 and probe 1 are used for detecting Mycobacterium tuberculosis,...

Embodiment 2

[0048] This embodiment is a method for detecting mycobacterium tuberculosis and non-tuberculosis mycobacteria using the kit described in Example 1, which includes the following steps:

[0049] 1) Nucleic acid extraction step of the sample to be tested

[0050] Using the standard strain of Mycobacterium tuberculosis, Mycobacterium abscessus, Mycobacterium kansasii and sputum samples (collected from sputum of tuberculosis patients in the Chest Hospital), the sputum was liquefied with 4% NaOH and identified by a bacterial culture (using conventional Hospital laboratory standard procedure), a nucleic acid identification of Mycobacterium tuberculosis and non-tuberculous mycobacteria infection.

[0051] Take 1 ml of liquefied sputum and add it to the nucleic acid extraction reagent in the kit (3M guanidine hydrochloride, 5M NaCl, 10mM EDTA and 1% SDS), ultrasonicate at 600W for 3 minutes, remove the liquid, add 200 microliters of eluent, and obtain the sample. Negative and positiv...

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Abstract

The invention relates to a kit and a method for detecting mycobacterium tuberculosis and non-mycobacterium tuberculosis. The kit comprises a primer pair 1 and a probe 1 for detecting the mycobacterium tuberculosis and a primer pair 2 and a probe 2 for detecting the non-mycobacterium tuberculosis; the primer pair 1 comprises sequences as shown in SEQ ID No.1 and SEQ ID No.2, and the primer pair 4 comprises sequences as shown in SEQ ID No.3 and SEQ ID No.4; the probe 1 comprises a sequence as shown in SEQ ID No.5 and the probe 2 comprises a sequence as shown in SEQ ID No.6. Fluorescent probes are designed by means of difference of gene sequences of different mycobacteria and mycobacterium tuberculosis and non-mycobacterium tuberculosis can be differentiated at one time. Therefore, the detection method is short in time and more accurate and reliable to classify, and solves the problem that in the prior art, the detection method is tedious and long in consumed time.

Description

technical field [0001] The invention relates to the field of biomedical detection, in particular to a kit and method for detecting mycobacterium tuberculosis and non-tuberculosis mycobacterium. Background technique [0002] Tuberculosis is a serious threat to human life and health in the world today. According to the estimates of the World Health Organization (WHO), about 2 billion people in the world are infected with Mycobacterium tuberculosis, and about 2 million people die of tuberculosis every year; my country is one of the 22 countries with a high burden of tuberculosis in the world, and there are currently 500 tuberculosis patients. Ten thousand. At the same time, the prevalence of non-tuberculous mycobacteria in my country is increasing year by year. Nontuberculous mycobacteria (nontuberculous mycobacteria, NTM) are other mycobacteria in the genus Mycobacterium except MTB complex and Mycobacterium leprae. So far, a total of 154 NTM species and 13 subspecies have be...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/32
CPCC12Q1/686C12Q1/689C12Q2600/166C12Q2563/107
Inventor 杨永辉齐飞虎方鹏
Owner HEBEI CHEST HOSPITAL
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