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Method and kit for detecting mycobacterium tuberculosis and drug-resistant gene mutation thereof

A Mycobacterium tuberculosis and gene technology, which is applied in the field of 199, can solve the problems of low detection accuracy, low efficiency, and inability to identify the detection site of Mycobacterium tuberculosis at the same time.

Active Publication Date: 2008-03-19
GUANGZHOU DARUI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in ordinary laboratories, there are few chip detection equipment such as fluorescence scanners, and all of them have the disadvantages of complex technology, high cost and low efficiency.
Biotin-labeled primers, PCR amplification, membrane strip hybridization detection system such as Mycobacterium tuberculosis drug resistance gene chip detection system (Chinese patent application number 200410009231.5); Mycobacterium tuberculosis drug resistance gene detection kit and its preparation method (Chinese patent application number 20041083826.5) and U.S. Patent Application No. US20020058422 (2002, 1, 30)) have greatly reduced the detection cost, but the multi-purpose samples of these inventions are the clinical tuberculosis isolates after the initial isolation and culture, and the biological samples before the culture are directly detected The accuracy is low, and there are shortcomings such as the inability to identify Mycobacterium tuberculosis at the same time or fewer detection sites
Due to the low extraction efficiency of Genomic DNA of Mycobacterium tuberculosis from sputum samples of tuberculosis patients, the sensitivity of routine PCR detection is high.
Therefore, for the actual use of clinical test samples, these existing technologies still have some shortcomings in clinical applicability

Method used

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  • Method and kit for detecting mycobacterium tuberculosis and drug-resistant gene mutation thereof
  • Method and kit for detecting mycobacterium tuberculosis and drug-resistant gene mutation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0244] Embodiment 1: Preparation of target nucleic acid of sputum sample

[0245] Add 4 times the volume of 4% NaOH (containing 1%, W / V) into the glass tube containing the sputum, shake it well and place it at room temperature for 15-30 minutes. Take 1ml of the liquefied sample and add it to a 1.5ml centrifuge tube, centrifuge at 12,000rpm for 5 minutes, remove the supernatant, add 1ml of sterilized saline to the precipitate, mix well, and centrifuge at 12,000rpm for 5 minutes. Remove the supernatant, keep about 50 μl of precipitation solution, add 10 μl of extraction solution A (mix well before use), then add 200 μl of extraction solution B, vortex and mix, and place at room temperature for 5 minutes. Centrifuge at 8,000rpm for 1 minute and carefully aspirate the supernatant. Then add 200 μl of extract B, mix well (by blowing with a pipette), centrifuge at 8,000 rpm for 1 minute, carefully absorb the supernatant, add 1000 μl of 75% ethanol and mix well (by blowing with a pip...

Embodiment 2

[0246] Embodiment 2: PCR amplification of target nucleic acid

[0247] Take several tubes of PCR reaction solution A for a single person, add 0.5 μl UDG enzyme to each tube, and then directly add 5 μl template (or negative and positive standards), mix well, and centrifuge briefly (3 seconds) to place each reaction tube. into the PCR machine. After pretreatment at 50°C for 3 minutes, amplification was carried out under the following conditions: 93°C for 5 minutes, followed by 93°C for 30 seconds, 65°C for 45 seconds, and 72°C for 45 seconds, a total of 30 cycles. Add 1 μl of the amplification product above to PCR reaction solutions B and C, centrifuge briefly (3 seconds), put each reaction tube into a PCR instrument, and amplify using the following conditions: 93°C for 5 minutes, then press 93°C for 30 seconds , 64°C for 45 seconds, 72°C for 30 seconds, a total of 40 cycles. The final product was detected by 2% agarose gel electrophoresis (see Figure 2).

Embodiment 3

[0248] Example 3: Reverse dot blot detection

[0249] Before hybridization, hybridization solution I (2×SSC-0.1% SDS) was mixed with hybridization solution II and preheated to 60°C for use. Take several 1.5ml centrifuge tubes according to the number of samples to be tested, add 0.5ml hybridization solution I to each tube and preheat to 60°C. The PCR amplification products B and C were mixed, denatured at 97°C for 5 minutes, and immediately placed in ice-water mixture for 2-5 minutes. Then take 1000 μl hybridization solution I + 2 μl solution I (1000:2) mixed solution as the binding solution and store it at 4°C for later use. Take the mixture of solution II: solution III: solution IV (1900:200:1) (19 ml solution II + 2 ml solution III + 10 μl solution IV) as a chromogenic solution protected from light for later use.

[0250] Before hybridization, fill the reaction chamber with distilled water, place the metal perforated plate, and turn on the water pump to drain the water on ...

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Abstract

The present invention relates to a method and a reagent kit which are used for detecting the existence of the mycobacterium tuberculosis in the clinical biological sample and the mutations of the drug-resistant genes, in particular to a method and a reagent kit which are used for quickly detecting the existence of the mycobacterium tuberculosis in the biological samples such as the clinical sputamentum, the bronchoalveolar lavage fluid, the blood, the ascites, and the cerebrospinal fluid, etc. with the reverse dot-blot hybridization technology.

Description

[0001] Field [0002] The invention relates to a method and a kit for detecting Mycobacterium tuberculosis and its drug-resistant gene mutation in clinical biological samples, in particular to rapid detection of clinical sputum, blood, bronchial lavage fluid, ascites and cerebrospinal fluid, etc. by reverse dot hybridization technology The method for the presence of Mycobacterium tuberculosis bacteria in a biological sample and the mutation of its drug-resistant gene and the kit used. Background of the invention [0003] Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (MTB). According to the estimates of the World Health Organization, about 2 billion people in the world are infected with Mycobacterium tuberculosis, and about 3 million people die from tuberculosis every year [Raviglione M C, et al. Global epidemiology of tuberculosis. Morbidity and mortality of a worldwide epidemic. JAMA, 1995, 273: 220-226.]. In recent years, due to the increase of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12P19/34
Inventor 张太松陈华云王伟毅程钢何蕴韶
Owner GUANGZHOU DARUI BIOTECH
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