Kit for detecting mycobacterium tuberculosis and mutation of drug resistant gene thereof, and application thereof

A technology of mycobacterium tuberculosis and kits, applied in the direction of microorganism-based methods, microorganism measurement/testing, microorganisms, etc., can solve the problems of cumbersome operation, high cost, complicated technology, etc., and achieve the omission of washing steps and shorten the time-consuming , significant washing effect

Pending Publication Date: 2016-03-02
JIANGSU HUNTARRAY BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The main methods for drug-resistant tuberculosis gene detection include polymerase chain reaction-single-strand conformation polymorphism analysis (PCR-SSCP), restriction fragment length polymorphism anal

Method used

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  • Kit for detecting mycobacterium tuberculosis and mutation of drug resistant gene thereof, and application thereof
  • Kit for detecting mycobacterium tuberculosis and mutation of drug resistant gene thereof, and application thereof
  • Kit for detecting mycobacterium tuberculosis and mutation of drug resistant gene thereof, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0123] Example 1:

[0124] Main raw materials and reagents

[0125] Nitrocellulose membrane: produced by Millipore, 0.45μm pore size;

[0126] The preparation process of 20×SSC buffer solution is as follows: Weigh 88.2g trisodium citrate (purchased from Shanghai Shenggong) and 175.3g NaCl, dissolve in 800ml pure water, mix well, adjust the pH value of the solution to 7.0± with concentrated HCl 0.1, add pure water to 1000ml;

[0127] Terminal deoxynucleotidyl transferase (TdT): 5U / μl, 100μl purchased from Shanghai Jianglai Biotechnology Co., Ltd.;

[0128] 10×TdT buffer: matched with TdT enzyme, provided by Shanghai Jianglai Biological Technology Co., Ltd.;

[0129] dTTP: 100mmol / L, purchased from Promega;

[0130] An oligonucleotide probe for detecting Mycobacterium tuberculosis, the sequence of which is shown in SEQ ID NO: 11;

[0131] The oligonucleotide probe for detecting Mycobacterium avium, the sequence of which is shown in SEQ ID NO: 14;

[0132] The oligonucleotide probe for detect...

Example Embodiment

[0198] Example 2:

[0199] Same as Example 1, except that the composition of the hybridization solution in Example 1 is adjusted to: 3×SSC, 0.1μg / mlSA-AP, 5mMZnCl 2 , 5mMMgCl 2 , 0.5% BSA, 0.03% Tween-20, 0.03% PLL and 2% polyethylene glycol 8000, the balance is water. At the same time, adjust the components of the color solution to: PH9.5, 0.1mol / LTris-HCl, 0.1mol / LNaCl, 50mMMgCl 2 , 0.33mg / mlNBT, 0.17mg / mlBCIP and 0.03% n-hexadecyl glucoside, the balance is water. Other conditions remain unchanged.

Example Embodiment

[0200] Example 3:

[0201] Same as Example 1, except that the composition of the hybridization solution in Example 1 is adjusted to: 3×SSC, 1.5μg / mlSA-AP, 50mMZnCl 2 , 50mMMgCl 2 , 7% BSA, 1.5% Tween-20, 0.3% CPAM and 4% polyethylene glycol 8000, the balance is water. At the same time, adjust the components of the color solution to: PH9.5, 0.1mol / LTris-HCl, 0.1mol / LNaCl, 50mMMgCl 2 , 0.33mg / mlNBT, 0.17mg / mlBCIP and 0.2% n-octyl glucoside, the balance is water. Other conditions remain unchanged.

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Abstract

The invention discloses a kit for detecting mycobacterium tuberculosis and mutation of a drug resistant gene thereof. The kit comprises a PCR reaction reagent, a solid phase carrier, a hybridization solution containing alkaline phosphatase labeled streptavidin, and a chromogenic reagent. The kit allows the alkaline phosphatase labeled streptavidin to be directly added to the hybridization solution in order to make nucleic acid hybridization and enzyme-linked immunosorbent assay be carried out in a combination mode, and also allows membrane cleaning and substrate chromogenic process to be carried out in a combination mode, so many washing operating steps and a large amount of a reaction solution are avoided, convenience is brought for operating personnel, a large amount of experiment time and reagents are saved, and the detection flux of target nucleic acid in a sample is greatly improved to make a detection result keep high specificity.

Description

technical field [0001] The invention relates to the field of detection of mycobacterium tuberculosis and its drug-resistant gene mutation, in particular to a kit for detecting mycobacterium tuberculosis and its drug-resistant gene mutation and its application. Background technique [0002] Tuberculosis is a disease caused by infection with the bacterium Mycobacterium tuberculosis (TB). Since the late 20th century, the incidence of tuberculosis has gradually increased, and it is still one of the diseases with the largest number of deaths caused by a single infection factor. According to the latest statistical report of the World Health Organization (WHO) in 2012, nearly one-third of the world's population has been or is currently infected with Mycobacterium tuberculosis. The results of the fifth national tuberculosis epidemiological sampling survey in 2010 showed that my country is still a country with a high burden of tuberculosis. Although the incidence of tuberculosis has...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/32
Inventor 杨华卫曾冀
Owner JIANGSU HUNTARRAY BIOTECH CO LTD
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