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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

AI Technical Summary

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 analysis (PCR-RFLP), dideoxy fingerprinting (ddF), direct However, these methods have not been widely used clinically due to the defects of cumbersome operation, high cost, complicated technology or low efficiency.

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

Embodiment 1

[0124] Main raw materials and reagents

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

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

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

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

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

[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 has a sequence as shown in SEQ ID NO: 14;

[0132] An oligonucleotide probe for detectin...

Embodiment 2

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

Embodiment 3

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