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Tubercle bacillus drug tolerance detection reagent kit and tubercle bacillus drug tolerance detection method

A detection kit and technology for Mycobacterium tuberculosis, applied in biochemical equipment and methods, microbial determination/examination, chemical library, etc., can solve the problems of undetectable patients, long turnaround time, accuracy limitations, etc. and sequencing, reducing operational steps, and improving accuracy

Pending Publication Date: 2020-05-19
GUANGZHOU KINGMED DIAGNOSTICS GRP CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First, AFB smears have low sensitivity (30-40%) and their accuracy is often limited
Second, while culture-based methods have high accuracy and have long been considered the gold standard for laboratory TB diagnosis, the long turnaround time (3-8 weeks) for mycobacterial cultures often leads to delays in diagnosis
The reverse dot hybridization method for molecular detection has low sensitivity, complex operation and unreliable results
Commercially available real-time polymerase chain reaction (PCR) assays such as Abbott Real-Time MTB Assay and GeneXpert MTB / RIF Ultra provide timely and accurate TB diagnosis but are expensive
However, most of these methods are used to detect Mycobacterium checkeriensis. GeneXpert MTB / RIF can detect drug-resistant tuberculosis, but it can only detect a drug-resistant gene of rifampicin, which leads to patients who are treated with other drugs. Drug resistance or other resistance genes to rifampicin drug resistance cannot be detected
Molecular reverse dot hybridization can also be detected, but currently only ten sites of five drug-resistant genes of four drugs (isoniazid, rifampicin, ethambutol, streptomycin) can be detected. Resistance genes beyond four drugs remain undetectable

Method used

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  • Tubercle bacillus drug tolerance detection reagent kit and tubercle bacillus drug tolerance detection method
  • Tubercle bacillus drug tolerance detection reagent kit and tubercle bacillus drug tolerance detection method
  • Tubercle bacillus drug tolerance detection reagent kit and tubercle bacillus drug tolerance detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 A kind of tubercle bacillus drug resistance detection kit

[0041] The present embodiment is a kind of tubercle bacillus drug resistance detection kit, comprises tubercle bacillus nucleic acid detection reagent and tubercle bacillus drug resistance detection reagent:

[0042] (1) Mycobacterium tuberculosis nucleic acid detection reagents include primer pair 1 for IS6110, primer pair 2 for IS6110, probe primers for IS6110, internal quality control template, probe primers for internal quality control template, QuantiNova ProbePCR Master Mix and DEPC water.

[0043] The primer pair 1 for IS6110 includes IS6110-FW1 shown in SEQ ID NO.1 and IS6110-RV1 shown in SEQ ID NO.2; the primer pair 2 for IS6110 includes the sequence shown in SEQ ID NO.3 IS6110-FW2 and IS6110-RV2 shown in SEQ ID NO.4; the probe primer sequence for IS6110 is shown in SEQ ID NO.5, the 5' end of the probe primer is modified with the fluorescent group FAM, and the 3' end Modified with quench...

Embodiment 2

[0059] Example 2 A library construction method for the detection of drug resistance of Mycobacterium tuberculosis

[0060] In this embodiment, a method for constructing a library for detecting drug resistance of Mycobacterium tuberculosis, using the kit described in Example 1 for library construction, includes the following steps:

[0061] (1) Collect sputum samples, extract DNA, and then perform PCR amplification detection on the nucleic acid of Mycobacterium tuberculosis:

[0062]A. Add 100 μL of digestive solution to 1 mL of sputum sample, vortex to mix, and then incubate at 65°C for 10 minutes; B. Centrifuge the mixture obtained in step A at 13200g for 10 minutes, discard the supernatant; C. Add 100 μL Tris-HCl solution to the precipitate obtained in B, vortex to mix, then centrifuge at 13200g for 10 minutes, discard the supernatant; D, add 100 μL lysate to the precipitate obtained in step C, and incubate at 65°C for 45 minutes; E. Add 100 μL Tris-HCl solution to the mixt...

Embodiment 3

[0108] Embodiment 3 A kind of method for detecting drug resistance of Mycobacterium tuberculosis

[0109] This embodiment is a method for detecting drug resistance of Mycobacterium tuberculosis. The library is constructed using the method described in Example 2, and the concentration of the constructed library is measured and then sequenced using a sequencer. The specific steps are as follows:

[0110] 1. Library concentration determination and 2100 determination

[0111] (1) use 3.0 Fluorometer for library concentration determination, use agilent 2100bioanalyzer for length determination;

[0112] (2) Record the concentration data in the test sample information record form;

[0113] (3) The library is stored at -20°C.

[0114] 2. On-board inspection

[0115] (1) Information recording and preparation of reagents on the machine

[0116] ①Record the reagent information used on the machine in the "Molecular Pathology Reagent Use Record Form";

[0117] ②Take out the SBS Reag...

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Abstract

The invention provides a tubercle bacillus drug tolerance detection reagent kit and method. The tubercle bacillus drug tolerance detection reagent kit comprises a tubercle bacillus drug tolerance detection reagent, wherein the tubercle bacillus drug tolerance detection reagent comprises a sequencing primer in accordance with a tubercle bacillus drug tolerance gene; the tubercle bacillus drug tolerance gene comprises one or more genes of rpoB, katG, inhA-promoter, inhA-structural, furA, embB, ubiA, pncA, rpsA, gyrA, gyrB, eis, rpsL, rrs, tlyA, rplC and rrl; and further, the reagent kit also contains a tubercle bacillus nucleic acid detection reagent, and the tubercle bacillus nucleic acid detection reagent comprises a primer pair 1 in accordance with IS6110, a primer pair 2 in accordance with the IS6110 and a probe primer in accordance with the IS6110. Through the adoption of the tubercle bacillus drug tolerance detection reagent kit disclosed by the invention, tubercle bacillus nucleicacid in samples can be quickly detected, and positive samples can be further subjected to drug tolerance detection; and the tubercle bacillus drug tolerance detection reagent kit has good sensitivity, good specificity and good accuracy, and can perform mutation detection on 48 sites of 17 drug tolerance genes of common antituberculosis drugs and fragment deficiency detection of an intergenic region, so that the tuberculosis medication can be more accurately and comprehensively guided.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and in particular relates to a drug resistance detection kit of Mycobacterium tuberculosis and a method for detecting drug resistance of Mycobacterium tuberculosis. Background technique [0002] Mycobacterium tuberculosis, commonly known as Mycobacterium tuberculosis, is the pathogenic bacteria that causes tuberculosis. Mycobacterium tuberculosis is a slender and slightly curved bacillus that can invade susceptible organisms through respiratory tract, digestive tract or skin damage, causing tuberculosis in various tissues and organs, among which pulmonary tuberculosis is the most common through the respiratory tract. Drug-resistant TB occurs when the Mycobacterium tuberculosis infected with the patient becomes resistant to one or more anti-TB drugs. According to the report of WHO in 2008, the total drug resistance rate of tuberculosis in the world is 20.0%, and the rate of multidr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6858C12Q1/04C12N15/11C40B50/06
CPCC12Q1/689C12Q1/6858C40B50/06C12Q2600/166C12Q2531/113C12Q2535/122C12Q2563/107C12Q2545/101
Inventor 韩琦王春晖孟宪法孙明明关宇佳刘菲菲陶磊贺亮陈敬贤赵薇薇刘勇严婷于世辉任永昌马超杰
Owner GUANGZHOU KINGMED DIAGNOSTICS GRP CO LTD
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