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Drug resistance gene mutation detection of tuberculous bacillus based on high-resolution melting curve technology

A Mycobacterium tuberculosis, high-resolution melting technology, applied to the detection of Mycobacterium tuberculosis (TB) drug resistance gene mutation PCR primer set, the detection kit of Mycobacterium tuberculosis (TB) drug resistance gene mutation and Microarray, detection of Mycobacterium tuberculosis (TB) drug resistance gene mutation in samples, detection of Mycobacterium tuberculosis (TB) drug resistance gene mutation field, can solve the problems of cumbersome operation, error, time-consuming and so on

Pending Publication Date: 2016-08-31
QIAGEN SHENZHEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The phage biological amplification method and conventional drug susceptibility test are cumbersome and time-consuming, and it is not easy to standardize
The preparation and labeling of gene chip samples are cumbersome, the instruments are expensive, and the detection cost is high
PCR-restriction fragment length polymorphism analysis can only analyze gene mutations at specific sites of known sequences
Rapid culture instrument detection system instruments and reagents rely on high import costs
At the same time, some non-sequencing methods need to rely on enzyme digestion or clustering to interpret the gene mutation status of patients, and there is a greater probability of typing errors
Although the accuracy of direct sequencing is high, the DNA fragments generated by the sequencing reaction need to be separated by capillary electrophoresis, and then the fluorescent signal is detected by the detection system, which takes a long time and costs a lot.

Method used

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  • Drug resistance gene mutation detection of tuberculous bacillus based on high-resolution melting curve technology
  • Drug resistance gene mutation detection of tuberculous bacillus based on high-resolution melting curve technology
  • Drug resistance gene mutation detection of tuberculous bacillus based on high-resolution melting curve technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: High-resolution melting curve analysis using the PCR primer set of the present invention

[0065] 1. Materials and Methods

[0066] 1.1. Materials

[0067] PCR reaction solution 1 (including PCR primer set 1), PCR reaction solution 2 (including PCR primer set 2), PCR reaction solution 3 (including PCR primer set 3) and HS Taq enzyme.

[0068] The concentration of each component in the PCR reaction solution is shown in Table 2.

[0069] Table 2

[0070] name

Final concentration

source

forward primer F

0.3uM

Invitrogen

reverse primer R

0.3uM

Invitrogen

dNTP

0.2mM

Roche

Fluorescent dye Eva Green

1x

Biotium

20x buffer

1x

QIAGEN

MgCl 2

2mM

PG

[0071] 1.2. Samples

[0072] Wild-type control plasmid wt (inhA-katG-rpoB gene wild-type plasmid (SEQ ID NO: 7), synthesized by Invitrogen Company) and hospital clinical sputum sample nucleic acid.

[0073] 1...

Embodiment 2

[0098] Embodiment 2: Comparison between different PCR primer sets

[0099]The materials, instruments, methods and conditions used in this example are the same as in Example 1, but in the PCR process, two PCR primer sets shown in Table 4 below were used to amplify the wild-type plasmid rpoB gene.

[0100] Table 4

[0101]

[0102] The PCR primer set 3 (the primer set of the present invention) and the PCR primer set 4 are completely identical in the reverse primer, and only a small part of bases are different in the forward primer. The quality of the detection results of the same wild-type plasmid sample using different PCR primer sets (3 and 4) is completely different, see Figure 4 . The results of PCR primer set 4 showed severe non-specific amplification of NTC ( Figure 4 A gray curve), while there is no non-specific amplification of PCR primer set 3.

[0103]

[0104]

[0105]

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Abstract

The invention relates to drug resistance gene mutation detection of tuberculous bacillus (TB) based on a high-resolution melting curve technology and particularly relates to a PCR (Polymerase Chain Reaction) primer set for detecting drug resistance gene mutation of the tuberculous bacillus (TB) based on the high-resolution melting curve technology. The invention further relates to a kit and a micro-array, which comprise the PCR set and are used for detecting the drug resistance gene mutation of the tuberculous bacillus (TB) based on the high-resolution melting curve technology. The invention further relates to application of the PCR set to preparation of the kit and the micro-array, which are used for detecting the drug resistance gene mutation of the tuberculous bacillus (TB) based on the high-resolution melting curve technology. The invention further relates to a method for detecting the drug resistance gene mutation of the tuberculous bacillus (TB) in a sample by utilizing the PCR primer set.

Description

technical field [0001] The present invention relates to Mycobacterium tuberculosis ( Mycobacterium tuberculosis , TB) drug resistance gene mutation detection. Specifically, the present invention relates to a PCR primer set for detecting drug-resistant gene mutations of Mycobacterium tuberculosis (TB) based on high-resolution melting curve technology. The invention also relates to a kit and a microarray for detecting drug-resistant gene mutations of Mycobacterium tuberculosis (TB) based on a high-resolution melting curve technique comprising a PCR primer set. The invention also relates to the use of the PCR primer set in preparing a kit and a microarray for detecting drug-resistant gene mutations of Mycobacterium tuberculosis (TB) based on high-resolution melting curve technology. The invention also relates to a method for detecting the drug resistance gene mutation of Mycobacterium tuberculosis (TB) in a sample by using the PCR primer set. Background technique [0002] ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/32
CPCC12Q1/6858C12Q1/689C12Q2600/156C12Q2531/113C12Q2527/107
Inventor 陈华杨蓉刘小青
Owner QIAGEN SHENZHEN CO LTD
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