Rapid identification method and kit for MTBC (mycobacterium tuberculosis complex)

A technology of mycobacterium tuberculosis and mycobacteria, applied in biochemical equipment and methods, material stimulation analysis, microbial measurement/testing, etc., can solve problems such as inability to meet rapid clinical diagnosis, time-consuming and labor-intensive

Inactive Publication Date: 2015-04-29
FUDAN UNIV
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  • Description
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Problems solved by technology

[0004] Usually, the traditional method of identifying MTBC and NTM is through culturing, staining, and biochemical identification of bacterial types, which has the disadvantage of time-consuming and labor-intensive
At present, in my country, the Roche medium growth test of p-nitrobenzoic acid (PNB) and thiophene-2-carboxyhydrazine (TCH) is widely used to identify MTBC and NTM. This method takes up to one month, so it cannot meet the requirements of rapid clinical diagnosis. needs

Method used

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  • Rapid identification method and kit for MTBC (mycobacterium tuberculosis complex)
  • Rapid identification method and kit for MTBC (mycobacterium tuberculosis complex)
  • Rapid identification method and kit for MTBC (mycobacterium tuberculosis complex)

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Experimental program
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Effect test

Embodiment 1

[0061] Embodiment 1. Primer and probe design

[0062] The 16S rRNA gene sequences of common mycobacteria (including MTBC and NTM) and common non-mycobacteria were searched in the NCBI database, and bioinformatics comparison analysis was performed to select the bacteria with the ability to identify and distinguish mycobacteria from non-mycobacteria. Bacillus and the conserved sequence fragments of MTBC and NTM are used as target sequences, among which, target sequence one (rrs1022-1046) is used to identify Mycobacterium bacteria and non-Mycobacterium bacteria. In this sequence region, Mycobacterium bacteria show Sequence identity, non-mycobacteria have different mutations in this region, so mycobacteria can be distinguished from non-mycobacteria by detecting this region; target sequence two (rrs1269-1291) is used to distinguish MTBC from NTM, In this sequence region, MTBC bacteria show sequence consistency, while NTM bacteria have different mutations in this region, and the ide...

Embodiment 2

[0068] Example 2: Construction and preparation of internal reference plasmids and positive control plasmids

[0069] 1. Construction of internal reference plasmids and positive control plasmids

[0070] Internal reference plasmid construction: artificially synthesize a DNA single-stranded fragment, which contains the binding sequence of the upstream and downstream primers (16sR, 16sL) and the sequence that is completely complementary to the probe Probe-Myco, and uses this sequence as a template to perform PCR amplification reaction to obtain The double-stranded product was cloned into the pMD19-T vector, and the DNA sequence was determined. The recombinant plasmid was used as an internal reference standard and named 16s-IC. The internal reference plasmid was added to each reaction system at a low concentration (50 copies / reaction), in order to monitor whether the entire reaction system is working properly: 1) When the test sample is non-mycobacterial DNA, the internal referen...

Embodiment 3

[0078] Example 3: Clinical Mycobacterium Genomic DNA Extraction

[0079] Extract mycobacterial genomic DNA by thermal lysis: 1) Scrape 1-2 inoculum loops of mycobacteria from the solid medium, resuspend in 100uL TE, inactivate at 80°C for 30 minutes, 2) inactivate the strain Take out the P3 laboratory and perform the following operations: boil at 100°C for 1 minute, immediately put it on ice for 2 minutes, centrifuge at 12000r / min for 10 minutes, take the supernatant and put it in another sterile EP tube, store it at -20°C, and put it on ice for 2 minutes. The supernatant is the template DNA for PCR amplification.

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Abstract

The invention belongs to the field of a detection reagent, and relates to a detection and identification kit for MTBC (mycobacterium tuberculosis complex) and an identification method of the MTBC. According to the method, fluorescence probes and amplification primers for detecting the MTBC are designed through sequence alignment, mycobacterium and the MTBC specific SNP (single nucleotide polymorphism) loci on an rrs gene are detected based on a real-time fluorescent quantitative PCR (polymerase chain reaction) platform and with an asymmetric PCR amplification technology and a probe melting curve analysis technology, so that mycobacteria are identified, and the MTBC and NTM (nontuberculosis mycobacteria) are further identified. The method has the characteristics of convenience in operation, short detection time and high specificity and sensitivity.

Description

technical field [0001] The invention belongs to the field of detection reagents, and relates to a detection and identification kit for mycobacterium nucleatum complex and a method for identification of mycobacterium tuberculosis complex, in particular to detecting the specific single nucleotide polymorphism position of mycobacterium 16S rRNA coding gene Point, identification of Mycobacterium bacteria, and identification kits and identification methods of Mycobacterium tuberculosis complex and non-tuberculous mycobacteria. Background technique [0002] The prior art discloses that tuberculosis is an infectious disease that threatens human health for a long time. Its pathogen is Mycobacterium tuberculosis, and about one-third of the world's population is infected with Mycobacterium tuberculosis. According to the statistics of the World Health Organization, in 2011, there were 8.7 million new cases of tuberculosis in the world, and about 1.4 million people died of tuberculosis....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
CPCC12Q1/6858
Inventor 罗涛柳清云高谦
Owner FUDAN UNIV
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