Method and detection kit for detecting mycobacterium tuberculosis complex cluster based on thermostatic technology
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A technology for Mycobacterium tuberculosis and detection method, which is applied in the directions of microorganism-based methods, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of short detection time, expensive instruments, and low reagent costs, and achieve short detection time. , the effect of strong specificity and low background
Active Publication Date: 2014-07-02
广州迪澳生物科技有限公司
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Problems solved by technology
The traditional biochemical identification method based on bacterial culture is laborious and time-consuming, the results are not provided in time, and sometimes the results are inaccurate
With the development of molecular biology, many identification methods based on molecular biology techniques have emerged, such as PCR-SSCP, PCR-RFLP, DNA probes, DNA chips, DNA direct sequencing, etc., with their advantages of high efficiency and specificity It is favored, but requires high technical level of testing personnel, expensive equipment, and long testing cycle. In order to meet the needs of rapid identification and classification, it provides a fast and convenient method for scientific research, shortens the identification time of Mycobacterium tuberculosis complex, and reduces the identification technology. Difficulty and cost, the present invention relies on constant temperature amplification technology to develop a method and detection kit that can quickly identify the Mycobacterium tuberculosis complex. Inexpensive, short detection time, low reagent cost, low technical requirements, etc., at the same time, this method can be applied to the rapid identification of other bacterial groups
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Embodiment 1
[0043] Example 1: Primer design
[0044] According to the GyrB gene of Mycobacterium tuberculosis, five SmartAmp constant-temperature amplification primers were designed. The sequences are as follows:
[0045] TP: GCCTTCCCGGATATCGTCACGGTGAACAAGTACGCCAA (SEQ ID NO:1)
[0046] FP: TTTATATATATATAAACGATTTGACCTCGGTGTT (SEQ ID NO: 2)
[0047] BP: GCCAGACCAAGACCAAGTT (SEQ ID NO: 3)
[0048] OP1: CTTCGCCAACACCATCAA (SEQ ID NO: 4)
[0049] OP2: GCTGTTCGTTACAGACCTT (SEQ ID NO: 5)
Embodiment 2
[0050] Example 2: Establishment of a kit for detecting Mycobacterium tuberculosis complex based on constant temperature technology
[0051] The kit includes: 5 SmartAmp constant temperature amplification primers shown in SEQ ID NO: 1 to 5, constant temperature amplification reaction solution, SYTO-9, Tris-HCl, Triton X-100, Bst DNA polymerase, wrong With binding protein Tap Muts.
[0052] The amplification reaction solution contains: dNTP, betaine, (NH 4 ) 2 SO 4 , MgCl 2 , KCl and MgSO 4 .
Embodiment 3
[0053] Example 3: Establishment of a method for rapid detection of Mycobacterium tuberculosis complex based on constant temperature technology
[0054] Reaction system: 25μL reaction system contains TP and EP primers 1.6 μmol / L each, BP primer 0.8 μmol / L, OP1 and OP2 primers 0.5 μmol / L, dNTP 0.5 mmol / L, betaine 0.6mol / L, (NH 4 ) 2 SO 4 10 mmol / L, MgCl 2 2.5 mmol / L, KCl 10 mmol / L, MgSO 4 2 mmol / L, 5 μmol / L SYTO-9, pH 8.8 Tris-HCl 20 mmol / L, 0.1% Triton X-100, 6U Bst DNA enzyme, 1μg Tap Muts, 1μL DNA template.
[0055] The reaction procedure is: 60~65℃ for 20~40 min.
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Abstract
The invention discloses a method and a detection kit for detecting a mycobacterium tuberculosis complex cluster based on a thermostatic technology. According to the method, 5 primers are designed aiming at a GyrB gene of mycobacterium tuberculosis, wherein TP is composed of an annealing sequence and a nucleotide sequence complementary with a target sequence; FP has a loop-stem structure, and can anneal with two complementary DNA single strands from two ends and stretch towards an opposite side; annealing sites of OP1 and OP2 are respectively located on the outer sides of the TP and the FP, anneal and stretch towards middle and simultaneously peel off double strands stretching from the TP and the FP; a single strand with the TP or FP, after being replaced, can serve as a template to continue the process, thus forming two key single-stranded intermediate products; the two intermediate products are then used for synthesizing DNA through self-guidance by taking special structures of the TP and the FP as starting points; through such circulation and repetition, a self-circulating chain displacement reaction is triggered under function of DNA polymerase, thus realizing massive amplification of the target sequence.
Description
technical field [0001] The invention belongs to the field of biotechnology detection, and in particular relates to a method and a detection kit for detecting mycobacterium tuberculosis complex based on constant temperature technology. Background technique [0002] Mycobacterium (Mycobacterium) includes more than 140 species, which are generally divided into three categories, Mycobacterium tuberculosis complex (MTBC), M. leprae (M.leprae) and non-tuberculous mycobacteria ( nontuberculous mycobacterial, NTM). MTBC includes Mycobacterium tuberculosis human, Mycobacterium bovis, Mycobacterium africanum, and Mycobacterium microti. They are highly pathogenic and are the main pathogens that cause tuberculosis in animals, but there are relatively few species. Compared with it, NTM has a large number (up to more than a hundred species) and a wide distribution, and some of them are pathogenic bacteria or conditional pathogens. Because the morphology of NTM is similar to that of MTB, ...
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