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Method for detecting mycobacterium tuberculosis complex through multi-crossover amplification and biosensing

A Mycobacterium tuberculosis, biosensing technology, applied in the field of microbiology, can solve the problems of expensive, difficult to judge the results, limit the promotion and application, etc., achieve high detection ability, high amplification specificity, and improve the effect of sensitivity

Inactive Publication Date: 2019-05-28
BEIJING CHILDRENS HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the former relies on expensive instruments and consumables, while the interpretation of the latter mainly depends on the color change of the reaction system. In specimens with a small amount of bacteria, it is easy to make the results difficult to judge.
Other techniques related to fluorescent PCR also rely on expensive equipment, probe synthesis and skilled operators
Therefore, the above factors limit the promotion and application of these technologies in underdeveloped areas

Method used

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  • Method for detecting mycobacterium tuberculosis complex through multi-crossover amplification and biosensing
  • Method for detecting mycobacterium tuberculosis complex through multi-crossover amplification and biosensing
  • Method for detecting mycobacterium tuberculosis complex through multi-crossover amplification and biosensing

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Experimental program
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Effect test

Embodiment 1

[0051] The feasibility verification of embodiment 1.MCDA primer

[0052] MCDA amplification (see patent CN106544434A for details).

[0053] Standard MCDA reaction system (a set of primers): the concentration of the cross primer 6110-CP1, 6110-CP2 (or 1081-CP1, 1081-CP2) is 60 pmol, and the replacement primer 6110-F1, 6110-F2 (or 1081-F1, The concentration of 1081-F2) is 10pmol, the concentration of amplification primers 6110-R1, 6110-R2, 6110-D1, 6110-D2 (or 1081-R1, 1081-R2, 1081-D1 and 1081-D2) is 30pmol, The concentration of amplification primers 6110-C1*, 6110-C2 (or 1081-C1* and 1081-C2) is 20pmol, 10mM Betain, 6mM MgSO4, 1mM dNTP, 12.5μL of 10×Bst DNA polymerase buffer , 10U of strand-displacing DNA polymerase, 1 μL of template, add deionized water to 25 μl. The entire reaction was kept at 67°C for 1 hour, and the reaction was terminated at 85°C for 5 minutes.

[0054] Improved MCDA reaction system (two sets of primers): cross primers 6110-CP1, 6110-CP2, 1081-CP1 and ...

Embodiment 2

[0057] The optimal reaction temperature determination of embodiment 2.MCDA technology

[0058] Under standard reaction system conditions, the Mycobacterium tuberculosis DNA template and the designed MCDA primer (IS6110 or IS1081) were added respectively, and the template concentration was 1 ng / μL. The reaction was carried out under constant temperature conditions (61-68°C), and the results were detected by a real-time turbidimeter, and different dynamic curves were obtained at different temperatures, see Figure 3A with Figure 3B , Figure 3A Indicates for IS6110, Figure 3BIt shows the temperature dynamic curve of MCDA primers designed for IS1081 gene sequence to detect MTBC. 65-68°C (Figure 3A5-3A8, Figure 3B5-B8) is recommended as the optimal reaction temperature for MCDA primers of IS6110 or IS1081. In the subsequent verification of the present invention, 67° C. was selected as the constant temperature condition for MCDA amplification.

Embodiment 3

[0059] Embodiment 3.MCDA-LFB detects the sensitivity determination of MTBC

[0060] After serially diluted Mycobacterium tuberculosis DNA was used for MCDA amplification reaction, LFB detection showed: (1) The detection range of MCDA-LFB (IS6110) was 10 ng to 10 fg, and LFB appeared red lines in the TL and CL regions ( Figure 4 Aa1-Aa7 in ). When the amount of genomic template in the reaction system is reduced to below 10fg, LFB only appears a red line in the CL area, indicating a negative result ( Figure 4 Aa8 in ). Figure 4 Aa in Aa uses LFB to visualize and read MCDA amplification results; Figure 4 Aa1 to Aa8 in represent the template amounts of Mycobacterium tuberculosis are 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg and 1fg, Figure 4 Aa9 in is the blank control (1 μL double distilled water). (2) The detection range of MCDA-LFB (IS1081) is 10ng ~ 100fg, and LFB appears red lines in the TL and CL areas ( Figure 4 Ba1-Ba6 in). When the amount of genomic template in...

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Abstract

The invention discloses a method for detecting a target gene through multi-crossover constant-temperature amplification and nano-biosensing (MCDA-LFB), which can simultaneously detect specific genes IS6110 and IS1081 of a mycobacterium tuberculosis complex (MTBC). The invention also discloses a specific primer combination which aims at the two genes and is applied to the method. The whole MCDA amplification can be completed within 30 minutes by the multi-crossover constant-temperature amplification method, the detection range for the two genes is 10 ng to 10 fg, and accordingly, extremely highsensitivity is achieved; only members belonging to the MTBC can have positive results through MTBC-MCDA-LFB tests, non-specific amplification of other common pathogens and opportunistic pathogens does not occur, and accordingly, the method has extremely high specificity.

Description

technical field [0001] The invention discloses a method for detecting a target gene by applying constant temperature amplification technology, which belongs to the field of microbiology. Background technique [0002] Mycobacterium tuberculosis complex (Mycobacterium tuberculosis complex, MTBC) includes Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium microti and BCG, among which more than 90% of human tuberculosis is caused by Mycobacterium tuberculosis Sincerely. Tuberculosis is an important infectious disease that seriously endangers human health. According to WHO estimates, there were 10 million new cases of tuberculosis in the world in 2017, of which 1.3 million died of tuberculosis, becoming a major public health problem in all countries. The early differential diagnosis of tuberculosis is an important factor in determining the clinical outcome of patients. Therefore, it is necessary to develop a rapid tuberculosis detection metho...

Claims

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Application Information

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IPC IPC(8): C12Q1/6825C12Q1/6844C12Q1/689C12Q1/04C12N15/11C12R1/32
CPCC12Q1/6825C12Q1/6844C12Q1/68Y02A50/30
Inventor 申阿东焦伟伟王毅孙琳肖婧李洁琼王亚翠祁雪綦辉申晨徐放王咏红郭雅洁马琦陈玉莹
Owner BEIJING CHILDRENS HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV
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