CRISPR detection primer set for MTBC (Mycobacterium Tuberculosis Complex) and use thereof

A technology for Mycobacterium tuberculosis and detection primers, which is applied in microorganism-based methods, microorganism determination/inspection, microorganisms, etc., can solve problems such as inability to improve sensitivity, and achieve the effect of getting rid of the dependence of precision instruments and having broad application prospects.

Active Publication Date: 2019-05-28
广州微远医疗器械有限公司 +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in principle, the essence of its single-stage amplification reaction has not changed. Compared with most real-ti

Method used

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  • CRISPR detection primer set for MTBC (Mycobacterium Tuberculosis Complex) and use thereof
  • CRISPR detection primer set for MTBC (Mycobacterium Tuberculosis Complex) and use thereof
  • CRISPR detection primer set for MTBC (Mycobacterium Tuberculosis Complex) and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Design and screening of primer sequences for CRISPR detection of Mycobacterium tuberculosis complex.

[0042] 1. Sequence design.

[0043] 1. Target sequence selection.

[0044] The inventor selected the sequence shown in SEQ ID NO:1 as Mycobacterium tuberculosis (M.tuberculosis), Mycobacterium bovis (M.bovis), African The target sequence of mycobacterium (M.africanum), mycobacterium canneri (M.canettii) and mycobacterium microti (M.microti), which is a conserved sequence of the above-mentioned bacteria, can detect the above-mentioned tuberculosis pathogenic bacteria out. The sequence of SEQ ID NO:1 is as follows:

[0045] AAAGACCGCGTCGGCTTTCTTCGCGGCCGAGCTCGACCGGCCAGCACGCTAATTACCCGGTTCATCGCCGATCATCAGGGCCACCGCGAGGGCCCCGATGGTTTGCGGTGGGGTGTCGAGTCGATCTGCACACAGCTGACCGAGCTGGGTGTGCCGATCGCCCCATCGACCTACTACGACCACATCAACCGGGAGCCCAGCCGCCGCGAGCTGCGCGATGGCGAACTCAAGGAGCACATCAGCCGCGTCCACGCCGCCAACTACGGTGTTTACGGTGCCCGCAAAGTGTGGCTAACCCTGAACCGTGAGGGCATCGAGGTGGCCAGATGCACCGTCGAACGGCTGATGAC...

Embodiment 2

[0082] A test kit for detecting Mycobacterium tuberculosis complex, comprising:

[0083] (1) RPA amplification system:

[0084] RPA amplification primer pair:

[0085] Forward direction: 5'-GGTCGGAAGCTCCTATGACAATGCACTAGCC-3'(SEQ ID NO:4), the concentration is 0.1~1μM

[0086] Reverse: 5'-TTGAGCGTAGTAGGCAGCCTCGAGTTCGAC-3' (SEQ ID NO: 5), the concentration is 0.1-1 μM.

[0087] RPA enzyme master mix: creatine phosphate (concentration 20-80mM), creatine kinase (concentration 50-150mM), dNTPs (concentration 100-300μM), ATP (concentration 20-80mM), DTT (concentration 1-10mM), acetic acid Potassium (concentration 50-200mM), recombinant enzyme uxsX (50-300ng / μL), uxsY (10-100ng / μL), single-chain binding protein (200-1000ng / μL), Bsu polymerase (10-100ng / μL) Wait.

[0088] Magnesium acetate solution: the concentration is 10-20mM

[0089] (2) CRISPR reaction system:

[0090] crRNA: 5'-UAAUUUCUACUAAGUGUAGAUAUCAGCUCGGUCUUGUAUAG-3' (SEQ ID NO:3), the concentration is 20-100nM

[009...

Embodiment 3

[0110] The detection of Mycobacterium tuberculosis complex in clinical samples was carried out with the above kit of Example 2.

[0111] 1. Method

[0112] (1) Sample source: Department of Infectious Diseases, Shanghai Huashan Hospital.

[0113] A total of 120 clinical samples were tested blindly with the GeneXpert detection system widely recognized in the market and the method of the present invention.

[0114] (2) Detection method:

[0115] With reference to the relevant detection steps of the present invention in Example 2, 120 samples were tested.

[0116] 120 samples were detected by referring to the relevant operation instructions of the GeneXpert detection system.

[0117] 2. Results

[0118] The detection results using the two methods are shown in the table below:

[0119] Table 2 The detection results of clinical samples by different methods

[0120]

[0121]

[0122] After calculation, it can be seen that:

[0123] Positive coincidence rate = 97.06%

[...

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Abstract

The invention relates to a CRISPR detection primer set for MTBC (Mycobacterium Tuberculosis Complex) and use thereof, and belongs to the technical field of gene detection. The primer set includes an amplification primer pair and a crRNA; the amplification primer pair is used to amplify a common conserved sequence of M. tuberculosis, M. bovis, M. africanum, M. canettii, and M. microti; the crRNA includes an anchor sequence and a guide sequence, wherein the anchor sequence is bound to a cas protein, and the guide sequence is matched with a targeted RNA fragment in the common conserved sequence.Through the primer set based on CRISPR detection technology, the detection of common conserved sequences of M. tuberculosis, M. bovis, M. africanum, M. canettii, and M. microti by CRISPR detection canquickly perform on-site detection of MTBC, and have the advantages of high specificity, high sensitivity and simplicity.

Description

technical field [0001] The invention relates to the technical field of gene detection based on CRISPR technology, in particular to a CRISPR detection primer set for Mycobacterium tuberculosis complex and its application. Background technique [0002] Tuberculosis (TB) is one of the ten leading causes of death worldwide. According to the World Health Organization (WHO) report, in 2016, there were an estimated 10.4 million new cases of tuberculosis worldwide, of which only 6.3 million were detected and reported, leaving a gap of 4.1 million cases (data from the 2017 Global Tuberculosis Report"). Therefore, the prevention and control of tuberculosis is related to the national public health security, and the key to controlling the spread of tuberculosis is early and accurate diagnosis. [0003] The pathogenic bacteria of tuberculosis is Mycobacterium tuberculosis complex (Mycobacterium tuberculosis complex, MTBC), including Mycobacterium tuberculosis (M.tuberculosis), Mycobact...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12N15/11C12Q1/6844C12R1/32
Inventor 许腾曾伟奇杨敏玲周叙全王小锐李永军苏杭
Owner 广州微远医疗器械有限公司
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