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Primer composition, corresponding kit and detection method for rt-lamp detection of live bacteria of Mycobacterium tuberculosis

A RT-LAMP, Mycobacterium tuberculosis technology, applied in the field of molecular detection, can solve the problems of high cost, inability to distinguish Mycobacterium tuberculosis from non-tuberculous mycobacteria, and inability to distinguish Mycobacterium tuberculosis from non-tuberculous mycobacteria, etc.

Active Publication Date: 2020-10-23
中国人民解放军联勤保障部队第九八〇医院
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Although the stained smear is simple and easy to perform, it has poor sensitivity and low detection rate, and cannot distinguish between dead bacteria and live bacteria, nor can it distinguish tuberculosis mycobacteria from non-tuberculous mycobacteria; the Roche solid culture method has a long culture period and requires 4 -8 weeks, the improved liquid rapid culture system shortens the time to about 10 days, but it is expensive and easily contaminated, only vigorous bacteria can be detected, and the culture method cannot distinguish Mycobacterium tuberculosis from non-tuberculous mycobacteria; Experimental techniques based on amplified nucleic acid are fast, sensitive, and specific, such as PCR, LAMP, etc. However, because PCR technology has high requirements for operator skills, the required thermal cycle equipment is expensive and difficult to obtain Promotion, loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) overcomes the above shortcomings, has the advantages of fast response and simple operation, a water bath and other constant temperature equipment can complete the operation, and the amplification result can be judged by naked eyes The operation can be completed with simple training for employees, and is suitable for application in primary medical institutions. However, molecular biology detection methods such as PCR and LAMP are based on primers designed for the target sequence of Mycobacterium tuberculosis DNA and amplified. Bacteria showed positive results, unable to evaluate the effect of anti-tuberculosis treatment clinically

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  • Primer composition, corresponding kit and detection method for rt-lamp detection of live bacteria of Mycobacterium tuberculosis
  • Primer composition, corresponding kit and detection method for rt-lamp detection of live bacteria of Mycobacterium tuberculosis
  • Primer composition, corresponding kit and detection method for rt-lamp detection of live bacteria of Mycobacterium tuberculosis

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1 A kind of primer composition for RT-LAMP detection of Mycobacterium tuberculosis live bacteria

[0036] This embodiment includes:

[0037] Forward outer primer F3: 5'-TCCTGGCTCAGGACGAAC-3' (SEQ ID NO: 1);

[0038] Reverse outer primer B3: 5'-CGCTTTTCCACCACAAGACAT-3' (SEQ ID NO: 2);

[0039] Forward internal primer FIP: 5'-TCGCCACTCGAGTATCTCCGAAGCGGCGTGCTTAACACAT-3' (SEQ ID NO: 3);

[0040] Reverse internal primer BIP: 5'-AGTAACACGTGGGTGATCTGCCATCCCGTGGTCCTATCCG-3' (SEQ ID NO: 4);

[0041] Reverse Loop Primer LB: 5 ， -ATAAGCCTGGGAAACTGGGT-3 ， (SEQ ID NO: 5).

[0042] The design process of the primer composition is as follows:

[0043] Download the complete 16S rRNA sequence of non-tuberculosis bacilli from the NCBI database, and use the Clustal Omega tool to compare the sequences of the standard strain of tuberculosis bacilli (Genbank number: NR_102810.1) with various non-tuberculosis bacilli, including: Mycobacterium intracellulare ( M.intracellμLare NR...

Embodiment 2

[0045] Example 2 A RT-LAMP kit for detecting live bacteria of Mycobacterium tuberculosis

[0046] This embodiment is an RT-LAMP kit for detecting viable Mycobacterium tuberculosis bacteria, and the kit includes an RT-LAMP amplification reaction solution.

[0047] RT-LAMP amplification reaction solution is 25 μL, including the following components:

[0048] 0.2 μM forward outer primer F3 (provided in Example 1), 0.2 μM reverse outer primer B3 (provided in Example 1), 1.6 μM forward inner primer FIP (provided in Example 1), 1.6 μM reverse inner Primer BIP (provided in Example 1), 0.8 μM reverse loop primer LB (provided in Example 1), 0.8 M betaine, 8 mM MgSO 4 , 1.4 mM dNTPs, 8 U Bst DNA polymerase, 20 U recombinant ribonuclease inhibitor, 100 U M-MLV reverse transcriptase, 2.5 μL 10×buffer, 1 μL template RNA, 5 mM / μL calcein, 10 mM / μL MnCl 2 , 11.7 μL deionized water.

[0049] In a specific experiment, in order to compare the effects, the kit also includes a control. The con...

Embodiment 3

[0051] Example 3 A method for detecting live Mycobacterium tuberculosis by RT-LAMP

[0052] In this embodiment, the kit provided in Example 2 is used in the detection process, and the specific detection method is as follows:

[0053] (31) Use the RNA extraction kit to extract the RNA of Mycobacterium tuberculosis from the extract to be tested containing Mycobacterium tuberculosis, and obtain template RNA as one of the components in the RT-LAMP amplification reaction solution (template RNA) ;

[0054] The extract to be tested in this example is taken from the sputum of patients diagnosed with pulmonary tuberculosis, which is to use the sputum as a sputum sample, add 2 times the volume of sputum RNA protective agent, and use 2 times the volume of 2% N-acetyl-L -Cysteine ​​(NALC)-NaOH solution for digestion, tighten the cap, vortex and shake on the vortex shaker for 2 minutes, until the sputum sample is fully liquefied, use the RNA extraction kit to extract the sample RNA accord...

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Abstract

A primer composition and kit for RT-LAMP detection of mycobacterium tuberculosis live bacteria are disclosed. The primer composition is reasonably designed and high in specificity. The kit comprising the primer composition is high in specificity, high in sensitivity, and convenient to operate, and an amplification result can be observed through naked eyes. The invention also provides a method of detecting mycobacterium tuberculosis live bacteria through RT-LAMP detection. The method is simple, high in sensitivity, convenient and rapid in detection, rapid in amplification, and time-saving, allows observation of the amplification result with naked eyes to be possible, and can specifically detect dead bacteria or live bacteria. The primer composition, the kit and the method are suitable for detection of the mycobacterium tuberculosis live bacteria.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and relates to a primer composition, a kit and a detection method, in particular to a primer composition, a corresponding kit and a detection method for RT-LAMP detection of viable Mycobacterium tuberculosis bacteria. Background technique [0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis that seriously affects human health. It is one of the ancient diseases that still plague humans. The existence of Mycobacterium tuberculosis has been found in ancient Egyptian mummies. Even in recent years, Around 9 million people also become infected with TB every year. With the emergence of drug-resistant and multidrug-resistant strains, the difficulty of tuberculosis control continues to increase, and the establishment of rapid, sensitive, and specific diagnostic methods is of great significance for the prevention and control of tuberculosis, and is also a p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2527/127C12Q2521/107
Inventor 孙殿兴吴丹丹亢继文李保胜牛莉娅
Owner 中国人民解放军联勤保障部队第九八〇医院
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