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Primers and probe for detecting mycobacterium tuberculosis mRNA and application of primers and probe

A technology of Mycobacterium tuberculosis and a probe, applied in the field of pathogen detection, can solve the problems of PCR amplification, inability to judge bacterial activity, etc.

Inactive Publication Date: 2014-05-07
SHENZHEN INT TRAVEL HEALTHCARE CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR method for detection of Mycobacterium tuberculosis DNA and 16S rRNA cannot judge the activity of the bacteria in the body, and often the PCR amplification is positive but the culture is negative (Lu Yu, Zhu Lizhen, Duan Lianshan, etc. mRNA is used as the viable bacteria of Mycobacterium tuberculosis Feasibility study of detection markers[J]. Chinese Journal of Tuberculosis and Respiratory Medicine, 2003,26(7):419-423.)

Method used

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  • Primers and probe for detecting mycobacterium tuberculosis mRNA and application of primers and probe
  • Primers and probe for detecting mycobacterium tuberculosis mRNA and application of primers and probe
  • Primers and probe for detecting mycobacterium tuberculosis mRNA and application of primers and probe

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Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1, design and synthesis of primers and probes for RT-PCR detection of Mycobacterium tuberculosis

[0032] Use the ClustalW software to compare the Mycobacterium tuberculosis TB gene sequence in the NCBI database, find out the conserved sequence, and analyze the gene sequence without homology with other bacteria and microorganisms by NCBI-Blast, apply Primer5.0 and Primer in this area The express software designed primers and probes. The 5' end of the probe was marked with the fluorescent reporter group JOE (the fluorescence emission peak is at 520nm), and the 3' end was marked with the fluorescent quencher group BHQ1.

[0033] Forward primer (TB Forward): 5'-TGCAATGATGCTCGTGCAA-3' (SEQ ID NO: 1)

[0034] Reverse primer (TB Reverse): 5'-TCTACGCCGGGAATTTCG-3' (SEQ ID NO: 2)

[0035] Probe (TB Probe): 5'-CTGCTCGACCGCGACCTTCGTG-3' (SEQ ID NO: 3)

[0036] The 5' end of the probe ToSLCVP was labeled with the reporter fluorophore JOE, and the 3' end was labeled wi...

Embodiment 2

[0037] Embodiment 2, establishment of real-time fluorescent RT-PCR detection method

[0038] Samples to be tested: 22 tuberculosis patients' sputum, sputum smear acid-fast staining microscopic examination results were 4+, 3+, 2+, 1+ samples in 5 cases each, sputum smear acid-fast staining negative (-) samples in 2 cases . The diagnostic criteria for pulmonary tuberculosis cases and the microscopic examination method of sputum smear staining were in line with the Ministry of Health WS288-2008 "Diagnostic Criteria for Tuberculosis".

[0039]Sputum sample pre-treatment: before RNA extraction, use sputosol sputum thinner (Oxoid company, product catalog number: SR0233A) for digestion, 37 ° C water bath shaking to homogenize the sputum, in which the volume of sputosol sputum thinner and sputum The ratio is 1:1.

[0040] 1. Extraction of sample mRNA

[0041] Use the column type mRNA extraction kit (catalogue number 71201-50) produced by Beijing Tianenze Gene Technology Co., Ltd., ...

Embodiment 3、 Embodiment 2

[0050] The specificity experiment of embodiment 3, embodiment 2 method

[0051] Samples to be tested: 10 sputum samples from healthy people without symptoms of tuberculosis and mycobacteria fortuitously (ATCC6841) after clinical testing.

[0052] 1. Extraction of sample mRNA

[0053] Sputum sample pre-treatment: before RNA extraction, use sputosol sputum thinner (Oxoid company, product catalog number: SR0233A) for digestion, 37 ° C water bath shaking to homogenize the sputum, in which the volume of sputosol sputum thinner and sputum The ratio is 1:1.

[0054] Use the column type mRNA extraction kit (catalogue number 71201-50) produced by Beijing Tianenze Gene Technology Co., Ltd., add 0.5ml solution A (Trizol, included with the kit) to 0.5ml sample (sputum sample after processing) and 0.1 g of acidified glass beads (Beijing Tianenze Gene Technology Co., Ltd., catalog number 100307B10), and after fully shaking and mixing, use a heating oscillator at 1200 rpm at 60°C to shake ...

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Abstract

The invention discloses primers and a probe for detecting mycobacterium tuberculosis mRNA and application of the primers and the probe. The invention provides a composition for detecting mycobacterium tuberculosis. The composition consists of a primer pair and one probe, wherein the primer pair is composed of two single-stranded DNA molecules shown in a sequence I and a sequence II in a sequence table, and the sequence of the probe is a sequence III in the sequence table. Experiments show that the detection result has good sensibility, specificity and repeatability when the method is used for detecting the mycobacterium tuberculosis mRNA gene, and the method has diagnostic value to phthisis patients with negative sputum smear.

Description

technical field [0001] The invention belongs to the field of pathogen detection, and relates to a primer and a probe for detecting mycobacterium tuberculosis and applications thereof. Background technique [0002] Tuberculosis is a chronic infectious disease that seriously endangers human health. The newly revised "Regulations for the Implementation of the Frontier Health and Quarantine Law of the People's Republic of China" in 2010 clearly stipulates that health and quarantine agencies should prevent foreigners with contagious tuberculosis from entering the country. Accurate detection of live infectious Mycobacterium tuberculosis is the key to intercepting infectious tuberculosis at border ports. PCR detection technology with high sensitivity, strong specificity and short detection time is playing an increasingly important role in the prevention and control of tuberculosis. The PCR method for detection of Mycobacterium tuberculosis DNA and 16S rRNA cannot judge the activit...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/04C12Q1/686C12Q2561/101
Inventor 董瑞玲朱玉兰王佃鹏刘胜牙李微叶健忠张树平
Owner SHENZHEN INT TRAVEL HEALTHCARE CENT