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

A technology of mycobacterium tuberculosis and probes, which is applied in the field of pathogen detection and can solve the problems of inability to judge bacterial activity and PCR amplification, etc.

Inactive Publication Date: 2015-04-15
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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1. Design and synthesis of primers and probes for RT-PCR detection of Mycobacterium tuberculosis

[0032] Use ClustalW software to compare the TB gene sequence of Mycobacterium tuberculosis in the NCBI database to find the conservative sequence, and use NCBI-Blast analysis to have no homology sequence with other bacteria and microorganisms. Primer5.0 and Primer are used in this area The express software designs primers and probes. The 5'end of the probe is labeled with the fluorescent reporter JOE (the fluorescence emission peak is at 520nm), and the 3'end is labeled with the fluorescence quenching group BHQ1.

[0033] Forward primer (TB Forward): 5'-TGCAATGATGCTCGTGCAA-3' (sequence 1)

[0034] Reverse primer (TB Reverse): 5'-TCTACGCCGGGAATTTCG-3' (sequence 2)

[0035] Probe (TB Probe): 5'-CTGCTCGACCGCGACCTTCGTG-3' (sequence 3)

[0036] The 5'end of the ToSLCVP probe is labeled with the reporter fluorophore JOE, and the 3'end is labeled with the quencher fluorophore BHQ1...

Embodiment 2

[0037] Example 2. Establishment of real-time fluorescent RT-PCR detection method

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

[0039] Pretreatment of sputum samples: Digestion with sputosol sputum diluent (Oxoid company, catalog number: SR0233A) before RNA extraction, and homogenize the sputum by shaking in a 37°C water bath. The volume of sputosol and sputum The ratio is 1:1.

[0040] 1. Extraction of sample mRNA

[0041] Using the column-type mRNA extraction kit produced by Beijing Tianenze Gene Technology Co., Ltd. (catalog number 71201-50), 0....

Embodiment 3、 Embodiment 2

[0050] Example 3, specific experiment of the method of Example 2

[0051] Samples to be tested: 10 sputum specimens from healthy people without tuberculosis symptoms and Mycobacterium fortuitum (ATCC6841).

[0052] 1. Extraction of sample mRNA

[0053] Pretreatment of sputum samples: Digestion with sputosol sputum diluent (Oxoid company, catalog number: SR0233A) before RNA extraction, and homogenize the sputum by shaking in a 37°C water bath. The volume of sputosol and sputum The ratio is 1:1.

[0054] Using the column-type mRNA extraction kit produced by Beijing Tianenze Gene Technology Co., Ltd. (catalog number 71201-50), 0.5ml of solution A (Trizol, included with the kit) is added to 0.5ml sample (after-treatment sputum sample) And 0.1g of acidified glass beads (Beijing Tianenze Gene Technology Co., Ltd., catalog number is 100307B10), shake and mix thoroughly, then use a heating oscillator at 1200rpm and 60℃ to shake and heat for 5 minutes. Extract mRNA according to the kit instr...

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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 "Implementation Rules for the Frontier Health and Quarantine Law of the People's Republic of China" in 2010 clearly stipulated that health and quarantine agencies should prevent foreigners with infectious 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 detecting Mycobacterium tuberculosis DNA and 16S rRNA cannot determine the activity of the bacteria...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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