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Isothermal nucleic acid amplification technique based kit for diagnosis of tuberculosis

A kit, a technology for pulmonary tuberculosis, is applied in recombinant DNA technology, microbial determination/examination, DNA/RNA fragments, etc. It can solve the problems of many affected factors, use restrictions, and high costs, and achieve major application prospects, simple equipment, and high cost. low cost effect

Inactive Publication Date: 2012-10-17
THE 309TH HOSPITAL OF CHINESE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is based on the secretion of a single cytokine, and many factors are affected. It has been reported that its sensitivity for diagnosing active tuberculosis is only 64%.
[0008] Although fluorescent real-time quantitative PCR (polymerase chain reaction) technology has high sensitivity and specificity in the diagnosis of tuberculosis, it requires special equipment, high cost and limited use

Method used

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  • Isothermal nucleic acid amplification technique based kit for diagnosis of tuberculosis
  • Isothermal nucleic acid amplification technique based kit for diagnosis of tuberculosis
  • Isothermal nucleic acid amplification technique based kit for diagnosis of tuberculosis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, the preparation of special primer

[0038] Each primer shown in Table 1 was synthesized. The dedicated primers consist of four primers (IS1081-F3, IS1081-B3, IS10811-FIP, IS1081-BIP). See Table 1 for the nucleotide sequences of IS1081-F3, IS1081-B3, IS1081-FIP, and IS1081-BIP.

[0039]The nucleotide sequence of each primer in table 1 special primer

[0040] Primer name

[0041] The transposase gene sequence of Mycobacterium tuberculosis H37Rv standard strain IS1081 (GenBank: BX842575) is shown in sequence 5 of the sequence table, and the primers are located in figure 1 . Special primers amplify a partial fragment (203bp) of the IS1801 target gene.

Embodiment 2

[0042] Embodiment 2, Sensitivity and specificity of special primer detection pathogenic mycobacteria

[0043] The samples to be tested were: 20 mycobacterial standard strains and 3 non-mycobacterial control strains (see Table 2). ATCC is the American type culture collection, and its website is http: / / www.atcc.org / .

[0044] The samples to be tested were subjected to the following experiments:

[0045] 1. Genomic DNA extraction

[0046] After aseptic treatment of the sample to be tested, add TE buffer solution containing 5mg / ml lysozyme, and incubate overnight at 37°C; then add protease A (final concentration is 2mg / ml) and SDS (final concentration is 1%; mass percentage ), incubated overnight in a 56°C water bath; then the genomic DNA was extracted by the phenol-chloroform method, dissolved in 10mM Tris-HCl buffer, and frozen at -20°C for future use.

[0047] 2. Loop-mediated gene isothermal amplification (LAMP)

[0048] LAMP reaction system (25 μl): containing 12.5 μl 2×L...

Embodiment 3

[0066] Embodiment 3, application-specific primer detection tuberculosis patient's sputum sample, is used for tuberculosis diagnosis

[0067] Samples to be tested: sputum from 18 cases of pulmonary tuberculosis patients (volunteers who gave informed consent) with positive sputum smear acid-fast staining; sputum from 2 cases of non-tuberculous pneumonia patients (volunteers who gave informed consent).

[0068] The samples to be tested were subjected to the following experiments:

[0069] 1. Genomic DNA extraction.

[0070] 2. Loop-mediated constant temperature amplification of genes

[0071] LAMP reaction system (25 μl): containing 12.5 μl 2×LAMP reaction buffer (Eiken Chemical, Tochigi, Japan), 1 μl Bst DNA polymerase (Eiken Chemical, Tochigi, Japan), 1 μl Fluorescent Detection Reagent (Eiken Chemical, Tochigi, Japan), 0.2 μmol IS1081-F3, 0.2 μmol IS1081-B3, 1.6 μmol IS1081-FIP, 1.6 μmol IS1081-BIP and 1 μl genomic DNA, and the rest was water; 1 μl 10 mM Tris-HCl buffer was u...

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Abstract

The invention discloses an isothermal nucleic acid amplification technique based kit for diagnosis of tuberculosis. The invention provides a special primer for assisting identification of pathogenic mycobacteria; and the special primer comprises DNA shown in the sequence 1 in the sequence table, DNA shown in the sequence 2 in the sequence table, DNA shown in the sequence 3 in the sequence table and DNA shown in the sequence 4 in the sequence table. The invention also protects a kit containing the special primer. The kit provided by the invention has the advantages of high sensitivity, strong specificity, easy operation, simple equipment and low cost, can be used for the detection of pathogenic mycobacteria to further assist diagnosis of tuberculosis, and has significant application prospect.

Description

technical field [0001] The invention relates to a kit for diagnosing tuberculosis based on constant temperature nucleic acid amplification technology. Background technique [0002] Mycobacterium (Mycobacterium) is a class of slender slightly curved microorganisms, sometimes branched or filamentous. The main characteristic of this genus of bacteria is that the cell wall contains a large amount of lipids, mainly mycolic acids. This is closely related to its dyeability, growth characteristics, pathogenicity, and resistance. Generally, it is not easy to be colored, but it can resist the decolorization of strong decolorizing agent hydrochloric acid ethanol after being colored by heating or prolonging the dyeing time, so it is also called acid-fast bacilli. The bacterium has no flagella, no spores, and does not produce endotoxins and exotoxins. Its pathogenicity is related to the composition of the bacteria. The diseases caused are all chronic and accompanied by granulomas. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 程小星杨秉芬
Owner THE 309TH HOSPITAL OF CHINESE PEOPLES LIBERATION ARMY
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