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Pretreating liquid for tubercle bacillus sputum specimen and its application

A technology of Mycobacterium tuberculosis and pretreatment solution, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc. It can solve the problems of low detection rate of clinical specimens, high nucleic acid loss rate, cumbersome steps, etc., and achieves a wide range of uses , high sensitivity, and easy operation

Inactive Publication Date: 2004-08-18
BEIJING HOSPITAL
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Problems solved by technology

Traditional nucleic acid extraction methods are based on the different denaturation effects of organic solvents (phenol or chloroform) on nucleic acids and proteins. Therefore, it is urgent to establish a nucleic acid probe detection method with high sensitivity and high specificity.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Mycobacterium tuberculosis clinical sputum collection:

[0027] Among the 83 clinical samples, 36 TB patients had sputum smear-positive samples, 32 TB patients had sputum smear-negative samples, and 18 non-tuberculosis samples came from the Department of Tuberculosis of the No. 309 Hospital of the People’s Liberation Army and the Department of Respiratory Medicine of Beijing Hospital.

[0028] Pretreatment of sputum specimens: Take 0.1ml of the specimen, add 0.2ml of primary treatment solution (4mol / l NaoH), liquefy at room temperature for 30 minutes, incubate the specimen in a water bath at 90°C for 1 hour, and centrifuge the specimen at 12000rpm for 15 minutes , Discard the supernatant, mix the centrifuged precipitate with 100μl lysate, place in a 60°C water bath, incubate for 1 hour, then place in a 92-96°C water bath for 15 minutes, centrifuge at 8000rpm for 5 minutes, take the supernatant for later use (lysate components are: NP40 (weight percent) 0.5%, Tris-HCl10m...

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Abstract

The pretreating liquid includes initial treating liquid and cracking liquid. The initial treating liquid is NaOH solution of 2-6 mol / L concentration. The cracking liquid consists of NP40, Tris-HCL, NaCl and MgCl2. The use of the pretreating liquid makes it possible for direct hybridization by use of DNA probe without PCR before horseradish peroxidase catalyzed reaction, such as chemical luminous self-developing detection. The present invention can obtain high sensitivity and high specificity and has simple operation, low cost and wide use.

Description

technical field [0001] The present invention relates to a kind of sample pretreatment liquid and its detection method in the gene detection of nucleic acid probe hybridization, especially a kind of Mycobacterium tuberculosis sputum sample pretreatment liquid and its application in horseradish peroxidase-labeled nucleic acid probe hybridization A method for detecting the genes of Mycobacterium tuberculosis specimens. Background technique [0002] At present, the prevalence of tuberculosis in the world is on the rise, and tuberculosis is still the number one killer of infectious diseases in my country. In the traditional detection methods of tuberculosis laboratories, the positive rate of acid-fast staining microscopic examination is only about 30%; the positive rate of tuberculin skin test is 75%, but the problem of false positives is serious; although the culture results are reliable, the growth of tuberculosis is extremely slow. It needs to be cultivated for 3-8 weeks. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/00C12Q1/68
Inventor 杨树德杨华卫
Owner BEIJING HOSPITAL
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