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Reverse transcription polymerase chain reaction (PCR) method for detecting tubercle bacillus infection from clinical specimen

A technology of tuberculosis and reverse transcription, which is applied in the direction of biochemical equipment and methods, and microbial determination/inspection, can solve the problems of long cultivation time, complicated operation, and poor specificity, and achieve short time-consuming, simple detection operation, and positive high rate effect

Active Publication Date: 2014-02-12
HARBIN MEDICAL UNIVERSITY
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Problems solved by technology

The disadvantage of the CSF acid-fast bacilli smear method is that the positive rate is low, about 10%, and the bacteria in the sample must be ≥ 10%. 4 / ml can only be found, poor specificity
The shortcomings of the Mycobacterium tuberculosis culture detection method are that the operation is complicated, the culture time is long, it takes 4 to 8 weeks, and the positive rate is low, mostly in the range of 20 to 30%.

Method used

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  • Reverse transcription polymerase chain reaction (PCR) method for detecting tubercle bacillus infection from clinical specimen
  • Reverse transcription polymerase chain reaction (PCR) method for detecting tubercle bacillus infection from clinical specimen
  • Reverse transcription polymerase chain reaction (PCR) method for detecting tubercle bacillus infection from clinical specimen

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Embodiment Construction

[0035] Obtain clinical specimens: collect 3-5 ml of peripheral blood from the patient, centrifuge horizontally at 1000-1500 rpm for 10 minutes. Carefully pipette 200 µl of the upper plasma layer into a sterile 1.5 ml centrifuge tube. Other liquid samples were centrifuged at 13,000 rpm for 10 minutes, and 200 microliters of the upper liquid was carefully drawn into a sterile 1.5ml centrifuge tube.

[0036] Step 1: Extract total RNA from clinical specimens.

[0037] Add 500 microliters of phenolguanidine RNA extract to 200 microliters of clinical specimens, mix well, and let stand at room temperature for 5 minutes. Add 300 microliters of chloroform, mix well and centrifuge at 12000 rpm for 10 minutes. Carefully pipette the supernatant liquid into a sterile 2 ml centrifuge tube. Add 900 microliters of acetone, mix well and absorb with silica gel membrane. Wash once with protein washing solution, wash once with 100% ethanol and 80% ethanol in turn, after the ethanol volatilize...

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Abstract

The invention relates to a reverse transcription polymerase chain reaction (PCR) method for detecting tubercle bacillus infection from a clinical specimen. The PCR detection method is used for detecting small RNA (Ribonucleic Acid) of tubercle bacillus in the clinical specimen. The method comprises the following steps: 1, extracting total RNA in the clinical specimen; 2, performing reverse transcription by taking the total RNA extracted in the step 1 to synthesize cDNA (complementary Deoxyribonucleic Acid); 3, performing PCR detection aiming at a tubercle bacillus small RNA detection primer and cDNA synthesized in the step 2.

Description

technical field [0001] The invention relates to a reverse transcription PCR method for detecting Mycobacterium tuberculosis infection from clinical specimens. Background technique [0002] In the prior art, commonly used detection methods for tuberculosis infection include CSF acid-fast bacilli smear method and Mycobacterium tuberculosis culture detection method. The disadvantage of the CSF acid-fast bacilli smear method is that the positive rate is low, about 10%, and the bacteria in the sample must be ≥ 10%. 4 / ml can only be found, and the specificity is poor. The shortcomings of the Mycobacterium tuberculosis culture detection method are complicated operation, long culture time, 4-8 weeks, and low positive rate, mostly 20-30%. Contents of the invention [0003] The object of the present invention is to provide a reverse transcription PCR method for detecting Mycobacterium tuberculosis infection from clinical specimens, which is simple in operation, short in time cons...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/04C12Q1/686C12Q2531/113
Inventor 张凤民付英梅宋武琦钱均李玉军李文静
Owner HARBIN MEDICAL UNIVERSITY
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