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Method for detecting viable bacteria of Mycobacterium tuberculosis through isothermal amplification of nucleic acid and kit

A technology of isothermal amplification and detection method, applied in biochemical equipment and methods, measurement/inspection of microorganisms, analysis through chemical reaction of materials, etc., to achieve wide application value, important clinical significance, detection sensitivity and specificity sex enhancing effect

Inactive Publication Date: 2010-06-16
SHANGHAI FOSUN PHARMA (GROUP) CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Based on this RNA nucleic acid isothermal transcription amplification and nano-gold probe detection method (Nucleic Acid Isothermal and Transcriptional Amplification with Gold Nano-particle Probes, NITAG), the 85-B mRNA template of Mycobacterium tuberculosis can be amplified and detected, and tuberculosis can be realized. The live bacteria detection of mycobacteria, this technology has the advantages of no need for special expensive equipment, sensitive and fast detection, and intuitive and visible results, etc. It is suitable for the promotion and application of primary medical institutions. At present, there is no report on the application of NITAG technology in the detection of live bacteria

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  • Method for detecting viable bacteria of Mycobacterium tuberculosis through isothermal amplification of nucleic acid and kit
  • Method for detecting viable bacteria of Mycobacterium tuberculosis through isothermal amplification of nucleic acid and kit

Examples

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Embodiment 1

[0044] NITAG detection of Mycobacterium tuberculosis cultures

[0045] The implementation of the present invention is exemplified by introducing NITAG detection of mRNA extracted from cultures of Mycobacterium tuberculosis.

[0046] Bacterial culture : Mycobacterium tuberculosis H37Rv strain (ATCC 27294) was cultured in Middlebrook 7H9 broth for 7 to 14 days, harvested and dispersed with 4mm glass beads for 5 to 10 minutes, diluted with normal saline for use.

[0047] RNA extraction : Take 250 μl of the culture of Mycobacterium tuberculosis diluted with normal saline, add 500 μl of Trizol extraction reagent, repeatedly blow and mix with a pipette, place at room temperature for 5 minutes, add 200 μl of chloroform, shake back and forth by hand for 15 seconds, and place at room temperature 5 minutes, centrifuge at 12,000 rpm at 4°C for 15 minutes, transfer the supernatant to a new centrifuge tube, add 200 μl isopropanol, vortex for 5 seconds, settle in an ice bath for 10 minu...

Embodiment 2

[0050] NITAG detection of sputum samples from patients with clinical pulmonary tuberculosis

[0051] Pretreatment method : After adding 1ml of 2.5% N-acetyl-L-cysteine ​​(NALC) to the deep cough sputum specimen of tuberculosis patients in the morning, transfer it to a test tube containing 4mm glass beads for oscillation, and the homogenized sputum specimen in Store at -70°C until RNA extraction.

[0052] RNA extraction: Take 500 μl homogenized sputum sample, add 1000 μl Trizol extraction reagent, repeatedly blow and mix with a pipette, place at room temperature for 5 minutes, add 200 μl chloroform, shake back and forth by hand for 15 seconds, and place at room temperature for 5 minutes, 4 Centrifuge at 12,000 rpm for 15 minutes, transfer the supernatant to a new centrifuge tube, add 200 μl of isopropanol, vortex for 5 seconds, settle in an ice bath for 10 minutes, centrifuge at 12,000 rpm at 4°C for 10 minutes, discard the supernatant, and add 500 μl of pre-cooled Wash wi...

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Abstract

The invention relates to a method for detecting viable bacteria of Mycobacterium tuberculosis through isothermal amplification of nucleic acid and a kit thereof. The method comprises the following detection steps: unlinking an mRNA template of the Mycobacterium tuberculosis at the temperature of between 60 and 70 DEG C and then reducing temperature; adding reverse transcriptase, RNaseH and RNA polymerase, and performing isothermal amplification at the temperature of between 37 and 42 DEG C under the guidance of a primer to obtain RNA amplicon; and then detecting the amplicon by utilizing a nanogold probe and a capture probe, and obtaining a detection result through chromatography hybridization color development reaction. That a color developing stripe appears on a hybrid membrane represents mRNA positive (the viable bacteria exist), no stripe represents negative. The kit adopting the method can detect the viable bacteria of the Mycobacterium tuberculosis, has the advantages of accuracy, sensitivity, simpleness, convenience, quickness and the like, can overcome the defects of long detection time, complex operation, low specificity and the like of the conventional methods, and can serve as an auxiliary experimental means for related researches such as diagnosis and prevention of tuberculosis, observation of treatment effect, screening of tuberculostatics, and sensitivity experiments.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to the nucleic acid amplification detection technology of mycobacterium tuberculosis living bacteria. Background technique [0002] Since Koch discovered Mycobacterium tuberculosis (MTB) in 1882 as the pathogenic bacterium of tuberculosis for more than 100 years, human beings have made great efforts to understand the biological characteristics, pathogenic mechanism, drug resistance mechanism, diagnostic methods and anti-tuberculosis drugs of Mycobacterium tuberculosis. Great achievements have been made in research and development. Today, tuberculosis is still one of the infectious diseases with the most widespread prevalence and the highest fatality rate affecting human health. Seeking rapid and accurate diagnostic methods has always been the primary research area of ​​tuberculosis. [0003] In the past 10 years, with the development of molecular biology, there hav...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04G01N21/78
Inventor 吴大治夏懿吕元
Owner SHANGHAI FOSUN PHARMA (GROUP) CO LTD
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