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Process for abstracting bacterial DNA from phlegm, kit and uses thereof

A bacterial nucleic acid and kit technology, applied in the direction of biochemical equipment and methods, microbial measurement/testing, resistance to vector-borne diseases, etc., can solve problems such as complex operations, long time-consuming, and difficulty in meeting clinical needs

Active Publication Date: 2011-07-20
CAPITALBIO CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This test is usually done on the basis of culture of sputum tubercle bacilli, so it takes longer
[0010] (4) Mycobacterium species identification, according to the physical and chemical characteristics of different mycobacteria, can accurately identify mycobacteria species, but the operation is complicated, and the drugs used in individual tests have certain risks
At present, the clinical detection of drug resistance of Mycobacterium tuberculosis is mainly based on culture plus drug sensitivity test. The method is reliable but time-consuming, generally takes 4-6 weeks, and it is difficult to meet the clinical needs

Method used

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  • Process for abstracting bacterial DNA from phlegm, kit and uses thereof
  • Process for abstracting bacterial DNA from phlegm, kit and uses thereof
  • Process for abstracting bacterial DNA from phlegm, kit and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1, the extraction of mycobacterium smegmatis nucleic acid in the sputum sample

[0076] 1. Take the sputum samples (provided by Beijing Municipal People's Hospital) of people whose acid-fast staining results are negative, and use them as test materials for future use.

[0077] 2. Into the above-mentioned sputum samples, the concentrations were respectively 10 2 -10 7 CFU / ml of M. smegmatis (American Type Culture Collection ATCC 700084) was used as a sputum sample for extraction of M. smegmatis nucleic acid.

[0078] 3. Add an equal volume of 20% (mass percentage) NaOH solution to the above-mentioned sputum samples mixed with different concentrations of Mycobacterium smegmatis, mix well, and liquefy at 37° C. for 30 minutes to obtain liquefied sputum.

[0079] 4. Take 1ml of the above liquefied sputum, centrifuge at 12000rpm for 5 minutes, and remove the supernatant; add 1ml of sterilized water containing 100mM EDTA, vortex and centrifuge at 12000rpm for 5 minu...

Embodiment 2

[0084] Embodiment 2, the identification of mycobacterial strain in sputum sample

[0085] 1. Extraction of mycobacterial nucleic acid in sputum samples

[0086]The mycobacterium-free sputum sample in Step 1 of Example 1 was used as the test material, and the concentration of 1×10 5 Mycobacterium tuberculosis (American Type Culture Collection ATCC27294) in CFU bacteria / ml, the nucleic acid of Mycobacterium tuberculosis in the sputum sample was extracted, and the specific nucleic acid extraction process was the same as in Example 1.

[0087] The mycobacterium-free sputum sample in Step 1 of Example 1 was used as the test material, and the sputum sample was mixed with a concentration of 1×10 5 CFU bacteria / ml Mycobacterium kansasii (American Type Culture Collection ATCC 12478), Mycobacterium intracellulare (American Type Culture Collection ATCC 13950), Mycobacterium chelonis (American Type Culture Collection ATCC 14472), Mycobacterium fortuitously (American Type Culture Collect...

Embodiment 3

[0104] Example 3, detecting whether the gene locus of Mycobacterium tuberculosis is mutant or wild type

[0105] 1. Extraction of Mycobacterium tuberculosis nucleic acid in sputum samples

[0106] Get the mycobacterium-free sputum sample in step 1 of Example 1 as the test material, and in this sputum sample, the concentration of 1 × 10 5 Mycobacterium tuberculosis (American Type Culture Collection ATCC27294) in CFU bacteria / ml, the nucleic acid of Mycobacterium tuberculosis in the sputum sample was extracted, and the specific nucleic acid extraction process was the same as in Example 1.

[0107] 2. Preparation of oligonucleotide primers and probes

[0108] The primers used for detecting whether the rpoB, katG, inhA, rrs and rpsL gene loci of Mycobacterium tuberculosis are mutant or wild type include primers for amplifying the rpoB, katG, inhA, rrs and rpsL genes of Mycobacterium tuberculosis, and the nucleotide The sequences are shown in Table 7. The probes used to detect w...

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Abstract

The invention discloses a method and a kit for extracting bacterial nucleic acid from sputa and applications thereof. The method is to use liquefaction reagent to liquefy a sputum sample and add solid particles to lyse cells to extract nucleic acid form the sputum. Overcoming the drawbacks of the prior method including complex procedures, a long period of time, complex operation and inadequate samples, the method of the invention for extracting nucleic acid samples is convenient and quick in operation, adequate in nucleic acid samples, low in cost and easy to implement. The method and kit forextracting bacterial nucleic acid can be used to identify strains of mycobacteria and to detect whether a gene locus of mycobacterium tuberculosis is of a wide type or a mutant type. With a chip for identifying strains and a chip for detecting whether the gene locus of mycobacterium tuberculosis is of a wide type or a mutant type, the method can identify the strains of mycobacteria and to detect whether a gene locus of mycobacterium tuberculosis is of a wide type or a mutant type in a quick, accurate and high-pass way.

Description

technical field [0001] The invention relates to a method, kit and application for extracting bacterial nucleic acid from sputum. Background technique [0002] In 1882, Kock discovered that the pathogenic bacteria of tuberculosis was Mycobacterium tuberculosis. In 1946, Hinshaw reported that 100 tuberculosis patients were treated with streptomycin (SM) for the first time, creating a new era of tuberculosis diagnosis and treatment. After more than 100 years of hard work, people have a complete set of experience in the onset, transmission, prevention, diagnosis, treatment and prognosis of tuberculosis. In the United States and other industrialized countries, tuberculosis decreased at a rate of 5.8-10% per year from 1953 to 1985. The American medical community was once optimistic that tuberculosis that could be prevented and treated would become a scene in American history and would no longer be a Public health issues, and believes that the spread of tuberculosis can be control...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/04C12Q1/68C12N15/10
CPCY02A50/30
Inventor 郭永祝令香程京高华方张琼杨华卫赵雁林吴雪琼王国青王璨张俊仙
Owner CAPITALBIO CORP
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