Membrane strip and kit for detecting drug resistant mutant genes of mycobacterium tuberculosis

A technology for Mycobacterium tuberculosis and drug-resistant mutation, which is applied in the directions of microorganism-based methods, microbial determination/inspection, biochemical equipment and methods, etc. , the effect of accurate flux

Active Publication Date: 2011-10-12
亚能生物技术(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the routine drug susceptibility testing of Mycobacterium tuberculosis mainly adopts the drug susceptibility culture method, which is not only complicated and time-consuming (3 to 4 weeks), but also difficult to culture, prone to false negatives, easy to cause misjudgment and delay the best treatment time for patients

Method used

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  • Membrane strip and kit for detecting drug resistant mutant genes of mycobacterium tuberculosis
  • Membrane strip and kit for detecting drug resistant mutant genes of mycobacterium tuberculosis
  • Membrane strip and kit for detecting drug resistant mutant genes of mycobacterium tuberculosis

Examples

Experimental program
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Effect test

Embodiment 1

[0067] A membrane strip for detecting drug-resistant mutant genes of Mycobacterium tuberculosis, comprising a nylon membrane, and specific oligonucleotide probes fixed on the nylon membrane, the specific oligonucleotide probes comprising:

[0068] 1) A specific oligonucleotide probe for detecting the rpoB drug-resistant mutant gene rpoB, the base sequence of which is shown in SEQ ID NO: 1-6;

[0069] 2) A specific oligonucleotide probe for detecting the isoniazid drug-resistant mutant gene KatG, the base sequence of which is shown in SEQ ID NO: 7;

[0070] 3) A specific oligonucleotide probe for detecting isoniazid resistance mutant gene inhA, the base sequence of which is shown in SEQ ID NO: 8;

[0071] 4) A specific oligonucleotide probe for detecting the streptomycin-resistant mutant gene rpsL, the base sequence of which is shown in SEQ ID NO: 9-10;

[0072] 5) A specific oligonucleotide probe for detecting the ethambutol-resistant mutant gene embB, the base sequence of wh...

Embodiment 2

[0080] A detection kit for the drug-resistant mutation gene of Mycobacterium tuberculosis, said kit comprising: a nylon membrane fixed with a specific oligonucleotide probe and

[0081] A primer for specifically amplifying the rpoB gene, the base sequence of which is shown in SEQ ID NO: 24-25;

[0082] A primer for specifically amplifying the KatG gene, the base sequence of which is shown in SEQ ID NO: 26-27;

[0083] A primer for specifically amplifying the inhA gene, the base sequence of which is shown in SEQ ID NO: 28-29;

[0084] A primer for specifically amplifying the rpsL gene, the base sequence of which is shown in SEQ ID NO: 30-31;

[0085] A primer for specifically amplifying the embB gene, the base sequence of which is shown in SEQ ID NO: 32-33;

[0086] Specific oligonucleotide probes immobilized on nylon membrane strips include:

[0087] A specific oligonucleotide probe for detecting the rpoB drug-resistant mutant gene rpoB, the base sequence of which is shown ...

Embodiment 3

[0098] Clinical samples were detected with the detection kit described in Example 2,

[0099] 1. Clinical sample acquisition: sputum sample

[0100] Take 3-5ml of deep cough sputum specimen in a sterilized glass container.

[0101] 2. Sample processing

[0102] 1) Collect the patient's sputum sample, add the same amount of phlegm-resolving reagent (4% NaOH), and treat the phlegm for 15-30 minutes until the sputum is completely dissolved.

[0103] 2) Transfer 1.2mL of sputum sample digestion solution to a 1.5mL centrifuge tube, centrifuge at 13000rpm for 5 minutes, and discard the supernatant.

[0104] 3) Add 1 mL sterile 1×PBS and mix well. Centrifuge at 13000 rpm for 5 minutes, discard the supernatant, and repeat once.

[0105] 4) Add 1 mL of 10 mM Tris solution, mix and suspend the sample, centrifuge at 13000 rpm for 5 minutes, and discard the supernatant.

[0106] 5) Add 50 μL of lysate, break up the precipitate, boil in a boiling water bath for 10 minutes (note that t...

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Abstract

The invention relates to a reagent for diagnosing drug resistance of mycobacterium tuberculosis, in particular to a membrane strip and a kit for detecting drug resistant mutant genes of the mycobacterium tuberculosis. The membrane strip for detecting the drug resistant mutant genes of the mycobacterium tuberculosis fixes a specific oligonucleotide probe which is used for detecting the drug resistant mutant genes of the mycobacterium tuberculosis on a substrate. The kit for detecting the drug resistant mutant genes of the mycobacterium tuberculosis comprises the membrane strip which is fixed with the specific oligonucleotide probe used for detecting the drug resistant mutant genes of the mycobacterium tuberculosis and a primer of drug resistant relevant genes of specific amplification mycobacterium tuberculosis. The kit for detecting the drug resistant mutant genes of the mycobacterium tuberculosis can simultaneously detect various usual drug resistant mutant gene types on five genes such as rpoB, katG, inhA, embB and rpsL, is suitable for the diagnosis of tubercle bacilli to the drug resistance of tuberculosis resistant drugs such as rifampicin, retozide, ethambutol, streptomycin,and the like, and has the advantages of quick and accurate detection, large flux, and the like.

Description

technical field [0001] The invention relates to a diagnostic reagent for drug resistance of mycobacterium tuberculosis, in particular to a detection membrane strip and a kit for the drug resistance mutation gene of mycobacterium tuberculosis. Background technique [0002] Tuberculosis is a chronic infectious disease that seriously endangers human health. In the past 10 years, due to the epidemic of AIDS, the increase of immigration and floating population, and the neglect of tuberculosis control in many countries and regions, etc., the multi-drug resistant MTB has increased, and the global tuberculosis situation has deteriorated sharply. In developing countries, the number of tuberculosis patients is increasing every year. In 1993, the World Health Organization (WHO) declared that "global tuberculosis is in a state of emergency" and listed tuberculosis as one of the infectious diseases under key control. In 1998, WHO once again pointed out that "it is urgent to curb tubercu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/32
Inventor 邹耀东任维廖生赟
Owner 亚能生物技术(深圳)有限公司
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