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Method and specimen for detecting sensitivity to isoniazid in tubercle bacillus

A technology of Mycobacterium tuberculosis and test strips, applied in biological testing, material inspection products, biochemical equipment and methods, etc., can solve problems such as undetectable, and achieve appropriate treatment and accurate detection effects

Inactive Publication Date: 2014-08-20
NIPRO CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, about 80% of INH-resistant bacteria were detected as resistant bacteria based on known mutations in the katG gene or inhA gene, and the remaining about 20% of INH-resistant bacteria could not be detected

Method used

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  • Method and specimen for detecting sensitivity to isoniazid in tubercle bacillus
  • Method and specimen for detecting sensitivity to isoniazid in tubercle bacillus
  • Method and specimen for detecting sensitivity to isoniazid in tubercle bacillus

Examples

Experimental program
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Effect test

Embodiment 1

[0090] (Example 1: Analysis of Mycobacterium tuberculosis genes of INH-resistant Mycobacterium tuberculosis)

[0091] In this example, 92 specimens were obtained, and these 92 specimens were obtained by confirming that they were in a solution containing INH (MGIT 960: manufactured by Becton Dickinson Japan Co., Ltd.) or an agar medium containing INH (Ogawa medium, 7H10) Genomic DNA was extracted and purified from these 92 specimens grown and judged to be INH-resistant Mycobacterium tuberculosis by the phenol extraction method.

[0092] 1. Analysis of fabG1 gene

[0093] The fabG1 gene was amplified using the primer pair shown below. The PCR reaction conditions are as follows: denaturation process at 94° C. for 30 seconds, annealing process at 55° C. for 20 seconds, and chain extension process at 72° C. for 20 seconds. This step is repeated 30 times to amplify the fabG1 gene.

[0094] fabG 1 primer 1 ggctacatcg acaccgatat gacc (SEQ ID NO: 6)

[0095] fabG 1 primer 2 gcgtcctt...

Embodiment 2

[0111] (Example 2: Preparation of test piece)

[0112] 1. Probe Design

[0113] As probes capable of detecting INH sensitivity of Mycobacterium tuberculosis, the following two probes (probes 1 and 2: fabG1 wild-type probe and furA wild-type probe, respectively) were designed. Both of these probes are wild-type probes that do not bind but only bind (hybridize) to the wild-type gene in the presence of mutations.

[0114] Probe 1 ggggcgctgc aatttatccc (SEQ ID NO: 3)

[0115] Probe 2 gctccggacg gccgacctgc g (SEQ ID NO: 10)

[0116] 2. Immobilization of probes

[0117] Using terminal transferase (Promega), poly T was added to each of the 5' ends of the above probes 1 and 2 (see SEQ ID NO: 3 and SEQ ID NO: 10, respectively). Specifically, 0.4 μL (30 units / μL) of terminal transferase, 2 μL (10 pmol / μL) of thymidine triphosphate, 2 μL (50 mM) of oligonucleotide probes, 2 μL of reaction buffer supplied with the product, and 3.6 μL of purified Water was mixed to prepare a reaction ...

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Abstract

According to the present invention, novel means for more reliably detecting sensitivity to INH in Mycobacterium tuberculosis, that is, detecting whether or not M . tuberculosis is an INH-resistant bacterium is provided. A method for detecting sensitivity to Isoniazid in M . tuberculosis, comprising the steps of obtaining a fabG1 gene of M . tuberculosis in a sample; and detecting a mutation involved in resistance to Isoniazid with respect to the obtained fabG1 gene. The method of the invention may, if necessary, comprise a step of detecting a mutation in a furA gene of M . tuberculosis, and may preferably be combined with another known method for detecting sensitivity to Isoniazid.

Description

technical field [0001] The present invention relates to a method and a test strip for detecting isoniazid susceptibility in Mycobacterium tuberculosis. Background technique [0002] According to reports, thousands of people in Japan and millions of people in the world die of tuberculosis every year. Tuberculosis is basically treated with medical therapy centered on chemotherapy, and surgical therapy is considered when medical therapy cannot achieve the curative purpose. [0003] Isoniazid (INH), rifampicin (RFP), streptomycin (SM), ethambutol (EB), pyrazinamide (PZA), etc. are known as tuberculosis treatment drugs used in medical therapy Multiple drugs. However, when administering drugs to patients, there is a problem of the development of drug-resistant bacteria that acquire resistance to administered drugs due to sudden mutations. [0004] It has been reported that drug-resistant bacteria acquire drug resistance due to mutations in specific genes. Therefore, assays hav...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/09G01N33/53G01N37/00
CPCG01N33/5695G01N2333/35G01N2800/44C12Q1/689C12Q2600/156
Inventor 切替照雄安藤弘树末竹寿纪中村友彦
Owner NIPRO CORP
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