Diagnosis kit for mycobacterium species identification and drug-resistance detection and mfg. method thereof

A diagnostic kit, mycobacteria technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, specific-purpose bioreactor/fermenter, etc., can solve the problem of time-consuming and limited enzyme treatment steps

Inactive Publication Date: 2003-09-24
BIOMEDLAB CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] However, the above methods all carry out a bacterial culture step before PCR amplification, which can only analyze limited bacterial spec...

Method used

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  • Diagnosis kit for mycobacterium species identification and drug-resistance detection and mfg. method thereof
  • Diagnosis kit for mycobacterium species identification and drug-resistance detection and mfg. method thereof
  • Diagnosis kit for mycobacterium species identification and drug-resistance detection and mfg. method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-1

[0049] Example 1-1: Mycobacterium Genomic DNA

[0050] The Department of Respiratory Diseases, Asan Medical Center (Seoul, South Korea) provided sequenced mycobacterial genomic DNA extracted from 10 clinical isolate cultures, and other mycobacterial genomic DNA was obtained from ATCC (Manassas, VA USA).

Embodiment 1-2

[0051] Example 1-2: Preparation of oligonucleotide probes and primers

[0052] Table 2 shows the sequences of the nucleotide probes and primers. Each of the biotin-TR8 and TR9 and probes used were custom-synthesized by Bionics (Seoul, South Korea), and all other reagents were first-class .

[0053] [Table 2]

[0054] According to the order shown in Table 2, select SEQ ID NO 1-40.

[0055] As shown in Table 2, the oligonucleotide chip designed for the study of rpoB gene point mutations included 7 wild-type probes, 17 RIF-resistant probes, and 8 RIB-sensitive probes.

[0056] The structure shows that 17 RIF resistance probes SEQ ID NO 20-36 can distinguish the mutations of the following codons: codon 511 (Leu>Pro), codon 513 (Gln>Pro), codon 516 (a:Asp> Val, b: Asp>Tyr, c: Asp>Glu), codon 518 (Asn>His), codon 521 (Leu>Met), codon 522 (Ser>Leu), codon 526 (a: His) Tyr, b: His) Asp, c: His) Arg, d: His) Asn, e: His) Ala, f: His>Cys, g: His) Gln, h: His> Gly), and codon 531...

Embodiment 1-3

[0062] Embodiment 1-3: the preparation of oligonucleotide chip

[0063] Fig. 1 is the flow chart of the preparation steps of the oligonucleotide chip that is used to construct the oligonucleotide chip of mycobacterium DNA identification among the present invention, as shown in Figure 1, the preparation step of this oligonucleotide chip comprises: the first step (10), Modify each mycobacterium composite probe: make the mycobacterium species identification probe containing the mycobacterium species-specific DNA sequence of the mycobacterium rpoB gene, the mycobacterium drug resistance detection probe containing one or more mycobacterium rpoB gene modification codons Needles, and control probes corresponding to the respective drug resistance detection probes composed of wild-type sequences, so that the 5' end contains an amine group; the second step (20), introduces an aldehyde group on the silanized glass slide and the third step (30), using Schiff base reaction to immobilize ...

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Abstract

The invention relates to a diagnostic kit for identification of mycobacterium strains and detection of drug resistance and a preparation method thereof. The kit can quickly and accurately distinguish between the identification of mycobacterium strains and the detection of drug resistance in large quantities by using an oligonucleotide chip. Point mutations in the rpoB gene of Mycobacterium tuberculosis associated with drug resistance. The diagnostic kit for the identification of mycobacterium strains and the detection of drug resistance of the present invention contains an oligonucleotide chip, and the chip contains a complex composed of mycobacterium tuberculosis mycobacterium strain identification probes and drug resistance detection probes. Probe, mycobacterium tuberculosis rpoB gene, and fluorescent material, wherein the fluorescent material contains biotin-binding protein, which is used as the label of biotin and the corresponding probe to detect the hybridization of the sample amplification product.

Description

technical field [0001] The invention relates to a diagnostic kit for identification of mycobacterium strains and detection of drug resistance and a preparation method thereof, in particular, the present invention relates to a kit for identification of mycobacterium strains and detection of drug resistance, the kit utilizes an oligo The nucleotide chip can quickly and accurately distinguish the point mutation of the mycobacterium rpoB gene related to drug resistance in large quantities, and the preparation method of the kit. Background technique [0002] About two million people worldwide die from tuberculosis each year, and increased immigration, the spread of HIV / AIDS, and the emergence of drug-resistant strains have increased the lethality of tuberculosis. AIDS patients and newborns with weakened immune systems will suffer from tuberculosis, which can be infected not only by Mycobacterium tuberculosis, but also by MOTT (Mycobacterium other than tuberculosis, mycobacteria o...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12M1/00C12Q1/04C12Q1/68
CPCC12Q1/689C12Q2600/156C12Q1/68
Inventor 金贤晶金娜瑛尹盛昱金祯美朴美宣
Owner BIOMEDLAB CORP
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