Kit for detecting and identifying mycobacteria and detection method thereof

A technology of mycobacteria and detection method, which is applied in the field of clinical microorganism detection and identification, can solve the problems of limited detection targets and limited application scope, and achieves the effects of convenient operation, low cost and efficient detection.

Pending Publication Date: 2021-01-29
HANGZHOU D A GENETIC ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the fluorescent quantitative PCR method needs to design specific primers and probes for specific bacterial species, and can only detect specific bacterial species, and the detection targets are limited, which limits its application to a certain extent.

Method used

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  • Kit for detecting and identifying mycobacteria and detection method thereof
  • Kit for detecting and identifying mycobacteria and detection method thereof
  • Kit for detecting and identifying mycobacteria and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: The preparation method of the kit for the detection and identification of mycobacteria is as follows:

[0036] (1) Inactivation reagent: absolute ethanol, purchased from Hangzhou Changzheng Chemical Reagent Co., Ltd.

[0037] (2) Lysozyme: 20 mg / ml, purchased from Shanghai Yisheng Biotechnology Co., Ltd.

[0038] (3) DNA extraction reagents:

[0039] 5% (w / v) g / mL Chelex 100 buffer: containing 0.03% (w / v) g / mL SDS (purchased from Hangzhou Fluoride Biotechnology Co., Ltd.), 1% (v / v) Tween-20 ( (purchased from Sigma, USA), 1% (v / v) NP-40 (purchased from Sigma, USA), 20 mg / mL proteinase K: (purchased from Amresco).

[0040] (4) Reaction solution:

[0041] PCR Buffer: 0.1% (v / v) NP-40, 0.02% (v / v) gelatin (purchased from Sigma, USA), 0.06% (w / v) g / mL BSA (purchased from Sigma, USA), 0.1 %(v / v) Tween-20 (purchased from Sigma Corporation, USA), 0.06MpH8.9Tricine (purchased from Merck Corporation), specific primer SEQ ID NO: 1~4, 2mM, MgCl2: purchased from Si...

Embodiment 2

[0046] Example 2: The detection method of the kit for the detection and identification of mycobacteria.

[0047] Instruments: Bio-Rad S1000 PCR instrument, Beckman Microfuge 22R desktop micro-refrigerated centrifuge, Beijing Liuyi agarose gel electrophoresis instrument, Shanghai Peiqing gel imaging system, QIAGEN PyroMark Q96ID sequencer.

[0048] (1) Sample pretreatment (inactivation)

[0049] (1a) Add 700 μL absolute ethanol to 300 μL clinical sample, shake to mix, and let stand at room temperature for 30 minutes;

[0050] (1b) Draw 1 mL of the inactivated clinical sample into a 1.5 mL sterilized centrifuge tube, centrifuge at 13000 rpm for 2 min, and remove the supernatant;

[0051] (1c) Add 1 mL of normal saline to the precipitate, shake and mix well, centrifuge at 13,000 rpm for 2 min, and remove the supernatant;

[0052] (1d) Repeat step (1c) once.

[0053] (2)

[0054] Extracting mycobacterial DNA specifically includes the following steps:

[0055] (2a) Add 200 μL ...

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Abstract

The invention relates to the field of clinical microorganism detection, and provides a kit and a method for rapidly detecting and identifying mycobacteria in a clinical sample. The kit comprises two universal primers used for amplifying mycobacterium 16S rRNA variable region sequences, a specific primer used for shielding Rogowski bacterium 16S rRNA variable region, a specific primer used for shielding corynebacterium bacterium 16SrRNA variable region, a sequencing primer used for determining the sequence of a forward segment of amplified mycobacterium specific fragment, and avidin-labeled magnetic beads. A sequencing result is compared with a database to judge species. The kit can be used for detecting and identifying mycobacteria in clinical samples, and can be used for clinical diagnosis of suspected mycobacteria infection in various clinical samples. The kit not only wins precious rescue time for clinical diagnosis and treatment, but also has the advantages of being low in cost, convenient to operate and high in specificity.

Description

technical field [0001] The invention belongs to the technical field of clinical microorganism detection and identification, and in particular relates to a kit for detection and identification of mycobacteria and a detection method thereof. Background technique [0002] The genus Mycobacterium includes Mycobacterium tuberculosis complex (MTB, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium microti, etc.), Mycobacterium leprae and Non-Tuberculosis Mycobacterium (NTM). Tuberculosis caused by MTB is a chronic zoonotic infectious disease. According to the statistics of the World Health Organization, the population infected with MTB in the world has exceeded 1 / 3, and about 2 million people die of tuberculosis every year. Currently, NTM contains 169 species and 13 subspecies, which are widely distributed in different environments, and are isolated from water, soil, air, food, protozoa, plants, animals and humans. Mycobacteria, which are opportunistic pathogens, are gene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C12Q1/689C12Q1/04G16B25/20G16B30/10G16B50/00C12R1/32
CPCC12Q1/6869C12Q1/689G16B25/20G16B30/10G16B50/00C12Q2565/301C12Q2563/107C12Q2531/113
Inventor 吕江峰任绪义潘彩霞杨燚超
Owner HANGZHOU D A GENETIC ENG
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