Quick identification kit for bacteria and detection method thereof

A detection method and technology of a kit are applied in the field of clinical microorganism identification, which can solve the problems of high cost and affect the effect of diagnosis and treatment, and achieve the effects of convenient operation, low cost and strong specificity.

Inactive Publication Date: 2010-12-22
南京迪安医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there have been no products and reports of sequencing to distinguish common clinical bacteria at home and abroad. Therefore, if you want to quickly obtain information on bacterial infections clinically, you need to wait 18 to 24 hours or longer and the cost is high, which affects the effectiveness of diagnosis and treatment.

Method used

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  • Quick identification kit for bacteria and detection method thereof
  • Quick identification kit for bacteria and detection method thereof
  • Quick identification kit for bacteria and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1: the preparation method of kit.

[0055] (1) DNA extraction reagents:

[0056] 5% (w / v, i.e. g / mL) Chelex 100 buffer: containing 0.03% (w / v, i.e. g / mL) SDS (purchased from Hangzhou Fluoride Biotechnology Co., Ltd.), 1% (v / v) Tween-20 (purchased from U.S. Sigma Company), 1% (v / v) NP-40 (purchased from U.S. Sigma Company),

[0057] 20 mg / mL proteinase K: (purchased from Amresco).

[0058] (2) Reaction solution:

[0059] PCRBuffer: 0.1% (v / v) NP-40, 0.02% (v / v) gelatin (purchased from Sigma, USA), 0.06% (w / v, ie g / mL) BSA (purchased from Sigma, USA), 0.1% (v / v) Tween-20 (purchased from Sigma, USA), 0.06MpH8.9Tricine (purchased from Merck),

[0060] Specific primers SEQ ID NO: 1-2 or SEQ ID NO: 3-4, 2mM,

[0061] MgCl 2 : Purchased from Sigma, USA,

[0062] 0.2mM dNTPs: purchased from Shanghai Dingguo Biotechnology Co., Ltd.,

[0063] 2U / μL Taq DNA polymerase: purchased from Fermentas, USA.

[0064] (3) Reagent for single-strand purification:

[0065]...

Embodiment 2

[0075] Embodiment 2: detection method.

[0076] 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.

[0077] (1) Extract bacterial DNA, specifically comprising the following steps:

[0078] (1a) 1000 μL of heparin-anticoagulated blood was placed at room temperature for about 10 minutes to precipitate blood cells, and 200 μL was drawn from the interface between serum and blood cells (ie buffy coat), and injected into a 1.5 mL sterilized centrifuge tube.

[0079] (1b) Add 1 mL of 0.87% (w / v, ie g / mL) NH4Cl, shake well, and place in a 37°C metal bath for 15 minutes.

[0080] (1c) Take out, centrifuge at 10000r / min for 5min, and remove the supernatant.

[0081] (1d) Repeat steps (1b) and (1c) 2 times.

[0082] (1e) Add 200 μL of 5% (w / v, ie g / mL) Chelex 100 buffer and 2 μL (20 mg / mL) of proteinase K to th...

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Abstract

The invention discloses a quick identification kit for bacteria. The kit comprises: (1) universal primers of a V1 variable area of amplified bacteria 16S rRNA, the nucleotide sequences of which are a primer pair expressed as SEQ ID NO: 1 and SEQ ID NO: 2; (2) a sequencing primer, the nucleotide sequence of which is expressed as SEQ ID NO: 2; and (3) avidin-marked magnetic beads, wherein the SEQ ID NO: 1 marks the avidin at the 5' ends of the magnetic beads; and the (1), (2) and (3) are used together in one-time detection. The invention also discloses a detection method using the quick identification kit for the bacteria. The kit can sequence and distinguish the clinical bacteria at one time, and can be used for clinical diagnosis of bacterial infection. The kit not only wins precious rescue time for clinical diagnosis and treatment, but also has the advantages of low cost, convenient operation and strong specificity.

Description

technical field [0001] The invention belongs to the technical field of clinical microorganism identification, and in particular relates to a kit and a detection method for rapidly identifying medical pathogenic bacteria. Background technique [0002] Traditional bacterial culture and biochemical analysis can no longer satisfy the diagnosis and epidemiological research of various pathogenic microorganisms. Researchers not only hope to study various bacteria and viruses at the molecular level, but also hope that the detection method will be faster and more accurate. The throughput is higher, and human influence factors should be reduced as much as possible to facilitate the construction of standardized operating procedures. PCR and quantitative PCR methods cannot obtain the gold standard of molecular diagnosis - gene sequence, and are limited by the detection of bacterial species. Each bacterial species requires specific PCR primers and or probes, which greatly limits its appl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 任绪义虞闰六吕江峰王栋梁
Owner 南京迪安医学检验所有限公司
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