Quick bacterium identification kit for sputum sample and detection method for the same

A detection method and kit technology, applied in the field of clinical microbial identification, to achieve the effect of convenient operation, low cost and strong specificity

Active Publication Date: 2010-10-20
南京迪安医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there have been no products and reports of sequencing to distinguish pathogenic bacteria in sputum samples at home and abroad. Ther

Method used

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  • Quick bacterium identification kit for sputum sample and detection method for the same
  • Quick bacterium identification kit for sputum sample and detection method for the same
  • Quick bacterium identification kit for sputum sample and detection method for the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1: the preparation method of kit.

[0065] (1) Liquefaction reagent: 0.2M NaOH, purchased from Shanghai Shisihewei Chemical Co., Ltd.

[0066] (2) Washing reagent: 0.9% NaCl solution, purchased from Shanghai Shisi Hewei Chemical Co., Ltd.

[0067] (3) DNA extraction reagents:

[0068] 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 Corporation in the U.S.), 1% (v / v) NP-40 (purchased from Sigma Corporation in the U.S.),

[0069] 20 mg / mL proteinase K: (purchased from Amresco).

[0070] (4) Reaction solution:

[0071] 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, USA), 0.06MpH8.9Tricine (purchased from Merck),

[0072] Specific primers SEQ ID NO: 1-4 or SEQ ID NO: 5-8, 2mM,

[0073] MgCl 2 : Purchased from Sigma, ...

Embodiment 2

[0087] Embodiment 2: detection method.

[0088] 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.

[0089] (1) Pretreatment of sputum samples

[0090] (1a) Add an equal amount of liquefaction reagent to the sputum sample, shake and mix well, and let stand at room temperature for 10-20 minutes;

[0091](1b) Pipette 1 mL of liquefied sputum into a 1.5 mL sterilized centrifuge tube, centrifuge at 13000 rpm for 10 min, and remove the supernatant;

[0092] (1c) Add 1 mL of washing reagent to the precipitate, shake and mix, centrifuge at 13,000 rpm for 10 min, and remove the supernatant;

[0093] (1d) Repeat step (1c) once.

[0094] (2) extracting bacterial DNA, specifically comprising the following steps:

[0095] (2a) Add 200 μL of 5% (w / v) g / mL Chelex 100 buffer solution and 2 μL of proteinase K (20 m...

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Abstract

The invention relates to the field of clinical microbiological assay and provides a quick bacterium identification kit for a sputum sample and a detection method for the same. The kit comprises two universal primers for propagating the V1 variable regions of the 16S rRNA of a bacterium, two specific primers for shielding the variable regions of the 16S rRNA of a neisseria bacterium, two specific primers for shielding the variable regions of the 16S rRNA of a veillonella parvula, a sequencing primer for sequencing the positive segment of the V1, and an avidin-marked magnetic ball, wherein the sequencing result is compared with a database to determine the species of the bacteria. By applying the kit, the bacteria in a sputum sample can be distinguished by once sequencing, thus, the kit can be applied in the clinical diagnosis on the bacterial infection in the sputum sample. The invention wins precious rescuing time for a clinical treatment and has the advantages of low cost, convenient operation and high 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 pathogenic bacteria in sputum samples. 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 limit...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 任绪义吕江峰虞闰六
Owner 南京迪安医学检验所有限公司
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