Bacteriaemia aspartame assay kit and assay method thereof

A detection kit and detection method technology, applied in the field of clinical microorganism identification, can solve the problems of prolonged hospitalization, low sensitivity, treatment failure, etc., and achieve the effects of shortened identification time, strong specificity, and convenient operation.

Inactive Publication Date: 2010-10-20
HANGZHOU D A GENETIC ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Low sensitivity due to limitations such as insufficient blood volume, the presence of antibiotics in the blood, and the need for special nutrition is also a limiting factor for traditional culture
In addition, traditional identification methods require Gram staining, biochemical reaction identification, etc. after obtaining positive results, and there are many potential problems in this: ①L

Method used

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  • Bacteriaemia aspartame assay kit and assay method thereof
  • Bacteriaemia aspartame assay kit and assay method thereof
  • Bacteriaemia aspartame assay kit and assay method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1: the preparation method of kit.

[0057] (1) DNA extraction reagents:

[0058] Nucleic acid purification column was purchased from Hangzhou Xinjie Biotechnology Co., Ltd.

[0059] Buffer L1, Buffer L2, Buffer WA, Buffer WB, and Buffer TE were purchased from Hangzhou Xinjie Biotechnology Co., Ltd.

[0060] (2) Reaction solution:

[0061] PCR Buffer: 0.1% (v / v) NP-40 (purchased from Sigma, USA), 0.02% (v / v) gelatin (purchased from Sigma, USA), 0.06% g / mL BSA (purchased from Sigma, USA) , 0.1% (v / v) Tween-20 (purchased from Sigma, USA), 0.06M pH8.9Tricine (purchased from Merck, Germany),

[0062] The universal primers SEQ ID NO: 1-2 or SEQ ID NO: 3-4 were synthesized by Shanghai Handsome Biotechnology Co., Ltd.

[0063] MgCl 2 : Purchased from Sigma, USA,

[0064] 0.2mM dNTPs: purchased from Shanghai Dingguo Biotechnology Co., Ltd.,

[0065] 2U / μL Taq DNA polymerase: purchased from Fermentas, USA.

[0066] (3) Reagent for single-strand purification:

...

Embodiment 2

[0077] Embodiment 2: detection method.

[0078] 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.

[0079] (1) Extract bacterial DNA, specifically comprising the following steps:

[0080] (1a) Add 300uL Buffer L1 to a 1.5mL centrifuge tube.

[0081] (1b) Add 400uL whole blood, cover the tube cap, and vortex for 30 seconds.

[0082] (1c) Add 300uL Buffer L2, shake the centrifuge tube 3-5 times vigorously, and then vortex for 30 seconds to mix.

[0083] (1d) Centrifuge at 13000 rpm for 2 minutes.

[0084] (1e) Pour the supernatant in step (1d) into a nucleic acid purification column, cover the tube cap, and centrifuge at 12000 rpm for 30 seconds.

[0085] (1f) Discard the filtrate in the 2mL centrifuge tube, put the nucleic acid purification column back into the 2mL centrifuge tube, add 500uL Buffer WA...

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Abstract

The invention relates to the field of clinical microorganism identification, and provides a kit for rapidly identifying a bacterium in a blood source and a method thereof. The kit comprises: two universal primers amplify the V1 variable region of a bacterium 16S r RNA, one sequencing primer measures the sequence of a V1 forward sector, HRP-avidin labels a magnetic bead, and genus is judged by comparing sequencing results and a database. The kit can sequence and distinguish the bacterium in the blood source at a time, can be used for clinical diagnosis of bacteriaemia and ichorrhemia, not only saves the valuable rescuing time for clinical diagnosis and treatment, and has 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 pathogenic bacteria in blood samples. Background technique [0002] Bacteremia in adults and children is a disease with high morbidity and mortality worldwide. With 20 million cases of bacteremia worldwide each year, bacteremia remains a major challenge even in medical practice in developed countries, especially in neonatal intensive care units, transplant wards, hospitalized patients, immunosuppressed and postoperative patients 135,000 deaths per year in Europe and 215,000 in the United States. In order to achieve a better therapeutic effect, targeted antibiotics should be used within 6 hours of symptom onset. Early identification of bacteria is especially important for immunosuppressed patients, who have a wider range of bacteria infections than ordinary patients and are more unpred...

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 HANGZHOU D A GENETIC ENG
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