Product and method for detecting clinic common pathogenic bacteria in blood culture bottle

A technology for pathogenic bacteria and products, applied in the field of microbial detection, can solve the problems of high cost, inability to meet clinical large-scale bacterial detection, complicated operation, etc., and achieve the effect of reducing the cost of consumables

Inactive Publication Date: 2018-04-20
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, its technical limitation lies in the complex operation and ...

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  • Product and method for detecting clinic common pathogenic bacteria in blood culture bottle
  • Product and method for detecting clinic common pathogenic bacteria in blood culture bottle
  • Product and method for detecting clinic common pathogenic bacteria in blood culture bottle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Primer Design and Synthesis

[0053] For Klebsiella pneumoniae khe gene, Acinetobacter baumannii OXA gene, Streptococcus pneumoniae LytA gene, Enterococcus faecium and Enterococcus faecalis ddl gene, Escherichia coli phoA3 gene, Staphylococcus aureus nuc gene, Staphylococcus epidermidis Design specific primers for 2312 gene, Pseudomonas aeruginosa ecfX gene and Proteus ure gene. The specific primer sequences are shown in Table 1 above.

[0054] Related primers were synthesized at Sangon Bioengineering (Shanghai) Co., Ltd.

Embodiment 2

[0055] Embodiment 2: sample DNA extraction

[0056] A total of 150 samples of clinical blood culture bottles were collected, and the bacterial genomic DNA in the blood culture bottles was extracted by the guanidine hydrochloride-benzyl alcohol extraction method.

[0057] (1) Aseptically take 100uL of the culture from the positive blood culture bottle and put it into a 1.5mL EP tube, then add guanidine hydrochloride buffer and vortex for 3min.

[0058] (2) Add 100uL double-distilled water and 400uL benzyl alcohol in turn, vortex for 1min, and then centrifuge for 5min.

[0059] (3) Transfer the upper aqueous phase to a new EP tube, add 40 uL of sodium acetate buffer solution and 440 uL of isopropanol in sequence, and then centrifuge at high speed for 15 min.

[0060] (4) Discard the upper layer liquid, add 1mL of 70% ethanol to wash, then centrifuge at high speed for 5min, then discard the upper layer of ethanol, after natural drying, add 100uL double distilled water to dissolv...

example 3

[0061] Example 3: Biological Experiment

[0062] 1. Multiplex PCR

[0063] System: 10 μL total volume

[0064] Hot start enzyme 0.05 μL;

[0065] 1 μL bacterial DNA template;

[0066] The upstream and downstream primers for each type of bacteria were mixed at a concentration of 1 μM, and 1 μL was taken;

[0067] Sterile ultrapure water 2.95μL;

[0068] PCR reaction buffer 5mL

[0069] (10 kinds of bacterial primers are divided into two groups, added in different reaction gates, each group has 5 different bacteria).

[0070] Reaction parameters:

[0071] Pre-denaturation at 95°C for 5 minutes;

[0072] Denaturation at 95°C for 30s;

[0073] Anneal at 59°C for 90s;

[0074] Extend at 72°C for 60s;

[0075] A total of 35 cycles were performed, and the cycle was extended at 72° C. for 10 min.

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Abstract

The invention discloses a method for detecting clinic common pathogenic bacteria in a blood culture bottle. The method is based on a multiplex polymerase chain type reaction technology, and comprisesthe steps of bacterium genome DNA extraction, PCR amplification, electrophoresis gel ultraviolet imaging and the like in the blood culture bottle. A product prepared by the method can realize the simultaneous detection of ten kinds of bacteria. Compared with technologies of sequencing, digital PCR and the like, the method provided by the invention has the advantages that the operation is simple and convenient; the cost is low; high sensitivity and specificity are realized. By using the product, the clinic bacterium detection and identification period can be greatly shortened; the bacterium inspection efficiency and accuracy can be improved.

Description

technical field [0001] The invention belongs to the field of microorganism detection, and relates to a primer system for detecting various common clinical pathogenic bacteria in blood culture bottles, a product and a method thereof, and can quickly and accurately identify clinical microorganisms. Background technique [0002] Bloodstream infection refers to the infection caused by pathogenic bacteria and their toxins invading the bloodstream. The main clinical manifestations are: sudden chills, high fever, tachycardia, shortness of breath, skin rash, hepatosplenomegaly, and a series of severe clinical symptoms such as mental and mental changes. Severe cases can cause shock, disseminated intravascular coagulation (DIC) and Multiple organ failure. Due to the characteristics of strong reproduction, high erosivity, and rapid mutation, pathogenic microorganisms can easily cause a pandemic of diseases. Therefore, rapid and sensitive analysis and identification are the first choic...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/14C12Q1/10C12Q1/04C12N15/11C12R1/22C12R1/46C12R1/19C12R1/445C12R1/45C12R1/385C12R1/01
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 王成彬陈琛何赏胡翀
Owner GENERAL HOSPITAL OF PLA
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