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Method for detecting pathogenic bacteria of listeria monocytogenes from clinical blood

A mononuclear cell hyperplasia and Listeria monocytogenes technology is applied in the field of detection of pathogenic bacteria of blood infection, which can solve the problems of low specificity, long detection period and low sensitivity, and achieve the effects of high specificity, high sensitivity and improved detection efficiency.

Inactive Publication Date: 2019-11-08
卓源健康科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The invention solves the technical problems such as long detection period, low specificity and low sensitivity of the traditional hospital culture method for detection of Listeria monocytogenes infection in clinical blood, and improves the detection of clinical Listeria monocytogenes infection detection at the same time Efficiency, the present invention has developed a simple, fast and efficient method for detecting Listeria monocytogenes pathogenic bacteria from clinical blood

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  • Method for detecting pathogenic bacteria of listeria monocytogenes from clinical blood
  • Method for detecting pathogenic bacteria of listeria monocytogenes from clinical blood

Examples

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Embodiment 1

[0038] Embodiment 1: PCR primer design

[0039] Utilize Listeria monocytogenes genomic DNA sequence to design a specific PCR primer, this specific PCR primer comprises the forward primer shown in SEQ ID NO.1 and the reverse primer shown in SEQ ID NO.2 ;in,

[0040] SEQ ID NO.1: 5'-GGGAAATCTGTCTCAGGTGATGT-3';

[0041] SEQ ID NO.2: 5'-CGATGATTTGAACTTCATCTTTTGC-3';

[0042] The length of the target fragment amplified by the primers is 106bp.

Embodiment 2

[0044] Listeria monocytogenes-specific primers were used to detect pathogenic bacteria such as Listeria monocytogenes, Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli added in blood. In this example, OD is added to every 100 μL of blood 600 0.1 of the above-mentioned various pathogenic bacteria. This example includes the extraction of each bacterial genome DNA in the blood and the PCR amplification detection of the specific gene of Klebsiella pneumoniae. This method comprises the following steps:

[0045] (1) Extraction of genomic DNA of Listeria monocytogenes in blood

[0046] (1) Collect 100 μL cultured OD 600 =0.1 for Listeria monocytogenes, Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli. Centrifuge at 12000 rpm for 1 minute to collect the precipitated bacteria.

[0047] (2) Take 100 μL of fresh blood from 6 groups, and add Listeria monocytogenes, Staphylococcus a...

Embodiment 3

[0064] 100 μL of Listeria monocytogenes-specific primers were used to detect Listeria monocytogenes added to the blood. In this example, 10 9 , 10 7 , 10 5 , 10 3 , 10CFU of Listeria monocytogenes. This example includes the extraction and PCR amplification detection of bacterial genomic DNA in blood. This method comprises the following steps:

[0065] (1) Collect 10 cultivated 9 , 10 7 , 10 5 , 10 3 , 10 CFU of Listeria monocytogenes. Centrifuge at 12000 rpm for 1 minute to collect the precipitated bacteria.

[0066] (2) Take 5 groups of 100 μL of fresh blood, add the different numbers of Listeria monocytogenes precipitated in step (1) to the 5 groups of 100 μL of fresh blood, resuspend the precipitated cells, and pipette fully well mixed.

[0067] (3) Add 100 μL enzyme lysate solution to each group of blood, and incubate at 37° C. for 15 minutes. The enzyme lysate contains: 0.1 mg / mL ribonuclease A, 1 mg / mL proteinase K, 0.05 mg / mL broad-spectrum lyase ClyH, 10 m...

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Abstract

The invention provides a method for detecting pathogenic bacteria of listeria monocytogenes from clinical blood. The method includes the following steps that S1, listeria monocytogenes genomic DNA inblood is extracted; S2, PCR is detected, specifically, S2.1, a specific PCR primer is designed by using the listeria monocytogenes genomic DNA sequence, and the PCR primer includes a forward primer asshown in SEQ ID NO. 1 and a reverse primer shown in SEQ ID NO. 2; S2.2, the extracted listeria monocytogenes genomic DNA is used as a template, the PCR primer is used for amplify PCR; S2.3, the PCR amplified products are agarose gel electrophoresis and detection, and it is determined that the pathogenic bacteria of the listeria monocytogenes are detected due to the amplified fragment with the length being 106bp. The detection limit for listeria monocytogenes infection in blood reaches 10 CFU / mL. According to the method, rapid and sensitive diagnosis of listeria monocytogenes infection in clinical blood is realized.

Description

technical field [0001] The invention relates to the technical field of detecting blood infection pathogenic bacteria, in particular to a method for detecting Listeria monocytogenes pathogenic bacteria from clinical blood. Background technique [0002] Bloodstream infection has become a serious infectious disease, and the mortality rate of bacteremia and sepsis caused by bloodstream infection is high. Listeria monocytogenes is a Gram-positive bacterium. Listeria monocytogenes meningitis caused by Listeria monocytogenes infection has a high mortality rate and is often accompanied by complications such as acute cerebral infarction. [0003] Clinically, early diagnosis and intervention of Listeria monocytogenes infection and anti-infection treatment are of great significance. The "gold standard" of traditional clinical culture and detection methods not only requires long-term bacterial culture and other enrichment processes, but also requires subsequent tedious physiological an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/6806C12Q1/04
CPCC12Q1/689C12Q1/686C12Q1/6806C12Q2565/125C12Q2527/125C12Q2521/537C12Q2521/327
Inventor 李敬王旭孙宝林郭建吴谨
Owner 卓源健康科技有限公司
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