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A set of nucleotide sequences and its application in the identification of Listeria monocytogenes

A nucleotide sequence and reaction technology, applied in the fields of molecular biology and microbiology, can solve the problems of low specificity and poor sensitivity, and achieve the effect of good specificity, fast research and judgment, and simple conditions

Active Publication Date: 2017-01-04
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In view of the shortcomings of poor sensitivity and low specificity in the detection of pathogenic microorganisms in conventional techniques such as PCR, how to use CPA technology to solve these problems in the differential diagnosis of some pathogenic microorganisms has become an urgent problem to be solved in the field of pathogenic microorganism detection technology. need

Method used

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  • A set of nucleotide sequences and its application in the identification of Listeria monocytogenes
  • A set of nucleotide sequences and its application in the identification of Listeria monocytogenes
  • A set of nucleotide sequences and its application in the identification of Listeria monocytogenes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: S-CPA detection

[0042] The genomic DNA of Listeria monocytogenes was extracted, and the extracted DNA was used as a template for S-CPA and D-CPA reactions. Finally, different detection methods were used to interpret the results.

[0043] 1. Genome extraction: DNA extraction kits from Qiagen (QIAamp DNA minikits; Qiagen, Hilden, Germany) were used to extract bacterial genomes, and the operation was performed according to the instructions. Utilize the ultraviolet spectrophotometer to measure the concentration and the purity of genomic DNA, listeria monocytogenes genomic DNA is serially diluted with GE buffer solution (from 25ng, 2.5ng, 250pg, 25pg, 2.5pg, 250fg, 125fg to 62.5fg). A small amount of various genomic DNAs were aliquoted and stored at -20°C for future use. Cross reaction system:

[0044] 2. The reaction system of S-CPA: the total volume is 20μl, including 2.4mM cross primer As (2a+1s), 1.44mM amplification primers 3a and 2a, 0.3mM displacemen...

Embodiment 2

[0051] Embodiment 2: D-CPA detection

[0052] 1. Genome Extraction

[0053] Same as Example 1

[0054] 2. The reaction conditions of D-CPA: the total volume is 20μl, including 2.4mM cross primer As (2a+1s), 1.44mM cross primer Sa (1s+2a), 1.44mM amplification primer 3a, 0.3mM displacement primer 4s and 5a, 20mM Tris-HCl (pH 8.8), 10mM KCl, 4mM MgSO 4 ,10mM (NH 4 ) 2 SO 4 , 0.1% Tween 20, 0.8M betaine, 1.4mM dNTPs, 1μl of BstDNA polymerase (8U / mL), 1μl Loopamp fluorescent dye (FD) and 1μl DNA template.

[0055] 3. Amplification reaction

[0056] The constant temperature reaction was carried out in a DK-450 B type electric thermostat (Shanghai Senxin Experimental Instrument Co., Ltd.), the reaction temperature was 64° C., and the reaction time was 90 minutes.

[0057] 4. Interpretation of results

[0058] After the reaction is completed, the reaction system is green and positive for amplification by visual inspection.

[0059] Take 5 μl of the amplified product for elec...

Embodiment

[0061] Example 3. Comparative Example: Common RCR Detection

[0062] 1. Genome extraction Same as Example 1

[0063] 2. Ordinary RCR reaction system:

[0064]

[0065] 3. Amplification reaction

[0066] PCR reaction conditions:

[0067]

[0068]

[0069] PCR primer sequences: SEQ ID NO:1 and SEQ ID NO:5

[0070] 4. Interpretation of results

[0071] Take 6 μl of the amplified product for electrophoresis on a 2.5% agarose gel, and observe the analysis results on a gel imaging system.

[0072] Interpretation and comparison of test results

[0073] 1. Demonstration of Availability of Designed Cross-Reactive Primers

[0074] Such as figure 2 with image 3 As shown, S-CPA ( figure 2 ) and CPA ( image 3 ) The positive reaction tube showed an obvious color change, from light gray to green (the black-and-white photo shows higher turbidity), and the color of the negative reaction tube remained unchanged, still light gray (the black-and-white photo showed lower tur...

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Abstract

The invention discloses a combination of nucleotide sequences and its application in cross constant temperature amplification reaction. The cross constant temperature amplification reaction can be used to identify Listeria monocytogenes, and has the characteristics of simple, fast, convenient, strong specificity and high sensitivity.

Description

technical field [0001] The invention relates to the application of cross constant temperature amplification in bacterial detection, especially the application in the differential diagnosis of Listeria monocytogenes, belonging to the fields of molecular biology and microbiology. Background technique [0002] Listeria monocytogenes (Listeria monocytogens, LM) is a Gram-positive, motile, facultative intracellular parasitic brevibacterium widely present in the environment, and is an important foodborne zoonotic pathogen that can cause human severe infection. Susceptible populations for listeriosis are mainly immunocompromised populations and immunocompromised populations. The former include the elderly, newborns, and pregnant women; the latter are cancer patients, AIDS patients, and organ transplant recipients. The main clinical symptoms of human listeriosis are sepsis, meningitis, miscarriage, stillbirth and febrile gastroenteritis in pregnant women. The bacteria can cross the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68C12Q1/04C12R1/01
Inventor 王毅王艳叶长芸
Owner ICDC CHINA CDC
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