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Listeria monocytogenes nucleic acid testing plasmid DNA reference sample as well as preparation method and application thereof

A technology of Listeria monocytogenes and reference samples, applied in the field of plasmid DNA reference sample preparation for nucleic acid detection of Listeria monocytogenes, can solve the lack of nucleic acid reference materials, the inability to provide a comparable reference, and the difficulty of multiple targets. Provide traceable quantitative reference and other issues to achieve the effect of clear sequence source

Pending Publication Date: 2020-09-29
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is a lack of nucleic acid reference products corresponding to PCR detection methods.
Genomic DNA is used as a reference, and it is difficult to provide traceable quantitative references for multiple targets at the same time
Small fragments of nucleic acid cannot provide comparable reference among various detection reagents and methods

Method used

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  • Listeria monocytogenes nucleic acid testing plasmid DNA reference sample as well as preparation method and application thereof
  • Listeria monocytogenes nucleic acid testing plasmid DNA reference sample as well as preparation method and application thereof
  • Listeria monocytogenes nucleic acid testing plasmid DNA reference sample as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Synthesis, fragment design and cloning of embodiment 1 plasmid

[0039] 1. Fragment Design

[0040] According to the hlyA (GenBank: KC770796.1), plcB (GenBank: JF712529.1), and inlA (GenBank: EF445938.1) gene sequences detected by Listeria monocytogenes, three genes were artificially synthesized as detection target fragments. The nucleic acid sequence of a single gene can be downloaded from the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), and sequence comparisons can be used to ensure that the selected genes have clear representativeness and universality.

[0041] 2. Plasmid Construction

[0042] The three genes were connected in series at a ratio of 1:1 (separated by the unrelated gene fragment aagtcg), and cloned into pUC57 through the method of whole gene artificial synthesis (produced by Shanghai Gemma Biotechnology Engineering Co., Ltd.) To form a recombinant plasmid (the length of the chemically synthesized double-stranded DNA fragment is 1561bp, the length of ...

Embodiment 2

[0047] The mensuration of embodiment 2 homogeneity, fixed value and shelf life

[0048] 1. Judgment of pDNA homogeneity

[0049] Homogeneity is a basic property of reference materials, which is used to describe the spatial distribution characteristics of reference materials. The quality of plasmid DNA standard substances with good uniformity will not be affected by factors such as aliquoting, so as to ensure the reliability of the test results. In order to meet the requirements of the national standard material technical specification, the present invention uses the ultraviolet method (minimum sampling volume is 1 μ L) to analyze the uniformity between bottles and inside bottles of pNDA, and analyzes the data by F test.

[0050] 1) Sampling method for uniformity between bottles: In order to test the homogeneity between bottles, 10 tubes of pDNA were randomly selected, and the 1 μL sample extracted from each tube was repeatedly measured by UV for 3 times, and the average value...

Embodiment 3

[0103] The preparation of embodiment 3 fluorescence quantitative standard curve

[0104] 1. Doubling dilution of pDNA

[0105] Pass A 260 The pDNA reference was extracted and quantified, and the copy number of pDNA Listeria was estimated based on 4271bp.

[0106] After quantification, use pDNA Listeria to prepare a 10-fold dilution series to prepare 2 × 10 6 , 2×10 5 , 2×10 4 , 2×10 3 , 2×10 2 and 2×10 1 copies / ml of diluted sample.

[0107] 2. qPCR reaction conditions

[0108] A five-point standard curve for qPCR was generated using 10-fold serial dilutions of 1 μl samples extracted from pDNA. The primer sequences and amplicon sizes are listed in Table 3. PCR Analysis ABI SYBR FASTqPCR Master Mix (2X) (NEWEngland Biolabs, UK) was used to perform PCR reactions in 8-tube strips with a volume of 25 μl, each reaction containing 10 pM primers. The PCR protocol was as follows: 40 cycles of 10 min at 94°C and 15 s at 94°C, 45 s at 55°C, respectively.

[0109] Table 3 Lis...

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Abstract

The invention discloses a Listeria monocytogenes nucleic acid testing plasmid DNA reference sample as well as a preparation method and application thereof, and belongs to the field of microbial detection. The reference sample is formed by inserting segments containing genes hlyA, plcB and inlA from different Listeria monocytogenes strains into plasmids, wherein the genes are connected in series according to a ratio of 1: 1 and are separated by irrelevant gene segments; the reference sample contains the three specific detection genes hlyA, plcB and inlA, the genes are clear in sequence source and are stored in the plasmids in a single copy manner, and the reference sample can be used for qualitative detection and can also be used as a reference sample with the traceable quantity value for reagent performance identification and capability evaluation between laboratories.

Description

technical field [0001] The invention relates to the field of microorganism detection, in particular to a reference sample of Listeria monocytogenes nucleic acid detection plasmid DNA, a preparation method and an application thereof. Background technique [0002] Listeria monocytogenes (L Monocytogenes), referred to as Listeria monocytogenes, belongs to the genus Listeria (Listeria), and is recognized worldwide as an important zoonotic foodborne pathogen. bacteria. Listeria monocytogenes has strong pathogenicity, mainly causing clinical manifestations such as mononucleosis, sepsis, meningitis, abortion and stillbirth in pregnant women. Listeria monocytogenes still has a strong ability to grow and reproduce in a low temperature environment of 4°C, and food that has been frozen in the refrigerator for a long time in daily life is easily contaminated by Listeria monocytogenes. Listeria monocytogenes can easily cause human infection through meat and dairy products. Therefore, r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6806C12Q1/6851C12N15/63C12Q1/06C12R1/01
CPCC12Q1/689C12Q1/6806C12Q1/6851C12N15/63C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 陈瑶许龙岩袁慕云郝文波刘二龙
Owner SOUTHERN MEDICAL UNIVERSITY
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