Polynucleotide, method and kit for detecting listeria monocytogenes

A technology for Listeria monocytogenes and polynucleotides, which is applied in the field of polynucleotides, methods and kits for the detection of Listeria monocytogenes, can solve the problem of lack of uniform determination methods, lack of comparability, and plasmid DNA molecular determination. Poor accuracy, etc.

Active Publication Date: 2015-12-23
SHANGHAI INST OF MEASUREMENT & TESTING TECH
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

At present, plasmid DNA molecules have been more and more deeply studied and applied as standard substances for genetic testing, but the plasmid DNA standard substances for the real-time fluorescent PCR detection method of Listeria monocytogenes are still blank.
In the actual real-time fluorescent PCR of Listeria monocytogenes, most of the units use plasmid DNA molecules designed and constructed by themselves as standard products, and the specific fragments of the target genes are different, and there is a lack of uniform value determination methods. , The accuracy of plasmid DNA molecular determination is poor, resulting in great differences in test results between laboratories and lack of comparability

Method used

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  • Polynucleotide, method and kit for detecting listeria monocytogenes
  • Polynucleotide, method and kit for detecting listeria monocytogenes
  • Polynucleotide, method and kit for detecting listeria monocytogenes

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preparation example Construction

[0115] The present invention also provides a set of methods for preparing the above-mentioned plasmid standard molecule of Listeria monocytogenes, comprising the following steps:

[0116] ① Artificially synthesizing the specific sequence of the Listeria monocytogenes, the sequence is shown in SEQ ID NO: 1 and / or 3;

[0117] ② Cloning the sequence of Listeria monocytogenes obtained in step ① into a cloning vector to obtain a plasmid standard molecule of Listeria monocytogenes.

[0118] The artificial synthesis method described in step ① is preferably: the sequence is obtained by whole gene synthesis or PCR primer amplification.

[0119] The plasmid standard molecular construction method of the present invention preferably comprises the following steps:

[0120] ① Query the internalizing gene InlA gene and / or transcriptional activation regulatory protein gene prfA gene of Listeria monocytogenes in Genbank of NCBI (National Center for Biotechnology Information);

[0121] ②Analy...

Embodiment 1

[0134] The construction of embodiment 1 plasmid standard molecule

[0135] Experimental reagents and experimental equipment:

[0136] A large number of plasmid extraction kits (OMEGA), other biochemical reagents are imported aliquots or domestic analytically pure biochemical reagents; experimental equipment includes centrifuges, constant temperature water baths, constant temperature culture shakers, pipette guns, etc.

[0137] The experimental method includes the following steps:

[0138] 1. Search the sequence of the internalizing gene InlA of Listeria monocytogenes in GenBank;

[0139] 2. Analyze the above sequence, select a suitable sequence and a suitable restriction site, the length of the internalization gene InlA gene sequence is 2403bp, and add KpnI and BamHI restriction sites at both ends;

[0140] 3. Send the processed sequence to Treasure Bioengineering (Dalian) Co., Ltd., which will be responsible for the artificial synthesis of the whole gene, including the synt...

Embodiment 2

[0159] The homogeneity test of embodiment 2 plasmid standard molecules LY01 and LY02

[0160] Homogeneity is the consistent state of structure or composition that characterizes one or more properties in a substance. By measuring samples of specified size taken from different packaging units (such as bottles, bags, etc.) or from different locations of the same packaging unit, if the measurement results fall within the specified uncertainty range, it can be considered that the reference material has a certain effect on the specified characteristic quantity. is even. Homogeneity is a basic property of reference materials, which is used to describe the spatial distribution characteristics of reference materials. Uniformity evaluation must be carried out during the development (production) of reference materials to prove that they have good uniformity. The value of plasmid standard molecules with good uniformity will not be affected by factors such as aliquoting, and there is littl...

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Abstract

The invention discloses polynucleotide, a method and a kit for detecting listeria monocytogenes. Specifically, the invention discloses polynucleotide, which can be used as a standard molecule in realtime fluorescent PCR detection of listeria monocytogenes, and DNA constructs (LY01 and LY02). The provided plasmid standard molecule can solve the problem that realtime fluorescence PCR detection of listeria monocytogenes is lack of standard substances, and moreover, can guarantee the comparability of the detection results of realtime fluorescence PCR detection. A reliable quality control method is provided for the realtime fluorescence PCR detection of listeria monocytogenes.

Description

technical field [0001] The invention relates to a set of plasmid molecules in the technical field of bioengineering, in particular to a set of polynucleotides, methods and kits for detecting Listeria monocytogenes. Background technique [0002] Listeria monocytogenes (L. monocytogenes) belongs to the genus Listeria. Listeria monocytogenes is the only pathogenic bacterium in the genus Listeria that is pathogenic to humans. It is an important zoonosis and food hygiene pathogen. The bacterium mainly causes human infection through meat, milk and its products. It can cause meningitis and sepsis in humans and animals and abortion in pregnant women, so it has attracted the attention of relevant departments in many countries in the world. [0003] At present, the detection methods of Listeria monocytogenes are divided into two categories, one is the traditional culture and its improvement method, which depends on the biochemical and morphological characteristics, the detection cycl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12N15/10
CPCC12Q1/689C12Q2600/166
Inventor 李妍刘刚许丽梁文闻艳丽李兰英徐勤任淑贞
Owner SHANGHAI INST OF MEASUREMENT & TESTING TECH
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