Serotype listeria monocytogenes specific new molecular targets and rapid detection method thereof

A Listeria and molecular target technology, applied in the field of microbial testing, can solve the problems that have not yet been found, and reports that no PCR method has been established, and achieve the effects of simple result judgment, practicability, and specific test results.

Active Publication Date: 2020-05-08
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are almost no research reports on PCR detection targets and primers for other lineage types, serogroups, and serotypes. Therefore, there are no reports on the establishment of PCR methods for detection of other lineage types, se

Method used

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  • Serotype listeria monocytogenes specific new molecular targets and rapid detection method thereof
  • Serotype listeria monocytogenes specific new molecular targets and rapid detection method thereof
  • Serotype listeria monocytogenes specific new molecular targets and rapid detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Mining of new molecular targets specific to common serotypes of Listeria monocytogenes

[0039] According to the GenBank database and the complete genome DNA sequences of different serotypes of Listeria monocytogenes self-tested by our team, through comparative genomics analysis, the common serotypes of Listeria monocytogenes Non-essential genes specific to terpene strains. The MP method in the prokaryotic pan-genome automated analysis software PGAP is used for analysis, and the analysis results are processed through the local Perl script to obtain the core gene and non-core gene information of all strains, and to screen out common serotypes of Listeria monocytogenes specific gene fragments. The gene sequence is shown in SEQ ID NO.1-SEQ ID NO.13. Extract the 13 specific gene protein sequences unique to the target serotype Listeria monocytogenes strains, and compare them back to the total protein library of Listeria monocytogenes and the NCBI non-redundant pr...

Embodiment 2

[0040] Example 2 Establishment of rapid detection method for Listeria monocytogenes lineage type I specific new molecular target

[0041] This embodiment provides a method for detecting Listeria monocytogenes lineage type I strains: design primers according to the new specific molecular targets of Listeria monocytogenes lineage type I, and form a rapid detection method, including the following steps:

[0042] Primer design: Design specific amplification primers according to the new specific molecular target base sequence SEQ ID NO.1. The primer sequences are as follows:

[0043] Table 1 Sequences of primers for Lineage I specific PCR detection of Listeria monocytogenes

[0044]

[0045] DNA template preparation: The Listeria monocytogenes lineage type I strain was enriched in BHI liquid medium, and the bacterial genomic DNA was extracted using a commercial bacterial genomic DNA extraction kit as a template to be tested.

[0046] PCR detection system and amplification proce...

Embodiment 3

[0056] Example 3 Establishment of a Rapid Detection Method for Listeria monocytogenes Lineage Type II Specific New Molecular Targets

[0057] This example provides a method for detecting Listeria monocytogenes lineage type II strains: design primers according to the new specific molecular targets of Listeria monocytogenes lineage type II, and form a rapid detection method, including the following steps:

[0058] Design of primers: Design specific amplification primers according to the base sequence of the new specific molecular target base sequence SEQ ID NO.2. The primer sequences are as follows:

[0059] Table 3 The sequence of primers for detection of Listeria monocytogenes lineage type Ⅱ specific PCR

[0060]

[0061] Preparation of DNA template: The Listeria monocytogenes lineage type II strain was enriched and cultured in BHI liquid medium, and the bacterial genomic DNA was extracted using a commercial bacterial genomic DNA extraction kit as a template to be tested. ...

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Abstract

The present invention provides a set of specific new molecular detection targets for identifying common serotype listeria monocytogenes. Nucleotide sequences of the specific new molecular detection targets are shown as SEQ ID NO.1-13. The targets can identify different types of the listeria monocytogenes, such as lineage type I, lineage type II, lineage type III, serogroup I.1, serogroup I.2, serogroup II.1, serogroup II.2, serotype 4a and serotype 4c. The present invention also provides a detection method for the common serotype listeria monocytogenes. The method designs specific primers forthe 13 specific target molecules, electrophoresis of PCR amplification products is conducted, and whether detection samples contain the common serotype listeria monocytogenes is directly determined byelectrophoresis results.

Description

technical field [0001] The invention belongs to the technical field of microbial inspection and relates to new specific molecular targets for identifying common serotypes of Listeria monocytogenes. Background technique [0002] Listeria monocytogenes (Listeria monocytogenes, LM) is an important food-borne pathogen, which is listed by WHO as one of the four major food-borne pathogens, which can cause severe listeriosis. Such as meningitis, sepsis, endocarditis, abscess and local purulent injury, etc., sensitive groups mainly include pregnant women, infants, the elderly and people with low immunity, and the fatality rate is 20%-30%. According to the US CDC data, there are about 1,600 cases of diseases caused by Listeria each year, and about 260 deaths (https: / / www.cdc.gov / listeria / ). Occasional cases of listeriosis have been reported in some cities in my country, and the main groups are pregnant women and newborns, which pose a certain threat to the physical and mental health...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/686C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 吴清平李凡叶青华陈谋通张菊梅丁郁吴诗李滢古其会吴慧清
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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