Multiplex PCR primer group and next-generation sequencing database building method for MLST (multilocus sequence typing) tracing of vibrio parahaemolyticus

A hemolytic vibrio and next-generation sequencing technology, applied in the field of microbial detection, can solve problems such as heavy workload and complicated operation

Active Publication Date: 2020-10-23
上海国际旅行卫生保健中心
View PDF4 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The currently promulgated industry standard "SNT 4525.3-2016 Molecular Typing of Pathogenic Bacteria in Food for Export MLST Method Part 3: Vibrio parahaemolyticus" specifies 7 housekeeping genes for MLST typing of Vibrio parahaemolyticus , but for the amplification of these 7 genes, the ordina

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Multiplex PCR primer group and next-generation sequencing database building method for MLST (multilocus sequence typing) tracing of vibrio parahaemolyticus
  • Multiplex PCR primer group and next-generation sequencing database building method for MLST (multilocus sequence typing) tracing of vibrio parahaemolyticus
  • Multiplex PCR primer group and next-generation sequencing database building method for MLST (multilocus sequence typing) tracing of vibrio parahaemolyticus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] 1. The basic information of the seven housekeeping genes and the primers for library construction are shown in Table 1.

[0057] 2. Database construction plan

[0058] 2.1 One round of primer synthesis

[0059] In addition to the sequence used for multiplex PCR amplification, the one-round primer will also have a general sequence for the second round of primer-binding amplification. Take the tnaA gene primer as an example:

[0060] F1: ACACTCTTTCCCTACACGACGCTCTTCCGATCT CGGTATCGATGGAAAACATGC (SEQ ID NO. 23);

[0061] R1: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC GCAATATTTTTCGCCGCATCAAC (SEQ ID NO. 24);

[0062] Among them, ACACTCTTTCCCTACACGACGCTCTTCCGATCT (SEQ ID NO.25) is the universal sequence of the F-terminal primer: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC (SEQ ID NO.26) is the general sequence of the R-terminal primer.

[0063] 2.2 DNA extraction method of the sample to be tested

[0064] The Vibrio parahaemolyticus isolates obtained from the port samples, numbered 1...

Embodiment 2

[0097] The four sets of libraries prepared in Example 1 were mixed and then sequenced, see Table 9 for details.

[0098] Table 9 High-throughput sequencing library information (equal-quality mixed sample sequencing of 4 groups of libraries)

[0099]

[0100] Analysis of sequencing results

[0101] 1. See Table 10 for data quality.

[0102] Table 10 Summary of library sequencing data quality prepared by different methods

[0103]

[0104]

[0105] 2. See Table 11 for gene sequence matching.

[0106] Table 11 Matching of library gene sequences prepared by different methods

[0107]

[0108] 3. Analysis of sequencing results

[0109] 1) 1269 strain, two sets of amplification schemes, all seven gene sequences were detected, all of which could be matched to the corresponding gene numbers, and the comparison results of the two sets of data were consistent.

[0110] 2) For the 1177 strain, two sets of amplification schemes, seven gene sequences were detected, all of wh...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention provides a multiplex PCR primer group and next-generation sequencing database building method for MLST (multilocus sequence typing) tracing of vibrio parahaemolyticus, and belongs to thetechnical field of microbiological detection. The multiplex PCR primer group for the MLST tracing of the vibrio parahaemolyticus comprises a primer for gyrB gene amplification, a primer for dnaE geneamplification, a primer for dtdS gene amplification, a primer for pyrC gene amplification, a primer for tnaA gene amplification, a primer for pntA gene amplification and one of a primer for recA-1 gene amplification and a primer for recA-2 gene amplification. The next-generation sequencing based database building method is established by multiplex PCR amplification for the seven genes, amplification and sequencing of the two types of RecA genes can be realized, and a sequencing result is relatively consistent with a first-generation sequencing result; and meanwhile, amplification of all genescan be realized by one-time PCR, the method can be directly applied to database building, and the operation is simple.

Description

technical field [0001] The invention belongs to the technical field of microbial detection, and in particular relates to a multiplex PCR primer set for MLST typing and traceability of Vibrio parahaemolyticus and a method for building a library by next-generation sequencing. Background technique [0002] With the development of traffic and trade, the flow of people and goods has become more frequent, and a large number of cross-border organisms have been intercepted and detected at ports, including a large number of food-borne pathogenic bacteria, among which Vibrio parahaemolyticus is a common diarrheal bacterium . [0003] Vibrio parahaemolyticus, Vibrio family Vibrio, is commonly found in coastal seawater, seabed sediments, seafood and salted foods; it is more common in Japan, Southeast Asia, the United States and Chinese Taipei, and is an important pathogen that causes foodborne diseases One; food poisoning caused by this bacterium accounts for 40-60% of bacterial food p...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12Q1/689C12Q1/04C12Q1/686C12Q1/6806C12Q1/6869C12N15/11C40B50/06C12R1/63
CPCC12Q1/689C12Q1/686C12Q1/6806C12Q1/6869C40B50/06C12Q2537/143C12Q2535/122Y02A50/30
Inventor 张子龙田桢干张威周娴李深伟杜鹃
Owner 上海国际旅行卫生保健中心
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap