Primer group for amplifying cronobacter kronoi MLST (multiple-locus typing) tracing housekeeping genes, next-generation sequencing library building method and application

A Cronobacter, next-generation sequencing library technology, applied in biochemical equipment and methods, ICT adaptation, microbial measurement/inspection, etc., can solve the problems of heavy workload and complicated operation, and reduce workload and operation The effect of simple and convenient sequencing work

Active Publication Date: 2020-12-18
上海国际旅行卫生保健中心
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Problems solved by technology

[0008] The MLST typing technology for Cronobacter has been studied for a long time, and its industry standard has been promulgated. However, the method of next-generation sequencing still has certain limitations, especially when there are multiple housekeeping genes. The method of first-generation sequencing requires multiple amplifications for the same bacterium, and the sequencing needs to be sent to a third-party company. The operation is complicated and the workload is heavy.

Method used

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  • Primer group for amplifying cronobacter kronoi MLST (multiple-locus typing) tracing housekeeping genes, next-generation sequencing library building method and application
  • Primer group for amplifying cronobacter kronoi MLST (multiple-locus typing) tracing housekeeping genes, next-generation sequencing library building method and application
  • Primer group for amplifying cronobacter kronoi MLST (multiple-locus typing) tracing housekeeping genes, next-generation sequencing library building method and application

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Embodiment 1

[0070] Using the information presented in Table 1, Table 2, and Table 3, perform the following experiments:

[0071] 1. One round of amplification

[0072] 1) In addition to the sequence used for gene synthesis, the first-round primer will also have a general sequence for the second-round primer combination amplification. Taking the aptD gene primer as an example, F1 (SEQ ID NO.19+SEQ ID NO.1): ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGTGTACGGCCAGATGAAC;

[0073] R1 (SEQ ID NO. 20+SEQ ID NO. 2): GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCGCTCCTGGCCTACTACCAG.

[0074] 2) One-round amplification system: 25 μL: Cronos strain gDNA 5ng, 2×Mix 12.5 μL, primer mixture 5 μL, the balance is H 2 O.

[0075] 3) Amplification program: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, 26 cycles; extension at 72°C for 5 min, and the product of one round was stored at 4°C.

[0076] 4) Purification of amplification products (1×beads) ...

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Abstract

The invention provides a primer group for amplifying cronobacter kronoi MLST (multiple-locus typing) tracing housekeeping genes, a next-generation sequencing library building method and application, and relates to the technical field of next-generation sequencing. The amplification primer group is simultaneously built for seven housekeeping genes specified in Cronobacter Kronoi in the Part 5 of MLST Method for Molecular Typing of Pathogenic Bacteria in Export Food of the industry standard SNT 4525.5-2016. The next-generation sequencing library building method for cronobacter kronoi MLST tracing is built on the basis of the primer group; meanwhile, the amplification of seven genes can be realized through one-time PCR (polymerase chain reaction); the method is directly used for library building; the operation is simple. The sequencing work can be more conveniently performed; the MLST typing speed is accelerated; the workload is reduced.

Description

technical field [0001] The invention belongs to the technical field of next-generation sequencing, and in particular relates to a set of primers for amplifying housekeeping genes traceable to Cronobacter MLST typing, and a method and application for building a library by next-generation sequencing. Background technique [0002] Since the beginning of the 21st century, with the acceleration of the global integration process, the cross-border flow of people and goods has grown by leaps and bounds, and the country and the people have higher and higher demands for safety and health. The entry of a large number of imported items has brought new risks to the health and safety of the people. Among them, milk powder contamination incidents caused by Cronobacter (Cronobacter) are also emerging in an endless stream. [0003] Cronobacter genus is an important foodborne opportunistic pathogen, which can cause neonatal meningitis, bacteremia, necrotizing enterocolitis and systemic sepsi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6869C12Q1/04C12N15/11C40B50/06
CPCC12Q1/689C12Q1/6869C40B50/06C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2535/122C12Q2565/125Y02A90/10
Inventor 张子龙田桢干张威周娴李深伟水晶
Owner 上海国际旅行卫生保健中心
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