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CN-121992123-A - Multi-site sequence typing primer and typing method for Serratia marcescens

CN121992123ACN 121992123 ACN121992123 ACN 121992123ACN-121992123-A

Abstract

The invention provides a multi-site sequence typing primer and a typing method for Serratia marcescens, belonging to the technical field of pathogenic microorganism molecule detection. The invention provides a specific primer group for multi-site sequence typing of Serratia marcescens, which comprises primer groups pitA, cyoB, glnA, ftsP, nadR, purR and potD designed for 7 housekeeping genes. The invention also provides an MLST typing method of Serratia marcescens, which comprises the steps of extracting genome DNA of a strain to be detected, respectively carrying out PCR amplification by using the primers, sequencing the amplified products, and comparing the amplified products with a pre-established MLST standard sequence database of Serratia marcescens to obtain the sequence type of the strain. The invention provides a standardized MLST typing primer and a method for Serratia marcescens, which have the advantages of accurate typing, good repeatability, digitalized result, convenience for data sharing among laboratories and the like.

Inventors

  • LIU XIONG
  • JIN JIN
  • XU YAQING
  • QIN RIHUI
  • ZHENG XIAOYU
  • LIU WEI
  • Zou dayang
  • WANG KEHUI
  • LI XIANHUANG
  • LI LINHAO
  • Du Xingyue
  • ZHOU RENHUI

Assignees

  • 中国人民解放军疾病预防控制中心

Dates

Publication Date
20260508
Application Date
20260212

Claims (10)

  1. 1. A multi-site sequence typing primer group of Serratia marcescens is characterized in that the multi-site sequence typing primer is designed aiming at housekeeping genes derived from Serratia marcescens, and the housekeeping genes comprise a set of pitA, cyoB, glnA, ftsP, nadR, purR and potD genes.
  2. 2. The multi-site sequence typing primer set of claim 1, wherein the nucleotide sequences of the multi-site sequence typing primer set are shown in SEQ ID No. 1-SEQ ID No.14 in sequence.
  3. 3. Use of the multi-site sequence typing primer group of claim 1 or 2 in serratia marcescens typing identification.
  4. 4. A method for typing and identifying Serratia marcescens is characterized by comprising the following steps of taking genomic DNA of Serratia marcescens to be detected as a template, and respectively carrying out PCR (polymerase chain reaction) amplification by using the multi-site sequence typing primer group of claim 1 or 2 to obtain 7 amplification products; Two-way sequencing is carried out after 7 amplification products are recovered, so that sequences of 7 amplification products are obtained; Comparing the sequences of the 7 amplification products with a serratia marcescens MLST standard sequence database to obtain ST typing of the serratia marcescens.
  5. 5. The genotyping method according to claim 4, wherein the PCR amplification system comprises, in terms of 50. Mu.L, 2 XTaq Master mix 25. Mu.L, 10. Mu.M forward primer 1. Mu.L, 10. Mu.M reverse primer 1. Mu.L, bacterial sample DNA template 2. Mu.L and the balance double distilled water.
  6. 6. The method according to claim 4 or 5, wherein the PCR amplification procedure comprises pre-denaturation at 95℃for 3 minutes, denaturation at 95℃for 30 seconds, annealing at 56-61℃for 30 seconds, extension at 72℃for 60 seconds, 34 cycles, and final extension at 72℃for 5 minutes.
  7. 7. The method of typing according to claim 4, further comprising performing an electrophoretic verification after the PCR amplification.
  8. 8. The genotyping method according to claim 4, wherein the construction method of the serratia marcescens MLST standard sequence database comprises numbering different sequence variants of each housekeeping gene fragment using Blast comparison and extraction of housekeeping gene sequences for serratia marcescens from a public database, and defining a combination of 7 allele numbers as a sequence type; The housekeeping genes include the following sets pitA, cyoB, glnA, ftsP, nadR, purR and potD.
  9. 9. The genotyping method according to claim 8, wherein the numbering is carried out sequentially by pitA, cyoB, glnA, ftsP, nadR, purR and potD.
  10. 10. A serratia marcescens genotyping kit comprising the multi-site sequence typing primer set of claim 1 or 2 and a PCR amplification reagent.

Description

Multi-site sequence typing primer and typing method for Serratia marcescens Technical Field The invention belongs to the technical field of pathogenic microorganism molecule detection, and particularly relates to a multi-site sequence typing primer and a typing method for Serratia marcescens. Background Serratia marcescens (SERRATIA MARCESCENS) is an important opportunistic pathogen and nosocomial infection pathogen, and development of molecular typing technology of Serratia marcescens has undergone three stages of phenotype typing, genotyping and genome typing. Currently, the main typing methods used in research and clinical practice are Pulsed Field Gel Electrophoresis (PFGE), whole Genome Sequencing (WGS) based typing methods, and the like. PFGE has become the "gold standard" technique for epidemiological investigation of bacterial molecules since the 90 s of the 20 th century, and is particularly suitable for the source of outbreak of nosocomial infections. The basic principle is that a rare cutting restriction endonuclease (such as XbaI and SpeI) is used for carrying out enzyme cutting on the whole genome DNA of bacteria to generate 10-800 kb large fragment DNA, and the DNA is separated by pulse electric field electrophoresis to form a strain specific DNA fingerprint. For example, milisavljevic et al (2004) used PFGE for cloning-related analysis in the outbreak investigation of Serratia marcescens in neonatal intensive care units. The method has higher resolution and repeatability, and is a typing technology recommended to be used in many countries and regions. With the decline in high throughput sequencing costs and the development of bioinformatics, WGS-based typing has become a "new gold standard" for bacterial molecular typing. The WGS-based typing method of Serratia marcescens mainly involves ① core genome multi-site sequence typing (cgMLST) by allelic typing of a core gene common to all strains (typically hundreds to thousands). KAMPMEIER et al (2022) established a cgMLST protocol for Serratia marcescens containing 2,692 core gene sites that exhibited extremely high resolution in outbreak surveys. ② The genome-wide multisite sequence typing (wgMLST) is to incorporate part of accessory genes based on cgMLST to further improve the resolution. Rosen et al (2019) have used wgMLST for epidemiological analysis of Serratia marcescens. ③ Single Nucleotide Polymorphism (SNP) analysis, namely, identifying SNP differences among strains through whole genome comparison, constructing a phylogenetic tree, which is the method with the highest resolution at present and is commonly used for accurate tracing. Multi-site sequence typing (MLST) is a standardized typing technique based on housekeeping gene sequences, first proposed by Maiden et al in 1998 and used for Neisseria meningitidis. It selects 5-10 housekeeping genes with conservation of function and stable evolution, and obtains Sequence Type (ST) after PCR amplification and sequencing of internal fragments of about 450-500bp, and assigning allele numbers according to sequence differences. This approach has established standardized protocols among hundreds of bacteria and has formed a global shared database such as PubMLST, BIGSdb. Among enterobacteriaceae and other pathogenic bacteria, MLST has become a conventional molecular epidemiological tool, for example, 7-gene MLST protocols such as adk, fumC, gyrB are used for escherichia coli (ESCHERICHIA COLI), and gapA, infB, mdh are used for klebsiella pneumoniae (Klebsiella pneumoniae). Although the MLST technique is mature in its application to numerous pathogenic bacteria, the standardized MLST regimen for Serratia marcescens is currently lacking. The existing MLST primers for other bacteria (such as klebsiella pneumoniae, staphylococcus aureus and the like) have no specificity on Serratia marcescens and cannot be directly applied. Although few studies have attempted to use housekeeping genes from other species of bacteria for local MLST analysis, the selected gene combinations are not uniform, primer sequences are not disclosed, large-scale validation and database support are lacking, resulting in data incompatibility between different studies and failure to form a standard MLST protocol. Disclosure of Invention The invention provides a multi-site sequence parting primer and a parting method for Serratia marcescens, which have the advantages of accurate parting, good repeatability, digitalized results, convenience for data sharing among laboratories and the like, and are suitable for molecular epidemiological investigation and genetic evolution research of Serratia marcescens. The invention provides a multi-site sequence typing primer group of Serratia marcescens, which is designed aiming at housekeeping genes derived from Serratia marcescens, wherein the housekeeping genes comprise the following sets of pitA, cyoB, glnA, ftsP, nadR, purR and potD. In a specific embodiment of the invention, the nucleotide se