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Cronobacter MLST Primers and Protocols

Genes

The Cronobacter spp. MLST scheme uses fragments of the following seven house-keeping genes:

PCR amplification

Reaction conditions for all the primers were as follows: initial denaturation at 96°C for 1 min; 30 cycles of denaturation at 96°C for 1 min, primer annealing at 58°C for 1 min, extension at 72°C for 2 min; followed by a final extension step of 72°C for 5 min. Each 50 µl amplification reaction mixture comprised: 10 µl Q solution (Qiagen), 4.0 µl chromosomal DNA (5-20 ng/µl), 2.0 µl forward primer (10 pmol/µl), 2.0 µl reverse primer (10 pmol/µl), 5.0 µl 10x PCR buffer (Qiagen, contains 15 mM MgCl2), 1.0 µl dNTP solution (Qiagen, 10 mM each dNTP), 0.25 µl Taq polymerase (Qiagen, 5 Units/µl) and 25.75 µl PCR-grade water. All Qiagen solutions from PCR CORE Kit (Cat No. 201225).

The amplification product was then purified using MinElute UF plates (Qiagen) following the manufacturers protocol before being used in a sequencing reaction. Sequencing was carried out on each DNA strand with BigDye Terminator Ready Reaction Mix v3.1 (PE Biosystems, Foster City, US) under standard sequencing conditions according to the manufacturer's protocol. Unincorporated dye terminators were removed by precipitation with 95% alcohol. The reaction products were separated and detected on an ABI PRISM genetic analyser 3100 (PE Biosystems) using a standard sequencing module with a Performance Optimised Polymer and 5 cm array.

The main primers used in MLST analysis of Cronobacter spp. are listed below.

Please try alternative primers, designed by Emily Jackson, if you experience problems with amplification or sequencing.

GeneDirectionPrimer Sequence
atpDFCGACATGAAAGGCGACAT
RTTAAAGCCACGGATGGTG
fusAFGAAACCGTATGGCGTCAG
RAGAACCGAAGTGCAGACG
glnSFGCATCTACCCGATGTACG
RTTGGCACGCTGAACAGAC
gltBFCATCTCGACCATCGCTTC
RCAGCACTTCCACCAGCTC
gyrBFTGCACCACATGGTATTCG
RCACCGGTCACAAACTCGT
infBFGAAGAAGCGGTAATGAGC
RCGATACCACATTCCATGC
ppsFGTCCAACAATGGCTCGTC
RCAGACTCAGCCAGGTTTG

Sequencing

GeneDirectionPrimer SequenceAllele size (bp)
atpDFCGAAATGACCGACTCCAA390
RGGATGGCGATGATGTCTT
fusAFGCTGGATGCGGTAATTGA438
RCCCATACCAGCGATGATG
glnSFGGGTGCTGGATAACATCA363
RCTTGTTGGCTTCTTCACG
gltBFGCGAATACCACGCCTACA507
RGCGTATTTCACGGAGGAG
gyrBFCTCGCGGGTCACTGTAAA402
RACGCCGATACCGTCTTTT
infBFTGACCACGGTAAAACCTC441
RGGACCACGACCTTTATCC
ppsFACCCTGACGAATTCTACG495
RCAGATCCGGCATGGTATC

Citing the database

The preferred format for citing this website in publications is:

This publication made use of the Cronobacter Multi Locus Sequence Typing website (https://pubmlst.org/ cronobacter/) developed by Keith Jolley and sited at the University of Oxford (Jolley & Maiden 2010, BMC Bioinformatics, 11:595). The development of this site has been funded by the Wellcome Trust.

Status

Sequence database
Sequences: 64056
Profiles:
Ext-MLST: 0
MLST: 625
O-serotype: 0

Isolate database
Isolates: 2391
Last updated: 2017-12-28