Multilocus Sequence Typing of Bacterial Strains

Based on the previous results from the adhesion/invasion assay, we selected 34 strains for sequence typing (Du et al., 2016 (link)). Based on the manufacturer’s instruction for the DNA Extraction Kit (Omega Bio-Tek, Norcross, GA, United States), genomic DNA from the 34 strains was obtained after overnight cultivation. Primers for seven housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, and pps) were used for PCR (Baldwin et al., 2009 (link)), and the amplified products were sequenced with the Sanger chain termination method by Suzhou Jinweizhi Biotechnology Co., Ltd. (Suzhou, China). The sequencing results of the seven housekeeping genes were concatenated in a certain order to match with the PubMLST database and used to construct a phylogenetic tree by MEGA 6.0. The relationships among the 34 strains were analyzed via the neighbor-joining statistical method coupled with Tamura’s 3-parameter model (Joseph and Forsythe, 2012 (link)).

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Li P., Dong X., Wang X.Y., Du T., Du X.J, & Wang S. (2020). Comparative Proteomic Analysis of Adhesion/Invasion Related Proteins in Cronobacter sakazakii Based on Data-Independent Acquisition Coupled With LC-MS/MS. Frontiers in Microbiology, 11, 1239.

Publication 2020

DNA Extraction Kit

Assay Genomic Housekeeping genes Primers Strains

Corresponding Organization : Nankai University

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Variable analysis

independent variables

Selection of 34 strains for sequence typing based on previous adhesion/invasion assay results

dependent variables

Genomic DNA obtained after overnight cultivation of the 34 strains
Sequences of seven housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, and pps) amplified by PCR
Phylogenetic tree constructed using the concatenated sequences of the seven housekeeping genes

control variables

DNA Extraction Kit (Omega Bio-Tek, Norcross, GA, United States) used according to manufacturer's instructions
Primers for the seven housekeeping genes (Baldwin et al., 2009)
Sanger chain termination method used for sequencing by Suzhou Jinweizhi Biotechnology Co., Ltd. (Suzhou, China)
PubMLST database used as the reference for matching the concatenated sequences
Neighbor-joining statistical method coupled with Tamura's 3-parameter model used for phylogenetic tree construction (Joseph and Forsythe, 2012)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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