The genetic relationship of S. aureus with one or more enterotoxins was analyzed with multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). MLST was performed as previously described by Saunders and Holmes (2014) [20 (link)], and seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and yqiL) purified using the GFX PCR DNA and Gel Band Purification Kit (Amersham Bioscience, Freiburg, Germany) were sequenced with an automatic sequencer (Cosmogenetech, Deajeon, Korea). Sequence types (STs) were obtained by combination using the S. aureus database (https://pubmlst.org/organisms/staphylococcus-aureus (accessed on 22 January 2022)). Moreover, PFGE was conducted by digesting genomic DNA using the SmaI enzyme (Takara Bio Inc., Shiga, Japan) according to a standard protocol of the Centers for Disease Control and Prevention (CDC, USA) [21 ], using a CHEF-MAPPER apparatus (Bio-Rad Laboratories, Hercules, CA), as described previously [22 (link)], and analyzed using the BioNumerics software (Applied Maths, Kortrijk, Belgium).
Smai enzyme
Manufactured by Takara Bio
Sourced in Japan
SmaI is a type II restriction endonuclease enzyme isolated from the bacterium Serratia marcescens. It recognizes and cleaves the palindromic DNA sequence 5'-CCCGGG-3' with high specificity.
Automatically generated - may contain errors
4 protocols using smai enzyme
1
Genetic Relationship of S. aureus and Enterotoxins
2
Molecular Typing of Enterococcus spp.
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Pulsed-field gel electrophoresis (PFGE) was performed using the SmaI enzyme (Takara Bio Inc., Shiga, Japan), as described previously [2 (link)]. PFGE banding profiles were analyzed using Bionumerics software version 4.0 (Applied Maths, Sint-Martens-Latem, Belgium) and relatedness was calculated using the unweighted pair-group method with arithmetic averages (UPGMA) algorithm, based on the Dice similarity index. Multi-locus sequence typing (MLST) was performed as recommended on the PubMLST website (https://pubmlst.org ), and allelic profiles and sequence types were determined using the E. faecium or E. faecalis MLST database (http://pubmlst.org/efaecalis/ or http://pubmlst.org/efaecium/ ), respectively.
3
Pulsed-field Gel Electrophoresis for Campylobacter
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Pulsed-field gel electrophoresis was performed based on the CDC PulseNet protocol for Campylobacter spp. (Ribot et al., 2001 (link)). Briefly, DNA was digested with 40 U of SmaI enzyme (TaKaRa, Japan) at 30°C for 4 h. The separation of restriction fragments was performed in 1% SeaKem gold agarose (Lonza, Switzerland) gels in 0.5× Tris–borate–EDTA (TBE) buffer (Millipore Sigma, Burlington, MA, United States) using the CHEF Mapper system (Bio-Rad), with the following parameters: initial switch time, 6.76 s; final switch time, 35.38 s for 18 h at 6 V/cm, and condensation temperature of 14°C. PFGE profiles were analyzed using BioNumerics software version 7.5 (Applied Maths, Kortrijk, Belgium). XbaI-digested Salmonella enterica serovar Braenderup H9812 was used as a molecular size marker. The dendrogram was created by UPGMA with the Dice similarity coefficient and a position tolerance of 1.5%. Clusters were defined based on an 85% similarity cutoff.
4
Genetic Profiling of S. aureus Enterotoxins
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The genetic relationship of S. aureus with one or more enterotoxin was analyzed by multilocus sequence typing (MLST) and pulsed-eld gel electrophoresis (PFGE). MLST was performed as previously described by Saunders and Holmes (2014) [20] (link), and seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and yqiL) puri ed using the GFX PCR DNA and Gel Band Puri cation Kit (Amersham Bioscience, Freiburg, Germany) were sequenced with an automatic sequencer (Cosmogenetech, Deajeon, Korea). Sequence types (STs) were obtained by combination at the S. aureus database (https://pubmlst.org/organisms/staphylococcus-aureus). Moreover, PFGE was conducted by digesting genomic DNA using the SmaI enzyme (Takara Bio Inc., Shiga, Japan) according to a standard protocol of the Centers for Disease Control and Prevention (CDC, USA) [21], using a CHEF-MAPPER apparatus (Bio-Rad Laboratories, Hercules, CA), as described previously [22] , and analyzed using the BioNumerics software (Applied Maths, Kortrijk, Belgium).
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