Multilocus VNTR Analysis for Vibrio cholerae

All PCRs were conducted by the same technician. Fluorescently labeled PCR primers were used to amplify 5 loci with variable-length tandem repeats; a new reverse primer was used for VCA0283 (5′-AGCCTCCTCAGAAGTTGAG-3′) (13 (link)). The reaction products were separated, detected, and sized by using a 3730xl automatic sequencer, internal lane standards (Liz600), and GeneScan program (all from Applied Biosystems). Genotypes were determined by using published formulas to calculate the number of repeats from the length of each allele except for VC0437, which is calculated by (x – 252)/7 (13 (link)). Five loci were ordered: VC0147, VC0437, VC1650, VCA0171, and VCA0283. A genotype (e.g., 9-4-6-19-11) was interpreted as an isolate having alleles of 9, 4, 6, 19, and 11 repeats at the 5 loci, respectively. Relatedness of isolates was assessed by using eBURST version 3 (http://eburst.mlst.net). Genetically related genotypes are defined as those possessing identical alleles at 4 of the 5 loci and groups of related genotypes (clonal complexes). Additional sensitivity analyses were conducted by using only the 3 more stable loci from the large chromosome (VC0147, VC0437, VC1650); relatedness was determined by identical alleles at 2 of the 3 loci (13 (link),21 (link)).

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Rashed S.M., Azman A.S., Alam M., Li S., Sack D.A., Morris JG J.r., Longini I., Siddique A.K., Iqbal A., Huq A., Colwell R.R., Sack R.B, & Stine O.C. (2014). Genetic Variation of Vibrio cholerae during Outbreaks, Bangladesh, 2010–2011. Emerging Infectious Diseases, 20(1), 54-60.

Publication 2014

3730xl automatic sequencer Liz600 GeneScan program

Alleles Chromosome 3 Clonal Formulas Genotypes Primer Sensitivity Variable tandem repeats

Corresponding Organization :

Other organizations : International Centre for Diarrhoeal Disease Research, Johns Hopkins University, University of Florida, University of Maryland, College Park

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Variable analysis

independent variables

Fluorescently labeled PCR primers were used to amplify 5 loci with variable-length tandem repeats
A new reverse primer was used for VCA0283 (5′-AGCCTCCTCAGAAGTTGAG-3′)

dependent variables

Genotypes were determined by using published formulas to calculate the number of repeats from the length of each allele
Relatedness of isolates was assessed by using eBURST version 3

control variables

All PCRs were conducted by the same technician
The reaction products were separated, detected, and sized by using a 3730xl automatic sequencer, internal lane standards (Liz600), and GeneScan program (all from Applied Biosystems)

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