Molecular Typing for MLVA

Multiple-Locus Variable-number tandem repeat Analysis

MLVA is the second major genotyping tool to be used in PulseNet network. This page contains detailed MLVA protocols for both laboratory and BioNumerics analysis, powerpoint presentations explaining the protocols, bionumerics anlaysis and troubleshooting, details of Equipment and Reagents required for MLVA, protocols currently in development, and a brief explanation of what MLVA is.

MLVA Protocols (Updated Feb 2014)

Standardized MLVA protocols are currently available for STEC O157 and S. enterica serotype Typhimurium. Separate protocols for Beckman Coulter CEQ™8000/8800/GeXP, Applied Biosystems Genetic Analyzer 3130, and the Applied Biosystems Genetic Analyzer 3500 platforms have been developed.

MLVA protocols for Beckman Coulter CEQ™8000/8800/GeXP
E. coli O157 (STEC), Salmonella enterica serotypes Typhimurium and Enteritidis

 Lab protocol (PNL19) [PDF, 1.7 MB] - Feb 2014, 1704kb

 PCR template (PNL19-2) [XLS, 50 KB] - Feb 2014, 50kb

 MasterMix worksheet - E.coli  (PNL19-3a) [XLS, 62 KB] - Feb 2014, 62kb

 MasterMix worksheet - S.Typhimurium (PNL19-3b) [XLS, 65 KB] - Feb 2014, 65kb

 MasterMix worksheet - S.Enteritidis (PNL19-3c [XLS, 47 KB]) - Feb 2014, 47kb

 

 BioNumerics Protocol (PND14) [PDF, 527 KB] 

       Feb 2014, 527kb

MLVA protocols for Applied Biosystems Genetic Analyzer 3130
E. coli O157 (STEC), Salmonella enterica serotypes Typhimurium and Enteritidis

 Lab protocol (PNL23) [PDF, 1.7 MB] - February 2014, 1771 kb

 PCR template (PNL23-2) [XLS, 45 KB] - Feb 2014, 45kb

 MasterMix worksheet - E.coli (PNL23-3a) [XLS, 54 KB] - Feb 2014, 54kb

 MasterMix worksheet - S.Typhimurium (PNL23-3b) [XLS, 63 KB] - Feb 2014, 63kb

 MasterMix worksheet - S.Enteritidis (PNL23-3c) [XLS, 58 KB] - Feb 2014, 58kb

 

 BioNumerics Protocol (PNDL16) [PDF, 591 KB]

          Feb 2014, 591kb

MLVA protocols for Applied Biosystems Genetic Analyzer 3500
E. coli O157 (STEC), Salmonella enterica serotypes Typhimurium and Enteritidis

 Lab protocol (PNL28) [PDF, 1.9 MB] - February 2014, 1771 kb

 PCR template (PNL28-2) [XLS, 45 KB] - Feb 2014, 45kb

 MasterMix worksheet - E.coli (PNL28-3a) [XLS, 54 KB] - Feb 2014, 54kb

 MasterMix worksheet - S.Typhimurium (PNL28-3b) [XLS, 63 KB] - Feb 2014, 63kb

 MasterMix worksheet - S.Enteritidis (PNL28-3c) [XLS, 58 KB] - Feb 2014, 58kb

 

 BioNumerics Protocol (PNDL16) [PDF, 591 KB]

          Feb 2014, 591kb

MLVA Protocols (Updated May 2013)

Standardized MLVA protocols are currently available for STEC O157 and S. enterica serotype Typhimurium. Separate protocols for Beckman Coulter CEQ™8000/8800, Applied Biosystems Genetic Analyzer 3130xl and 3730xl, and the Applied Biosystems Genetic Analyzer 3500 platforms have been developed. The newer sequencer versions Beckman Coulter GeXP has not been validated yet so the data generated by that version may or may not be comparable with the data from the older versions.

MLVA protocols for Beckman Coulter CEQ™8000/8800
E. coli O157 (STEC)

 Lab protocol (PNL19) - Sept 2010, 764kb

 PCR template (PNL19-2) - April 2013, 38kb

 MasterMix worksheet (PNL19-3) - April 2013, 62kb

 BioNumerics Protocol (PND14) 

       Sept 2010, 417kb

S. enterica serotype Typhimurium

 Lab protocol (PNL21) - October 2009, 983kb

 PCR template (PNL21-2) - April 2013, 37kb

 MasterMix worksheet (PNL21-3) - April 2013, 65kb

 BioNumerics Protocol (PND15) 

        May 2007, 433kb

S. enterica serotype Enteritidis

 Lab protocol (PNL27) - June 2010, 911kb

 PCR template (PNL27-2) - April 2013, 37kb

 MasterMix worksheet (PNL27-3) - April 2013, 47kb

 BioNumerics Protocol (PND19)

        June 2010, 400kb

MLVA protocols for Applied Biosystems Genetic Analyzer 3130xl and 3730xl
E. coli O157 (STEC)

 Lab protocol (PNL23) - February 2009, 990 kb

 PCR template (PNL23-2) - April 2013, 37kb

 MasterMix worksheet (PNL23-3) - April 2013, 54kb

 BioNumerics Protocol (PNDL16)

          March 2010, 447kb

S. enterica serotype Typhimurium

 Lab protocol (PNL24) - October 2009, 965kb

 PCR template (PNL24-2) - April 2013, 38kb

 MasterMix worksheet (PNL24-3) - April 2013, 63kb

 BioNumerics Protocol (PNDL17)

          October 2009, 436kb

S. enterica serotype Enteritidis

 Lab protocol (PNL26) - March 2010, 994kb 

 PCR template (PNL26-2) - April 2013, 37kb

 MasterMix worksheet (PNL26-3) - April 2013, 58kb

 BioNumerics Protocol (PNDL18)

          August 2010, 412kb

MLVA protocols for Applied Biosystems Genetic Analyzer 3500
E. coli O157 (STEC)

 Lab protocol (PNL28) - May 2013, 1061kb

 PCR template (PNL28-2) - April 2013, 37kb

 MasterMix worksheet (PNL28-3) - April 2013, 54kb

 BioNumerics Protocol (PNDL16)

          March 2010, 447kb

S. enterica serotype Typhimurium

 Lab protocol (PNL29) - May 2013, 1030kb

 PCR template (PNL29-2) - April 2013, 38kb

 MasterMix worksheet (PNL29-3) - April 2013, 63kb

 BioNumerics Protocol (PNDL17)

          October 2009, 436kb

S. enterica serotype Enteritidis

 Lab protocol (PNL30) - May 2013, 1063kb 

 PCR template (PNL30-2) - May 2013, 37kb

 MasterMix worksheet (PNL30-3) - May 2013, 58kb

 BioNumerics Protocol (PNDL18)

          August 2010, 412kb

 

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MLVA Protocols Presentations

These presentations give an overview of the MLVA Protocol. Click on the image or the link itself.

MLVA Overview

 MLVA Protocols - General Overview - PDF 834 KB

Download MLVA Analysis in BioNumerics PDF

 Analysis of MVLA data in BioNumerics - PDF 618 KB

MLVA Troubleshooting PDF

 Troubleshooting hints on MVLA - PDF 594 KB

 

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MLVA protocols under development

MLVA protocols for Salmonella serotype Enteritidis (Group D) and Listeria monocytogenes are currently under validation. When completed, the completed protocols will be posted on this site. Participants in the network are also working on MLVA protocols for Salmonella serotype Newport, Shigella sonnei and Non-O157 STEC.

For additional information on these activities, please email Eija Trees at CDC.

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Equipment and Reagents Required for MLVA

These MLVA protocols have been developed on Beckman Coulter CEQ™8000/8800 and Applied Biosystems Genetic Analyzer 3130xl and 3730xl platforms. The newer sequencer versions Beckman Coulter GeXP and Applied Biosystems Genetic Analyzer 3500 and 3500xl have not been validated yet so the data generated by those versions may or may not be comparable with the data from the older versions.

Click here to download a list of equipment and reagents: MLVA_equipmentsupplies.pdf (74 KB)

The control strains are in the process of being submitted to ATCC. In the meantime requests may be made to Eija Trees.

PulseNet have developed BioNumerics scripts which can be requested from Eija Trees.

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MLVA Explained

Virtually all pro- and eukaryotic genomes contain short repeat sequences that are dispersed throughout the genome. In each repeat sequence locus the repeat copy number can vary between different strains, hence these sequences are often referred to as “variable-number tandem repeats” or VNTRs (Figure 1). 

Two loci

Figure 1. Examples of two loci from different strains of an organism. The repeat sequences are in color and in locus 1 is a four base repeat (ACgC), while in locus 2 a three base repeat is targeted (ACC).

When multiple different VNTRs are targeted for analysis, the technology is called “multiple-locus VNTR analysis” or MLVA. In MLVA, the VNTR array is amplified using PCR (Figure 2).

Two loci

Figure 2.PCR amplification of VNTR array in MLVA

Forward primers are fluorescently labeled so that the PCR product can be detected and sized using high resolution capillary electrophoresis (automatic sequencer) (Figure 3). Use of multiple different fluorescent tags enables multiplexing targets with overlapping fragment sizes. At each loci, the number of copies of each repeat is assigned based on the observed fragment sizes (allele type).

Two loci

Figure 3. An example of a chromatogram from the Beckman Coulter CEQ platform. The red peaks are the size standard peaks and the blue, green and yellow peaks are the VNTR peaks. The CEQ software labels each peak with the observed size on the chromatogram. 

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