On this website

On the whole CNRS Web

Home page > MLVA Web service > Frequently asked questions

Frequently asked questions

I can get the PCR assay to work, but am having problems analyzing the bands to get accurate sizes from my gels. I wonder how to actually calculate an exact band size and the repeat number. I am using the size markers to make a standard curve based on distance migrated on the gel. This old fashioned method is working, but once I get a band size, I cannot figure out how to get a repeat copy number.

When setting up the assay, a first step is to identify a reference strain. Any strain in current use in the laboratory is adequate. Vaccine strains are a good choice since typing data for them is given in the Le Flèche 2006 report (see supplementary data file). Then eventually it will be useful to sequence the alleles from this reference strain, to make sure no discrepancy will have been introduced. As described by Garcia-Yoldi et al., 2007, some loci may have mutated upon culturing in (...)

Read more

Do I need to do a PCR on the control strain each time a PCR for an "unknown" strain is setup? Or could I simply use leftover product from a previous PCR on the control strain to run on a gel with the products from the strains I am trying to type ?

Do not reuse old PCR for your control DNA : the control DNA controls many things, i.e. mix-up of primers, mix up of samples, gel loading errors, PCR quality, in addition to gel to gel variations.

Read more

May I run different loci on the same agarose gel ?

As a rule, avoid mixing different loci on the same gel : some markers need to be run longer than the others (see the spacing in the 100 bp ladder bands in Le Flèche 2006 Figure 2). Some are very easily typed, so that short runs are sufficient. Some markers require a 20 bp size marker, others a 100 bp size marker.

Read more

I do not have the 16M reference strain. Is it possible to use an other control strain ?

Any strain can be used. A vaccine strain is convenient, Rev1, S19, RB51, since typing data for representatives is available from Le Flèche 2006 supplementary file. For instance, if willing to use the abortus S19 vaccine strain as a reference : first find what genotype is expected for strain S19. Go to Le Flèche 2006, get supplementary data (text) file, search for S19, (genotype 161) the panel 1 typing result is : 4-5-4-12-2-2-3-3 Then using the allele conversion table and the published (...)

Read more

Is it better to use agarose or capillary electrophoresis for MLVA typing?

The rule is to do what is most convenient for you. If you have access to a capillary electrophoresis sequencing machine, and anticipate to have a significant number of strains to type, then this is clearly the best option : you can multiplex the amplifications so that even a 20 loci MLVA assay can be run in less than five PCR amplifications per strain. Also allele calling is automated. Whatever applicable method you use, the end results will be identical. Applicable means that of course you (...)

Read more