PROTOCOL FOR MLVA TYPING, application to Legionella MLVA typing

 

Summary : this overview describes the MLVA assay from the PCR reaction set-up to the visual analysis of gel images. This text has been adapted by CP from the tutorial written for the Brucella MLVA workshop, COST845 project Orsay March 2006.

 

1- DNA

 

It is recommended to use good PCR-quality DNA.

A reference strain (such as Legionella pneumophila Philadelphia-1 or one of the sequenced strains) for which the expected size is known for each VNTR locus is needed.

Typical set-up to genotype 30 strains : it is recommended to make a DNA master plate using a 96 wells PCR plate (or V-type wells) :

Figure 1 : DNA master plate (1 to 5 ng/l) including the reference DNA

 

2- VNTR PCR amplification and genotyping

 

2.1 PCR conditions :

Reactions are made in a 15l final volume including : 

-1 to 5ng of DNA

-10x PCR Reaction Buffer

-1U of Taq DNA polymerase

-200M of each deoxynucleotide triphosphate (2 mM each dNTP master solution)

-0.3 M of each flanking primer.

 

 

1 PCR reaction

30 PCR reactions

+10%

Primers mix

1.5l

45l

50 l

10x PCR buffer

1.5l

45l

50 l

dNTP (2 mM each)

1.5l

45l

50 l

Taq (5U/l)

0.2 l

6 l

7 l

H2O

8.5 l

255l

280l

Distribute 13 l of above mix per well

DNA

Distribute 2l per well

Final volume

15l per well

Table 1 : PCR mix for 30 reactions

 

 

Figure 2 : distributing reaction mix and DNAs into PCR plate for VNTR locus 1

 

 

 

2.2 Amplification :

-Initial denaturation step at 94C for 5 minutes

-followed by 35 cycles of

denaturation at 94C for 30s,

primer annealing at 60C for 30 s,

extension at 72C for 45 s.

-Final extension step : 72C for 10 min.

2.3 Electrophoresis

 Add 1l loading solution and load 3l of the PCR reaction products on an agarose gel in 0.5x TBE buffer. Run at  8 V/cm until the bromophenol blue has reached 20 cm (2 combs on a 40 cm long gel).

For all VNTRs except Lpms37, use a 2% standard agarose gel. For Lpms37 use a 4% gel comprising 2% Metaphor (FMC Bioproducts-Cambrex) plus 2% regular agarose.

The size markers used are a 100-bp ladder (Bio-Rad, EZ Load 100pb PCR Molecular Ruler) or 20-bp ladder (Bio-Rad, EZ Load 20pb Molecular Ruler) according to the VNTR unit length. 

Three microliters of amplification product load on gel (3 by 3) with a multi-channel pipette.

 

Figure 3 : Loading of PCR products on the agarose gel

 

 

2.4 Ethidium bromide staining and photograph

Gels are stained with ethidium bromide ([0.5 to 1 g/ml] ; 20l to 40l of 10 mg/ml stock solution in 400ml 0.5x TBE) for 15-30 minutes and photographed under U.V. light.

 

 

 

Figure 4. Analysis of Lpms1_b amplicons by 2% agarose gel electrophoresis. Ten EUL strains were analysed together with the Philadelphia-1 (PhiI) and Paris (plus Lens in image C) reference strains as control by the three centers. Gel images from Center I, II and III are shown in (A), (B) and (C), respectively. The Lpms1_b repeat unit is 45-bp, and the expected size in Philadelphia-1, Paris and Lens strains is 565, 520, and  475-bp respectively (8, 7, and 6 units) (Table 1). Strains EUL no. 137 and 025 have 9 repeats (allele 9), strains EUL no. 153, 154 and 155 have 8 repeats (allele 8), strains EUL no. 157 and 121 have 7 repeats (allele 7), while strains EUL no. 048 and 056 have an intermediate repeat size coded 7.5 (allele 7.5) and strain EUL no.156 a repeat size coded 9.5 (Table 3). The DNA size marker is a  100-bp ladder. (D), BioNumerics-assisted analysis of gel image C performed by Center I. As illustrated, the cursor was positioned at the top of the bands in order to fit with the expected size in the reference strain Philadelphia-1, and then similarly positioned in the subsequent lanes.

 

Figure 5 : gel image, locus Lpms35.

Migration on 2% agarose gel

 

3- Data analysis

The number of repeat units can be deduced from the allele size using reference strain for comparison.

 

 

 

 

 

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