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) :
2.1 PCR conditions :
Reactions
are made in a 15µl final volume including :
-1 to
5ng of DNA
-10x
PCR Reaction Buffer
-1U
of Taq DNA polymerase
-200µM
of each deoxynucleotide triphosphate (2 mM each dNTP master solution)
-0.3
µM of each flanking primer.
|
1 PCR reaction |
30 PCR reactions |
+10% |
Primers
mix |
1.5µl |
45µl |
50 µl |
10x
PCR buffer |
1.5µl |
45µl |
50 µl |
dNTP
(2 mM each) |
1.5µl |
45µl |
50 µl |
Taq
(5U/µl) |
0.2 µl |
6 µl |
7 µl |
H2O |
8.5 µl |
255µl |
280µl |
Distribute
13 µl of above mix per
well |
|||
DNA |
Distribute
2µl per well |
||
Final
volume |
15µl 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 94°C for 5 minutes
-followed
by 35 cycles of
denaturation
at 94°C for 30s,
primer
annealing at 60°C for 30 s,
extension
at 72°C for 45 s.
-Final extension step : 72°C for 10 min.
Add 1µl loading
solution and load 3µl 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 µg/ml] ; 20µl to 40µl 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
The
number of repeat units can be deduced from the allele size using reference
strain for comparison.
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