Multilocus Sequence Typing (MLST): Markers for the Traceability of Pathogenic Leptospira Strains


Locus/genea

Primer

Sequence (5′ to 3′)b

MgCl2 (mM)c

Size of PCR product (bp)

Location of the sequence used to define MLST locusd, e

Size of MLST locus (bp)d

adk

adkF

GGGCTGGAAAAGGTACACAA

(alternative: ACATTATCTTCATGGGACCTCC) f

1.5

531

557

3458789–3458361

429
 
adkR

ACGCAAGCTCCTTTTGAATC

(alternative: TTACACAAGCTCCCTTTGAAT) f
    
icdA

icdAF

GGGACGAGATGACCAGGAT

1.5

674

3980926–3980372

555
 
icdAR

TTTTTTGAGATCCGCAGCTTT

(alternative: CTTTTTTGAGATCTCCGGCTTT) f
 
674
  
lipL41

LipL41F

TAGGAAATTGCGCAGCTACA

1.5

520

3603644–3604120

477
 
LipL41R

GCATCGAGAGGAATTAACATCA
    
rrs

rrsF

CATGCAAGTCAAGCGGAGTA

1.5

541

1862535–1862984

450
 
rrsR

AGTTGAGCCCGCAGTTTTC
    
secY

secYF

ATGCCGATCATTTTTGCTTC

1.5

549

3459402–3458902

501
 
secYR

CCGTCCCTTAATTTTAGACTTCTTC

(alternative: CCTTCCTTTAATTTTAGACTTTTTC) f
 
548
  
lipL32

LipL32F

ATCTCCGTTGCACTCTTTGC

1.5

474

1667072–166641

432
 
LipL32R

ACCATCATCATCATCGTCCA
    

a adk (adenylate kinase), icdA (isocitrate dehydrogenase), lipL41 (outer membrane lipoprotein LipL41), rrs (16S rRNA), secY (preprotein translocase SecY protein), and lipL32 (outer membrane lipoprotein LipL32)

bOriginal primers according to Ahmed et al. [1] and additional alternative primers for some loci according to Ahmed et al. [5]

cConcentration of MgCl2 in the PCR mixture as adjusted for each primer pair

dThe location of the sequence used to define MLST locus and the size of MLST locus (bp) as modified in Ahmed et al. [5]

eNucleotide positions based in the published genome sequence of L. interrogans serovar Copenhageni strain Fiocruz L1-130 chromosome I (GenBank Accession Number AE016823)

fWhen using these alternative primers, the annealing temperature should be adjusted to 54 °C in the PCR cycling conditions




Table 2
Loci and respective flanking oligonucleotide primers for the Leptospira 7 L MLST scheme




































































































































Locus/genea

Primer
Sequence (5′ to 3′)b

MgCl2 (mM)c

Size of PCR product (bp)
Location of MLST locusd

Size of MLST locus (bp)

glmU

glmU-FM

AGGATAAGGTCGCTGTGGTA
1.5
650
3784955–3784512
444
 
glmU-RM

AGTTTTTTTCCGGAGTTTCT
        

pntA

pntA-FM
TAGGAAARATGAAACCRGGAAC
1.5
621
56347–56871
525
 
pntA-RM
AAGAAGCAAGATCCACAAYTAC
        

sucA

sucA-FM
TCATTCCACTTYTAGATACGAT
2.5
640
1227474–1227920
447
 
sucA-RM
TCTTTTTTGAATTTTTGACG
        

tpiA

tpiA-FM
TTGCAGGAAACTGGAAAATGAAT
3.5
639
1694673–1694248
426
 
tpiA-RM
GTTTTACRGAACCHCCGTAGAGAAT
        

pfkB

pfkB-FM
CGGAGAGTTTTATAARAAGGACAT
1.5
588
1386553–1386984
432
 
pfkB-RM
AGAACACCCGCCGCAAAACAAT
        

mreA

mreA-FM
GGCTCGCTCTYGACGGAAA
2.0
719
2734550–2734116
435
 
mreA-RM
TCCRTAACTCATAAAMGACAAAGG
        

caiB

caiB-F
CAACTTGCGGAYATAGGAGGAG
1.5
650
1562845–1563246
402
 
caiB-R
ATTATGTTCCCCGTGAYTCG
        


a glmU (UDP-N-acetylglucosamine pyrophosphorylase), pntA (NADP transhydrogenase subunit alpha), sucA (2-oxoglutarate dehydrogenase decarboxylase component), tpiA (triosephosphate isomerase), pfkB (ribokinase), mreA (rod-shape-determining protein rodA), and caiB (carnitine dehydratase)

bPrimers according to Boonsilp et al. [4]

cConcentration of MgCl2 in the PCR mixture as adjusted for each primer pair

dNucleotide positions based in the published genome sequence of L. interrogans serovar Lai strain 56601 chromosome I (GenBank Accession Number NC004342)



 


2.

Reagents for conventional PCR reactions: DNA polymerase enzyme and respective 10× buffer, MgCl2 (25 mM), dNTPs (25 mM), and distilled PCR grade water.

 

3.

Negative (sample without added DNA from leptospires) and positive (sample spiked with leptospiral DNA approximating 20 ng genomic DNA per reaction) PCR reaction controls.

 

4.

PCR thermocycler.

 

5.

Tris-borate EDTA (TBE) buffer (pH 8.0): 50 mM Tris, 50 mM boric acid, 0.5 mM EDTA.

 

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Mar 17, 2017 | Posted by in GENERAL | Comments Off on Multilocus Sequence Typing (MLST): Markers for the Traceability of Pathogenic Leptospira Strains

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