Genotyping of <i>Leptospira interrogans</i> isolates from Mexican patients

García-González, Rafael; Reyes-Torres, Angélica; Reyes-Montes, María del Rocío; Duarte-Escalante, Esperanza; Frías-De-León, María Guadalupe; Rivas-Sánchez, Beatriz; Velasco-Castrejón, Oscar

doi:10.1590/S1678-9946201961026

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Original Article • Rev. Inst. Med. trop. S. Paulo 61 • 2019 • https://doi.org/10.1590/S1678-9946201961026 copy

Genotyping of Leptospira interrogans isolates from Mexican patients

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

The aim of this study was genotypically characterize Leptospira sp. clinical isolates from Mexico which were previously identified as Leptospira interrogans serovar Pomona (POM) by phenotypic methods. The Random Amplified Polymorphic DNA (RAPD) method was used for DNA amplification with five oligonucleotides. A dendrogram was constructed using the Unweighted Pair Group Method Analysis (UPGMA). During the genotypic characterization, the studied isolates constituted a group which was associated with the reference strain L. interrogans serovar Pomona. The Minimum Spanning Networks (MST) analysis revealed the same cluster between Mexican isolates and the reference strain POM. Clinical isolates identified as L. interrogans serovar POM have a clonal reproduction type, suggesting that this clone is distributed in different regions of Mexico.

KEYWORDS:
L. interrogans serovar POM; RAPD; UPGMA; Minimum Spanning Networks

Reference strains	Species	Serovar
ICT	Leptospira interrogans	Icterohaemorrhagiae
CAN	Leptospira interrogans	Canicola
POM	Leptospira interrogans	Pomona
AUT	Leptospira interrogans	Autumnalis
BRA	Leptospira interrogans	Bratislava
TA	Leptospira interrogans	Tarassoni
LAI	Leptospira interrogans	Icterohaemorrhagiae lai lai
PYR	Leptospira interrogans	Pyrogenes
BAL	Leptospira interrogans	Balum
SHER	Leptospira interrogans	Shermani
PTC	Leptospira interrogans	Ptc Patoc

Oligonucleotide	05	B11-B12	M16	PB1
Reaction volume	25 μL	25 μL	25 μL	25 μL
DNA	20 ng	20 ng	20 ng	20 ng
Taq buffer 10X (Tris-HCI 100 mM, KCI 500 mM, pH 9.0)	1X	1X	1X	1X
dNTPs	0.1 mM of each	0.1 mM of each	250 μM of each	250 μM of each
MgCl₂	3.5 mM	4.5 mM	1.5 mM	3.5 mM
Oligonucleotide	300 pmol	300 pmol	200 pmol	300 pmol
Taq DNA polimerase	0.5 U	0.5 U	0.5 U	0.5 U
Amplification program	One initial cycle of one min at 94 °C, followed by 40 cycles of 1 min at 94 °C, 1 min at 36 °C, and 2 min at 72 °C, with a final extension cycle of 3 min at 72 °C	Two cycles of 5 min at 95 °C, 5 min at 40 °C, and 5 min at 72 °C, followed by 35 cycles of 1 min at 95 °C, 1 min at 60 °C, and 3 min at 72 °C, with one final extension cycle of 72 °C for 10 min	One cycle of 3 min at 94 °C, 1 min at 55 °C, and 2 min at 72 °C, followed by 38 cycles of 1 min at 94 °C, 1 min at 55 °C, and 2 min at 72 °C, with a final extension cycle of 1 min at 94 °C, 1 min at 55 °C, and 9 min at 72 °C	One cycle of 7 min at 94 °C, 1 min at 40 °C, and 1 min at 72 °C, and four cycles of 1 min at 94 °C, 1 min at 40 °C, and 1 min at 72 °C, which continued with 24 cycles of 1 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C, with a final extension cycle of 1 min at 94 °C, 1 min at 55 °C, and 7 min at 72 °C

Isolate	Abbrevation
Rivepal	RIV
Verimol	SM
Veriluma	LMJT
Verimer	GGMC
Beribéri	BRS
Rivemar	LMFA
Verimat	MGR
Beriveca	BCB
Vecorisa	VECO
Verijua	HCHJ
Verichan	CHAN
Veritsa	TSAB

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E-mail: revimtsp@usp.br

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[1] Correspondence to: Rafael García-González, Universidad Nacional Autónoma de México (UNAM), Facultad de Medicina, Departamento de Microbiología y Parasitología, Avenida Universidad, N° 3000, 04510, Cd. Mx., México E-mail:rafagargon@hotmail.com