Can cloning and sequencing help to genotype positive Toxoplasma gondii clinical samples? Results and validation using SAG3 as a model.

Autor: Rico-Torres CP; Laboratorio de Inmunología Experimental, Instituto Nacional de Pediatría, Insurgentes Sur 3700-C, Colonia Insurgentes-Cuicuilco, Delegación Coyoacán, 04530 Ciudad de México, Mexico., Valenzuela-Moreno LF; Laboratorio de Inmunología Experimental, Instituto Nacional de Pediatría, Insurgentes Sur 3700-C, Colonia Insurgentes-Cuicuilco, Delegación Coyoacán, 04530 Ciudad de México, Mexico., Méndez-Cruz ST; Laboratorio de Bioquímica Genética, Instituto Nacional de Pediatría, Insurgentes Sur 3700-C, Colonia Insurgentes-Cuicuilco, Delegación Coyoacán, 04530 Ciudad de México, Mexico., Cedillo-Peláez C; Laboratorio de Inmunología Experimental, Instituto Nacional de Pediatría, Insurgentes Sur 3700-C, Colonia Insurgentes-Cuicuilco, Delegación Coyoacán, 04530 Ciudad de México, Mexico., Caballero-Ortega H; Laboratorio de Inmunología Experimental, Instituto Nacional de Pediatría, Insurgentes Sur 3700-C, Colonia Insurgentes-Cuicuilco, Delegación Coyoacán, 04530 Ciudad de México, Mexico. Electronic address: hcaballero_2000@yahoo.com.mx.
Jazyk: angličtina
Zdroj: Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases [Infect Genet Evol] 2022 Jul; Vol. 101, pp. 105283. Date of Electronic Publication: 2022 Apr 11.
DOI: 10.1016/j.meegid.2022.105283
Abstrakt: Genotyping of T. gondii in human cases is relevant to understand the transmission patterns and epidemiology of this parasitosis. However, this genetic characterization can be hampered by the difficulty of isolating the parasite from mild or asymptomatic cases and by the detection efficiency of molecular assays such as the multilocus nested-polymerase chain reaction-restriction fragment length polymorphism (Mn-PCR-RLFP). To propose an alternative for the genotyping of positive clinical samples of T. gondii with a low amount of the parasite DNA mixed within the host DNA or mixed infections, we carried out this study to validate the sequences of the SAG3 gene of T. gondii obtained after two rounds of amplification cloned into a bacterial model, thereby achieving the separation and identification of more than one genotype of T. gondii. Also, the detection limit of the parasite DNA and the fidelity of the reagents used in the nested PCR-RFLP in artificial clinical samples by sequencing were determined. T. gondii DNA was detected from 6.25 ng of DNA and 200 parasites/mL of blood. The fidelity of the AmpliTaq Gold™ polymerase after 65 cycles of amplification was 100%. Denaturation of the products obtained after two rounds of nested PCR amplification showed no evidence of chimera or artifact production. The cloning efficiency was 97.5% (39/40 clones), and none of the experiments produced recombinant sequences. Thus, the generation of chimeras with this methodology could be ruled out. Genotyping of clinical samples is important because there is no strain selection bias, as can occur in the bioassay (where more virulent strains can be selected over nonvirulent strains), and therefore, mixed infections can be detected through cloning and sequencing. Furthermore, these two techniques could be useful tools to genotype weak amplicons of any T. gondii gene obtained during nested PCR.
(Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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