RESUMO
INTRODUCTION: Toxoplasmosis is caused by the parasite Toxoplasma gondii. The infection is generally asymptomatic and the most severe cases occur in immunosuppressed patients. The main route of transmission is the ingestion of water or food contaminated with cysts of the parasite. The objective of this work was the standardization of the PCR for the detection of the T. gondii B1 gene in meat and water samples and cloning of the product for use as a control. METHODS: The optimal reaction conditions of the different components of the PCR were determined and the technique was used to detect DNA from meat and water samples. Bands were purified and cloned into a pGEM-T-Easy vector and used as a control in the PCR. RESULTS: Optimal PCR conditions were; 100 µM dNTP, 0.4 µM primers, and 0.5 U Taq polymerase. The product obtained from the PCR was cloned with a simple cloning strategy with efficient results. With the standardized PCR and using the cloned DNA as a control, T. gondii DNA was detected in 90% of the positives samples of meat and water and there was no amplification in the negative samples. CONCLUSIONS: The PCR assay standardized in this study was demonstrated to be an effective technique to detect T. gondii DNA in meat and water samples. The cloning of PCR product and its application as a control in molecular diagnosis of toxoplasmosis might improve the reproducibility of this method and avoid the use of patient samples or cultures, which present several limitations.
Assuntos
Toxoplasma , Toxoplasmose , Clonagem Molecular , DNA de Protozoário/genética , Humanos , Carne , Reação em Cadeia da Polimerase , Padrões de Referência , Reprodutibilidade dos Testes , Toxoplasma/genética , Toxoplasmose/diagnóstico , ÁguaRESUMO
RESUMEN La determinación de Chlamydia psittaci (Cp) en aves psitácidas en parques zoológicos de Venezuela representa una estrategia de conservación y preservación para este grupo de aves, donde múltiples especies se encuentran amenazadas de extinción y otras han perdido su capacidad de reincorporación a su hábitat natural. A través de la reacción en cadena de la polimerasa anidada (PCR) fue amplificada la subunidad 16S del ADNr de Cp en 50 muestras de hisopado cloacal de aves psitácidas, reportando una frecuencia de 62 %. El trabajo fue realizado en el Parque Zoológico Las Delicias (PZD) 8 % y el Aquarium de Valencia (AV) 54 %. La elevada frecuencia fue asociada a un genotipo de baja concentración y virulencia debido a la ausencia de signos clínicos de clamidiosis aviar. Estos resultados demuestran la necesidad de promover la detección de Cp, principalmente para el AV que actúa como centro de recepción de ejemplares de decomiso, y, al igual que el PZD, poseen otras especies vulnerables a la extinción con riesgo de infección a Cp.
ABSTRACT The determination of Chlamydia psittaci (Cp) in psittacida birds in zoological parks in Venezuela represents a strategy of conservation and preservation for this group of birds, where multiple species are threatened with extinction and others have lost their capacity of reincorporation to their natural habitat. Through the nested polymerase chain reaction (PCR) the 16S subunit of Cp DNAr was amplified in 50 cloacal swab samples from psittacine birds, reporting a frequency of 62 %. The work was carried out in the Zoo Park Las Delicias (PZD) 8% and the Aquarium of Valencia (AV) 54%. The high frequency was associated with a genotype of low concentration and virulence due to the absence of clinical signs of avian chlamydiosis. These results demonstrate the need to promote the detection of Cp, mainly for the AV that acts as a center of reception of specimens of confiscation, and, like the PZD, have other species vulnerable to extinction with risk of infection to Cp.