Elianee Useche1, Angélica Jiménez1, Katherine Armada1, Bárbara Castillo1, Mercedes Viettri1, Anabel Bandes2, Elizabeth Ferrer3,4. 1. Instituto de Investigaciones Biomédicas "Dr. Francisco J. Triana Alonso" (BIOMED), Facultad de Ciencias de la Salud, Universidad de Carabobo Sede Aragua, Calle Cecilio Acosta, Urb. La Rinconada, Las Delicias, estado Aragua, Maracay, Venezuela. 2. Instituto Nacional de Higiene "Rafael Rangel", Ministerio del Poder Popular Para la Salud, Caracas, Venezuela. 3. Instituto de Investigaciones Biomédicas "Dr. Francisco J. Triana Alonso" (BIOMED), Facultad de Ciencias de la Salud, Universidad de Carabobo Sede Aragua, Calle Cecilio Acosta, Urb. La Rinconada, Las Delicias, estado Aragua, Maracay, Venezuela. elizabeth.ferrer@gmail.com. 4. Departamento de Parasitología, Facultad de Ciencias de la Salud, Universidad de Carabobo Sede Aragua, Maracay, Venezuela. elizabeth.ferrer@gmail.com.
Abstract
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.
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.
Authors: C Bourdin; A Busse; E Kouamou; F Touafek; B Bodaghi; P Le Hoang; D Mazier; L Paris; A Fekkar Journal: J Clin Microbiol Date: 2014-09-10 Impact factor: 5.948
Authors: Jean Joel Bigna; Joel Noutakdie Tochie; Dahlia Noelle Tounouga; Anne Olive Bekolo; Nadia S Ymele; Paule Sandra Simé; Jobert Richie Nansseu Journal: BMJ Open Date: 2019-10-19 Impact factor: 2.692