Development of a Quantitative Real-Time PCR Assay for Detection of Toxoplasma gondii in Brain Samples

Background: Toxoplasmosis is a worldwide-distributed infection that can cause serious diseases, mainly in congenitally infected and immunodeficient individuals. PCR assays play an indispensable role in the detection of Toxoplasma gondii in different biological samples. Methods: This study was con...

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Autores principales:	Mahboubeh Berizi, Jalal Babaie, Pezhman Fard esfahani, Marjan Enshaeieh, Rahmah Noordin, Geita Saadatnia, Majid Golkar
Formato:	article
Lenguaje:	EN
Publicado:	Tehran University of Medical Sciences 2021
Materias:	toxoplasma gondii detection polymerase chain reaction Infectious and parasitic diseases RC109-216
Acceso en línea:	https://doaj.org/article/88d25db1c2c84ddfa1931b26f8a02f78
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spelling	oai:doaj.org-article:88d25db1c2c84ddfa1931b26f8a02f782021-11-29T07:18:54ZDevelopment of a Quantitative Real-Time PCR Assay for Detection of Toxoplasma gondii in Brain Samples1735-70202008-238Xhttps://doaj.org/article/88d25db1c2c84ddfa1931b26f8a02f782021-11-01T00:00:00Zhttps://ijpa.tums.ac.ir/index.php/ijpa/article/view/3027https://doaj.org/toc/1735-7020https://doaj.org/toc/2008-238XBackground: Toxoplasmosis is a worldwide-distributed infection that can cause serious diseases, mainly in congenitally infected and immunodeficient individuals. PCR assays play an indispensable role in the detection of Toxoplasma gondii in different biological samples. Methods: This study was conducted in the Parasitology Department at Pasteur Institute of Iran (Tehran) during 2016-2018. We designed a highly sensitive quantitative real-time PCR (RT-qPCR) targeted REP-529, a noncoding repetitive DNA. We cloned the amplicon in a plasmid (pTZREP-529) and used it to generate the standard curve. The Toxoplasma RT-qPCR characteristics, i.e., detection limit, specificity, linear dynamic range, linearity, intra-, and inter-assay precisions, were determined. The detection limit of the assay was one plasmid copy number (PCN) per reaction (about 0.004 T. gondii genome), and the linear dynamic range was equal to 6 logs (1× 101 to 1× 107 PCN per reaction). Results: The assay showed no signal when genomic DNA of Plasmodium falciparum, Leishmania major, and Trichomonas vaginallis were used. The standard curve was drawn using dilutions of pTZREP-529 plasmid spiked with genomic DNA from a mouse brain, and test characteristics were shown unaffected. Applying the Toxoplasma RT-qPCR, we showed brain cysts were significantly decreased in mice vaccinated with GRA2 antigen of Toxoplasma formulated in Monophosphoryl Lipid A (MPL) adjuvant. Conclusion: We have developed a quantitative, specific, and highly sensitive PCR for detecting T. gondii in biological samples.Mahboubeh BeriziJalal BabaiePezhman Fard esfahaniMarjan EnshaeiehRahmah NoordinGeita SaadatniaMajid GolkarTehran University of Medical Sciencesarticletoxoplasma gondiidetectionpolymerase chain reactionInfectious and parasitic diseasesRC109-216ENIranian Journal of Parasitology, Vol 16, Iss 4, Pp 621-630 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	toxoplasma gondii detection polymerase chain reaction Infectious and parasitic diseases RC109-216
spellingShingle	toxoplasma gondii detection polymerase chain reaction Infectious and parasitic diseases RC109-216 Mahboubeh Berizi Jalal Babaie Pezhman Fard esfahani Marjan Enshaeieh Rahmah Noordin Geita Saadatnia Majid Golkar Development of a Quantitative Real-Time PCR Assay for Detection of Toxoplasma gondii in Brain Samples
description	Background: Toxoplasmosis is a worldwide-distributed infection that can cause serious diseases, mainly in congenitally infected and immunodeficient individuals. PCR assays play an indispensable role in the detection of Toxoplasma gondii in different biological samples. Methods: This study was conducted in the Parasitology Department at Pasteur Institute of Iran (Tehran) during 2016-2018. We designed a highly sensitive quantitative real-time PCR (RT-qPCR) targeted REP-529, a noncoding repetitive DNA. We cloned the amplicon in a plasmid (pTZREP-529) and used it to generate the standard curve. The Toxoplasma RT-qPCR characteristics, i.e., detection limit, specificity, linear dynamic range, linearity, intra-, and inter-assay precisions, were determined. The detection limit of the assay was one plasmid copy number (PCN) per reaction (about 0.004 T. gondii genome), and the linear dynamic range was equal to 6 logs (1× 101 to 1× 107 PCN per reaction). Results: The assay showed no signal when genomic DNA of Plasmodium falciparum, Leishmania major, and Trichomonas vaginallis were used. The standard curve was drawn using dilutions of pTZREP-529 plasmid spiked with genomic DNA from a mouse brain, and test characteristics were shown unaffected. Applying the Toxoplasma RT-qPCR, we showed brain cysts were significantly decreased in mice vaccinated with GRA2 antigen of Toxoplasma formulated in Monophosphoryl Lipid A (MPL) adjuvant. Conclusion: We have developed a quantitative, specific, and highly sensitive PCR for detecting T. gondii in biological samples.
format	article
author	Mahboubeh Berizi Jalal Babaie Pezhman Fard esfahani Marjan Enshaeieh Rahmah Noordin Geita Saadatnia Majid Golkar
author_facet	Mahboubeh Berizi Jalal Babaie Pezhman Fard esfahani Marjan Enshaeieh Rahmah Noordin Geita Saadatnia Majid Golkar
author_sort	Mahboubeh Berizi
title	Development of a Quantitative Real-Time PCR Assay for Detection of Toxoplasma gondii in Brain Samples
title_short	Development of a Quantitative Real-Time PCR Assay for Detection of Toxoplasma gondii in Brain Samples
title_full	Development of a Quantitative Real-Time PCR Assay for Detection of Toxoplasma gondii in Brain Samples
title_fullStr	Development of a Quantitative Real-Time PCR Assay for Detection of Toxoplasma gondii in Brain Samples
title_full_unstemmed	Development of a Quantitative Real-Time PCR Assay for Detection of Toxoplasma gondii in Brain Samples
title_sort	development of a quantitative real-time pcr assay for detection of toxoplasma gondii in brain samples
publisher	Tehran University of Medical Sciences
publishDate	2021
url	https://doaj.org/article/88d25db1c2c84ddfa1931b26f8a02f78
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Development of a Quantitative Real-Time PCR Assay for Detection of Toxoplasma gondii in Brain Samples

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