DEVELOPMENT OF REAL-TIME MULTIPLEX PCR FOR THE QUANTITATIVE DETERMINATION OF TREC'S AND KREC'S IN WHOLE BLOOD AND IN DRIED BLOOD SPOTS

Primary immunodeficiencies (PID) such as severe combined immunodeficiency (SCID) and X-linked agammaglobulinemia are characterized by the lack of functional Tand B-cells, respectively. Without early diagnosis and prompt treatment children with PID suffer from severe infectious diseases, leading to t...

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Autores principales:	M. A. Gordukova, I. P. Oskorbin, O. V. Mishukova, S. B. Zimin, N. V. Zinovieva, N. V. Davydova, A. S. Smirnova, I. A. Nikitina, I. A. Korsunsky, M. L. Filipenko, A. P. Prodeus
Formato:	article
Lenguaje:	RU
Publicado:	SPb RAACI 2015
Materias:	primary immunodeficiency diagnostics trec krec real-time pcr Immunologic diseases. Allergy RC581-607
Acceso en línea:	https://doaj.org/article/c3f1e10d7232434ca5cd629a3edb747d
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spelling	oai:doaj.org-article:c3f1e10d7232434ca5cd629a3edb747d2021-11-18T08:03:45ZDEVELOPMENT OF REAL-TIME MULTIPLEX PCR FOR THE QUANTITATIVE DETERMINATION OF TREC'S AND KREC'S IN WHOLE BLOOD AND IN DRIED BLOOD SPOTS1563-06252313-741X10.15789/1563-0625-2015-5-467-478https://doaj.org/article/c3f1e10d7232434ca5cd629a3edb747d2015-10-01T00:00:00Zhttps://www.mimmun.ru/mimmun/article/view/935https://doaj.org/toc/1563-0625https://doaj.org/toc/2313-741XPrimary immunodeficiencies (PID) such as severe combined immunodeficiency (SCID) and X-linked agammaglobulinemia are characterized by the lack of functional Tand B-cells, respectively. Without early diagnosis and prompt treatment children with PID suffer from severe infectious diseases, leading to their death or disability. Our purpose was developing of simple, inexpensive, high throughput technique based on the quantitative determination of TREC and KREC molecules by real-time PCR, and its validation in a group of children with a verified diagnosis of SCID and X-linked agammaglobulinemia.In this study, we developed and validated multiplex real-time PCR for the TREC’s and KREC’s quantitative analysis. We have shown that linear range of Ct changes depending on the concentrations of targets with a correlation coefficient R2 not worse than 0.98 was observed at concentrations from 109 to 5 × 104 copies per ml. The lowest amount of targets reliably detected in a reaction volume was 10 TREC’s copies, 5 KREC ‘s copies and 5 copies of internal control (IL17RA). We determined the age-depended reference values of TRECs and KRECs in whole blood in 29 boys and 27 girls with normal immunological parameters. The normal cut-offs for TRECs and KRECs were defined in dry blood spots depending on the method of extraction.The proposed method showed 100% diagnostic sensitivity and specificity in the studied group. The method can be proposed as a screening tool for the diagnosis of SCID and X-linked agammaglobulinemia both in whole blood and in the dry blood spots. The further investigation is required with larger number of samples.M. A. GordukovaI. P. OskorbinO. V. MishukovaS. B. ZiminN. V. ZinovievaN. V. DavydovaA. S. SmirnovaI. A. NikitinaI. A. KorsunskyM. L. FilipenkoA. P. ProdeusSPb RAACIarticleprimary immunodeficiencydiagnosticstreckrecreal-time pcrImmunologic diseases. AllergyRC581-607RUMedicinskaâ Immunologiâ, Vol 17, Iss 5, Pp 467-478 (2015)
institution	DOAJ
collection	DOAJ
language	RU
topic	primary immunodeficiency diagnostics trec krec real-time pcr Immunologic diseases. Allergy RC581-607
spellingShingle	primary immunodeficiency diagnostics trec krec real-time pcr Immunologic diseases. Allergy RC581-607 M. A. Gordukova I. P. Oskorbin O. V. Mishukova S. B. Zimin N. V. Zinovieva N. V. Davydova A. S. Smirnova I. A. Nikitina I. A. Korsunsky M. L. Filipenko A. P. Prodeus DEVELOPMENT OF REAL-TIME MULTIPLEX PCR FOR THE QUANTITATIVE DETERMINATION OF TREC'S AND KREC'S IN WHOLE BLOOD AND IN DRIED BLOOD SPOTS
description	Primary immunodeficiencies (PID) such as severe combined immunodeficiency (SCID) and X-linked agammaglobulinemia are characterized by the lack of functional Tand B-cells, respectively. Without early diagnosis and prompt treatment children with PID suffer from severe infectious diseases, leading to their death or disability. Our purpose was developing of simple, inexpensive, high throughput technique based on the quantitative determination of TREC and KREC molecules by real-time PCR, and its validation in a group of children with a verified diagnosis of SCID and X-linked agammaglobulinemia.In this study, we developed and validated multiplex real-time PCR for the TREC’s and KREC’s quantitative analysis. We have shown that linear range of Ct changes depending on the concentrations of targets with a correlation coefficient R2 not worse than 0.98 was observed at concentrations from 109 to 5 × 104 copies per ml. The lowest amount of targets reliably detected in a reaction volume was 10 TREC’s copies, 5 KREC ‘s copies and 5 copies of internal control (IL17RA). We determined the age-depended reference values of TRECs and KRECs in whole blood in 29 boys and 27 girls with normal immunological parameters. The normal cut-offs for TRECs and KRECs were defined in dry blood spots depending on the method of extraction.The proposed method showed 100% diagnostic sensitivity and specificity in the studied group. The method can be proposed as a screening tool for the diagnosis of SCID and X-linked agammaglobulinemia both in whole blood and in the dry blood spots. The further investigation is required with larger number of samples.
format	article
author	M. A. Gordukova I. P. Oskorbin O. V. Mishukova S. B. Zimin N. V. Zinovieva N. V. Davydova A. S. Smirnova I. A. Nikitina I. A. Korsunsky M. L. Filipenko A. P. Prodeus
author_facet	M. A. Gordukova I. P. Oskorbin O. V. Mishukova S. B. Zimin N. V. Zinovieva N. V. Davydova A. S. Smirnova I. A. Nikitina I. A. Korsunsky M. L. Filipenko A. P. Prodeus
author_sort	M. A. Gordukova
title	DEVELOPMENT OF REAL-TIME MULTIPLEX PCR FOR THE QUANTITATIVE DETERMINATION OF TREC'S AND KREC'S IN WHOLE BLOOD AND IN DRIED BLOOD SPOTS
title_short	DEVELOPMENT OF REAL-TIME MULTIPLEX PCR FOR THE QUANTITATIVE DETERMINATION OF TREC'S AND KREC'S IN WHOLE BLOOD AND IN DRIED BLOOD SPOTS
title_full	DEVELOPMENT OF REAL-TIME MULTIPLEX PCR FOR THE QUANTITATIVE DETERMINATION OF TREC'S AND KREC'S IN WHOLE BLOOD AND IN DRIED BLOOD SPOTS
title_fullStr	DEVELOPMENT OF REAL-TIME MULTIPLEX PCR FOR THE QUANTITATIVE DETERMINATION OF TREC'S AND KREC'S IN WHOLE BLOOD AND IN DRIED BLOOD SPOTS
title_full_unstemmed	DEVELOPMENT OF REAL-TIME MULTIPLEX PCR FOR THE QUANTITATIVE DETERMINATION OF TREC'S AND KREC'S IN WHOLE BLOOD AND IN DRIED BLOOD SPOTS
title_sort	development of real-time multiplex pcr for the quantitative determination of trec's and krec's in whole blood and in dried blood spots
publisher	SPb RAACI
publishDate	2015
url	https://doaj.org/article/c3f1e10d7232434ca5cd629a3edb747d
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DEVELOPMENT OF REAL-TIME MULTIPLEX PCR FOR THE QUANTITATIVE DETERMINATION OF TREC'S AND KREC'S IN WHOLE BLOOD AND IN DRIED BLOOD SPOTS

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