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Development of Real-Time PCR Array for Simultaneous Detection of Eight Human Blood-Borne Viral Pathogens
BACKGROUND: Real-time PCR array for rapid detection of multiple viral pathogens should be highly useful in cases where the sample volume and the time of testing are limited, i.e. in the eligibility testing of tissue and organ donors. FINDINGS: We developed a real-time PCR array capable of simultaneo...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Public Library of Science
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422334/ https://www.ncbi.nlm.nih.gov/pubmed/22912836 http://dx.doi.org/10.1371/journal.pone.0043246 |
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author | Pripuzova, Natalia Wang, Richard Tsai, Shien Li, Bingjie Hung, Guo-Chiuan Ptak, Roger G. Lo, Shyh-Ching |
author_facet | Pripuzova, Natalia Wang, Richard Tsai, Shien Li, Bingjie Hung, Guo-Chiuan Ptak, Roger G. Lo, Shyh-Ching |
author_sort | Pripuzova, Natalia |
collection | PubMed |
description | BACKGROUND: Real-time PCR array for rapid detection of multiple viral pathogens should be highly useful in cases where the sample volume and the time of testing are limited, i.e. in the eligibility testing of tissue and organ donors. FINDINGS: We developed a real-time PCR array capable of simultaneously detecting eight human viral pathogens: human immunodeficiency virus types 1 and 2 (HIV-1 and -2), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia virus-1 and -2 (HTLV-1 and -2), vaccinia virus (VACV) and West Nile virus (WNV). One hundred twenty (120) primers were designed using a combination of bioinformatics approaches, and, after experimental testing, 24 primer sets targeting eight viral pathogens were selected to set up the array with SYBR Green chemistry. The specificity and sensitivity of the virus-specific primer sets selected for the array were evaluated using analytical panels with known amounts of viruses spiked into human plasma. The array detected: 10 genome equivalents (geq)/ml of HIV-2 and HCV, 50 geq of HIV-1 (subtype B), HBV (genotype A) and WNV. It detected 100–1,000 geq/ml of plasma of HIV-1 subtypes (A – G), group N and CRF (AE and AG) isolates. Further evaluation with a panel consisting of 28 HIV-1 and HIV-2 clinical isolates revealed no cross-reactivity of HIV-1 or HIV-2 specific primers with another type of HIV. All 28 viral isolates were identified with specific primer sets targeting the most conserved genome areas. The PCR array correctly identified viral infections in a panel of 17 previously quantified clinical plasma samples positive for HIV-1, HCV or HBV at as low as several geq per PCR reaction. CONCLUSIONS: The viral array described here demonstrated adequate performance in the testing of donors’ clinical samples. Further improvement in its sensitivity for the broad spectrum of HIV-1 subtypes is under development. |
format | Online Article Text |
id | pubmed-3422334 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-34223342012-08-21 Development of Real-Time PCR Array for Simultaneous Detection of Eight Human Blood-Borne Viral Pathogens Pripuzova, Natalia Wang, Richard Tsai, Shien Li, Bingjie Hung, Guo-Chiuan Ptak, Roger G. Lo, Shyh-Ching PLoS One Research Article BACKGROUND: Real-time PCR array for rapid detection of multiple viral pathogens should be highly useful in cases where the sample volume and the time of testing are limited, i.e. in the eligibility testing of tissue and organ donors. FINDINGS: We developed a real-time PCR array capable of simultaneously detecting eight human viral pathogens: human immunodeficiency virus types 1 and 2 (HIV-1 and -2), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia virus-1 and -2 (HTLV-1 and -2), vaccinia virus (VACV) and West Nile virus (WNV). One hundred twenty (120) primers were designed using a combination of bioinformatics approaches, and, after experimental testing, 24 primer sets targeting eight viral pathogens were selected to set up the array with SYBR Green chemistry. The specificity and sensitivity of the virus-specific primer sets selected for the array were evaluated using analytical panels with known amounts of viruses spiked into human plasma. The array detected: 10 genome equivalents (geq)/ml of HIV-2 and HCV, 50 geq of HIV-1 (subtype B), HBV (genotype A) and WNV. It detected 100–1,000 geq/ml of plasma of HIV-1 subtypes (A – G), group N and CRF (AE and AG) isolates. Further evaluation with a panel consisting of 28 HIV-1 and HIV-2 clinical isolates revealed no cross-reactivity of HIV-1 or HIV-2 specific primers with another type of HIV. All 28 viral isolates were identified with specific primer sets targeting the most conserved genome areas. The PCR array correctly identified viral infections in a panel of 17 previously quantified clinical plasma samples positive for HIV-1, HCV or HBV at as low as several geq per PCR reaction. CONCLUSIONS: The viral array described here demonstrated adequate performance in the testing of donors’ clinical samples. Further improvement in its sensitivity for the broad spectrum of HIV-1 subtypes is under development. Public Library of Science 2012-08-17 /pmc/articles/PMC3422334/ /pubmed/22912836 http://dx.doi.org/10.1371/journal.pone.0043246 Text en https://creativecommons.org/publicdomain/zero/1.0/ This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. |
spellingShingle | Research Article Pripuzova, Natalia Wang, Richard Tsai, Shien Li, Bingjie Hung, Guo-Chiuan Ptak, Roger G. Lo, Shyh-Ching Development of Real-Time PCR Array for Simultaneous Detection of Eight Human Blood-Borne Viral Pathogens |
title | Development of Real-Time PCR Array for Simultaneous Detection of Eight Human Blood-Borne Viral Pathogens |
title_full | Development of Real-Time PCR Array for Simultaneous Detection of Eight Human Blood-Borne Viral Pathogens |
title_fullStr | Development of Real-Time PCR Array for Simultaneous Detection of Eight Human Blood-Borne Viral Pathogens |
title_full_unstemmed | Development of Real-Time PCR Array for Simultaneous Detection of Eight Human Blood-Borne Viral Pathogens |
title_short | Development of Real-Time PCR Array for Simultaneous Detection of Eight Human Blood-Borne Viral Pathogens |
title_sort | development of real-time pcr array for simultaneous detection of eight human blood-borne viral pathogens |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422334/ https://www.ncbi.nlm.nih.gov/pubmed/22912836 http://dx.doi.org/10.1371/journal.pone.0043246 |
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