Development and clinical evaluation of an in-house human immunodeficiency virus-1 real-time RT-PCR quantitation assay
Quantification of Human Immunodeficiency Virus (HIV-1) RNA plays a vital role in diagnosing and monitoring HIV virological behavior in patients. Currently, the HIV antiretroviral therapy aims to suppress the HIV replication to its minimal, thus quantifying HIV RNA is an indicative tool for assessing...
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| Format: | Thesis |
| Language: | English |
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2012
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| Online Access: | https://hdl.handle.net/10356/48079 |
| _version_ | 1826111871888916480 |
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| author | Wee, Sue Yuen |
| author2 | Lim Chu Sing |
| author_facet | Lim Chu Sing Wee, Sue Yuen |
| author_sort | Wee, Sue Yuen |
| collection | NTU |
| description | Quantification of Human Immunodeficiency Virus (HIV-1) RNA plays a vital role in diagnosing and monitoring HIV virological behavior in patients. Currently, the HIV antiretroviral therapy aims to suppress the HIV replication to its minimal, thus quantifying HIV RNA is an indicative tool for assessing the therapeutics response. Despite its importance in HIV treatment, most patients have limited access to the viral load tests due to cost and resource constraints. Additionally, great variability among HIV genotypes and wide dynamic range of clinical HIV RNA levels has presented tough challenges for developing Polymerase Chain Reaction (PCR)-based amplification assays.
In this study, we describe the development, evaluation, and validation of a real-time RT-PCR assay designed to provide accurate quantification of viral RNA patients’ specimens. This assay can perform a wide dynamic range of at least 8log10 copies/mL. It has a high intra-assay precision with coefficient of variation (CV) of 0.825% and standard deviation (SD) of 0.04 log10 at 7.0log10 copies/μL of external standards (ES); CV of 2.2% and SD of 0.23 log10 at 1.0log10 copies/μL. It produces inter-assay precision of CV% of Ct value less than 4.2% and SD less than 0.9 PCR cycles for serially diluted ES of from 10 to 108 copies/reaction. The high precision and repeatability of this assay allow the derivation of a model for the viral load quantification result for determining antiretroviral drug therapy effectiveness. Probit analysis has determined the 95% confidence detection level is 61.5copies/ml. The genotype inclusivity of the Group M and N, combined with a small sample volume requirement and low cost, makes this novel quantitative HIV real-time RT-PCR assay particularly well suited for application to high volume of clinical samples. |
| first_indexed | 2024-10-01T02:57:57Z |
| format | Thesis |
| id | ntu-10356/48079 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T02:57:57Z |
| publishDate | 2012 |
| record_format | dspace |
| spelling | ntu-10356/480792023-03-11T17:50:22Z Development and clinical evaluation of an in-house human immunodeficiency virus-1 real-time RT-PCR quantitation assay Wee, Sue Yuen Lim Chu Sing School of Mechanical and Aerospace Engineering DRNTU::Engineering::Bioengineering Quantification of Human Immunodeficiency Virus (HIV-1) RNA plays a vital role in diagnosing and monitoring HIV virological behavior in patients. Currently, the HIV antiretroviral therapy aims to suppress the HIV replication to its minimal, thus quantifying HIV RNA is an indicative tool for assessing the therapeutics response. Despite its importance in HIV treatment, most patients have limited access to the viral load tests due to cost and resource constraints. Additionally, great variability among HIV genotypes and wide dynamic range of clinical HIV RNA levels has presented tough challenges for developing Polymerase Chain Reaction (PCR)-based amplification assays. In this study, we describe the development, evaluation, and validation of a real-time RT-PCR assay designed to provide accurate quantification of viral RNA patients’ specimens. This assay can perform a wide dynamic range of at least 8log10 copies/mL. It has a high intra-assay precision with coefficient of variation (CV) of 0.825% and standard deviation (SD) of 0.04 log10 at 7.0log10 copies/μL of external standards (ES); CV of 2.2% and SD of 0.23 log10 at 1.0log10 copies/μL. It produces inter-assay precision of CV% of Ct value less than 4.2% and SD less than 0.9 PCR cycles for serially diluted ES of from 10 to 108 copies/reaction. The high precision and repeatability of this assay allow the derivation of a model for the viral load quantification result for determining antiretroviral drug therapy effectiveness. Probit analysis has determined the 95% confidence detection level is 61.5copies/ml. The genotype inclusivity of the Group M and N, combined with a small sample volume requirement and low cost, makes this novel quantitative HIV real-time RT-PCR assay particularly well suited for application to high volume of clinical samples. MASTER OF ENGINEERING (MAE) 2012-03-06T01:19:51Z 2012-03-06T01:19:51Z 2012 2012 Thesis Wee, S. Y. (2012). Development and clinical evaluation of an in-house human immunodeficiency virus-1 real-time RT-PCR quantitation assay. Master’s thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/48079 10.32657/10356/48079 en 148 p. application/pdf |
| spellingShingle | DRNTU::Engineering::Bioengineering Wee, Sue Yuen Development and clinical evaluation of an in-house human immunodeficiency virus-1 real-time RT-PCR quantitation assay |
| title | Development and clinical evaluation of an in-house human immunodeficiency virus-1 real-time RT-PCR quantitation assay |
| title_full | Development and clinical evaluation of an in-house human immunodeficiency virus-1 real-time RT-PCR quantitation assay |
| title_fullStr | Development and clinical evaluation of an in-house human immunodeficiency virus-1 real-time RT-PCR quantitation assay |
| title_full_unstemmed | Development and clinical evaluation of an in-house human immunodeficiency virus-1 real-time RT-PCR quantitation assay |
| title_short | Development and clinical evaluation of an in-house human immunodeficiency virus-1 real-time RT-PCR quantitation assay |
| title_sort | development and clinical evaluation of an in house human immunodeficiency virus 1 real time rt pcr quantitation assay |
| topic | DRNTU::Engineering::Bioengineering |
| url | https://hdl.handle.net/10356/48079 |
| work_keys_str_mv | AT weesueyuen developmentandclinicalevaluationofaninhousehumanimmunodeficiencyvirus1realtimertpcrquantitationassay |