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Sample-ready multiplex qPCR assay for detection of malaria
BACKGROUND: Microscopy and antigen detecting rapid diagnostic tests are the diagnostic tests of choice in management of clinical malaria. However, due to their limitations, the need to utilize more sensitive methods such as real-time PCR (qPCR) is evident as more studies are now utilizing molecular...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4026594/ https://www.ncbi.nlm.nih.gov/pubmed/24767409 http://dx.doi.org/10.1186/1475-2875-13-158 |
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author | Kamau, Edwin Alemayehu, Saba Feghali, Karla C Juma, Dennis W Blackstone, George M Marion, William R Obare, Peter Ogutu, Bernhards Ockenhouse, Christian F |
author_facet | Kamau, Edwin Alemayehu, Saba Feghali, Karla C Juma, Dennis W Blackstone, George M Marion, William R Obare, Peter Ogutu, Bernhards Ockenhouse, Christian F |
author_sort | Kamau, Edwin |
collection | PubMed |
description | BACKGROUND: Microscopy and antigen detecting rapid diagnostic tests are the diagnostic tests of choice in management of clinical malaria. However, due to their limitations, the need to utilize more sensitive methods such as real-time PCR (qPCR) is evident as more studies are now utilizing molecular methods in detection of malaria. Some of the challenges that continue to limit the widespread utilization of qPCR include lack of assay standardization, assay variability, risk of contamination, and the need for cold-chain. Lyophilization of molecular assays can overcome some of these limitations and potentially enable widespread qPCR utilization. METHODS: A recently published multiplex malaria qPCR assay was lyophilized by freezing drying into Sample-Ready™ format (MMSR). MMSR assay contained all the required reagents for qPCR including primers and probes, requiring only the addition of water and sample to perform qPCR. The performance of the MMSR assay was compared to the non-freeze dried, “wet” assay. Stability studies were done by maintaining the MMSR assays at four different ambient temperatures of 4°C, room temperature (RT), 37°C and 42°C over a period of 42 days, tested at seven-day intervals. Plasmodium falciparum and Plasmodium vivax DNAs were used for analysis of the MMSR assay either as single or mixed parasites, at two different concentrations. The C(T) values and the standard deviations (SD) were used in the analysis of the assay performance. RESULTS: The limit of detection for the MMSR assay was 0.244 parasites/μL for Plasmodium spp. (PLU) and P. falciparum (FAL) assay targets compared to “wet” assay which was 0.39 and 3.13 parasites/μL for PLU and FAL assay targets, respectively. The MMSR assay performed with high efficiencies similar to those of the “wet” assay and was stable at 37°C for 42 days, with estimated shelf-life of 5 months. When used to analyse field clinical samples, MMSR assay performed with 100% sensitivity and specificity compared to the “wet” assay. CONCLUSION: The MMSR assay has the same robust performance characteristics as the “wet” assay and is highly stable. Availability of MMSR assay allows flexibility and provides an option in choosing assay for malaria diagnostics depending on the application, needs and budget. |
format | Online Article Text |
id | pubmed-4026594 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-40265942014-05-21 Sample-ready multiplex qPCR assay for detection of malaria Kamau, Edwin Alemayehu, Saba Feghali, Karla C Juma, Dennis W Blackstone, George M Marion, William R Obare, Peter Ogutu, Bernhards Ockenhouse, Christian F Malar J Methodology BACKGROUND: Microscopy and antigen detecting rapid diagnostic tests are the diagnostic tests of choice in management of clinical malaria. However, due to their limitations, the need to utilize more sensitive methods such as real-time PCR (qPCR) is evident as more studies are now utilizing molecular methods in detection of malaria. Some of the challenges that continue to limit the widespread utilization of qPCR include lack of assay standardization, assay variability, risk of contamination, and the need for cold-chain. Lyophilization of molecular assays can overcome some of these limitations and potentially enable widespread qPCR utilization. METHODS: A recently published multiplex malaria qPCR assay was lyophilized by freezing drying into Sample-Ready™ format (MMSR). MMSR assay contained all the required reagents for qPCR including primers and probes, requiring only the addition of water and sample to perform qPCR. The performance of the MMSR assay was compared to the non-freeze dried, “wet” assay. Stability studies were done by maintaining the MMSR assays at four different ambient temperatures of 4°C, room temperature (RT), 37°C and 42°C over a period of 42 days, tested at seven-day intervals. Plasmodium falciparum and Plasmodium vivax DNAs were used for analysis of the MMSR assay either as single or mixed parasites, at two different concentrations. The C(T) values and the standard deviations (SD) were used in the analysis of the assay performance. RESULTS: The limit of detection for the MMSR assay was 0.244 parasites/μL for Plasmodium spp. (PLU) and P. falciparum (FAL) assay targets compared to “wet” assay which was 0.39 and 3.13 parasites/μL for PLU and FAL assay targets, respectively. The MMSR assay performed with high efficiencies similar to those of the “wet” assay and was stable at 37°C for 42 days, with estimated shelf-life of 5 months. When used to analyse field clinical samples, MMSR assay performed with 100% sensitivity and specificity compared to the “wet” assay. CONCLUSION: The MMSR assay has the same robust performance characteristics as the “wet” assay and is highly stable. Availability of MMSR assay allows flexibility and provides an option in choosing assay for malaria diagnostics depending on the application, needs and budget. BioMed Central 2014-04-25 /pmc/articles/PMC4026594/ /pubmed/24767409 http://dx.doi.org/10.1186/1475-2875-13-158 Text en Copyright © 2014 Kamau et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/4.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Kamau, Edwin Alemayehu, Saba Feghali, Karla C Juma, Dennis W Blackstone, George M Marion, William R Obare, Peter Ogutu, Bernhards Ockenhouse, Christian F Sample-ready multiplex qPCR assay for detection of malaria |
title | Sample-ready multiplex qPCR assay for detection of malaria |
title_full | Sample-ready multiplex qPCR assay for detection of malaria |
title_fullStr | Sample-ready multiplex qPCR assay for detection of malaria |
title_full_unstemmed | Sample-ready multiplex qPCR assay for detection of malaria |
title_short | Sample-ready multiplex qPCR assay for detection of malaria |
title_sort | sample-ready multiplex qpcr assay for detection of malaria |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4026594/ https://www.ncbi.nlm.nih.gov/pubmed/24767409 http://dx.doi.org/10.1186/1475-2875-13-158 |
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