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Multiplex qPCR for detection and absolute quantification of malaria.

Kamau E, Alemayehu S, Feghali KC, Saunders D, Ockenhouse CF - PLoS ONE (2013)

Bottom Line: We describe development of an absolute multiplex quantitative real-time PCR for detection of Plasmodium spp., P. falciparum and P. vivax targets in order to produce an assay amenable to high throughput but with reduced costs.Inhibition studies were performed to test and compare co-purification of PCR inhibitors in samples extracted from whole blood using either the manual or automated methods.To establish the most optimal qPCR reaction volume, volume titration of the reaction master mix was performed starting at 10 µl to 1 µl reaction master mix with 1 µl of template DNA in each reaction.

View Article: PubMed Central - PubMed

Affiliation: Military Malaria Research Program, Malaria Vaccine Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA. edwin.kamau@us.army.mil

ABSTRACT
We describe development of an absolute multiplex quantitative real-time PCR for detection of Plasmodium spp., P. falciparum and P. vivax targets in order to produce an assay amenable to high throughput but with reduced costs. Important qPCR experimental details and information that is critical to performance and reliability of assay results were investigated. Inhibition studies were performed to test and compare co-purification of PCR inhibitors in samples extracted from whole blood using either the manual or automated methods. To establish the most optimal qPCR reaction volume, volume titration of the reaction master mix was performed starting at 10 µl to 1 µl reaction master mix with 1 µl of template DNA in each reaction. As the reaction volume decreased, qPCR assays became more efficient with 1 µl reaction master mix being the most efficient. For more accurate quantification of parasites in a sample, we developed plasmid DNAs for all the three assay targets for absolute quantification. All of absolute qPCR assays performed with efficiency of more than 94%, R(2) values greater than 0.99 and the STDEV of each replicate was <0.167. Linear regression plots generated from absolute qPCR assays were used to estimate the corresponding parasite density from relative qPCR in terms of parasite/µl. One copy of plasmid DNA was established to be equivalent to 0.1 parasite/µl for Plasmodium spp. assay, 0.281 parasites for P. falciparum assay and 0.127 parasite/µl for P. vivax assay. This study demonstrates for the first time use of plasmid DNA in absolute quantification of malaria parasite. The use of plasmid DNA standard in quantification of malaria parasite will be critical as efforts are underway to harmonize molecular assays used in diagnosis of malaria.

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Linear regression plots for absolute qPCR assays.Real-time PCR assays were performed using plasmid DNAs for each assay. Plasmid DNA was 10-fold serially diluted at each point and ran in 4–8 replicates. A linear regression plot was generated using GraphPad Prism. The slope, the Y-intercept and the r2 value were determined. Data shown confirms that these assays perform with high efficiencies.
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pone-0071539-g002: Linear regression plots for absolute qPCR assays.Real-time PCR assays were performed using plasmid DNAs for each assay. Plasmid DNA was 10-fold serially diluted at each point and ran in 4–8 replicates. A linear regression plot was generated using GraphPad Prism. The slope, the Y-intercept and the r2 value were determined. Data shown confirms that these assays perform with high efficiencies.

Mentions: In absolute quantification, sample concentration is expressed in terms of genomic equivalence (GE) or copy numbers. However, for malaria, parasite density is mostly expressed as parasite/µl, based on parasite density as determined by microscopy. It is important therefore that when performing absolute qPCR for malaria, parasite density is expressed in terms that makes clinical sense [and/or other application] and is based on the gold standard for malaria diagnosis which is microscopy. Here, an objective was laid out to determine the amount of GE that is equivalent to parasites/µl. The CT values obtained from absolute and relative qPCR assays were correlated to determine the amount of GE [plasmid DNA] that is equivalent to parasite/µl. NAT assays was used for relative quantification of Plasmodium spp. and P. falciparum assays whereas P. vivax clinical samples were used for relative quantification of the P. vivax assay. The parasite density of the NAT assays was determined as described above. For analysis of P. vivax parasite density using the VIV assay, 5 clinical samples with known parasite densities as determined by expert microscopists were used. Real-time PCR assays were performed for all the three assays using either serially diluted plasmid DNA (absolute qPCR, as shown in Table 4) or genomic DNA (relative qPCR). To estimate the amount of GE that is equivalent to parasites/µl from the relative qPCR assay, the CT values obtained from relative qPCR assays were interpolated as unknowns from the linear regression standard curve of the absolute qPCR assays to obtain equivalent GE (Figure 2). The amount of GE that corresponds to or is equivalent to parasite density in parasites/µl was estimated based on averages obtained from multiple dilutions for NAT assays and 5 P. vivax clinical isolates that had been serially diluted. For the PLU absolute qPCR assay, 10.05 GE corresponds to 1 parasites/µl or 1 GE is equivalent to 0.1 parasites/µl; for the FAL absolute qPCR assay, 3.55 GE correlates to 1 parasites/µl or 1 GE is equivalent to 0.281 parasites/µl; and for the VIV absolute qPCR assay, 7.88 GE corresponds to 1 parasites/µl or 1 GE is equivalent to 0.127 parasites/µl.


Multiplex qPCR for detection and absolute quantification of malaria.

Kamau E, Alemayehu S, Feghali KC, Saunders D, Ockenhouse CF - PLoS ONE (2013)

Linear regression plots for absolute qPCR assays.Real-time PCR assays were performed using plasmid DNAs for each assay. Plasmid DNA was 10-fold serially diluted at each point and ran in 4–8 replicates. A linear regression plot was generated using GraphPad Prism. The slope, the Y-intercept and the r2 value were determined. Data shown confirms that these assays perform with high efficiencies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756973&req=5

pone-0071539-g002: Linear regression plots for absolute qPCR assays.Real-time PCR assays were performed using plasmid DNAs for each assay. Plasmid DNA was 10-fold serially diluted at each point and ran in 4–8 replicates. A linear regression plot was generated using GraphPad Prism. The slope, the Y-intercept and the r2 value were determined. Data shown confirms that these assays perform with high efficiencies.
Mentions: In absolute quantification, sample concentration is expressed in terms of genomic equivalence (GE) or copy numbers. However, for malaria, parasite density is mostly expressed as parasite/µl, based on parasite density as determined by microscopy. It is important therefore that when performing absolute qPCR for malaria, parasite density is expressed in terms that makes clinical sense [and/or other application] and is based on the gold standard for malaria diagnosis which is microscopy. Here, an objective was laid out to determine the amount of GE that is equivalent to parasites/µl. The CT values obtained from absolute and relative qPCR assays were correlated to determine the amount of GE [plasmid DNA] that is equivalent to parasite/µl. NAT assays was used for relative quantification of Plasmodium spp. and P. falciparum assays whereas P. vivax clinical samples were used for relative quantification of the P. vivax assay. The parasite density of the NAT assays was determined as described above. For analysis of P. vivax parasite density using the VIV assay, 5 clinical samples with known parasite densities as determined by expert microscopists were used. Real-time PCR assays were performed for all the three assays using either serially diluted plasmid DNA (absolute qPCR, as shown in Table 4) or genomic DNA (relative qPCR). To estimate the amount of GE that is equivalent to parasites/µl from the relative qPCR assay, the CT values obtained from relative qPCR assays were interpolated as unknowns from the linear regression standard curve of the absolute qPCR assays to obtain equivalent GE (Figure 2). The amount of GE that corresponds to or is equivalent to parasite density in parasites/µl was estimated based on averages obtained from multiple dilutions for NAT assays and 5 P. vivax clinical isolates that had been serially diluted. For the PLU absolute qPCR assay, 10.05 GE corresponds to 1 parasites/µl or 1 GE is equivalent to 0.1 parasites/µl; for the FAL absolute qPCR assay, 3.55 GE correlates to 1 parasites/µl or 1 GE is equivalent to 0.281 parasites/µl; and for the VIV absolute qPCR assay, 7.88 GE corresponds to 1 parasites/µl or 1 GE is equivalent to 0.127 parasites/µl.

Bottom Line: We describe development of an absolute multiplex quantitative real-time PCR for detection of Plasmodium spp., P. falciparum and P. vivax targets in order to produce an assay amenable to high throughput but with reduced costs.Inhibition studies were performed to test and compare co-purification of PCR inhibitors in samples extracted from whole blood using either the manual or automated methods.To establish the most optimal qPCR reaction volume, volume titration of the reaction master mix was performed starting at 10 µl to 1 µl reaction master mix with 1 µl of template DNA in each reaction.

View Article: PubMed Central - PubMed

Affiliation: Military Malaria Research Program, Malaria Vaccine Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA. edwin.kamau@us.army.mil

ABSTRACT
We describe development of an absolute multiplex quantitative real-time PCR for detection of Plasmodium spp., P. falciparum and P. vivax targets in order to produce an assay amenable to high throughput but with reduced costs. Important qPCR experimental details and information that is critical to performance and reliability of assay results were investigated. Inhibition studies were performed to test and compare co-purification of PCR inhibitors in samples extracted from whole blood using either the manual or automated methods. To establish the most optimal qPCR reaction volume, volume titration of the reaction master mix was performed starting at 10 µl to 1 µl reaction master mix with 1 µl of template DNA in each reaction. As the reaction volume decreased, qPCR assays became more efficient with 1 µl reaction master mix being the most efficient. For more accurate quantification of parasites in a sample, we developed plasmid DNAs for all the three assay targets for absolute quantification. All of absolute qPCR assays performed with efficiency of more than 94%, R(2) values greater than 0.99 and the STDEV of each replicate was <0.167. Linear regression plots generated from absolute qPCR assays were used to estimate the corresponding parasite density from relative qPCR in terms of parasite/µl. One copy of plasmid DNA was established to be equivalent to 0.1 parasite/µl for Plasmodium spp. assay, 0.281 parasites for P. falciparum assay and 0.127 parasite/µl for P. vivax assay. This study demonstrates for the first time use of plasmid DNA in absolute quantification of malaria parasite. The use of plasmid DNA standard in quantification of malaria parasite will be critical as efforts are underway to harmonize molecular assays used in diagnosis of malaria.

Show MeSH
Related in: MedlinePlus