Limits...
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.

Show MeSH

Related in: MedlinePlus

Titration of reaction master mix volume in qPCR reaction.Multiplex qPCR reactions were set-up that contained descending reaction master mix from 10 µl to 1 µl and 1 µl DNA template in each reaction. Experiments were performed in total replicates of 8. All the four targets in the multiplex qPCR assay were analyzed simultaneously. There was a negative correlation between reaction master mix volume and assay sensitivity. As the volume of reaction master mix increased, the sensitivity of the qPCR decreased with 1 µl reaction master mix reaction being the most sensitive for PLU, FAL and VIV assays.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3756973&req=5

pone-0071539-g001: Titration of reaction master mix volume in qPCR reaction.Multiplex qPCR reactions were set-up that contained descending reaction master mix from 10 µl to 1 µl and 1 µl DNA template in each reaction. Experiments were performed in total replicates of 8. All the four targets in the multiplex qPCR assay were analyzed simultaneously. There was a negative correlation between reaction master mix volume and assay sensitivity. As the volume of reaction master mix increased, the sensitivity of the qPCR decreased with 1 µl reaction master mix reaction being the most sensitive for PLU, FAL and VIV assays.

Mentions: In our previous study [5], qPCR assay was performed by adding 1 µl of template DNA to 9 µl of reaction master mix. The reaction master mix was prepared to a final volume of 20 µl or multiples thereof as needed. To further investigate if the volumes of reaction master mix could be further optimized, a volume titration was performed starting at 10 µl to 1 µl reaction master mix with 1 µl of template DNA used in each reaction. The template DNA used in these experiments contained P. falciparum and P. vivax genomic DNAs. This sample was prepared by mixing P. falciparum and P. vivax clinical sample into uninfected fresh whole blood which was then extracted as described using EZ method. Real-time PCR experiments were performed in replicates of 4, and repeated on two separate occasions bringing the number of total replicates performed to 8. All the four targets in the multiplex qPCR assay were analyzed. Surprisingly, for PLU, FAL and VIV assays, the 1 µl reaction master mix (2 µl total reaction volume) was the most efficient with exception of RNaseP assay which did not work (Figure 1). The 2 µl reaction master mix assays performed superiorly as well, with an overall CT values slightly better than the rest of the reactions. In general, data showed a trend where as the reaction volume increased, qPCR assays became slightly less efficient for all the assays with a plateau being reached at reaction master mix of 8 µl. The amplification plots of all the reactions were smooth and looked similar in all the different reaction volumes tested. To further test the importance of molar concentrations of the reactions, starting at 5 µl down to 1 µl reaction master mixes, water was added to bring the final reaction master mix to 10 µl. One microliter DNA template was used in each reaction. Real-time PCR assays were completely compromised with most of the reactions failing to amplify (data not shown).


Multiplex qPCR for detection and absolute quantification of malaria.

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

Titration of reaction master mix volume in qPCR reaction.Multiplex qPCR reactions were set-up that contained descending reaction master mix from 10 µl to 1 µl and 1 µl DNA template in each reaction. Experiments were performed in total replicates of 8. All the four targets in the multiplex qPCR assay were analyzed simultaneously. There was a negative correlation between reaction master mix volume and assay sensitivity. As the volume of reaction master mix increased, the sensitivity of the qPCR decreased with 1 µl reaction master mix reaction being the most sensitive for PLU, FAL and VIV assays.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0071539-g001: Titration of reaction master mix volume in qPCR reaction.Multiplex qPCR reactions were set-up that contained descending reaction master mix from 10 µl to 1 µl and 1 µl DNA template in each reaction. Experiments were performed in total replicates of 8. All the four targets in the multiplex qPCR assay were analyzed simultaneously. There was a negative correlation between reaction master mix volume and assay sensitivity. As the volume of reaction master mix increased, the sensitivity of the qPCR decreased with 1 µl reaction master mix reaction being the most sensitive for PLU, FAL and VIV assays.
Mentions: In our previous study [5], qPCR assay was performed by adding 1 µl of template DNA to 9 µl of reaction master mix. The reaction master mix was prepared to a final volume of 20 µl or multiples thereof as needed. To further investigate if the volumes of reaction master mix could be further optimized, a volume titration was performed starting at 10 µl to 1 µl reaction master mix with 1 µl of template DNA used in each reaction. The template DNA used in these experiments contained P. falciparum and P. vivax genomic DNAs. This sample was prepared by mixing P. falciparum and P. vivax clinical sample into uninfected fresh whole blood which was then extracted as described using EZ method. Real-time PCR experiments were performed in replicates of 4, and repeated on two separate occasions bringing the number of total replicates performed to 8. All the four targets in the multiplex qPCR assay were analyzed. Surprisingly, for PLU, FAL and VIV assays, the 1 µl reaction master mix (2 µl total reaction volume) was the most efficient with exception of RNaseP assay which did not work (Figure 1). The 2 µl reaction master mix assays performed superiorly as well, with an overall CT values slightly better than the rest of the reactions. In general, data showed a trend where as the reaction volume increased, qPCR assays became slightly less efficient for all the assays with a plateau being reached at reaction master mix of 8 µl. The amplification plots of all the reactions were smooth and looked similar in all the different reaction volumes tested. To further test the importance of molar concentrations of the reactions, starting at 5 µl down to 1 µl reaction master mixes, water was added to bring the final reaction master mix to 10 µl. One microliter DNA template was used in each reaction. Real-time PCR assays were completely compromised with most of the reactions failing to amplify (data not shown).

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