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Comparative evaluation of published real-time PCR assays for the detection of malaria following MIQE guidelines.

Alemayehu S, Feghali KC, Cowden J, Komisar J, Ockenhouse CF, Kamau E - Malar. J. (2013)

Bottom Line: The use of malaria-specific quantitative real-time PCR (qPCR) is increasing due to its high sensitivity, speciation and quantification of malaria parasites.All the analyses were automatically established using the default settings.The data demonstrate the need for increased consensus and guidelines that will encourage better experimental practices, allowing more consistent and unambiguous interpretation of qPCR results.

View Article: PubMed Central - HTML - PubMed

Affiliation: Military Malaria Research Program, Malaria Vaccine Branch, Walter Reed Army Institute of Research, 503 Robert Grant Ave, Silver Spring, MD, USA.

ABSTRACT

Background: The use of malaria-specific quantitative real-time PCR (qPCR) is increasing due to its high sensitivity, speciation and quantification of malaria parasites. However, due to the lack of consensus or standardized methods in performing qPCR, it is difficult to evaluate and/or compare the quality of work reported by different authors for a cross-study and/or cross-platform assay analysis.

Methods: The performances of seven published qPCR assays that detect Plasmodium spp or Plasmodium falciparum were compared using standard DNA and samples from a clinical trial. Amplification and qPCR measurements were performed using the Applied Biosystems 7500 Fast Real-Time PCR System. All the analyses were automatically established using the default settings. For the TaqMan probe format, the assays were performed in the background of QuantiFast Probe Master Mix whereas in SYBR Green format, the assays were performed in the background of QuantiFast SYBR Green Master Mix and QuantiTect SYBR Green Master Mix background.

Results: Assays with high PCR efficiencies outperformed those with low efficiencies in all categories including sensitivity, precision and consistency regardless of the assay format and background. With the exception of one assay, all assays evaluated showed lower sensitivity compared to what have been published. When samples from a malaria challenge study were analysed, the qPCR assay with the overall best performance detected parasites in subjects earliest and with most consistency.

Conclusion: The data demonstrate the need for increased consensus and guidelines that will encourage better experimental practices, allowing more consistent and unambiguous interpretation of qPCR results.

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Related in: MedlinePlus

Comparative evaluation of PCR limit of detection from published malaria PCR assays. Data showing TaqMan probe assays performed in the background of QuantiFast Master Mix. Assays were performed in triplicate. Only Cq values of 40 and below were considered and only two out of three values were required to call the results a positive. LoD was established which contained the lowest parasite concentration expressed as parasites/mL.
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Figure 1: Comparative evaluation of PCR limit of detection from published malaria PCR assays. Data showing TaqMan probe assays performed in the background of QuantiFast Master Mix. Assays were performed in triplicate. Only Cq values of 40 and below were considered and only two out of three values were required to call the results a positive. LoD was established which contained the lowest parasite concentration expressed as parasites/mL.

Mentions: The fluorescence emission of any molecule is dependent on environmental factors including the pH of the solution, salt concentration and much more. To obtain more accurate and representative performance of all the assays tested, assays were performed using TaqMan probe and SYBR Green formats. For the TaqMan probe format, the assays were performed in the background of QuantiFast Master Mix whereas in SYBR Green format, the assays were performed in the background of QuantiFast SYBR Green Master Mix and QuantiTect Sybr green Master Mix. All assays were performed in triplicate and the LoD was established as the highest Cq value where the lowest parasite concentration was detected, expressed as parasites/mL. Only Cq values 40 and below were considered and only two out of three assays were required to call the results a positive. It is important to note that at very low copy numbers, the normal distribution of the template in the sample is not expected. Instead, Poisson distribution is followed where only a certain percentage of copy number of the template is detected which is likely to vary each time resulting in a larger standard deviation (SD). For the TaqMan probe format, the PLU3 and MACH assays were the most sensitive with LoD of 313 parasites/mL whereas the CTYB and FAL were the least sensitive with LoD of 2,500 parasites/mL (FigureĀ 1). The sensitivities of these assays were previously reported as follows: PLU3 at 50 parasites/mL, MACH at 100 parasites/mL, CTYB at 50 parasites/mL, WHO at 16,200 parasites/mL, FAL at 700 parasites/mL, and TURBO at 20 parasites/mL. The sensitivity of PLASMO assay was not reported.


Comparative evaluation of published real-time PCR assays for the detection of malaria following MIQE guidelines.

Alemayehu S, Feghali KC, Cowden J, Komisar J, Ockenhouse CF, Kamau E - Malar. J. (2013)

Comparative evaluation of PCR limit of detection from published malaria PCR assays. Data showing TaqMan probe assays performed in the background of QuantiFast Master Mix. Assays were performed in triplicate. Only Cq values of 40 and below were considered and only two out of three values were required to call the results a positive. LoD was established which contained the lowest parasite concentration expressed as parasites/mL.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750446&req=5

Figure 1: Comparative evaluation of PCR limit of detection from published malaria PCR assays. Data showing TaqMan probe assays performed in the background of QuantiFast Master Mix. Assays were performed in triplicate. Only Cq values of 40 and below were considered and only two out of three values were required to call the results a positive. LoD was established which contained the lowest parasite concentration expressed as parasites/mL.
Mentions: The fluorescence emission of any molecule is dependent on environmental factors including the pH of the solution, salt concentration and much more. To obtain more accurate and representative performance of all the assays tested, assays were performed using TaqMan probe and SYBR Green formats. For the TaqMan probe format, the assays were performed in the background of QuantiFast Master Mix whereas in SYBR Green format, the assays were performed in the background of QuantiFast SYBR Green Master Mix and QuantiTect Sybr green Master Mix. All assays were performed in triplicate and the LoD was established as the highest Cq value where the lowest parasite concentration was detected, expressed as parasites/mL. Only Cq values 40 and below were considered and only two out of three assays were required to call the results a positive. It is important to note that at very low copy numbers, the normal distribution of the template in the sample is not expected. Instead, Poisson distribution is followed where only a certain percentage of copy number of the template is detected which is likely to vary each time resulting in a larger standard deviation (SD). For the TaqMan probe format, the PLU3 and MACH assays were the most sensitive with LoD of 313 parasites/mL whereas the CTYB and FAL were the least sensitive with LoD of 2,500 parasites/mL (FigureĀ 1). The sensitivities of these assays were previously reported as follows: PLU3 at 50 parasites/mL, MACH at 100 parasites/mL, CTYB at 50 parasites/mL, WHO at 16,200 parasites/mL, FAL at 700 parasites/mL, and TURBO at 20 parasites/mL. The sensitivity of PLASMO assay was not reported.

Bottom Line: The use of malaria-specific quantitative real-time PCR (qPCR) is increasing due to its high sensitivity, speciation and quantification of malaria parasites.All the analyses were automatically established using the default settings.The data demonstrate the need for increased consensus and guidelines that will encourage better experimental practices, allowing more consistent and unambiguous interpretation of qPCR results.

View Article: PubMed Central - HTML - PubMed

Affiliation: Military Malaria Research Program, Malaria Vaccine Branch, Walter Reed Army Institute of Research, 503 Robert Grant Ave, Silver Spring, MD, USA.

ABSTRACT

Background: The use of malaria-specific quantitative real-time PCR (qPCR) is increasing due to its high sensitivity, speciation and quantification of malaria parasites. However, due to the lack of consensus or standardized methods in performing qPCR, it is difficult to evaluate and/or compare the quality of work reported by different authors for a cross-study and/or cross-platform assay analysis.

Methods: The performances of seven published qPCR assays that detect Plasmodium spp or Plasmodium falciparum were compared using standard DNA and samples from a clinical trial. Amplification and qPCR measurements were performed using the Applied Biosystems 7500 Fast Real-Time PCR System. All the analyses were automatically established using the default settings. For the TaqMan probe format, the assays were performed in the background of QuantiFast Probe Master Mix whereas in SYBR Green format, the assays were performed in the background of QuantiFast SYBR Green Master Mix and QuantiTect SYBR Green Master Mix background.

Results: Assays with high PCR efficiencies outperformed those with low efficiencies in all categories including sensitivity, precision and consistency regardless of the assay format and background. With the exception of one assay, all assays evaluated showed lower sensitivity compared to what have been published. When samples from a malaria challenge study were analysed, the qPCR assay with the overall best performance detected parasites in subjects earliest and with most consistency.

Conclusion: The data demonstrate the need for increased consensus and guidelines that will encourage better experimental practices, allowing more consistent and unambiguous interpretation of qPCR results.

Show MeSH
Related in: MedlinePlus