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

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.

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 C_q 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.