An Optimized Real-Time qPCR Method for the Effective Detection of Human Malaria Infections

Abstract

Polymerase chain reaction, although an expensive method for the detection of human Plasmodium spp., is still considered the finest for the diagnosis of malaria. The conventional diagnostic PCR is an inexpensive process but consumes a lot of time, reagents and lacks sensitivity. On the other hand, real-time PCR assays currently being used are mostly probe-based expensive methods and sometimes not feasible to detect all the species in a single amplification reaction condition. Here we have established a real-time PCR method that is time and cost effective with a single protocol to detect and distinguish five human Plasmodium species using the existing primers efficiently. The primers used here are being used in the conventional method and the sensitivity as well as specificity of this method has also been immensely improved (100%). The lower limit of detection for Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae are 0.064 parasites/µL, 1.6 parasites/µL, and 0.32 parasites/µL respectively and no cross reactivity was observed. Besides, we have analyzed melt curves that can be used for further species confirmation and validation purposes using multiplex systems. This method, therefore, can be considered as an alternative to the existing lineup for molecular diagnosis of malaria in endemic countries.

Keywords: Plasmodium; asymptomatic; diagnosis; malaria; nested PCR; real time PCR.

Figure 1

Melt curve analysis of Plasmodium spp. The melt curve shows different T_m for Pf (73.5 °C), Pv (76 °C), Pm (71 °C), and Po (79.5 °C) except for Pk (73.5 °C) which overlaps with the Pf.

Figure 2

Melt curve analysis of clinical fifty Pf and forty Pv samples depicted by blue and orange color respectively. The melting temp (T_m) for Pf and Pv of clinical samples were 73.5 °C ± 0.5 °C and 75 °C ± 0.5 °C respectively.

Figure 3

Melting curve analysis of clinical twenty clinical Pm samples with melting temperature (T_m) of 71 °C ± 0.5 °C.

Figure 4

Melting curve analysis of Pm, Pf, and Pv samples in multiplex depicted by blue, orange, and pink colors respectively. The melting temperature (T_m) for Pm, Pf, and Pv of samples were 71 °C, 74 °C, and 75 °C respectively. The multiplex melting curve analysis could only verify mono-infection with distinct melting temperatures for each species.

Figure 5

Technical performance and range of detection of the Pf real-time SYBR green PCR. DNA was extracted from MRA-102 3D7 strain cultured in vitro, ranging from 5 × 10³ to 6.4 × 10⁻² parasites/µL, and subjected to real-time SYBR green based PCR. Amplification curves are depicted by different colors each containing a different parasite load. The standard curve has been made by the ct values obtained from the assay plotted against the logarithmic parasite concentration in a linear regression curve fit analysis (R² = 0.997, E = 90.8%).

Figure 6

Technical performance and range of detection of the Pv real-time SYBR green PCR. Amplification curves of archived Pv samples with concentration ranging from 25 × 10³ to 1.6 parasites/µL, have been depicted by different colors each containing a different parasite load. The standard curve has been made by the ct values obtained from the assay plotted against the logarithmic parasite concentration in a linear regression curve fit analysis (R² = 0.997, E = 94.6%).

Figure 7

Technical performance and range of detection of the Pm, real-time SYBR green PCR. Amplification curves of archived Pm samples with concentration ranging from 1 × 10³ to 3.2 × 10⁻¹ parasites/µL, have been depicted by different colors each containing a different parasite load. The standard curve has been made by the ct values obtained from the assay plotted against the logarithmic parasite concentration in a linear regression curve fit analysis (R² = 0.994, E = 98.0%).