Abstract
Molecular detection has overcome limitations of microscopic examination by providing greater sensitivity and specificity in Plasmodium species detection. The objective of the present study was to develop a quantitative real-time polymerase chain reaction coupled with high-resolution melting (qRT-PCR-HRM) assay for rapid, accurate and simultaneous detection of all five human Plasmodium spp. A pair of primers targeted the 18S SSU rRNA gene of the Plasmodium spp. was designed for qRT-PCR-HRM assay development. Analytical sensitivity and specificity of the assay were evaluated. Samples collected from 229 malaria suspected patients recruited from Sabah, Malaysia were screened using the assay and results were compared with data obtained using PlasmoNexTM, a hexaplex PCR system. The qRT-PCR-HRM assay was able to detect and discriminate the five Plasmodium spp. with lowest detection limits of 1–100 copy numbers without nonspecific amplifications. The detection of Plasmodium spp. in clinical samples using this assay also achieved 100% concordance with that obtained using PlasmoNexTM. This indicated that the diagnostic sensitivity and specificity of this assay in Plasmodium spp. detection is comparable with those of PlasmoNexTM. The qRT-PCR-HRM assay is simple, produces results in two hours and enables high-throughput screening. Thus, it is an alternative method for rapid and accurate malaria diagnosis.
Highlights
Symptoms of malaria are nonspecific and similar to other diseases
A number of nested and semi-nested polymerase chain reaction (PCR) methods have been developed over the years[6,7,11,13,14,15,16], which allow for detection of up to four human Plasmodium spp. (P. falciparum, P. vivax, P. malariae and P. ovale), and these methods were replicated in other studies and epidemiological surveillance of malaria cases in numerous countries[14,15,17,18,19]
We developed a quantitative real-time PCR method for the detection of the five human-infecting Plasmodium spp., but instead of SYBR green dye, high resolution melting (HRM) analysis was incorporated
Summary
Symptoms of malaria are nonspecific and similar to other diseases. Without prompt and proper treatment, mild and moderate malaria may be lethal in children and some adult patients especially for those infected with Plasmodium falciparum. Since various limitations which include the requirement of skilled personnel, low sensitivity (100 to 200 parasites/μ l of blood), time-consuming and unreliability in species identification especially of P. knowlesi due to its similarities with P. falciparum in the early ring form stage and P. malariae in the latter stages, the microscopic technique needs to be coupled with other alternative diagnostic methods to heighten the accuracy of the identification[4,5]. QRT-PCR-SYBR green assay which can detect quantitatively up to five species of Plasmodium has been developed by Oddoux and colleagues[31]. They used a total of nine primers to amplify P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi. We developed a quantitative real-time PCR method for the detection of the five human-infecting Plasmodium spp., but instead of SYBR green dye, high resolution melting (HRM) analysis was incorporated.
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