Universal two-dimensional labelled probe-mediated melting curve analysis based on multiplex PCR for rapid typing of plasmodium in a single closed tube

Abstract

Nowadays, malaria is still one of the major public health problems which commonly caused by four plasmodium species, especially in the epidemic of COVID-19 harboring similar symptoms of fever or fatigue, which easily result in misdiagnosis. The disadvantages of previous traditional detection methods, such as time-consuming, costly, complicated operation, strong professionalism, indistinguishable typing and so on, lead to the dilemma of difficulty to meet the clinical requirements of rapid, easy and accurate typing of common plasmodiums. Herein, we developed and maximally optimized a universal two-dimensional labeled probe-mediated melting curve analysis (UP-MCA) assay based on multiplex PCR for rapid and accurate typing of five plasmodiums, including novel human plasmodium, Plasmodium knowlesi (Pk), in a single closed tube following genome extraction. The assay showed the limit of detection (LOD) of 10 copies per reaction and can accurately distinguish plasmodium species from intra-plasmodium and other pathogens. In addition, we also proposed and verified different methods of fluorescence-quenching and two dimensional labeled tag for probes that are suitable for UP-MCA assay. Furthermore, its clinical performance was evaluated by 184 samples and showed sensitivity of 100% (164/164) and specificity of 100% (20/20) at 99% confidence interval, respectively, with the microscopy method as gold standard. Taken together, the UP-MCA system showed excellent sensitivity, specificity and accuracy for genotyping of plasmodium, and it meets the requirements of rapidity and convenience for plasmodium detection in clinical routine and has great potential for clinical translation.