A rapid multiplex assay of human malaria parasites by digital PCR

•The multiplex dPCR system newly established here was the first reported quadruple dPCR assay for four common human Plasmodium species, which enables a four-in-one detection.•The analytical sensitivity of multiplex dPCR was as low as 0.557 copies/μL (95% CI 0.521 to 0.607), and it had a sensitivity of 98.0% and a specificity of 100% in clinical samples to make up for the false negative of microscopic examination.•The multiplex dPCR system has great potential in diagnosing mixed-infection and monitoring pharmacotherapy effectiveness.•The multiplex dPCR system presents a promising candidate as an absolute quantitative tool in clinical practice.