Multiplex LAMP for Rickettsia and Malaria

Rickettsia monacensis from in vitro culture was spiked with human blood at 5000 bacteria copies per µL of blood. The Rickettsia spiked with blood was then spotted on the classic Whatman FTA Classic card (GE Healthcare, Buckinghamshire, UK) and dried. Similarly, red blood cells infected with P. falciparum at 1% parasitemia were dried on the Whatman FTA Classic card (GE Healthcare, UK). A negative template control was prepared by applying uninfected blood onto the Whatman FTA Classic card (GE Healthcare, UK) and dried it. The DNA from dried blood spots was extracted by the modified boiling method [42 (link)]. In brief, two dried blood spots of 3 mm were punched out and soaked in 40 µL of nuclease-free water in the PCR tube. The mixed template was prepared by mixing two dried blood spot punches from R. monacensis and P. falciparum in a single tube. The samples were then incubated at 60 °C for 30 min and boiled at 99 °C for 10 min. The multiplex LAMP was performed on a heating block, powered by a portable rechargeable battery, at 62 °C for 60 min. The amplicon was then visualized using a portable fluorometer [36 (link)] and dipstick chromatography.