Breakthrough has been recorded in malaria detection as the new FREM platform delivers 98 per cent accuracy for rapid screening, genotyping.
In a recent study published in eBioMedicine, researchers developed the Flexible, Robust, Equipment-free Microfluidic (FREM) platform for malaria screening and Plasmodium species genotyping.
Malaria, a worldwide health concern caused by Plasmodium species, needs precise detection and genotyping to be effectively controlled. The primary diagnostic technique is nucleic acid amplification testing; however, their widespread deployment poses hurdles in resource-limited settings.
CRISPR/Cas systems have transformed molecular diagnostics, but they frequently need two independent stages, complicating procedures and preventing general implementation.
Using limited systems raises biocompatibility difficulties and the possibility of reducing detection efficiency. Accurate genetic typing is critical for malaria treatment and control efforts.
In the present study, researchers evaluated the efficacy of the microfluidic platform for simultaneous malaria screening and plasmodium genotyping.
For malaria infection screening and plasmodium genotyping, the microfluidic platform combined recombinase polymerase amplification (RPA) with clustered regularly interspaced short palindromic repeats (CRISPR)-based detection.
The researchers created universal and species-specific CRISPR RNA (crRNA) candidates for five Plasmodium species, each with a protospacer adjacent motif (PAM) to recognise Cas12a.
Blood samples were collected from all subjects, and RPA reagents were mixed with sucrose in a tube before being administered to the CRISPR devices.
The crRNA targeting conserved Plasmodium sequences was used for malaria infection testing. In each Cas12a-mediated experiment, five CRISPR RNAs targeting distinct loci of Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, and Plasmodium knowlesi were used. A microfluidic system capable of evaluating six targets in tandem was used to accomplish malaria diagnosis.
A microfluidic chip, measuring 7.2 mm (height) by 26 mm (diameter), was created, using a three-dimensional printer to accomplish multiplex detection. Cas12a systems (n=6) were placed into their respective chambers, each harbouring distinct CRISPR RNAs for Plasmodium and other species.
Ninety litres of recombinase polymerase amplification solution was injected into the central intake port and distributed uniformly over the periphery chambers through capillary tubes.
Cas12a cleavage activity was engaged upon specific identification of the CRISPR RNA to recombinase polymerase amplification amplicons, giving a fluorescent or colorimetric signal to differentiate positive malaria cases.
The microfluidic chip was photographed using smartphone cameras after blue light-emitting diode (LED) light exposure.