Diagnosis and genotyping of Plasmodium falciparum by a DNA biosensor based on quartz crystal microbalance (QCM)
Tiparat Potipitak1, Warunee Ngrenngarmlert2, Chamras Promptmas3
1Faculty of Medical Technology, Department of Clinical Microscopy, Mahidol University, Nakhon Pathom, Thailand.
Background:
Malaria infection with Plasmodium falciparum is an important basic health problem in the tropical and sub-tropical countries. The standard diagnostic method is blood film examination to visualize parasite morphology. However, in cases of low parasitemia or mixed infection with very low cryptic species, microscopy is not sensitive enough. Therefore, molecular techniques have been widely employed.
Methods:
A label-free DNA biosensor based on quartz crystal microbalance (QCM) to diagnose and genotype P. falciparum was developed. Avidin-biotin interaction was used to coat the specific biotinylated probe on the gold surface of QCM. The gene encoding merozoite surface protein 2 (msp2) was amplified and the PCR products were then cut with restriction enzyme (MwoI). Enzymatic cutting made the PCR products suitable for QCM development. Hybridization between probe and enzymatic cutting DNA fragments resulted in frequency changes of the QCM.
Results:
The newly developed QCM was tested for its diagnosis ability using both malaria laboratory strains and clinical isolates. The biosensor was sensitive at the sub-nanogram level, specific for only P. falciparum detection, no cross-reaction with P. vivax, and stable at room temperature for up to 6 months. Selection of msp2 as a target gene and a geno-typing marker made the QCM potentially useful for falciparum diagnosis simultaneously with genotyping. Potency was tested by genotyping two allelic families of P. falciparum, FC27 and IC1, using malaria laboratory strains, K1 and 3D7, respectively.
Conclusions:
The dual function QCM was successfully developed with high sensitivity and specificity, and was cost-effective, stable and field adaptable.
