Genome Mining–Based Identification of Identical Multirepeat Sequences in Plasmodium falciparum Genome for Highly Sensitive Real-Time Quantitative PCR Assay and Its Application in Malaria Diagnosis

Publication date: Available online 31 May 2019Source: The Journal of Molecular DiagnosticsAuthor(s): Lolabattu S. Raju, Shwetha Kamath, Manjunatha C. Shetty, Sanghamitra Satpathi, Akshaya K. Mohanty, Susanta K. Ghosh, Nikunja Kolluri, Catherine M. Klapperich, Mario Cabodi, Govindarajan Padmanaban, Viswanathan A. NagarajMalaria continues to impact global health, and developing ultrasensitive methods capable of detecting submicroscopic parasitemia—a challenge that persists in low transmission areas, asymptomatic carriers, and patients showing recrudescence—is vital to achieving malaria eradication. Nucleic acid amplification techniques offer improved analytical sensitivity but are limited by the number of copies of the amplification targets. Herein, we perform a novel genome mining approach to identify a pair of identical multirepeat sequences (IMRSs) that constitute 170 and 123 copies in the Plasmodium falciparum genome and explore their potential as primers for PCR. Real-time quantitative PCR analyses have shown the ability of P. falciparum IMRSs to amplify as low as 2.54 fg of P. falciparum genomic DNA (approximately 0.1 parasite), with a striking 100-fold increase in detection limit when compared with P. falciparum 18S rRNA (251.4 fg; approximately 10 parasites), the most common target for diagnosis. Validation with clinical samples from malaria-endemic regions has shown 6.70 ± 1.66 cycle better detection threshold in terms of Ct value for P. falciparum IMRSs, with a...
Source: The Journal of Molecular Diagnostics - Category: Pathology Source Type: research