Raju, LS and Kamath, S and Shetty, MC and Satpathi, S and Mohanty, AK and Ghosh, SK and Kolluri, N and Klapperich, CM and Cabodi, M and Padmanaban, G and Nagaraj, VA (2019) 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. In: Journal of Molecular Diagnostics, 21 (5). pp. 824-838.
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Abstract
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). 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 approximately 100% sensitivity and specificity. Plasmodium falciparum IMRS assays are also capable of detecting submicroscopic infections in asymptomatic samples. To summarize, this approach of initiating amplification at multiple loci across the genome and generating more products with increased analytical sensitivity is different from classic approaches amplifying multicopy genes or tandem repeats. This can serve as a platform technology to develop advanced diagnostics for various pathogens.
| Item Type: | Journal Article |
|---|---|
| Publication: | Journal of Molecular Diagnostics |
| Publisher: | Elsevier B.V. |
| Additional Information: | The copyright for this article belongs to the Authors. |
| Keywords: | genomic DNA; protozoal DNA; protozoal RNA; RNA 18S; protozoal DNA, Article; data mining; gene amplification; gene sequence; genome analysis; identical multirepeat sequence; limit of detection; malaria; microbial genome; molecular diagnosis; nonhuman; Plasmodium falciparum; quantitative analysis; real time polymerase chain reaction; sensitivity and specificity; biology; blood; data mining; gene; genetics; genome; human; isolation and purification; malaria falciparum; molecular diagnosis; nucleic acid amplification; nucleotide repeat; parasitemia; parasitology; Plasmodium falciparum; procedures; real time polymerase chain reaction, Computational Biology; Data Mining; DNA, Protozoan; Genes, Protozoan; Genome, Protozoan; Humans; Malaria, Falciparum; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; Parasitemia; Plasmodium falciparum; Real-Time Polymerase Chain Reaction; Repetitive Sequences, Nucleic Acid |
| Department/Centre: | Division of Biological Sciences > Biochemistry |
| Date Deposited: | 13 Oct 2022 05:29 |
| Last Modified: | 13 Oct 2022 05:29 |
| URI: | https://eprints.iisc.ac.in/id/eprint/77366 |
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