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
| Autor: | Akshaya Kumar Mohanty, Sanghamitra Satpathi, Shwetha Kamath, Mario Cabodi, Viswanathan Arun Nagaraj, Nikunja Kolluri, Lolabattu Srinivasa Raju, Govindarajan Padmanaban, Susanta K. Ghosh, Catherine M. Klapperich, Manjunatha Chandana Shetty |
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| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Genes Protozoan Plasmodium falciparum Computational biology Biology Parasitemia Real-Time Polymerase Chain Reaction Genome Pathology and Forensic Medicine 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Tandem repeat parasitic diseases Data Mining Humans Malaria Falciparum Gene Repetitive Sequences Nucleic Acid Computational Biology Nucleic acid amplification technique DNA Protozoan biology.organism_classification genomic DNA 030104 developmental biology Real-time polymerase chain reaction Molecular Diagnostic Techniques chemistry 030220 oncology & carcinogenesis Molecular Medicine Genome Protozoan Nucleic Acid Amplification Techniques DNA |
| Zdroj: | The Journal of Molecular Diagnostics. 21:824-838 |
| ISSN: | 1525-1578 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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