Selection and exploitation of prevalent, tandemly repeated genomic targets for improved real-time PCR-based detection of Wuchereria bancrofti and Plasmodium falciparum in mosquitoes

Abstract

Background Optimization of polymerase chain reaction (PCR)-based diagnostics requires the careful selection of molecular targets that are both highly repetitive and pathogen-specific. Advances in both next-generation sequencing (NGS) technologies and bioinformaticsbased analysis tools are facilitating this selection process, informing target choices and reducing labor. Once developed, such assays provide disease control and elimination programs with an additional set of tools capable of evaluating and monitoring intervention successes. The importance of such tools is heightened as intervention efforts approach their endpoints, as accurate and complete information is an essential component of the informed decision-making process. As global efforts for the control and elimination of both lymphatic filariasis and malaria continue to make significant gains, the benefits of diagnostics with improved analytical and clinical/field-based sensitivities and specificities will become increasingly apparent. Methodology/Principal findings Coupling Illumina-based NGS with informatics approaches, we have successfully identified the tandemly repeated elements in both the Wuchereria bancrofti and Plasmodium falciparum genomes of putatively greatest copy number. Utilizing these sequences as quantitative real-time PCR (qPCR)-based targets, we have developed assays capable of exploiting the most abundant tandem repeats for both organisms. For the detection of P. falciparum, analysis and development resulted in an assay with improved analytical and field-based sensitivity vs. an established ribosomal sequence-targeting assay. Surprisingly, analysis of the W. bancrofti genome predicted a ribosomal sequence to be the genome's most abundant

Development of a novel real-time polymerase chain reaction assay for the sensitive detection of Schistosoma japonicum in human stool

“…Although comparisons made using genomic DNA as template cannot be used to evaluate the comparative clinical sensitivities of the examined assays, these sizable differences in mean Cq values strongly suggest that the SjTR1 is considerably more abundant in copy number within the S . japonicum genome than previously published targets [ 17 – 19 , 21 ]. When testing stool spiked with S .…”

“…japonicum genome than the target regions of the other qPCR assays tested. This provides additional support for improved analytical sensitivity via the use of satellite repeats instead of traditional targets such as ribosomal or mitochondrial DNA targets [ 20 – 21 ]. Evidence of the increased clinical sensitivity for our assay compared to previously published assays is supported by the results of the spiking study in which the SjTR1 assay was the only real-time PCR assay capable of reliably detecting 1 egg per gram in all samples ( Table 4 ).…”

“…While these assays have exhibited greater sensitivity than microscopy-based techniques, targeting coding sequences inevitably raises concerns about target specificity as such genes frequently exhibit greater conservation among closely related species [20]. In contrast, non-coding tandemly repeated genomic regions make ideal real-time PCR targets, often providing high sensitivity and species-level specificity [20][21]. Here we report the development of a highly sensitive, specific real-time PCR assay for the detection of S. japonicum in human stool by targeting an abundant, tandemly-repeated genomic DNA sequence, which we have named SjTR1 (Schistosoma japonicum Tandem Repeat 1) (GenBank:MW631938).…”

Molecular xenomonitoring as a complement to human antigen seroprevalence for lymphatic filariasis surveillance in Samoa

“…Extractions occurred using a modi ed version of the manufacturer's suggested protocol which has been previously described 8 . Following extraction, all samples were tested for the presence of W. bancrofti using the real-time PCR assay 25 utilizing all recommended recipes and protocols. S4 for survey timeline).…”

Comparison of real time PCR and conventional PCR by identifying genomic DNA of bovine and porcine

“…The genomic method with the PCR technique has been used by to detect the species content in the product accurately. Furthermore, living organisms have their own DNA' molecules that very unique for each organism (Williams et al 2020;Zulch et al 2020). PCR technique is strongly selective and sensitive by multiplying nucleotide sequence-specific nucleotides exponentially in vitro (Toohey -Kurth et al 2020).…”