Dataset Information

A real-time PCR-based assay for detection of Wuchereria bancrofti DNA in blood and mosquitoes.

ABSTRACT: We developed and evaluated real-time polymerase chain reaction (PCR) assays for detecting Wuchereria bancrofti DNA in human blood and in mosquitoes. An assay based on detection of the W. bancrofti "LDR" repeat DNA sequence was more sensitive than an assay for Wolbachia 16S rDNA. The LDR-based assay was sensitive for detecting microfilarial DNA on dried membrane filters or on filter paper. We also compared real-time PCR with conventional PCR (C-PCR) for detecting W. bancrofti DNA in mosquito samples collected in endemic areas in Egypt and Papua New Guinea. Although the two methods had comparable sensitivity for detecting filarial DNA in reference samples, real-time PCR was more sensitive than C-PCR in practice with field samples. Other advantages of real-time PCR include its high-throughput capacity and decreased risk of cross-contamination between test samples. We believe that real-time PCR has great potential as a tool for monitoring progress in large-scale filariasis elimination programs.

SUBMITTER: Rao RU

PROVIDER: S-EPMC2196401 | biostudies-literature | 2006 May

REPOSITORIES: biostudies-literature

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A real-time PCR-based assay for detection of Wuchereria bancrofti DNA in blood and mosquitoes.

Rao Ramakrishna U RU Atkinson Laura J LJ Ramzy Reda M R RM Helmy Hanan H Farid Hoda A HA Bockarie Moses J MJ Susapu Melinda M Laney Sandra J SJ Williams Steven A SA Weil Gary J GJ

The American journal of tropical medicine and hygiene 20060501 5

We developed and evaluated real-time polymerase chain reaction (PCR) assays for detecting Wuchereria bancrofti DNA in human blood and in mosquitoes. An assay based on detection of the W. bancrofti "LDR" repeat DNA sequence was more sensitive than an assay for Wolbachia 16S rDNA. The LDR-based assay was sensitive for detecting microfilarial DNA on dried membrane filters or on filter paper. We also compared real-time PCR with conventional PCR (C-PCR) for detecting W. bancrofti DNA in mosquito samp ...[more]

PMID: 16687688

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Project description: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 bioinformatics-based 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 tandem repeat. While resulting cycle quantification values comparing a qPCR assay targeting this ribosomal sequence and a commonly targeted repetitive DNA sequence from the literature supported our finding that this ribosomal sequence was the most prevalent tandemly repeated target in the W. bancrofti genome, the resulting assay did not significantly improve detection sensitivity in conjunction with field sample testing. CONCLUSIONS/SIGNIFICANCE:Examination of pathogen genomes facilitates the development of PCR-based diagnostics targeting the most abundant and specific genomic elements. While in some instances currently available tools may deliver equal or superior performance, systematic analysis of potential targets provides confidence that the selected assays represent the most advantageous options available and that informed assay selection is occurring in the context of a particular study's objectives.

Dataset Information

A real-time PCR-based assay for detection of Wuchereria bancrofti DNA in blood and mosquitoes.

Publications

A real-time PCR-based assay for detection of Wuchereria bancrofti DNA in blood and mosquitoes.

Similar Datasets

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