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

BackgroundOptimization 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.

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

PLoS ONE ◽

10.1371/journal.pone.0232325 ◽

2020 ◽

Vol 15 (5) ◽

pp. e0232325 ◽

Cited By ~ 1

Author(s):

Michael F. Zulch ◽

Nils Pilotte ◽

Jessica R. Grant ◽

Corrado Minetti ◽

Lisa J. Reimer ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Real Time ◽

Real Time Pcr ◽

Wuchereria Bancrofti

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DETECTION SENSITIVITY AND QUANTITATION OF PLASMODIUM FALCIPARUM VAR GENE TRANSCRIPTS BY REAL-TIME RT-PCR IN COMPARISON WITH CONVENTIONAL RT-PCR

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.2006.75.212 ◽

2006 ◽

Vol 75 (2) ◽

pp. 212-218 ◽

Cited By ~ 10

Author(s):

MICHELLE L. GATTON ◽

ELIZABETH V. FOWLER ◽

KARRYN GRESTY ◽

QIN CHENG ◽

NANHUA CHEN ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Real Time ◽

Detection Sensitivity ◽

Rt Pcr ◽

Gene Transcripts ◽

Var Gene

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Potential pitfalls in the quantitative molecular detection ofEscherichia coliO157:H7 in environmental matrices

Canadian Journal of Microbiology ◽

10.1139/w05-149 ◽

2006 ◽

Vol 52 (5) ◽

pp. 482-488 ◽

Cited By ~ 25

Author(s):

Rebekka R.E Artz ◽

Lisa M Avery ◽

Davey L Jones ◽

Ken Killham

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Real Time Pcr ◽

Environmental Samples ◽

Detection Sensitivity ◽

Escherichia Coli O157 ◽

Soil System ◽

E Coli ◽

Animal Wastes ◽

Pcr Assays

The detection sensitivity and potential interference factors of a commonly used assay based on real-time polymerase chain reaction (PCR) for Escherichia coli O157:H7 using eae gene-specific primers were assessed. Animal wastes and soil samples were spiked with known replicate quantities of a nontoxigenic strain of E. coli O157:H7 in a viable or dead state and as unprotected DNA. The detection sensitivity and accuracy of real-time PCR for E. coli O157:H7 in animal wastes and soil is low compared to enrichment culturing. Nonviable cells and unprotected DNA were shown to produce positive results in several of the environmental samples tested, leading to potential overestimates of cell numbers due to prolonged detection of nonviable cells. This demonstrates the necessity for the specific calibration of real-time PCR assays in environmental samples. The accuracy of the eae gene–based detection method was further evaluated over time in a soil system against an activity measurement, using the bioluminescent properties of an E. coli O157:H7 Tn5luxCDABE construct. The detection of significant numbers of viable but nonculturable (VBNC) as well as nonviable and possibly physically protected cells as shown over a period of 90 days further complicates the use of real-time PCR assays for quick diagnostics in environmental samples and infers that enrichment culturing is still required for the final verification of samples found positive by real-time PCR methods.Key words: Escherichia coli O157:H7, real-time PCR, animal waste, soil, VBNC.

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A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.00505-18 ◽

2018 ◽

Vol 56 (8) ◽

Cited By ~ 3

Author(s):

Nawal El Houmami ◽

Guillaume André Durand ◽

Janek Bzdrenga ◽

Anne Darmon ◽

Philippe Minodier ◽

...

Keyword(s):

Real Time ◽

Malate Dehydrogenase ◽

Real Time Pcr ◽

Detection Sensitivity ◽

Rt Pcr ◽

Pcr Assay ◽

Content Type ◽

Clinical Specimens ◽

Specificity And Sensitivity ◽

Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

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Rapid identification of transgenic cotton (Gossypium hirsutum L.) plants by loop-mediated isothermal amplification

Czech Journal of Genetics and Plant Breeding ◽

10.17221/7/2011-cjgpb ◽

2011 ◽

Vol 47 (No. 4) ◽

pp. 140-148 ◽

Cited By ~ 7

Author(s):

N. Rostamkhani ◽

A. Haghnazari ◽

M. Tohidfar ◽

A. Moradi

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Lamp Assay ◽

Transgenic Cotton ◽

Rapid Identification ◽

Isothermal Amplification ◽

Detection Sensitivity ◽

Lamp Method ◽

Loop Mediated Isothermal Amplification ◽

Leaf Tissues

In an attempt to speed up the process of screening of transgenic cotton (G. hirsutum L.) plants, a visual and rapid loop-mediated isothermal amplification (LAMP) assay was adopted. Genomic DNA was extracted from fresh leaf tissues of T2 transgenic cotton containing chitinase (chi) and cry1A(b) genes. Detection of genes of interest was performed by polymerase chain reaction (PCR), LAMP and real-time PCR methods. In LAMP assay the amplification was performed after 30 min at 65°C when loop primers were involved in the reaction. The involvement of loop primers decreased the time needed for amplification. By testing serial tenfold dilutions (10–1 to 10–8) of the genes of interest, the detection sensitivity of LAMP was found to be 100-fold higher than that of PCR. The rapid DNA extraction method and LAMP assay can be performed within 30 min and the derived LAMP products can be directly observed as visually detectable based on turbidity in the reaction tube. The accuracy of LAMP method in the screening of transgenes was confirmed by PCR and real-time PCR. The developed method was efficient, rapid and sensitive in the screening of cotton transgenic plants. This method can be applied to any other crops.

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DNA recovery from archived RDTs for genetic characterization of Plasmodium falciparum in a routine setting in Lambaréné, Gabon

Malaria Journal ◽

10.1186/s12936-019-2972-y ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 5

Author(s):

The Trong Nguyen ◽

Brice Nzigou Mombo ◽

Albert Lalremruata ◽

Erik Koehne ◽

Rella Zoleko Manego ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Real Time ◽

Real Time Pcr ◽

Rural Areas ◽

Large Scale ◽

Genetic Material ◽

Chloroquine Resistance ◽

Dna Recovery ◽

Parasite Populations ◽

Pcr Targeting

Abstract Background Rapid diagnostic tests (RDTs) have been described as a source of genetic material to analyse malaria parasites in proof-of-concept studies. The increasing use of RDTs (e.g., in focal or mass screening and treatment campaigns) makes this approach particularly attractive for large-scale investigations of parasite populations. In this study, the complexity of Plasmodium falciparum infections, parasite load and chloroquine resistance transporter gene mutations were investigated in DNA samples extracted from positive RDTs, obtained in a routine setting and archived at ambient temperature. Methods A total of 669 archived RDTs collected from malaria cases in urban, semi-urban and rural areas of central Gabon were used for P. falciparum DNA extraction. Performance of RDTs as a source of DNA for PCR was determined using: (i) amplification of a single copy merozoite surface protein 1 (msp1) gene followed by highly sensitive and automated capillary electrophoresis; (ii) genotyping of the pfcrt gene locus 72–76 using haplotype-specific-probe-based real-time PCR to characterize chloroquine resistance; and, (iii) real-time PCR targeting 18S genes to detect and quantify Plasmodium parasites. Results Out of the 669 archived RDTs, amplification of P. falciparum nucleic materials had a success rate of 97% for 18S real-time PCR, and 88% for the msp1 gene. The multiplicity of infections (MOI) of the whole population was 2.6 (95% CI 2.5–2.8). The highest number of alleles detected in one infection was 11. The MOI decreased with increasing age (β = − 0.0046, p = 0.02) and residence in Lambaréné was associated with smaller MOIs (p < 0.001). The overall prevalence of mutations associated with chloroquine resistance was 78.5% and was not associated with age. In Lambaréné, prevalence of chloroquine resistance was lower compared to rural Moyen-Ogooué (β = − 0.809, p-value = 0.011). Conclusion RDT is a reliable source of DNA for P. falciparum detection and genotyping assays. Furthermore, the increasing use of RDTs allows them to be an alternative source of DNA for large-scale genetic epidemiological studies. Parasite populations in the study area are highly diverse and prevalence of chloroquine-resistant P. falciparum remains high, especially in rural areas.

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