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Sources of blood meals of sylvatic Triatoma guasayana near Zurima, Bolivia, assayed with qPCR and 12S cloning.

Lucero DE, Ribera W, Pizarro JC, Plaza C, Gordon LW, Peña R, Morrissey LA, Rizzo DM, Stevens L - PLoS Negl Trop Dis (2014)

Bottom Line: We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive.However, not all samples positive by qPCR were positive by cloning.We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Vermont, Burlington, Vermont, United States of America; Vector-borne Diseases Section, Tennessee Department of Health, Nashville, Tennessee, United States of America.

ABSTRACT

Background: In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia.

Methodology/principal findings: We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors).

Conclusions/significance: We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.

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Related in: MedlinePlus

Human qPCR amplification of 14 samples (green), 5 serial dilutions (red) and NTC (no template control, black).
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pntd-0003365-g006: Human qPCR amplification of 14 samples (green), 5 serial dilutions (red) and NTC (no template control, black).

Mentions: The limit of detection (LOD), or template concentration that can be detected within 95% certainty [20], is 10−4 ng/uL for all three qPCR assays (Figure 3). Although positive controls were detected at more dilute concentrations, we could only yield consistent and reproducible results until 10−4 ng/uL. Averages of three replicates per concentration are 3.9 (range 3.7–4.6) cycles apart for chicken (Figure 4), 3.8 (range 3.1–4.3) cycles apart for Canis sp. (Figure 5) and 3.3 cycles (range 2.2–3.8) apart for human (Figure 6), respectively, a little higher than the expected 3.3 cycles [37]. The more dilute samples (<10−5 ng/uL) sometimes amplified after the 40 cycles at which one DNA molecule should be detectable (Figure 7) [37]. Four to five positive controls for chicken, Canis sp. and human qPCR products were sequenced, including the 10−4 ng/uL sample, and correctly identified in a BLAST query.


Sources of blood meals of sylvatic Triatoma guasayana near Zurima, Bolivia, assayed with qPCR and 12S cloning.

Lucero DE, Ribera W, Pizarro JC, Plaza C, Gordon LW, Peña R, Morrissey LA, Rizzo DM, Stevens L - PLoS Negl Trop Dis (2014)

Human qPCR amplification of 14 samples (green), 5 serial dilutions (red) and NTC (no template control, black).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4256209&req=5

pntd-0003365-g006: Human qPCR amplification of 14 samples (green), 5 serial dilutions (red) and NTC (no template control, black).
Mentions: The limit of detection (LOD), or template concentration that can be detected within 95% certainty [20], is 10−4 ng/uL for all three qPCR assays (Figure 3). Although positive controls were detected at more dilute concentrations, we could only yield consistent and reproducible results until 10−4 ng/uL. Averages of three replicates per concentration are 3.9 (range 3.7–4.6) cycles apart for chicken (Figure 4), 3.8 (range 3.1–4.3) cycles apart for Canis sp. (Figure 5) and 3.3 cycles (range 2.2–3.8) apart for human (Figure 6), respectively, a little higher than the expected 3.3 cycles [37]. The more dilute samples (<10−5 ng/uL) sometimes amplified after the 40 cycles at which one DNA molecule should be detectable (Figure 7) [37]. Four to five positive controls for chicken, Canis sp. and human qPCR products were sequenced, including the 10−4 ng/uL sample, and correctly identified in a BLAST query.

Bottom Line: We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive.However, not all samples positive by qPCR were positive by cloning.We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Vermont, Burlington, Vermont, United States of America; Vector-borne Diseases Section, Tennessee Department of Health, Nashville, Tennessee, United States of America.

ABSTRACT

Background: In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia.

Methodology/principal findings: We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors).

Conclusions/significance: We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.

Show MeSH
Related in: MedlinePlus