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Real-time PCR of the mammalian hydroxymethylbilane synthase (HMBS) gene for analysis of flea (Ctenocephalides felis) feeding patterns on dogs.

Wang C, Mount J, Butler J, Gao D, Jung E, Blagburn BL, Kaltenboeck B - Parasit Vectors (2012)

Bottom Line: Sensitivity of the assay was established by limiting dilution and detection of single copies of HMBS DNA equivalent to 0.043 nL blood.Application of the assay indicated that after 15 minutes on a dog, male and female fleas had ingested low, but similar amounts of approximately 1.1. nL blood.The HMBS PCR method developed here offers the advantages of both exquisite sensitivity and specificity that make it superior to other approaches for quantification of blood ingested by fleas.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849-5519, USA.

ABSTRACT

Background: Precise data on quantitative kinetics of blood feeding of fleas, particularly immediately after contact with the host, are essential for understanding dynamics of flea-borne disease transmission and for evaluating flea control strategies. Standard methods used are inadequate for studies that simulate early events after real-life flea access to the host.

Methods: Here, we developed a novel quantitative polymerase chain reaction targeting mammalian DNA within fleas to quantify blood consumption with high sensitivity and specificity. We used primers and fluorescent probes that amplify the hydroxymethylbilane synthase (HMBS) gene, an evolutionary divergent gene that is unlikely to be detected in insects by mammalian-specific primers and probes. To validate this assay, fleas were placed on dogs, allowed to distribute in the hair, and removed at specific time points with single-use combs. Fleas were then immediately homogenized by vigorous shaking with ceramic beads in guanidinium-based DNA preservation buffer for DNA extraction.

Results: The specificity of this assay was ascertained by amplification of canine, feline and equine blood with differential product melting temperatures (Tm), and lack of amplification of bovine and porcine blood and of adult fleas reared from larvae fed with bovine blood. Sensitivity of the assay was established by limiting dilution and detection of single copies of HMBS DNA equivalent to 0.043 nL blood. Application of the assay indicated that after 15 minutes on a dog, male and female fleas had ingested low, but similar amounts of approximately 1.1. nL blood. Saturation uptake of 118 and 100 nL blood per flea was found at 30 and 60 min on the dog, respectively.

Conclusions: The HMBS PCR method developed here offers the advantages of both exquisite sensitivity and specificity that make it superior to other approaches for quantification of blood ingested by fleas. The capability to detect minute quantities of blood in single fleas, particularly immediately after colonization of the host, will provide a superior tool for studying flea-host interactions, flea-borne disease transmission, and flea control strategies.

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PCR amplification curves of HMBS standards and canine blood specimens. HMBS standards and DNA of a canine whole blood specimen were logarithmically diluted (103 to 10-1 HMBS template copies or PCR input equivalents of 102 to 10-2 nL canine blood) were amplified in a background of 200 ng pUC19 plasmid DNA. Fluorescence channel F2 normalized by F1 (F2/F1) shows the robust amplification curves of HMBS standards and canine whole blood specimens. The 103 copies or 102 nL blood amplification curves cross the fluorescence threshold at approximately cycle 10, while the 100 copy or 10-1 nL curves (single copy or 0.1 nL blood) cross at approximately cycle 19. In contrast, the 10-1 copy or 10-2 nL curves (0.1 theoretical copy or 0.01 nL blood) do not cross the threshold and are indistinguishable from the negative control. On average for all amplifications, the nL amount of blood was equivalent to 23 × the amount of HMBS copies detected, defining the theoretical detection limit (without consideration of Poisson sampling error) as 0.043 nL blood per 1 HMBS copy.
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Figure 2: PCR amplification curves of HMBS standards and canine blood specimens. HMBS standards and DNA of a canine whole blood specimen were logarithmically diluted (103 to 10-1 HMBS template copies or PCR input equivalents of 102 to 10-2 nL canine blood) were amplified in a background of 200 ng pUC19 plasmid DNA. Fluorescence channel F2 normalized by F1 (F2/F1) shows the robust amplification curves of HMBS standards and canine whole blood specimens. The 103 copies or 102 nL blood amplification curves cross the fluorescence threshold at approximately cycle 10, while the 100 copy or 10-1 nL curves (single copy or 0.1 nL blood) cross at approximately cycle 19. In contrast, the 10-1 copy or 10-2 nL curves (0.1 theoretical copy or 0.01 nL blood) do not cross the threshold and are indistinguishable from the negative control. On average for all amplifications, the nL amount of blood was equivalent to 23 × the amount of HMBS copies detected, defining the theoretical detection limit (without consideration of Poisson sampling error) as 0.043 nL blood per 1 HMBS copy.

Mentions: The HMBS copy number was determined in FRET-PCR performed on a LightCycler® 1.5 real-time PCR platform with Software version 3.53 (Roche Molecular Biochemicals, Indianapolis, IN, USA) with PCR conditions as previously described [12]. The sensitivity of the PCRs was confirmed by amplification of logarithmic dilutions of HMBS standard and DNA extracted from canine blood (Figure 2). For specificity assurance, the HMBS PCR was performed on nucleic acids extracted from adult fleas reared from larvae fed on bovine blood, and from bovine, canine, equine, feline, and porcine blood followed by melting curve analysis. PCR products were verified by automated DNA sequencing of both strands at the Genomic Sequencing Laboratory (Auburn University, Auburn, AL).


Real-time PCR of the mammalian hydroxymethylbilane synthase (HMBS) gene for analysis of flea (Ctenocephalides felis) feeding patterns on dogs.

Wang C, Mount J, Butler J, Gao D, Jung E, Blagburn BL, Kaltenboeck B - Parasit Vectors (2012)

PCR amplification curves of HMBS standards and canine blood specimens. HMBS standards and DNA of a canine whole blood specimen were logarithmically diluted (103 to 10-1 HMBS template copies or PCR input equivalents of 102 to 10-2 nL canine blood) were amplified in a background of 200 ng pUC19 plasmid DNA. Fluorescence channel F2 normalized by F1 (F2/F1) shows the robust amplification curves of HMBS standards and canine whole blood specimens. The 103 copies or 102 nL blood amplification curves cross the fluorescence threshold at approximately cycle 10, while the 100 copy or 10-1 nL curves (single copy or 0.1 nL blood) cross at approximately cycle 19. In contrast, the 10-1 copy or 10-2 nL curves (0.1 theoretical copy or 0.01 nL blood) do not cross the threshold and are indistinguishable from the negative control. On average for all amplifications, the nL amount of blood was equivalent to 23 × the amount of HMBS copies detected, defining the theoretical detection limit (without consideration of Poisson sampling error) as 0.043 nL blood per 1 HMBS copy.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3275464&req=5

Figure 2: PCR amplification curves of HMBS standards and canine blood specimens. HMBS standards and DNA of a canine whole blood specimen were logarithmically diluted (103 to 10-1 HMBS template copies or PCR input equivalents of 102 to 10-2 nL canine blood) were amplified in a background of 200 ng pUC19 plasmid DNA. Fluorescence channel F2 normalized by F1 (F2/F1) shows the robust amplification curves of HMBS standards and canine whole blood specimens. The 103 copies or 102 nL blood amplification curves cross the fluorescence threshold at approximately cycle 10, while the 100 copy or 10-1 nL curves (single copy or 0.1 nL blood) cross at approximately cycle 19. In contrast, the 10-1 copy or 10-2 nL curves (0.1 theoretical copy or 0.01 nL blood) do not cross the threshold and are indistinguishable from the negative control. On average for all amplifications, the nL amount of blood was equivalent to 23 × the amount of HMBS copies detected, defining the theoretical detection limit (without consideration of Poisson sampling error) as 0.043 nL blood per 1 HMBS copy.
Mentions: The HMBS copy number was determined in FRET-PCR performed on a LightCycler® 1.5 real-time PCR platform with Software version 3.53 (Roche Molecular Biochemicals, Indianapolis, IN, USA) with PCR conditions as previously described [12]. The sensitivity of the PCRs was confirmed by amplification of logarithmic dilutions of HMBS standard and DNA extracted from canine blood (Figure 2). For specificity assurance, the HMBS PCR was performed on nucleic acids extracted from adult fleas reared from larvae fed on bovine blood, and from bovine, canine, equine, feline, and porcine blood followed by melting curve analysis. PCR products were verified by automated DNA sequencing of both strands at the Genomic Sequencing Laboratory (Auburn University, Auburn, AL).

Bottom Line: Sensitivity of the assay was established by limiting dilution and detection of single copies of HMBS DNA equivalent to 0.043 nL blood.Application of the assay indicated that after 15 minutes on a dog, male and female fleas had ingested low, but similar amounts of approximately 1.1. nL blood.The HMBS PCR method developed here offers the advantages of both exquisite sensitivity and specificity that make it superior to other approaches for quantification of blood ingested by fleas.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849-5519, USA.

ABSTRACT

Background: Precise data on quantitative kinetics of blood feeding of fleas, particularly immediately after contact with the host, are essential for understanding dynamics of flea-borne disease transmission and for evaluating flea control strategies. Standard methods used are inadequate for studies that simulate early events after real-life flea access to the host.

Methods: Here, we developed a novel quantitative polymerase chain reaction targeting mammalian DNA within fleas to quantify blood consumption with high sensitivity and specificity. We used primers and fluorescent probes that amplify the hydroxymethylbilane synthase (HMBS) gene, an evolutionary divergent gene that is unlikely to be detected in insects by mammalian-specific primers and probes. To validate this assay, fleas were placed on dogs, allowed to distribute in the hair, and removed at specific time points with single-use combs. Fleas were then immediately homogenized by vigorous shaking with ceramic beads in guanidinium-based DNA preservation buffer for DNA extraction.

Results: The specificity of this assay was ascertained by amplification of canine, feline and equine blood with differential product melting temperatures (Tm), and lack of amplification of bovine and porcine blood and of adult fleas reared from larvae fed with bovine blood. Sensitivity of the assay was established by limiting dilution and detection of single copies of HMBS DNA equivalent to 0.043 nL blood. Application of the assay indicated that after 15 minutes on a dog, male and female fleas had ingested low, but similar amounts of approximately 1.1. nL blood. Saturation uptake of 118 and 100 nL blood per flea was found at 30 and 60 min on the dog, respectively.

Conclusions: The HMBS PCR method developed here offers the advantages of both exquisite sensitivity and specificity that make it superior to other approaches for quantification of blood ingested by fleas. The capability to detect minute quantities of blood in single fleas, particularly immediately after colonization of the host, will provide a superior tool for studying flea-host interactions, flea-borne disease transmission, and flea control strategies.

Show MeSH
Related in: MedlinePlus