Detecting Human DNA in Sand Flies

Female sand flies identified as P. argentipes by PCR-RFLP were analysed for the presence of human DNA using a qPCR protocol with primers targeting the cytochrome b (cytb) gene as described previously [25 (link)]. Quantitative detection of human DNA was performed on an Applied Biosystems 7500 Fast Real-Time PCR System according to the KAPA SYBR FAST Universal Master Mix recommended protocol. For standard curves, human DNA obtained from purchased donor blood (Cambridge Bioscience) was serially diluted to provide a range of 1–0.0001ng/μl. DNA free water was used as No Template Control (NTC) in each assay. A total of 5μl of DNA was used from each female P. argentipes. Samples were considered positive if Ct values were lower than the lower limit of detection of the assay (Ct< 30). All samples and controls were run in duplicate.

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McIntyre-Nolan S., Kumar V., Mark-Carew M., Kumar K., Nightingale E.S., Dalla Libera Marchiori G., Rogers M.E., Kristan M., Campino S., Medley G.F., Das P, & Cameron M.M. (2023). Comparison of collection methods for Phlebotomus argentipes sand flies to use in a molecular xenomonitoring system for the surveillance of visceral leishmaniasis. PLOS Neglected Tropical Diseases, 17(9), e0011200.

Publication 2023

7500 Fast Real-Time PCR System KAPA SYBR FAST Universal Master Mix

A dna Assay Cytochrome b Donor blood Female Gene Human Primers Rflp Sand flies

Corresponding Organization : London School of Hygiene & Tropical Medicine

Other organizations : Rajendra Memorial Research Institute of Medical Sciences, National Institute of Cholera and Enteric Diseases

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Variable analysis

independent variables

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dependent variables

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Female sand flies identified as P. argentipes by PCR-RFLP
Primers targeting the cytochrome b (cytb) gene
Applied Biosystems 7500 Fast Real-Time PCR System
KAPA SYBR FAST Universal Master Mix
DNA free water as No Template Control (NTC)
Human DNA obtained from purchased donor blood (Cambridge Bioscience) for standard curves

controls

Human DNA obtained from purchased donor blood (Cambridge Bioscience) for standard curves
DNA free water as No Template Control (NTC)

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