John B Keven1,2, Georgia Artzberger3, Mary L Gillies3, Rex B Mbewe4,5, Edward D Walker6,4. 1. Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA. kevenjoh@msu.edu. 2. Vector Borne Diseases Unit, Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea. kevenjoh@msu.edu. 3. Department of Biomedical Laboratory Diagnostics, Michigan State University, East Lansing, MI, USA. 4. Department of Entomology, Michigan State University, East Lansing, MI, USA. 5. Department of Physics and Biochemical Sciences, University of Malawi, The Polytechnic, Blantyre, Malawi. 6. Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA.
Abstract
BACKGROUND: Determination of blood-meal hosts in blood-fed female Anopheles mosquitoes is important for evaluating vectorial capacity of vector populations and assessing effectiveness of vector control measures. Sensitive molecular methods are needed to detect traces of host blood in mosquito samples, to differentiate hosts, and to detect mixed host blood meals. This paper describes a molecular probe-based quantitative PCR for identifying blood-meal hosts in Anopheles malaria vectors from Papua New Guinea. METHODS: TaqMan oligonucleotide probes targeting specific regions of mitochondrial or nuclear DNA of the three primary Anopheles blood-meal hosts, humans, pigs and dogs, were incorporated into a multiplex, quantitative PCR which was optimized for sensitivity and specificity. RESULTS: Amplification of serially diluted DNA showed that the quantitative PCR detected as low as 10-5 ng/μl of host DNA. Application to field-collected, blood-fed Anopheles showed that the quantitative PCR identified the vertebrate hosts for 89% (335/375) of mosquitoes whereas only 55% (104/188) of blood-meal samples tested in a conventional PCR were identified. Of the 104 blood-fed Anopheles that were positive in both PCR methods, 16 (15.4%) were identified as mixed blood meals by the quantitative PCR whereas only 3 (2.9%) were mixed blood meals by the conventional PCR. CONCLUSIONS: The multiplex quantitative PCR described here is sensitive at detecting low DNA concentration and mixed host DNA in samples and useful for blood-meal analysis of field mosquitoes, in particular mixed-host blood meals.
BACKGROUND: Determination of blood-meal hosts in blood-fed female Anopheles mosquitoes is important for evaluating vectorial capacity of vector populations and assessing effectiveness of vector control measures. Sensitive molecular methods are needed to detect traces of host blood in mosquito samples, to differentiate hosts, and to detect mixed host blood meals. This paper describes a molecular probe-based quantitative PCR for identifying blood-meal hosts in Anophelesmalaria vectors from Papua New Guinea. METHODS: TaqMan oligonucleotide probes targeting specific regions of mitochondrial or nuclear DNA of the three primary Anopheles blood-meal hosts, humans, pigs and dogs, were incorporated into a multiplex, quantitative PCR which was optimized for sensitivity and specificity. RESULTS: Amplification of serially diluted DNA showed that the quantitative PCR detected as low as 10-5 ng/μl of host DNA. Application to field-collected, blood-fed Anopheles showed that the quantitative PCR identified the vertebrate hosts for 89% (335/375) of mosquitoes whereas only 55% (104/188) of blood-meal samples tested in a conventional PCR were identified. Of the 104 blood-fed Anopheles that were positive in both PCR methods, 16 (15.4%) were identified as mixed blood meals by the quantitative PCR whereas only 3 (2.9%) were mixed blood meals by the conventional PCR. CONCLUSIONS: The multiplex quantitative PCR described here is sensitive at detecting low DNA concentration and mixed host DNA in samples and useful for blood-meal analysis of field mosquitoes, in particular mixed-host blood meals.
Authors: John B Keven; Michelle Katusele; Rebecca Vinit; Daniela Rodríguez-Rodríguez; Manuel W Hetzel; Leanne J Robinson; Moses Laman; Stephan Karl; Edward D Walker Journal: Malar J Date: 2022-01-05 Impact factor: 2.979
Authors: Rex B Mbewe; John B Keven; Themba Mzilahowa; Don Mathanga; Mark Wilson; Lauren Cohee; Miriam K Laufer; Edward D Walker Journal: Malar J Date: 2022-03-03 Impact factor: 2.979