Development and evaluation of real‐time PCR assays for bloodmeal identification in <i>Culicoides</i> midges

Saag, M. R. Van Der; Gu, Xingnian; Ward, Michael P.; Kirkland, PD

doi:10.1111/mve.12163

Cited by 12 publications

(10 citation statements)

References 42 publications

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“…Identification of arthropod blood meals by qPCR has been applied to sand flies, biting midges, kissing bugs, fleas and mosquitoes [22,23,[31][32][33][34][35]. Most of these were SYBR green-based systems; only three were probe-based.…”

Section: Discussionmentioning

confidence: 99%

“…b Comparison for mixed blood meals (detected, n = 19; undetected, n = 22) not grouped according to host species. Asterisk (*) indicates significant difference (P < 0.05) between groups (Studentʼs t-test) and did not include humans, pigs or dogs [23], another was for identifying blood-meal hosts of biting midges and included humans and pigs but not dogs [33] and another was for identifying flea blood meals and included humans and dogs but not pigs [35]. Notably, the human probe in the latter flea blood-meal study was tested here and found to cross-react with dog DNA.…”

Section: Discussionmentioning

confidence: 99%

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Probe-based multiplex qPCR identifies blood-meal hosts in Anopheles mosquitoes from Papua New Guinea

et al. 2020

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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 bloodmeal 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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Probe-based multiplex qPCR identifies blood-meal hosts in Anopheles mosquitoes from Papua New Guinea

et al. 2020

View full text Add to dashboard Cite

show abstract

Section: Discussionmentioning

confidence: 99%

Probe-based multiplex qPCR identifies blood-meal hosts in Anopheles mosquitoes from Papua New Guinea

Keven

Artzberger

Gillies

et al. 2020

Preprint

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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/ml 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.

show abstract

“…Identification of arthropod bloodmeals by qPCR has been applied to sand flies, biting midges, kissing bugs, fleas and mosquitoes [22,23,[31][32][33][34][35]. Most of these were SYBR green-based systems; only three were probe-based.…”

Section: Discussionmentioning

confidence: 99%

“…Most of these were SYBR green-based systems; only three were probe-based. Of the three probe-based qPCR, one was for identifying Australian mammals in Culex mosquito bloodmeals and did not include humans, pigs or dogs [23], another was for identifying bloodmeal hosts of biting midges and included humans and pigs but not dogs [33], and another was for identifying flea bloodmeals and included humans and dogs but not pigs [35]. Notably, the human probe in the latter flea bloodmeal study was tested here and found to cross-react with dog DNA.…”

Section: Discussionmentioning

confidence: 99%

Probe-based multiplex qPCR identifies bloodmeal hosts in Anopheles mosquitoes from Papua New Guinea

Keven

Artzberger

Gillies

et al. 2020

Preprint

View full text Add to dashboard Cite

Background: Determination of bloodmeal 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 bloodmeal 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 bloodmeal 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 335/375 (89%) of mosquitoes whereas only 104/188 (55%) of bloodmeal 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 bloodmeals by the quantitative PCR whereas only 3 (2.9%) were mixed bloodmeals 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 bloodmeal analysis of field mosquitoes, in particular mixed-host bloodmeals.

show abstract

Development and evaluation of real‐time PCR assays for bloodmeal identification in Culicoides midges

Cited by 12 publications

References 42 publications

Probe-based multiplex qPCR identifies blood-meal hosts in Anopheles mosquitoes from Papua New Guinea

Probe-based multiplex qPCR identifies blood-meal hosts in Anopheles mosquitoes from Papua New Guinea

Probe-based multiplex qPCR identifies blood-meal hosts in Anopheles mosquitoes from Papua New Guinea

Probe-based multiplex qPCR identifies bloodmeal hosts in Anopheles mosquitoes from Papua New Guinea

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