Real-time PCR Tests in Dutch Exotic Mosquito Surveys; Implementation of Aedes aegypti and Aedes albopictus Identification Tests, and the Development of Tests for the Identification of Aedes atropalpus and Aedes japonicus japonicus (Diptera: Culicidae).
Since 2009, The Netherlands Food and Consumer Product Safety Authority carries out surveys focusing on, amongst others, the presence of invasive mosquito species (IMS). Special attention is given to exotic container-breeding Aedes species Aedes aegypti (L.), Aedes albopictus (Skuse), Aedes atropalpus (Coquillett), and Aedes japonicus japonicus (Theobald). This study describes the implementation of real-time PCR tests described by Hill et al. (2008) for the identification of Ae. aegypti and Ae. albopictus, and the development of two novel real-time PCR tests for the identification of Ae. atropalpus and Ae. j. japonicus. Initial test showed that optimization of elements of the Ae. aegypti and Ae. albopictus tests was needed. Method validation tests were performed to determine if the implemented and newly developed tests are fit for routine diagnostics. Performance criteria of analytical sensitivity, analytical specificity, selectivity, repeatability, and reproducibility were determined. In addition, experiments were performed to determine the influence of environmental conditions on the usability of DNA extracted from mosquito specimens trapped in BG-Sentinel traps. The real-time PCR tests were demonstrated to be sensitive, specific, repeatable, reproducible, and are less prone to false negative results compared to partial cytochrome c oxidase I gene sequencing owing to the DNA fragmentation caused by environmental influences.
Netherlands Food and Consumer Product Safety Authority, Dutch National Plant Protection Organization, Molecular Biology P.O. Box 9102, 6700 HC Wageningen, The Netherlands. b.t.l.h.vandevossenberg@nvwa.nl.
B T L H
BT
van de Vossenberg
Netherlands Food and Consumer Product Safety Authority, Centre for Monitoring of Vectors, P.O. Box 9102, 6700 HC Wageningen, The Netherlands.
A
A
Ibáñez-Justicia
Netherlands Food and Consumer Product Safety Authority, Dutch National Plant Protection Organization, Molecular Biology P.O. Box 9102, 6700 HC Wageningen, The Netherlands.
E
E
Metz-Verschure
Netherlands Food and Consumer Product Safety Authority, Dutch National Plant Protection Organization, Molecular Biology P.O. Box 9102, 6700 HC Wageningen, The Netherlands.
E J
EJ
van Veen
Netherlands Food and Consumer Product Safety Authority, Dutch National Plant Protection Organization, Molecular Biology P.O. Box 9102, 6700 HC Wageningen, The Netherlands.
M L
ML
Bruil-Dieters
Netherlands Food and Consumer Product Safety Authority, Centre for Monitoring of Vectors, P.O. Box 9102, 6700 HC Wageningen, The Netherlands.
E J
EJ
Scholte
D000330:Aedes / Q000145:classification* / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D003576:Electron Transport Complex IV / Q000235:genetics; D019476:Insect Proteins / Q000235:genetics; D058865:Introduced Species; D008969:Molecular Sequence Data; D009032:Mosquito Control; D009426:Netherlands; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D016415:Sequence Alignment
D019476:Insect Proteins; D003576:Electron Transport Complex IV
DNA fragmentation; environmental influence; invasive mosquito species; validation; vector identification