In order to optimize resources, an assay allowing the detection of several etiological agents would be useful. This paper describes a polymerase chain reaction (PCR)-based assay for the parallel detection of different pathogens in mosquitoes (Diptera: Culicidae). The assay combines the analysis of both RNA and DNA, hence allowing the detection of both flaviviruses (Flaviviridae: Flavivirus) and filarial nematodes (Spirurida: Onchocercidae). The sensitivity of diagnostic assay for both flavivirus and nematode infections was tested by means of spiked samples and resulted adequate for the purpose of screening mosquito samples. The assay was implemented to check pathogen infections in 637 mosquitoes field-collected in Romagna, northern Italy. The integrity of RNA extracted from mosquito pools was checked by control amplifications targeting the 18 S rRNA of mosquitoes. Control amplifications were successful in 118 out of 119 pools. Flavivirus RNA was not found in any of these 118 pools, whereas DNA of Dirofilaria immitis (Leidy) was detected in one pool of Aedes albopictus (Skuse).
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