Detection of Wuchereria bancrofti L3 larvae in mosquitoes: a reverse transcriptase PCR assay evaluating infection and infectivity.
BACKGROUND
Detection of filarial DNA in mosquitoes by PCR cannot differentiate infective mosquitoes from infected mosquitoes. In order to evaluate transmission risk an assay is needed that can specifically detect infective L3 stage parasites. We now report the development of an assay that specifically detects the infective stage of Wuchereria bancrofti in mosquitoes. The assay detects an L3-activated mRNA transcript by reverse-transcriptase PCR (RT-PCR).


RESULTS
W. bancrofti cuticle-related genes were selected using bioinformatics and screened as potential diagnostic target genes for L3 detection in mosquitoes. Expression profiles were determined using RT-PCR on RNA isolated from mosquitoes collected daily across a two-week period after feeding on infected blood. Conventional multiplex RT-PCR and real-time multiplex RT-PCR assays were developed using an L3-activated cuticlin transcript for L3 detection and a constitutively expressed transcript, tph-1, for 'any-stage' detection.


CONCLUSIONS
This assay can be used to simultaneously detect W. bancrofti infective stage larvae and 'any-stage' larvae in pooled vector mosquitoes. This test may be useful as a tool for assessing changes in transmission potential in the context of filariasis elimination programs.
Laney
Sandra J
SJ
Department of Biological Sciences, Smith College, Northampton, Massachusetts, USA. slaney@smith.edu
Ramzy
Reda M R
RM
Helmy
Hanan H
HH
Farid
Hoda A
HA
Ashour
Ameen A
AA
Weil
Gary J
GJ
Williams
Steven A
SA
D000818:Animals; D001483:Base Sequence; D009033:Culicidae / Q000469:parasitology*; D017931:DNA Primers / Q000235:genetics; D004199:Disease Vectors; D007814:Larva; D008969:Molecular Sequence Data; D010274:Parasitology / Q000379:methods*; D018510:RNA, Helminth / Q000235:genetics / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014958:Wuchereria bancrofti / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers; D018510:RNA, Helminth; D012333:RNA, Messenger