MOLECULAR METHODS FOR DETECTION AND QUANTITATION OF VIRUS IN APHIDS

摘要

The detection of virus-associated targets in aphids that are capable of transmitting viruses is crucial not only for the study of viral replication but also for the optimization of control strategies. Molecular methods targeting nucleic acids are required for a sensitive detection of the viruliferous state of aphid species. RT-PCR methods has been applied to a wide variety of virus families (Naidu et al., 1998; Vercruysse et al., 2000; Singh et al., 2004), nevertheless more sensitive methods are required for accurate detection of semipersistently and nonpersistenly transmitted viruses. Variants of RT-nested-PCR have been successfully applied for the detection of minute quantities of viral targets (Olmos et al., 1999; Marroquin et al., 2004).rnHowever, the information available to date has been only qualitative. Realtime quantitative RT-PCR is now known to be particularly sensitive for the detection of viral nucleic acid in aphid species (Fabre et al., 2003; Olmos et al., 2005). It has been demonstrated the possibility of quantitation from fresh individual aphids as well as from aphids previously captured on traps and squashed on paper, without the need of previous RNA extraction. These combined technologies (direct squash capture and real-time target amplification) open possibilities for a better understanding of the role of vectors in spreading nonpersistently transmitted viruses.