Journal of Virological Methods 137 (2006) 7–13 Improved detection of episomal Banana streak viruses by multiplex immunocapture PCR Gr´ egoire Le Provost a,1,2 , Marie-Line Iskra-Caruana a,2 , Isabelle Acina b , Pierre-Yves Teycheney b,∗ a CIRAD, UMR BGPI, Campus International de Baillarguet, F-34398 Montpellier Cedex 5, France b CIRAD, UPR75, Station de Neufchˆ ateau, F-97130 Capesterre Belle-Eau, Guadeloupe Received 28 March 2006; received in revised form 17 May 2006; accepted 25 May 2006 Available online 20 July 2006 Abstract Banana streak viruses (BSV) are currently the main viral constraint to Musa germplasm movement, genetic improvement and mass propagation. Therefore, it is necessary to develop and implement BSV detection strategies that are both reliable and sensitive, such as PCR-based techniques. Unfortunately, BSV endogenous pararetrovirus sequences (BSV EPRVs) are present in the genome of Musa balbisiana. They interfere with PCR-based detection of episomal BSV in infected banana and plantain, such as immunocapture PCR. Therefore, a multiplex, immunocapture PCR (M-IC-PCR) was developed for the detection of BSV. Musa sequence tagged microsatellite site (STMS) primers were selected and used in combination with BSV species-specific primers in order to monitor possible contamination by Musa genomic DNA, using multiplex PCR. Furthermore, immunocapture conditions were optimized in order to prevent Musa DNA from interfering with episomal BSV DNA during the PCR step. This improved detection method successfully allowed the accurate, specific and sensitive detection of episomal DNA only from distinct BSV species. Its implementation should benefit PCR-based detection of viruses for which homologous sequences are present in the genome of their hosts, including transgenic plants expressing viral sequences. © 2006 Elsevier B.V. All rights reserved. Keywords: Banana streak virus (BSV); Badnavirus; Endogenous pararetrovirus (EPRV); Episomal; Multiplex immunocapture PCR (M-IC-PCR); Diagnosis 1. Introduction Banana streak viruses (BSV) are mealybug-transmitted members of the plant pararetrovirus genus Badnavirus, infect- ing banana and plantain worldwide (Hull et al., 2000; Fauquet et al., 2005). BSV infections cause characteristic chlorotic and necrotic leaf streak symptoms. Depending on infecting BSV species, highly susceptible banana cultivars can develop more severe symptoms, such as pseudostem splitting and necrosis, eventually leading to the death of infected plants (Lockhart and Jones, 2000; Daniells et al., 2001). Although originally not considered an economically important virus, BSV has raised strong concern over the past 15 years due to an increasing ∗ Corresponding author. Tel.: +590 86 17 71. E-mail address: pierre-yves.teycheney@cirad.fr (P.-Y. Teycheney). 1 Present address: INRA, UMR BIOGECO, Equipe de g´ en´ etique, F-33612 Cestas, France. 2 They contributed equally to this work. record of infections among new banana and plantain breeding lines and micropropagated hybrids. Interspecific Musa acumi- nata × Musa balbisiana genotypes, including a number of newly created hybrids, showed a tendency to produce BSV-infected propagules from virus-free source plants propagated by tis- sue culture (Dallot et al., 2001). Likewise, infected progeny were repeatedly obtained following genetic crosses involving virus-free M. acuminata and M. balbisiana parents (Lheureux et al., 2003). Such infections were correlated to the presence of BSV sequences integrated into the genome of M. balbisiana, a widespread progenitor of natural and created hybrid banana and plantain species. Similar pararetroviral sequences called endogenous pararetroviruses (EPRVs) have been identified in the nuclear genome of several other plants, including petu- nia, tobacco, potato, rice and tomato (Jakowitsch et al., 1999; Lockhart et al., 2000; Mao et al., 2000; Budiman et al., 2000; Richert-P¨ oggeler et al., 2003; Gregor et al., 2004; Kunii et al., 2004; Hansen et al., 2005). It was recently shown that some EPRVs have the potential to express infectious viral genomes 0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2006.05.021