Improved detection of episomal Banana streak viruses by multiplex immunocapture PCR.
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.
Le Provost
Grégoire
G
CIRAD, UMR BGPI, Campus International de Baillarguet, F-34398 Montpellier Cedex 5, France.
Iskra-Caruana
Marie-Line
ML
Acina
Isabelle
I
Teycheney
Pierre-Yves
PY
D019195:Badnavirus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D007158:Immunologic Techniques; D018895:Microsatellite Repeats / Q000235:genetics; D028521:Musa / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004279:DNA, Viral