Evaluation of linked PCR-transcription amplification procedure for bean yellow mosaic virus detection in gladioli.

Abstract Bean yellow mosaic virus (BYMV) concentration in in-vitro cultured Gladiolus cormlets was low and impossible to detect by the commonly used diagnostic methods. The polymerase chain reaction (PCR) detected viral RNA in most infected cormlets but not in all. Additional amplification of the PCR products by transcription, using T7 RNA polymerase (PCR/T), resulted in virus detection in cases which otherwise went undetected. PCR products having a single polymerase promoter at one end served as a better template for T7 RNA polymerase, and yielded more transcripts of a particular orientation than a template containing promoters at both ends. Repeated cycles of PCR/T resulted in the production of a heterogeneous amplified material which correlated with a progressive decline in amplification rate. Therefore, only the first PCR/T cycle proved to be effective. The PCR/T procedure was shown to be better than other commonly used diagnostic methods including PCR.