Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control.

Two multiplex RT-PCR assays with specific coamplification of plant mRNA as an internal control from total nucleic acids are described for the parallel detection of Apple chlorotic leaf spot virus, Apple stem pitting virus, Apple mosaic virus and Apple stem grooving virus. All are important economically and common pathogens in commercial apple and pear cultivars, except for Apple mosaic virus. Four virus specific primer pairs and one primer pair which allows the specific amplification of mRNA of the mitochondrial nad5 gene are described. Specificity of all primer pairs was confirmed by sequencing the RT-PCR products. A range of different virus isolates from various geographic origins could be detected by these multiplex RT-PCR assays all year round. Viruses were detected reliably in composite extracts at a ratio of one part total nucleic acid extract from an infected sample mixed with 39 parts of extract from healthy samples. The use of the internal control minimizes the risk of obtaining false negative RT-PCR results, which is desirable for routine testing, and avoids the need to eliminate contaminating DNA in extracts. To our knowledge, this is the first report on the use of a specific internal RNA control from total nucleic acids. The multiplex RT-PCR assays described are reliable, rapid and sensitive methods for the detection of these viruses, and may replace techniques need commonly like indexing by woody indicators or ELISA.