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Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control
W Menzel1, W Jelkmann, E Maiss
1Institute of Plant Diseases and Plant Protection, University of Hanover, Herrenhäuser Strasse 2, 30419, Hanover, Germany .
Abstract:
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.
Insights
New multiplex RT-PCR assays enable parallel detection of four key apple and pear viruses. These rapid, sensitive methods use an internal RNA control to ensure reliable results, potentially replacing traditional techniques.
Area of Science:
- Plant Pathology
- Molecular Biology
- Virology
Background:
- Apple chlorotic leaf spot virus, Apple stem pitting virus, and Apple stem grooving virus are economically significant pathogens affecting apple and pear cultivars.
- Current detection methods like woody indicator indexing and ELISA can be time-consuming and less sensitive.
Purpose of the Study:
- To develop and validate multiplex RT-PCR assays for the simultaneous detection of four major apple and pear viruses.
- To incorporate a specific internal RNA control for enhanced reliability and to minimize false negatives.
Main Methods:
- Development of four virus-specific primer pairs and one primer pair for amplifying the mitochondrial nad5 gene as an internal RNA control.
- Validation of multiplex RT-PCR assays using various virus isolates from different geographic origins.
- Testing the sensitivity of the assays with mixed nucleic acid extracts from infected and healthy samples.
Main Results:
- Successful parallel detection of Apple chlorotic leaf spot virus, Apple stem pitting virus, Apple mosaic virus, and Apple stem grooving virus.
- Demonstrated reliable detection of viruses in composite extracts at a 1:39 ratio, indicating high sensitivity.
- Confirmed specificity of primer pairs through sequencing of RT-PCR products.
Conclusions:
- The developed multiplex RT-PCR assays are reliable, rapid, and sensitive for detecting key apple and pear viruses.
- The use of an internal RNA control from total nucleic acids is a novel approach to improve diagnostic accuracy.
- These assays offer a promising alternative to conventional methods for routine virus detection in fruit tree cultivars.
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