Reverse transcription PCR (RT-PCR) is a popular method for detecting RNA viruses in plants. RT-PCR is usually performed in a classical two-step procedure: in the first step, cDNA is synthesized by reverse transcriptase (RT), followed by PCR amplification by a thermostable polymerase in a separate tube in the second step. However, one-step kits containing multiple enzymes optimized for RT and PCR amplification in a single tube can also be used. Here, we describe an RT-PCR single-enzyme assay based on an RTX DNA polymerase that has both RT and polymerase activities. The expression plasmid pET_RTX_(exo-) was transferred to various E. coli genotypes that either compensated for codon bias (Rosetta-gami 2) or contained additional chaperones to promote solubility (BL21 (DE3) with plasmids pKJE8 or pTf2). The RTX enzyme was then purified and used for the RT-PCR assay. Several purified plant viruses (TMV, PVX, and PVY) were used to determine the efficiency of the assay compared to a commercial one-step RT-PCR kit. The RT-PCR assay with the RTX enzyme was validated for the detection of viruses from different genera using both total RNA and crude sap from infected plants. The detection endpoint of RTX-PCR for purified TMV was estimated to be approximately 0.01 pg of the whole virus per 25 µL reaction, corresponding to 6 virus particles/µL. Interestingly, the endpoint for detection of TMV from crude sap was also 0.01 pg per reaction in simulated crude plant extracts. The longest RNA fragment that could be amplified in a one-tube arrangement was 2379 bp long. The longest DNA fragment that could be amplified during a 10s extension was 6899 bp long. In total, we were able to detect 13 viruses from 11 genera using RTX-PCR. For each virus, two to three specific fragments were amplified. The RT-PCR assay using the RTX enzyme described here is a very robust, inexpensive, rapid, easy to perform, and sensitive single-enzyme assay for the detection of plant viruses.

• Keywords
• Capillovirus, Foveavirus, Luteovirus, Nepovirus, Polerovirus, Potexvirus, Potyvirus, RTX-PCR, Tobamovirus, Trichovirus, Tritimovirus, one-step RT-PCR, virus detection,
• MeSH
• DNA-Directed DNA Polymerase metabolism   MeSH
• Phylogeny MeSH
• Plant Diseases virology   MeSH
• Polymerase Chain Reaction instrumentation   methods   MeSH
• RNA Viruses classification   genetics   isolation & purification   MeSH
• Plant Viruses classification   genetics   isolation & purification   MeSH
• Sensitivity and Specificity MeSH
• Crops, Agricultural virology   MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Directed DNA Polymerase MeSH

A SYBR Green(®)-based one step RT-qPCR assay was developed for the detection and quantification of Apple stem grooving virus (ASGV) and Apple mosaic virus (ApMV). The RT-qPCR assay employed seven plant-expressed genes-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA, ubiquitin, ribosomal protein S19, Rubisco, RNA polymerase subunit II and β-actin-as internal reference housekeeping genes in a relative quantification system in three apple cultivars (i.e. Idared, Champion, Fragrance). The average expression stability (M) found by GeNorm software suggest that GAPDH and S19 were the most stable reference genes. We propose employing GAPDH and S19 as housekeeping genes for accurate quantification of ASGV and ApMV in apple leaf samples. The detection limit for both viruses was found around 70 copies of viral genome by one-step RT-qPCR.

• Keywords
• ASGV, ApMV, Reference genes, qPCR,
• Publication type
• Journal Article MeSH

A rapid method for detection, discrimination and quantification of wheat and barley strains of wheat dwarf virus (WDV) was successfully developed. The sensitivity of quantification of the wheat and barley strains of WDV ranged from an average of 1.2 × 10(7)-1.2 × 10(2) and from an average of 1.4 × 10(7)-1.4 × 10(4) copies of viral genome, respectively. These standard serial dilutions were applied to plant and vector tissues for virus titer calculations. Both strains of WDV were clearly discriminated by specific probes and melting curve analysis. Both TaqMan(®) and SYBR(®) Green technologies provided accurate and reliable methods for monitoring, detection, discrimination, and quantification of WDV.

• MeSH
• Geminiviridae classification   genetics   isolation & purification   MeSH
• Hordeum virology   MeSH
• Real-Time Polymerase Chain Reaction methods   MeSH
• Plant Diseases virology   MeSH
• Triticum virology   MeSH
• Sensitivity and Specificity MeSH
• Viral Load methods   MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH