Automated DNA extraction for large numbers of plant samples.

The method described here is a rapid, total DNA extraction procedure applicable to a large number of plant samples requiring pathogen detection. The procedure combines a simple and quick homogenization step of crude extracts with DNA extraction based upon the binding of DNA to magnetic beads. DNA is purified in an automated process in which the magnetic beads are transferred through a series of washing buffers. The eluted DNA is suitable for efficient amplification in PCR reactions.

A real-time PCR detection system for the bois noir and flavescence dorée phytoplasmas and quantification of the target DNA.

The real-time PCR detection system for grapevine yellows phytoplasmas described here is composed of two assays for group-specific detection of flavescence dorée (FD) and bois noir (BN) phytoplasmas and a universal phytoplasma assay. It uses hydrolysis minor groove binder probes (TaqMan-MGB). The addition of an assay for amplification of plant DNA co-extracted with phytoplasma DNA provides a further quality control for the DNA extraction and PCR amplification for each sample. The detection system described is reliable, specific, sensitive, and easily applicable to fast, high-throughput diagnosis of grapevine yellows phytoplasmas. In addition to the detection system, an approach for the quantification of phytoplasmas in the sample is described.

Sensitive detection of multiple rotavirus genotypes with a single reverse transcription-real-time quantitative PCR assay.

Rotaviruses are one of the major causes of diarrhea in infants and children under 5 years old, especially affecting developing countries. In natural disasters, fecal matter and potable waters can mix, allowing low, yet infective, concentrations of rotavirus to be present in water supplies, constituting a risk for the population. Any of the most commonly detected rotavirus genotypes could originate an outbreak. The development of a fast and sensitive method that could detect the broadest possible range of rotavirus genotypes would help with efficient diagnosis and prevention. We have designed a reverse transcription (RT)-real-time quantitative PCR approach targeted to the rotaviral VP2 gene, based on a multiple-sequence alignment of different human rotaviral strains. To overcome the high nucleotide sequence diversity, multiple forward and reverse primers were used, in addition to a degenerate probe. The performance of the assay was tested on isolates representing the most prevalent human genotypes: G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8]. The developed method improved classical rotavirus detection by enzyme-linked immunosorbent assay and nested RT-PCR by 5 and at least 1 order of magnitude, respectively. A survey of 159 stool samples indicated that the method can efficiently detect a broad range of rotavirus strains, including different G-P genotype combinations of human, porcine, and bovine origin. No cross-reactivity was observed with other enteric viruses, such as astrovirus, sapovirus, and norovirus.

Short monolithic columns--a breakthrough in purification and fast quantification of tomato mosaic virus.

Drawbacks of conventional virus purification methods have led to the development of new, mostly chromatography-based methods. Short monolithic columns are stationary phases intended for purification of large molecules. In this work efficient chromatographic purification of tomato mosaic virus (ToMV) from plant material is described. Based on short monolithic column, the purification process was shortened from 5 days to 2 hours. High viral purity was achieved and recovery of chromatographic step was up to 90%. In addition, these columns enabled preliminary quantification of the virus in just a few minutes, much faster than other quantification methods (e.g. enzyme-linked immunosorbent assay or real-time polymerase chain reaction) which take 1-2 days. These results demonstrate the potential of short monolith column technology for purification and analysis of different viruses.