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Polymerase chain reaction (PCR) is used to detect groups of viruses with the use of group-specific degenerate primers. Inosine residues are sometimes used in the primers to match variable positions within the complementary target sequences, but there is little data on their effects on cDNA synthesis and amplification. A quantitative reverse-transcription PCR was used to measure the rate of amplification with primers containing inosine residues substituted at different positions and in increasing numbers. Experiments were conducted using standard quantities of cloned DNA copied from Potato virus Y genomic RNA and RNA (cRNA) transcribed from the cloned DNA. Single inosine residues had no affect on the amplification rate in the forward primer, except at one position close to the 3' terminus. Conversely, single inosine residues significantly reduced the amplification rate when placed at three out of four positions in the reverse primer. Four or five inosine substitutions could be tolerated with some decline in rates, but amplification often failed from cRNA templates with primers containing larger numbers of inosines. Greater declines in the rate of amplification were observed with RNA templates, suggesting that reverse transcription suffers more than PCR amplification when inosine is included in the reverse primer.

LAMP assays are targeted molecular tests for the rapid detection of species in the laboratory and field. We developed a LAMP assay for an economically important fruit fly species, Queensland fruit fly, Bactrocera tryoni. This assay was assessed against a broad panel of target and non-target species and found to be specific, only amplifying the target species and closest relatives, in a portable real-time fluorometer (Genie III) in under 15 minutes with an anneal derivative temperature of 82.5 oC. The assay is sensitive to low levels of target DNA (>0.016 ng/µl), performing equally to the existing qPCR test. To enable retention of a physical voucher specimen, for potential morphological confirmation of LAMP results, a novel whole-specimen non-destructive DNA extraction method was developed, suitable for LAMP in the field. The stability of DNA extraction and LAMP reagents was tested under simulated and actual field conditions and shown to be robust. Our new assay now provides a portable molecular tool for the detection of this significant tephritid fruit fly pest species of biosecurity/quarantine concern. This has already proven invaluable for in-field diagnostics, providing real-time support influencing immediate actions, with negative results allowing the release of fruit produce, and positive results initiating fruit fly control measures. [ABSTRACT FROM AUTHOR]

A number of viruses from the genera Furovirus, Hordeivirus and Rymovirus are known to infect and damage the four major temperate cereal crops, wheat, barley, sorghum and oats. Currently, there is no active testing in Australia for any of these viruses, which pose a significant biosecurity threat to the phytosanitary status of Australia's grains industry. To address this, broad spectrum PCR assays were developed to target virus species within the genera Furovirus, Hordeivirus and Rymovirus . Five sets of novel genus-specific primers were designed and tested in reverse-transcription polymerase chain reaction assays against a range of virus isolates in plant virus diagnostic laboratories in both Australia and New Zealand. Three of these assays were then chosen to screen samples in a three-year survey of cereal crops in western Victoria, Australia. Of the 8900 cereal plants screened in the survey, all were tested free of furoviruses, hordeiviruses and rymoviruses. To date, there were no published genus-specific primers available for the detection of furoviruses, hordeiviruses and rymoviruses. This study shows for the first time a broad-spectrum molecular test being used in a survey for exotic grain viruses in Australia. Results from this survey provide important evidence of the use of this method to demonstrate the absence of these viruses in Victoria, Australia. The primer pairs reported here are expected to detect a wide range of virus species within the three genera. [ABSTRACT FROM AUTHOR]