The presence of two different target-site resistance mechanisms in individual plants of Alopecurus myosuroides Huds., identified using a quick molecular test for the characterisation of six ALS and seven ACCase SNPs.

BACKGROUND: Target-site resistance to ALS- and ACCase-inhibiting herbicides in the grass weed Alopecurus myosuroides is associated with well-characterised allelic variants encoding ALS- and ACCase-based resistance. The potential for combined ALS and ACCase resistance presents a threat to future control, given the extent to which these herbicides are used. The authors present a primer extension method for rapid detection of known resistance-conferring substitutions. RESULTS: Individuals showing combined resistance to field-rate mesosulfuron + iodosulfuron and cycloxydim were identified in four field-collected populations, with proportions ranging from 30 to 100%. Genotyping with the SNaPshot primer extension kit showed the T197 and L574 ALS and L1781 ACCase isoforms to be associated with ALS and ACCase resistance whenever they occurred. CONCLUSION: Combined ALS and ACCase target-site resistance threatens future control of A. myosuroides. The SNaPshot extension assay provides a reliable new multiplexable method for characterising known allelic variants of the ALS and ACCase genes of A. myosuroides. The method offers significant advantages over both CAPS/dCAPS and PASA in that full genotyping can be accomplished at any nucleotide position using a single extension primer.

Molecular beacons as diagnostic tools: technology and applications.

Molecular beacons are single-stranded, fluorophore-labeled nucleic acid probes that are capable of generating a fluorescent signal in the presence of target, but are dark in the absence of target. Molecular beacons allow multiplex detection of PCR products in real time in a homogeneous assay format. Real time detection is inherently quantitative and affords a greater dynamic range than end-point detection methods. Reactions in a homogeneous assay format are sealed before amplification takes place, providing improved contamination control. A single cycler/reader instrument, coupled with automated sample preparation, results in higher throughput and greater ease of use. A multiplex qualitative assay that detects Chlamydia trachomatis and Neisseria gonorrhoeae, along with an internal control, has been developed. High specificity is achieved through careful selection of primers, probes and assay conditions. Quantitative HIV, HCV, and HBV viral load assays, with sensitivities of 50 copies/ml, 20 IU/ml, and 50 copies/ml, respectively, are achievable. The viral load assays are designed to quantitate all subtype and genotype specimens equivalently. A molecular beacon assay has been designed to detect a single nucleotide polymorphism in the beta2 adrenergic receptor gene.