Quantitative multiplex detection of plant pathogens using PRI-lock probes and universal, ultra-high-throughput real-time PCR on OpenArraysTM
Current technologies for multiplex, quantitative analyses frequently suffer from compromises between the level of multiplexing, throughput and accuracy of quantification. In general, for the detection of nucleic acids, microarrays provide very high levels of multiplexing, but less accurate quantification and usually low throughput. Real-time PCR provides the most reliable means of target quantification and is suitable for the analysis of a relatively high number of samples. However the achievable level of multiplexing is low. Nano-scale technology provides high-density, low-volume microchambers, which could accommodate very high number of reactions, performed under standard conditions. Yet many of these systems are still experimental and are incapable of real time fluorescent monitoring required for quantification. However recently, a conceptually new, ultra-highthroughput platform has become available for real-time PCR; capable of accommodating more than 3000 reactions per array. The OpenArrayTM has 48 subarrays, allowing parallel testing of up to 48 samples, with each subarray contains 64 microscopic (33 nL) throughholes. The primers are pre-loaded into the holes, while the sample and reagents are autoloaded due to surface tension. PRI has developed PRI-Lock probes for multiplex detection; providing flexibility and bridging the gap between target-specific recognition and highthroughput amplification. These probes are long oligonucleotides (similar in structure to Padlock probes) and contain artificially selected primer sites and a TaqMan probe region, flanked by target complementary regions. In this paper, we report the development of a high-throughput, quantitative multiplex diagnostic assay based on above technologies.
| Main Authors: | , , , |
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| Format: | Article in monograph or in proceedings biblioteca |
| Language: | English |
| Published: |
The Royal Veterinary and Agricultural University / EFPP & BSPP
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| Subjects: | Life Science, |
| Online Access: | https://research.wur.nl/en/publications/quantitative-multiplex-detection-of-plant-pathogens-using-pri-loc |
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