| Experimental objective: Considering the diversity, various species, complexity, compositions, and attributes of target analytes in organisms, it is desirable to employ a unified detection tool instead of applying various specific methods to accomplish the same job. In this study, a technology platform is aimed to establish for a specific, high sensitive, multiplexing and universal detection of a wide variety of pathogens at the same condition by using alfalfa mosaic virus (AMV), cucumber mosaic virus (CMV), potato leafroll virus (PLRV), potato virus A (PVA), potato virus M (PVM), potato virus S (PVS), potato virus X (PVX), potato virus Y (PVY), tobacco mosaic virus (TMV), and tobacco ring spot virus (TRSV) as an experimental object.Experimental methods: Viral RNAs were extracted from potato leaves respectively infected by each of ten potato viruses, followed by RNA reverse transcription for the cDNA with random hexamer primers or specific reverse primer, and amplified by using virus-specific primers. The amplification products were cloned to the plasmids and verified by gel electrophoresis and DNA sequencing, respectively. The plasmids containg the target viral sequences were used as standard sample for optimization of the the following assay. The upstream and downstream adjacent LDR (ligase detection reaction) probes were designed between each viruse specific primers. The upstream viruse specific sequence appended an upstream universal sequence at its 3′end, both of which constituted upstream LDR (ligase detection reaction) probe. Zip-code sequence respectively appending downstream universal sequence and downstream virus specific probe sequence at its 5′and 3′end, formed the downstream LDR probe. Each ligated product (about 100 nt) has identical universal tail sequences and one Zip-code sequence corresponding to one virus. The universal primers amplification and Zip-code hybridization were used to detect and identify each virus. Based on specific detection of each virus by single LDR-PCR microarray, the following experiments were performed: optimization of multiple LDR-PCR microarray, sensitivity and specificity assay of one and two mixed viruses in the multiple LDR-PCR microarrays, multiplex assay of ten mixed viruses in the multiple LDR-PCR microarray, and application of the developed assay to field samples detection.Experimental results: The results of these experiments mainly were displayed in the following five aspects:①The gel electrophoresis and hybridization of single LDR-PCR for each virus showed that the designed viral LDR probes and Zip-code sequence possessed good specificity;②The optimized parameters in multiple LDR-PCR electrophoresis experiment were as following, 0.1 U of Taq DNA ligase concentration, 0.1 pmol of each LDR probes pairs, 35 cycles number, 4 min annealing time, and 58°C annealing temperature;③The detection limit of the multiple LDR-PCR microarray for one and two viruse were both 3 copies per reaction;④The multiple LDR-PCR microarray assay possessed good multiplexing for ten viruses detection;⑤The positive detection rates of above ten potato viruses ranged from 2.53% to 44.30% when seventy-nine field samples were detected by the developed assay, higher than that of 0-39.24% by traditional PCR.Conclusion: In the present study, LDR, PCR and hybridization technology of universal oligonucleotide microarray were combined by introducing universal tails and Zip-code hybridization sequence, establishing a strong specifici, high sensitive, good multiplexing and universal technology platform for parallel ten potato viruses'detection. It can also be recommended for simultaneously detecting a large number of different target types such as clinical, veterinary, food, biodefense, environmental microorganisms or genome genetic variants in molecular diagnostics. |
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