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Developing a qPCR-based molecular technique for nematode community analysis
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|Title:||Developing a qPCR-based molecular technique for nematode community analysis|
qPCR-based molecular tool
|Issue Date:||May 2012|
|Publisher:||[Honolulu] : [University of Hawaii at Manoa], [May 2012]|
|Abstract:||Nematodes are good indicators for soil health. However performing nematode community analysis is laborious and technically challenging. This research seeks to develop a qPCR-based molecular tool for nematode community analysis. qPCR detecting 18S rDNA offered one approach to identify and quantify free-living nematodes. Owing to too many unpredictable nematode genera across soil ecosystems, one strategy is to develop universal qPCR markers selective for key nematode guilds (Ba1, Ba2, F2, P4, Om4, Om5, and P5) that are most critical for nematode faunal analysis. Universal qPCR primers were successfully being identified for all of these guilds except for Ba1. Two primers were needed for F2; Om4, Om5, and P5 cannot be differentiated and were thus being combined as Om4/ Om5/ P5 by one primer. These primers were then verified by BLAST and then run through artificial nematode mixture sample composed with known nematode guilds. The results confirmed the validity of these universal primers. The next logical step was to run these qPCR to nematodes collected from four natural ecosystems: forest, organic, pineapple field, and beach sites. Visual nematode identification on these four systems was being conducted to compare results. Two qPCR standard curves (plasmid DNA and genomic DNA) were used to obtain nematode abundance of the four ecosystems. Since both DNA standard curves did not estimate nematode abundance comparable to the visual count, ranking of nematode community indices of the four ecosystems were compared between molecular and the visual methods. While the ranking calculated by the plasmid DNA standard curve of qPCR assay were not consistent with most of the nematode community indices calculated by visual method, 4 out of 8 nematode indices estimated by the gDNA standard curve were relatively consistent. This research provided universal nematode guild qPCR primer sets and initial protocol of qPCR-based molecular tool for soil nematode community analysis. Further research need to be conducted on better estimation of nematode abundance, richness and diversity. More universal primers selective for Ba1, Ba3, F3, P3, also Om4, Om5, and P5 individual primers are needed.|
|Description:||Ph.D. University of Hawaii at Manoa 2012.|
Includes bibliographical references.
|Appears in Collections:||Ph.D. - Tropical Plant Pathology|
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