Application of PCR-DGGE into community structure analysis of soil nematodes
2006
Sato, E.(Tokyo Univ. of Agriculture and Technology, Fuchu (Japan)) | Toyota, K.
To develop nematode community structure analysis using Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE), nematode extraction methods were examined. Nematodes were extracted from four soils using the Baermann method and the double layer centrifugation technique (DLCT). In the latter method, optimum time of sonication was tested as a means of soil dispersion. One minute sonication yielded the highest number of nematodes. In all the soils, significant numbers of nematodes were extracted using DLCT from the soils that had been extracted using the Baermann method, suggesting that a combination of the two methods increased the extraction efficiency of soil nematodes. DNA was extracted from 50-300 nematodes and PCR-amplified targeted for their universal 18S rDNA sequences. The banding patterns were quite similar among the range of nematode numbers, suggesting that the number of nematodes used for DNA extraction was not critical for DGGE analysis. Next, PCR-DGGE analysis was done using 200 nematodes extracted with the Baermann method followed by DLCT and the results showed that nematode community structure was markedly different between the two methods, suggesting that a combination of the two methods may present a more diverse community of soil nematodes. The present study demonstrated that a combination of the Baermann method and double layer centrifugation technique may be a more effective method to obtain a larger number of nematodes and more diverse nematodes as a means to represent the whole soil nematode community.
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