Comparison of nematode community similarities assessed by polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) and by morphological identification

Abstract

Molecular biology techniques for nematode community analysis that are high throughput and operable by non-experts are in high demand for soil biological assessments. In the development of such techniques, the closeness of the analytical results to those obtained by conventional methods is of key importance. In this context, we compared similarity relationships of nematode community structures between polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) and individual-based morphological identification (CONV) using 12 nematode samples recovered from crop field soils in four localities in Japan. First, we determined whether the primer set chosen to amplify the 18s rRNA genes of nematode taxa was suitable for DGGE by comparing community structures of four selected samples between clone libraries (CLs) and those revealed by CONV. We considered the primer set to be suitable because we found significant correlations in the structures between CLs and CONV in three of the four samples examined. Then we determined the community structure of 12 samples by both DGGE and CONV, and calculated distance matrices to examine if analytical results were similar between the two methods. The correlations in matrices were 0.400-0.603, depending on the types of distance measures, and were always significant between the two methods. In comparing dendrograms drawn based on the matrices, DGGE and CONV were actually similar to some extent in that samples from a single locality tended to group together, although some localities were split in DGGE. We also conducted DGGE with four additional selected samples to confirm that the band pattern (i.e., number, position and relative intensity) was consistent in each sample across subsamples and gels. Given these results, we expect DGGE to become a useful and efficient tool for nematode community analysis, especially for non-experts, but our method of evaluation has limitations and more tests are needed.

Quantitative detection of nematodes by using PCR-DGGE for evaluation of community similarities

“…Previously, we tried to evaluate the use of PCR-DGGE for nematode community analysis, by comparing the resulting band patterns of DNA fragments with the results of morphological analyses of the nematode samples isolated from the field. We found that the correlation in similarity relationships among communities, i.e., dissimilarity matrices, between the two methods was significant, but not high (r = 0.400 0.603, Okada and Oba, 2008). One of the reasons might be that a single morphological taxon, representing a family in our study, produced two or more DNA bands.…”

“…For each of these communities, we performed PCR-DGGE, as described previously (Okada and Oba, 2008;Takemoto et al, 2010). Briefly, DNA was extracted and purified from nematodes by using the Wizard SV Genomic DNA Purification System ® (Promega, Madison, WI, USA) according to the manufacturer's instructions.…”

“…An essential process in such trials is the performance evaluation of the tools. One of the approaches is to compare the community profiles obtained with those profiles revealed by morphological analyses based on individual identification (Griffiths et al, 2006;Okada and Oba, 2008;Wu et al, 2009). Previously, we tried to evaluate the use of PCR-DGGE for nematode community analysis, by comparing the resulting band patterns of DNA fragments with the results of morphological analyses of the nematode samples isolated from the field.…”

Suppressed-Priming PCR, a Novel Concept of DNA Quantification Based on PCR Kinetics

“…Acro was regarded as an internal standard against Tyl, the target. Nematode DNA samples were prepared following Okada and Oba, 6) except that 300 nematodes were used per sample. Fragments of 18S rDNA were amplified from the DNA with primers SSU9R-GC (5 0 -CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC GAG CTG GAA TTA CCG CGG CTG-3 0 ) and SSU18A (5 0 -AAA GAT TAA GCC ATG CAT G-3 0 ), originally designed by Blaxter et al 7) and modified with a GC clamp.…”

“…PCR-DGGE was performed following Okada and Oba, 6) except that a dilution series of primer sets was used to limit PCR amplification differently. A set of reaction mixtures contained 0.6 units of Prime Star Polymerase HS (Takara, Otsu, Japan), 1Â PCR buffer, 0.2 mM dNTPs, and 0:40 Â 10 À7 ng/ml the each of the template DNAs and each of the primers at 0.096, 0.144, 0.192, 0.240, and 0.288 mM.…”