Technical Report on the PCR-DGGE Analysis of Soil
Nematode Community

Ver. 2.3

Revised on June 14, 2010

National Institute for Agro-Environmental Sciences

Table of ContentsPage
1. Preparation of soil samples

1

2. Extraction of nematodes from soil and pretreatment ofnematode samples

1

3. DNA extraction

5

4. Polymerase chain reaction (PCR)

7

5. Denaturing gradient gel electrophoresis (DGGE)

8

6. Examples

9

7. Troubleshooting

10

8. References

11For inquiries regarding this manual, please contact:
Hiroaki Okada
Environmental Biofunction Division
National Institute for Agro-Environmental SciencesE-mail: hokada@affrc.go.jp
Tel.: +81-29-838-8307

1. Preparation of soil samples【Collection of soil samples from a local agricultural field】
In order to ensure that the samples represent the entire field that is to be investigated, collectsoil samples*1 to the depth of approximately 15 cm from multiple locations by using an auger,soil probe, etc. Mix each sample thoroughly in a plastic bag. Avoid exposing the samples to hightemperatures, and bring them to the lab under room temperature*2.
*1 Avoid sample collection immediately after a rainfall since it may be difficult to extract
nematodes from wet soil.
*2 To prevent nematodes from being damaged by heavy weight, do not pile the plastic bags
containing the soil samples. Place the bags in shade before they are brought indoors.
Refer to other textbooks such as “Experimental Technique in Nematology”*1) or“Methods of soil analysis”*4) for methods of collecting soil samples for nematodeanalysis.

【Sieving】
Pass the soil through a sieve with a mesh size of approximately 5 mm to remove gravel, plantroots, etc.*3. Chip any soil mass with hands, wearing gloves. Remove all soil masses that are too
hard to chip, together with gravel etc. After mixing the soil, transfer 300 g of each soil sample toa relatively large plastic bag (No. 15, width 30 cm × length 45 cm × thickness 0.03 mm) foranalysis of nematodes. Store the samples at 10°C before and after sieving, and perform
nematode extraction as described in the next section within 3 weeks of soil sample collection.
*3 Since nematodes are damaged by the pressure of heavy weight, do not place the sieve on
the sieved soil, but place it on the collection vessel during sieving.

2. Extraction of nematodes from soil and pretreatment of nematode samples

Extract nematodes using the Baermann tray method (see figure below). Compared to the
Baermann funnel method, the Baermann tray method uses an instrument that is simpler and has
higher collection efficiency.

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【Materials required】-Stainless steel strainer (kitchen use, diameter approximately 15 cm)-Tray (plastic pot tray for gardening, diameter approximately 15 cm)-JK Wiper® (Kimberly-Clark)-Round paper plate (diameter approximately 18 cm)-Instruments for collecting nematode suspension (glass beakers of approximately 100 ml and500 ml, 50-ml centrifuging tube, etc. These should be large enough to hold the suspension
and the water used to rinse the tray and funnel.)-Wash bottle-Distilled water-Room in which temperature can be maintained at approximately 25°C
-Refrigerators with temperatures set at approximately 5°C and 10°C

【Preparation】(1) Wear gloves before assembling the instrument. Cut double sheets of JK Wiper®. Ensure thatthe sheets have a diameter approximately 15 mm greater than the outer diameter of thestrainer, and place them inside the strainer.

(2) Set the room temperature to 25°C.

(3) Use soil samples collected within 3 weeks and stored at 10°C. Mix a soil sample in a plastic
bag immediately before use*4.
*4 Take care not to mix too vigorously since doing so will kill the nematodes.

(4) Put the strainer on the tray containing a small amount (approximately 200–300 ml) ofdistilled water, and flood the wiper with water. Add some more water and remove large airbubbles.

2Soil

JK WiperStrainerTrayFig. Example of Baermann tray: a) Strainer set with JK Wiper®, b) Strainer (bottom view), c)
Nematode extraction, d) Extracted nematodes (*), e) Pattern diagram.

【Procedure】(1) Sprinkle 80 g of mixed virgin soil over the wiper, and add distilled water to the tray by using
the wash bottle*5. Spread the soil over the tray using a spatula etc.*6 Add more water to thetray until the soil sample is completely submerged.
*5 Avoid pouring distilled water directly on the soil.
*6 Take care not to mix the soil more than necessary since doing so will make it difficult torelease the nematodes.

(2) Ensure that the tray and the strainer have nearly leveled off and the entire soil sample has
been submerged, and then fold the edge of the wiper toward the inside (to prevent waterfrom leaking out of the tray). Cover the strainer with a paper plate.

(3) Allow it to stand still for 48 h, and then collect the nematode samples according to thefollowing procedure:
1) Lift the strainer and immediately remove the content without waiting for the water todrain*7.
*7 Take care not to let the dripping water enter another tray.
2) Inject about 70% of the water in the tray into the collection vessel. Rinse the tray
contents by stirring with the remaining water, and pour it into the same collection
vessel.
3) Add new distilled water to the tray, rinse again, and then pour it into the collection
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vessel. Repeat the same operation, and then store the vessel at 5°C. This will be used asa sample.

(4) Let the collection vessel stand still until the nematodes settle out, and concentrate thenematode suspension by removing the clear part of the supernatant. Repeat this operation
until the suspension is concentrated enough to transfer into a 15–50 ml centrifuge tube.
Settling of nematodes is achieved by letting the centrifuge tube stand still for half a day orovernight (depending on the amount of suspension). The samples can be stored at 5°C, but
the following operation should be performed as soon as possible.

(5) Estimation of the nematode concentration. Dilute or concentrate the sample suspension asnecessary, add Tween 20 (final concentration 0.05%) to prevent nematodes from adhering toplastic products, stir the sample suspension*8, and then take a subsample*9 to count thenematodes using a stereomicroscope. Repeat this operation at least twice per sample, andestimate the average nematode concentration in the sample.
*8 Do not vigorously pipette the sample suspension or stir the suspension by shaking the tube,
because this will damage the nematodes. It is recommended that the tube be rotated gently
so that the sample suspension is whirled.
*9 If an auto-pipette is used to collect the sample suspension, use a pipette tip with a large
head diameter (approximately 1–2 mm) or one with the tip of the head cut off so thatlarge-sized nematodes can pass through it.

(6) Removal of protozoans, enchytraeid worms, etc. After Step (5), allow the nematodes in thesample to settle out by letting the sample stand still for a night (at 5°C) or using some othermethod. Then, return the sample to normal temperature to release the protozoans, andremove them by taking away the supernatant by suction. Transfer the sample (reduced toapproximately 5 ml) to a petri dish etc., and remove the enchytraeid worms and large debris
under a microscope.

(7) Collect the sample in a 10–15 ml centrifuge tube. To collect as many of the remaining
nematodes as possible, rinse the petri dish (used in Step 6) with approximately 5 ml ofdistilled water containing Tween 20, and transfer the rinse solution to the centrifuge tube.

4(8) Repeat the centrifugation (5,800 ×g, 5 min) and concentration steps until 5–20 ml of thesuspension is left. Transfer an aliquot of the suspension containing approximately 300
nematodes to a 1.5-ml tube using a pipette, and then centrifuge (5,800 ×g, 5 min). To
concentrate the nematode suspension, gently pipette the supernatant until approximately 40μl is left. If DNA extraction is not performed immediately, store the sample at -20°C orlower. If possible, store a part of the sample for morphological identification. Kill thenematodes by heating, add TAF solution (a mixture of 40 ml triethanolamine, 140 ml 40%
formalin, and 820 ml distilled water) to an equal amount of nematode suspension, and afterstabilization, store it at room temperature.

3. DNA extraction

Use crushing tubes with optimized crushing performance to extract DNA from nematodes.
Use a commercially available kit for DNA purification.

【Materials required】- Tubes for bead mill homogenization
Prepare samples in 2-ml microtubes (Model 72.693, Assist Co., Ltd., ) containing 0.1 gof glass beads (diameter 0.1 mm; Model GB-05, Tomy Seiko Co., Ltd., , sterilized)
and 4 g of zirconia-silica beads (diameter 1.2 mm; Model ZS12-0001, BioMedical
Science Co., Ltd., sterilized).
-1.5-ml microtubes (sterilized)-20% (w/v) skim milk
Even after separating the nematodes using the tray, small soil particles are left in thesample. In this case, add skim milk to enable DNA extraction. After dissolving theskim milk (for culture substrate) in ultrapure water, heat-treat the solution at 115°C for
5 min using an autoclave to prevent invasion of DNA. The product is colored slightly
brown, but it is still useful for DNA extraction. Pour approximately 2 ml of theproduct into screw-capped tubes, and store the tubes at -20°C.
-Wizard® SV Genomic DNA Purification System (Promega, model A2360)-Nuclei Lysis Solution (Promega, model A7941. Purchase this if the amount included with the5kit is not enough.)-Special grade 95% ethanol (Add to the wash solution included in the above kit.)-Cell breaker [FastPrep 100A (BIO101), Bead-Beater (Central Science Co.), etc.]【Extraction procedure】(1) Add 200 μl of Nuclei Lysis Solution (NLS, included in the Promega Wizard® SV Genomic
DNA Purification System) to the nematode suspension, containing approximately 300
nematodes, in a 1.5-ml tube, and transfer to a crushing tube using a micropipette. Then, rinse
the 1.5-ml tube with 200 μl of NLS, and pour the suspension into the crushing tube.
(2) Add 50 μl of 20% skim milk and 50 μl of 0.5 M EDTA (included in the kit). After stirring
well, freeze it at -80°C for 15 min or longer.

(3) Set the tube on FastPrep 100A, and shake at 6.5 m/s for 155 s (45 s × 3 times, 20 s × 1 time).

(4) Centrifuge at 13,000 ×g for 1 min to remove air bubbles.
(5) Add 500 μl of Wizard SV Lysis Buffer, included in the kit, and stir.

(6) Centrifuge at 13,000 ×g for 1 min.
(7) Set an SV membrane column on a new 2-ml tube (included in the kit), add 800 μl of thesupernatant of Step (6) to the column, and centrifuge at 13,000 ×g for 3 min.

(8) Discard the eluate in the 2-ml tube using a decanter or pipette.
(9) Add 650 μl of wash solution containing ethanol , and centrifuge at 13,000 ×g for 1 min.

(10) Repeat Steps (8) and (9) four times, ending at Step (8), and then centrifuge at 13,000 ×gfor 3 min to completely remove the liquid remaining in the membrane.

(11) Transfer the membrane column to a new 1.5-ml microtube.

6(12) Add 250 μl of Nuclease-Free Water (included in the kit), let it stand still for 1 min, andthen centrifuge at 13,000 ×g for 1 min.

(13) Add another 250-μl aliquot of Nuclease-Free Water, and centrifuge at 13,000 ×g for 3 min.

(14) Discard the membrane column, cap the 1.5-ml tube, and store at -20°C in a freezer.

4. PCR

Perform polymerase chain reaction (PCR) using the following primer set, which targets theanterior part of the 18S rRNA gene. This primer set*2), 3) can be used to amplify DNA from all
kinds of nematodes, but it also amplifies DNA from filamentous fungi, protozoans, and someenchytraeids. However, since nematodes are usually dominant in the samples obtained by theBaermann method, the probability of appearance of bands from other organisms is low whendenaturing gradient gel electrophoresis (DGGE) is performed.

Forward primer: SSU18A
5′-aaa gat taa gcc atg cat g-3′

Reverse primer: SSU9R/GC5′-cgc ccg ccg cgc ccc gcg ccc ggc ccg ccg ccc ccg ccc gag ctg gaa tta ccg cgg ctg-3′
The underlined segment indicates a GC clamp.

【PCR conditions】
Use Takara Bio Inc. “Prime® STAR HS” under the following conditions:- The composition of the reaction mixture (Total volume, 25 μl)
AmountFinal concentration

Sterile distilled water

5.5 μl

5× Buffer

5.0 μl

1×dNTPs (2.5 mM)

2.0 μl0.2 mM
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SSU18A (20 μM)

0.6 μl

0.5 μM

SSU9R/GC (20 μM)

0.6 μl

0.5 μMPrime Star HS

0.25 μl

2.5 U/100 μM

Template DNA

10.0 μl- Reaction cycle
98°C (3 min)  [98°C (10 s), 54°C (15 s), 72°C (40 s)] × 27  72°C (10 min)【Detection and purification of PCR product】
Perform agarose gel electrophoresis using a 5-μl aliquot of the PCR product to confirm that aproduct of the expected size (approximately 590 bp) has been obtained. Purify the remaining
product using a commercially available PCR product purification kit, and then measure theDNA concentration. If DGGE is not performed immediately, store the purified product at -20°Cor lower.

5. DGGE
Use the Bio-Rad DCodeTM system for DGGE. The denaturant gel composition (see Table)
and electrophoresis conditions should be appropriately changed for nematode analysis, but thefundamental procedure for casting the gel slabs, electrophoresis, and staining is the same asthat for microbial analysis. Refer to the “Technical Report on the PCR-DGGE Analysis of
Bacterial and Fungal Soil Communities” for details. The electrophoresis conditions fornematodes are as follows: gel concentration, 6%; denaturing concentration gradient, 20%–50%
at 60°C and 75 V for 16 h. A DGGE marker optimized for the analysis conditions specified inthis manual is available from Nippon Gene Co., Ltd. (DGGE Marker V, model 319-06941).
Table Composition of gel stock solutions for DGGE for nematode analysis*10Composition

20% denatured

50% denatured

40% acrylamide/bis 37.5:1

15 ml

15 ml

50× TAE

2 ml

2 ml

Formamide (deionized)

8 ml

20 ml

Urea

8.4 g

21 gup to 100 ml

up to 100 ml

Ultrapure water*10 100% denaturation concentration is equivalent to a combination of 7 M urea and 40% formamide.
86. Examples

Highly humifiedandosol

Yellow soil

Gray lowland
soilLight-coloredandosol【DGGE analysis ofnematode community】
The soil samples
were provided by Dr.
Kazunari Nagaoka of
the National
Agricultural ResearchCenter. The numbers
indicate the differencein fertilization
management. After
extracting nematodeDNA and purifying
the PCR product of
18S rRNA gene
following theinstructions in thismanual, an amountequivalent to 100 ng
of each sample(20-well comb wasused) waselectrophoresed. The

6% acrylamide gel electrophoresis at 60°C and 75 V for 16 hproduct was firststained with SYBR® Green I, andthen imaged using Pharos FX(Bio-Rad). M is a DGGE marker fornematodes.
9Generally, the DNA bands of the nematodes in the soil of a tilled field tend to appear at orlower than the position of the 4th band of the marker.

7. TroubleshootingThis section describes problems related exclusively to the PCR-DGGE analysis of nematodes.
For more general problems related to PCR and DGGE, refer to the “Technical Report on thePCR-DGGE Analysis of Bacterial and Fungal Soil Communities.”【Problems related to nematode extraction】- The number of nematodes is less.
 The extraction efficiency may decrease if the soil used was collected immediately after arainfall. Collect soil samples at another time. The extraction efficiency also decreases
when heavy clay soil, such as soil from a paddy field, is used. In this case, use more trays(i.e., more amount of soil) for each sample, or use the density gradient centrifugation
method*1), 4) etc.
- The nematode densities vary between the same samples.
 Put soil on the wiper inside the strainer in advance, and then submerge the strainer in the
tray of water. In this procedure, there is little disintegration of soil aggregate, thereby
making nematode release from soil smooth and stabilizing the extraction efficiency.
However, care must be taken since it is not possible to confirm whether there are any air
bubbles under the strainer.
【Problems related to DNA extraction】- DNA cannot be obtained.
 Nematodes may have been lost during the concentration process. Collect some part of thewaste solution and check whether a large number of nematodes are present by using astereomicroscope.
 The shaking intensity and duration of the process of crushing the nematodes by using acell breaker may not be optimal. Check the settings of the equipment, and observe theresidue after the sample was applied to the column by using a stereomicroscope toconfirm that the nematode bodies were crushed. If the crush is incomplete, extend thetreatment time or increase the shaking intensity. However, take care not to crush thenematode bodies too much since doing so will fragment DNA.
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【Problems related to DGGE】- Bands appear as a smear.
 The DNA of nematodes that belong to the genus Caenorhabditis and the genus
Diploscapter do not form bands but appear as a smear under the current experiment
system. To confirm the existence of these nematodes, it is necessary to perform specific
PCR etc.
- The movement of the marker bands is irregular.
 Since the second band from the top may show Irregular movement, care must be taken
when comparing different gels.

8. References1) Edited by the Japanese Nematological Society (2004) Experimental Technique in
Nematology, Japanese Nematological Society, p. 247, Tsukuba (in Japanese).
2) Blaxter M, Ley P, Gareys J, Liu L, Scheldeman P, Vierstraete A, Vanfleteren J, Mackey L,Dorris M, Frisse L, Vida J and Thomas K (1998) A molecular evolutionary framework forthe phylum Nematoda. Nature, 392, 71-75.
3) Okada H and Oba H (2008) Comparison of nematode community similarities assessed bypolymerase chain reaction–denaturing gradient gel electrophoresis (DGGE) and bymorphological identification. Nematology, 10, 689-700.
4)Ingham RE (1994) Nematodes. p. 459–490. In R.W. Weaver et al. (ed.) Methods of soilanalysis. Part 2. SSSA Book Series 5. SSSA, Madison, WI.

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