Fig. 1
Step-by-step protocol for the molecular diagnosis of strongylid nematode infections from small ruminant fecal samples. Following coproscopy (1). the fecal suspension is washed and nematode parasite eggs are recovered (2). Genomic DNA is isolated from eggs (3). and regions within the second internal transcribed spacer of nuclear ribosomal DNA specifically amplified by real-time PCR (4). After completion of PCR and melting-curve analysis (5). an estimate of the contribution of individual nematode species or genera to the egg count is estimated
2 Materials
2.1 For Fecal Flotation and the Enumeration of Nematode Eggs
1.
Saturated sodium nitrate flotation solution: Fill a beaker with 1 l of hot water and add 400 g of sodium nitrate salt. Continuously stir the suspension until the salt is fully dissolved. Check specific gravity of 1.300 with a hydrometer (see Note 1).
2.
McMaster parasite egg counting chambers.
3.
Metal spatula.
4.
Metal sieve (aperture of 250 μm).
5.
60 ml plastic container.
6.
Pipette with sieve top.
7.
Weight (±0.05 g accuracy).
8.
50 ml tubes with conical bottom.
2.2 For the Isolation of Nucleic Acids from Nematode Eggs
1.
Standard pipettes and tips (1 ml, 200 μl and 20 μl).
2.
Vortex.
3.
RNase/DNase-free microcentrifuge tubes (1.5 ml).
4.
Refrigerator (4 °C).
5.
Centrifuge for 50 ml tubes; minimum of 2,000 × g at room temperature.
6.
Microfuge.
7.
Adequate DNA extraction kit (e.g., PowerSoil® DNA Isolation Kit, MO BIO Laboratories Inc., USA).
2.3 For the Amplification of Nucleic Acids by Real-Time PCR
1.
Oligonucleotide primers targeting the ITS-2 region of the most relevant nematode parasites (Table 1) and the conserved primers NC1 (5′-ACG TCT GGT TCA GGG TTG TT-3′) and NC2 (5′-TTA GTT TCT TTT CCT CCG CT-3′) [8, 11].
Table 1
Parasite-specific primers used in the real-time PCR assays
Primera | Sequence (5′–3′) | Start position in ITS-2 regionb | Amplicon length (bp)c | Melting tempera-ture of target sequence (°C) | Target species |
|---|---|---|---|---|---|
HAE | CAA ATG GCA TTT GTC TTT TAG | 41 | 265 | 78.81 | H. contortus |
TEL | TAT GCA ACA TGA CGT ACG ACG G | 98 | 218 | 78.15 | Te. circumcincta |
TRI | TCG AAT GGT CAT TGT CAAA | 40 | 267–268 | 77.87 | Trichostrongylus spp. |
CHO | GTG ATG ACC TCG TTG TCA CCG TG | 143 | 162 | 82.12 | C. ovina |
OEV | TGA AAT GAG ACA ACC GTA GTC G | 222 | 105 | 79.38 | O. venulosum |
2.
Real-time PCR thermal cycler and respective software (see Note 2).
3.
Commercial PCR mixture containing standard reaction buffer, heat-activated DNA polymerase, dNTPs, and 6 mM MgCl2 (see Note 3).
4.
SYTO®9 green fluorescent nucleic acid stain (Life Technologies, USA) (see Note 4).
3 Methods
3.1 Preparation of Fecal Samples and Enumeration of Nematode Eggs
1.
Collect feces from the rectum of sheep using a disposable plastic glove. Invert the glove to capture the sample, expel air, tie a knot in the glove and label with a permanent marker (see Note 5).
2.
Homogenize the entire fecal sample and transfer 4 g to a 60 ml container (see Note 6).
3.
Add 50 ml of sodium nitrate solution and homogenize (see Note 7).
4.
Pour suspension through a sieve and collect run-through in a clean container.
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