A Real-Time PCR Assay for the Diagnosis of Gastrointestinal Nematode Infections of Small Ruminants



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


aAccording to Bott et al. [8]

bUsing the sequences with the GenBank access numbers for H. contortus (accession no. X78803), Te. circumcincta (accession no. X86026), Trichostrongylus axei, Tr. colubriformis, Tr. vitrinus, Tr. rugatus (accession nos. X78065, X78063, X78064 and Y14818) Oe. venulosum (accession no. Y10790) and C. ovina (accession no. Y10789) as references

cWhen combined with the conserved NC2 reverse primer at the 5′ axei end of the large subunit of rDNA

 

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).

 

Mar 17, 2017 | Posted by in GENERAL | Comments Off on A Real-Time PCR Assay for the Diagnosis of Gastrointestinal Nematode Infections of Small Ruminants

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