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Received 14 April 2004; received in revised form 23 September 2004; accepted 29 September 2004
Abstract
We describe a rapid, sensitive and reproducible real-time PCR assay for detecting and quantifying canine parvovirus type 2
(CPV-2) DNA in the feces of dogs with diarrhea. An exogenous internal control was added to control the assay performance from
extraction to amplification. The method was demonstrated to be highly specific and sensitive, allowing a precise CPV-2 DNA
quantitation over a range of eight orders of magnitude (from 102 to 109 copies of standard DNA). The reproducibility of the CPV2 real-time PCR assay was assessed by calculating the coefficients of variation (CV) intra-assay and inter-assay for samples
containing amounts of CPV-2 DNA spanning the whole range of the real-time PCR standard curve. Then, fecal specimens from
diarrheic dogs were analyzed by hemagglutination (HA), conventional PCR and real-time amplification. Comparison between
these different techniques revealed that real-time PCR is more sensitive than HA and conventional gel-based PCR, allowing to
detect low viral titers of CPV-2 in infected dogs.
# 2004 Elsevier B.V. All rights reserved.
Keywords: Dog; Parvovirus; Real-time PCR; Diagnosis
1. Introduction
Canine parvovirus type 2 (CPV-2) is a pathogen of
dogs which causes acute gastroenteritis and lymphopenia in young pups. CPV-2 belongs to the feline
* Corresponding author. Tel.: +39 080 467 9833;
fax: +39 080 467 9843.
E-mail address: n.decaro@veterinaria.uniba.it (N. Decaro).
0378-1135/$ see front matter # 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2004.09.018
20
21
CPV-2 by HA and conventional PCR (and subsequently also by real-time PCR). Aliquots of each
dilution were frozen at 70 8C and used only once.
2.4. Real-time PCR
Real-time PCR was performed in an i-Cycler iQTM
Real-Time Detection System (Bio-Rad Laboratories
Srl) and the data were analyzed with the appropriate
sequence detector software (version 3.0). Duplicates
of the CPV standard dilutions and DNA templates
were simultaneously subjected to real-time analysis.
The 25-ml PCR mixture for one reaction contained
12.5 ml of IQTM Supermix (Bio-Rad Laboratories Srl),
600 nM of primer CPV-For (50 -AAACAGGAATTAACTATACTAATATATTTA-30 ) and CPV-Rev (50 AAATTTGACCATTTGGATAAACT-30 ), 200 nM of
probe CPV-Pb (50 -TGGTCCTTTAACTGCATTAAATAATGTACC-30 ) and 10 ml of DNA. The thermal
cycle protocol used was the following: activation of
iTaq DNA polymerase at 95 8C for 10 min and 40
cycles consisting of denaturation at 95 8C for 15 s,
primer annealing at 52 8C for 30 s and extension at
60 8C for 1 min.
2.5. Internal control
In order to verify the complete removal of the PCR
inhibitors after the easy and fast method of DNA
preparation, so that a precise quantitation of CPV-2
DNA could be ensured for all the fecal samples, an
internal control (IC) was added to each sample.
Recently, it has been proposed to introduce the seal
virus phocid herpesvirus type 1 (PhHV-1) as universal
internal viral control for real-time PCR (Niesters,
2004). Analogously, we used a DNA virus, ovine
herpesvirus type 2 (OvHV-2), the etiologic agent of
malignant catarrhal fever (MCF) of cattle, for which
real-time PCR is routinely carried out in our
laboratory. A sample of spleen, obtained from a
cattle affected by MCF (Decaro et al., 2004b), was
found to contain 2.26 107 OvHV-2 DNA copies/
mg, using the protocol established by Hu ssy et al.
(2001). A fixed amount of this virus was added to
each sample prior to DNA preparation, in order to
obtain an OvHV-2 DNA concentration of approximately 5000 DNA copies/ml of fecal suspension (able
to give a mean CT value in the real-time PCR assay of
22
3. Results
3.1. Internal control performance
All the examined samples were positive for the IC,
giving CT values below the threshold value of 34.25.
Therefore, PCR inhibition did not occur in any sample
during the real-time PCR assay, enabling a precise
23
S.D.
CV (%)
(a)
1
2
3
4
5
6
7
8
3.62 102
6.57 103
1.43 104
4.65 105
4.52 106
2.11 107
3.03 108
6.78 109
1.72 102
1.85 103
4.23 103
1.34 105
7.89 105
8.19 106
6.26 107
2.51 109
47.5
28.1
29.6
28.8
17.4
38.8
20.7
37.0
(b)
1
2
3
4
5
6
7
8
1.73 102
3.81 103
2.77 104
2.11 105
2.23 106
1.69 107
3.91 108
4.51 109
1.11 102
8.53 102
1.02 104
8.21 105
4.70 105
6.80 106
9.37 107
1.94 109
64.2
26.8
36.8
41.3
21.0
40.2
23.9
43.0
24
negative fecal samples instead of TE buffer, confirming that PCR inhibitors are reduced to ineffective
concentrations by means of the DNA preparation
procedure, consisting of boiling and diluting in
distilled water the fecal samples.
The standard curve, that was generated by the
standard dilutions, spanned eight orders of magnitude
and showed linearity over the entire quantitation range
(slope = 3.610), providing an accurate measurement
over a very large variety of starting target amounts.
The coefficient of linear regression (R2) was equal to
0.9995.
The CV between runs and within-run for samples
containing CPV-2 DNA amounts spanning the whole
range expected are reported in Table 1.
3.3. Endpoint titration by IF, HA and real-time PCR
As shown in Table 2, the virus titer was
105.50 TCID50/100 ml when determined by IF and
103.50 TCID50/100 ml (cryolysates of infected wells)
when determined by HA. Real-time analysis was able to
detect CPV-2 DNA in the cryolysates of all four
replicates at dilution 108 and even in the cryolysate
of one well of the dilution 109 (virus titer =
108.75 TCID50/100 ml).
3.4. Analysis of the fecal samples of dogs naturally
infected with CPV-2
By means of MAb characterization and restriction
analysis, 24 (48%) out of the 50 HA-positive samples,
Table 2
Viral titer by IF, HA and real-time PCR on cell cultures inoculated
with CPV-2
Dilutiona
1
10
102
103
104
105
106
107
108
109
1010
IF
+
+
+
+
+
HA
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Real-time
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Table 3
Analysis of HA-positive fecal samples from diarrheic dogs by
conventional and real-time PCR
Sample
Origin
Year
HA
titera
PCRb
Real-time
titera
CPV-2a
102/01 (C)
103/01 (B)
104/01 (A)
255/01
273/01 (1)
273/01 (2)
288/01
297/01 (A)
307/01 (A)
307/01 (B)
133/02 (B)
256/02 (1)
18/03 (G)
133/03 (7)
197/03
300/03
312/03
366/03
28/04
51/04
62/04
67/04 (B)
74/04
105/04
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Turkey
Apulia
Apulia
Apulia
Apulia
Hungary
Apulia
Apulia
Apulia
Apulia
Apulia
2001
2001
2001
2001
2001
2001
2001
2001
2001
2001
2002
2002
2003
2003
2003
2003
2003
2003
2004
2004
2004
2004
2004
2004
640
320
320
5120
320
2560
40
2560
80
2560
320
640
160
640
2560
40
2560
2560
1280
640
40
320
1280
320
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
9.82 1010
3.11 1010
1.85 1010
3.47 1010
2.29 1010
1.87 1011
2.06 108
7.43 1011
8.55 109
2.33 109
9.88 108
2.11 1010
1.12 109
5.02 109
2.25 1010
5.37 109
3.42 1010
8.19 1010
3.76 1010
4.89 108
3.24 109
1.77 109
2.24 1010
2.02 1010
CPV-2b
197/98
201/98
212/98
224/98
242/98 (B)
111/99
130/99 (A)
42/01
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
1998
1998
1998
1998
1998
1999
1999
2001
1280
320
80
2560
80
1280
320
2560
+
+
+
+
+
+
+
+
7.98 1010
6.87 109
9.62 108
3.59 1010
6.28 1010
9.13 108
8.11 109
9.68 1010
(B)
(11)
(12)
(28)
(38)
(19)
(29)
Glu-426 mutant
157/02 (2)
161/02
202/02 (1)
202/02 (2)
105/03
135/03
148/03
180/03
220/03
347/03
111/04 (2)
Origin
Year
HA
titera
PCRb
Real-time
titera
Apulia
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
2001
2003
2003
2003
2003
2003
2003
80
5120
2560
80
40
640
640
+
+
+
+
+
+
+
1.01 1010
9.44 109
4.19 1010
3.53 109
8.08 107
7.11 1010
1.31 1011
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Basilicata
Apulia
Apulia
2002
2002
2002
2002
2003
2003
2003
2003
2003
2003
2004
2560
80
40
40
10
40
80
320
20
640
40
+
+
+
+
+
+
+
+
+
+
+
1.61 1011
4.01 108
1.63 107
9.98 108
9.79 108
5.71 108
3.44 108
2.10 1010
9.52 107
1.55 1010
4.33 107
(+) positive.
a
HA and real-time titers are referred to 1 mg of feces. HA titers
are expressed as the reciprocal of the fecal dilutions; real-time titers
are expressed as number of copies.
b
Buonavoglia et al. (2001).
25
Table 4
Analysis of HA-negative fecal samples from diarrheic dogs by
conventional and real-time PCR
Sample
158/01 (A)
158/01 (B)
158/01 (1)
158/01 (4)
158/01 (5)
158/01 (6)
158/01 (7)
158/01 (8)
158/01 (10)
158/01 (12)
6/02 (A)
24/02
117/02 (7)
144/03 (B)
111/04 (1)
111/04 (3)
111/04 (4)
111/04 (5)
111/04 (7)
116/04 (1)
116/04 (3)
116/04 (4)
116/04 (5)
Origin
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Year
2001
2001
2001
2001
2001
2001
2001
2001
2001
2001
2002
2002
2002
2003
2004
2004
2004
2004
2004
2004
2004
2004
2004
HA
PCRa
+
+
+
w
+
+
w
+
+
+
+
+
w
w
w
w
Real-time titer
6.91 10
7.33 103
1.92 105
1.89 104
1.44 104
3.88 104
5.21 104
6.06 103
5.41 104
5.88 103
2.83 105
5.32 103
2.33 104
8.21 103
6.50 104
8.11 104
4.79 104
1.27 105
1.55 104
3.65 103
1.23 104
1.85 104
2.06 104
Table 5
Summary of the results of CPV-2 detection in the fecal samples from
diarrheic dogs by HA, conventional and real-time PCR
Tested samples
89
a
Positive samples
HA
PCRa
Real-time
50 (56.18%)
68 (76.40%)
73 (82.02%)
4. Discussion
Real-time quantitative PCR is based on continuous
optical monitoring of a fluorogenic PCR reaction
(Holland et al., 1991; Heid et al., 1996). In this study,
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