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International Journal of Molecular Veterinary Research
2012, Vol.2, No.1, 1
-
5
http://ijmvr.sophiapublisher.com
4
4.3 Real-time PCR
EDTA blood samples were taken for RNA extraction,
Prior to RNA extraction, Canine Enteric Coronavirus
(12.5µL CECov, 1×10
5
TCID50/mL) were added to
blood samples (100 µL) as an external RNA reference
(Young et al., 2006), the viral RNA was extracted by
using a commercial kit (QIAGEN Viral RNA
extraction kit) as described by the manufacturer.
The reverse transcription was performed in two steps.
For the first step, nine µl of RNA template was mixed
with 1µl Random Hexamers (Promega) and incubated
at 70
℃
for 5 min followed by cooling to 4
℃
using a
thermal cycler (AB). For reverse transcriptase (RT)
step, a total volume of 20 µL reaction mixture was
prepared consisting of 10 µL RNA/primer mixture
from the first step, 4 µL 5
×
RT buffer, 2.4 µL 25 mM
MgCl
2
, 1 µL dNTPs (Qiagen), 1.6 µL nuclease-free
water (Qiagen), 1 µL reverse transcriptase (Improm II,
Promega). The mixture was returned to the thermal
cycler and incubated at 20
℃
for 5 min, 42
℃
for 30 min
and 70
℃
for 15 min before being cooled to 4
℃
and
kept at -70 until required.
The primers used for the Real-Time PCR was from a
published literature (Baxi et al., 2006); 107 bp
Forward: 5' CTAGCCATGCCCTTAGTAG and Reverse:
5' CGTCGAACCAGTGACGACT (5UTR region) and
a 280 bp region of CECov nucleocapsid gene (young
et al., 2006) (sense: 5'
-
CTCGTGGYCGGAAGAGTAA
T
-
3'; antisense: 5'
-
GCAACCCAGAMRACTCCA
TC
-
3').
Before beginning real time experiments with these
sets of primers, primers efficiency test has been done
to insure that the primers will work appropriately and
to determine what concentration of cDNA to use in the
experiment, for this Canine Enteric Coronavirus used
as an internal control. The reactions set up using a
series of concentrations of cDNA in nuclease-free
water (Stock, 1\1, 1\2, 1\4, 1\8) and different quantity
of primers (5 pmol/µL, 10 pmol/µL, 15 pmol/µL,
20 pmol/µL, 25 pmol/µL), a standard curve run in
triplicate has been done. Primer efficiency was
confirmed for each experiment using the formula:
Primer efficiency=10
−1
/slope (Pfaffl, 2001).
To determine the sensitivity of the PCR for naturally
infected blood samples, blood from carrier animals
were diluted in negative blood samples. Melting curve
analysis of the PCR products was also carried out for
each experiment.
For the real-time PCR, a total volume of 20 µL
reaction mixture was prepared consisting of 10 µL (1
unit) Hotstar
Taq
Plus Master Mix (Qiagen), 0.75 µL
25 mM MgCl
2
(Qiagen), 1 µL BVDV F primer, 1 µL
BVDV R primer, 1 µL CECov F primer, 1 µL CECov
R primer, 0.5 µL SYBR Green (1 in 1000 dilution),
0.75 µL nuclease free water and 5 µL cDNA. The
mixture was placed in a thermal cycler and the
polymerase activated by incubation at 95
℃
for 5 min.
The mixture was then cycled at 95
℃
for 10 sec, 60
℃
for 15 sec for 45 cycles. In order to determine the
melting curve, the thermal cycler was programmed to
read the fluorescence from 60
℃
to 100
℃
in 1
℃
increments every 10 sec.
Acknowledgement
I would like thank to Damascus Molecular technology
laboratory in Syria and Istanbul Veterinary Faculty for their
supporting.
References
Alkan F., Ozkul A., Bilge-Dagalp S., Yesilbag K., Oguzoglu T.C., Akca Y.,
and Burgu I., 2000, Virological and serological studies on the role of
PI-3 virus, BRSV, BVDV and BHV-1 on respiratory infections of
cattle.I. The detection of etiological agents by direct immunofluorescence
technique, Dtsch. tierärztl. Wschr., Mai, 107(5): 193-195
Baxi M., McRae D., Baxi S., Greiser-Wilke I., Vilcek S., Amoako K., and
Deregt D., 2006, A one-step multiplex real-time RT-PCR for detection
and typing of bovine viral diarrhea viruses, Veterinary Microbiology,
116(1-3): 37-44 http://dx.doi.org/10.1016/j.vetmic.2006.03.026 PMid:16687219
Belak S., and Ballagi Pordany A., 1991, Bovine viral diarrhea virus
infection: rapid diagnosis by the polymerase chain reaction, Archives
of Virology, Supplementum, 3: 181-190 http://dx.doi.org/10.1007/978-3-7
091-9153-8_22
Bhudevi B., and Weinstock D., 2001, Fluorogenic RT-PCR assay (TaqMan)
for detection and classification of bovine viral diarrhea virus, Vet.
Microbiol, 83(1): 1-10 http://dx.doi.org/10.1016/S0378-1135(01)00390-X
Castelain S., Descamps V., Thibault V., Franc¸ois C., Bonte D., Morel V.,
Izopet J., Capron D., Zawadzki P., Duverlie G., 2004, TaqMan
amplification system with an internal positive control for HCV RNA
quantitation, Journal of Clinical Virology, 31(1): 227-234 http://dx.doi.org/
10.1016/j.jcv.2004.03.009 PMid:15465417
Cleland A., Nettleton P., Jarvis L.M., and Simmonds P., 1999, Use of bovine
viral diarrhoea virus as an internal control for amplification of hepatitis
C virus, Vox Sang., 76(3): 170-174 http://dx.doi.org/10.1046/j.1423-0410.
1999.7630170.x PMid:10341333