Basic HTML Version
Genomics and Applied Biology, 2010, Vol.1 No.4
http://gab.sophiapublisher.com
- 32 -
RT-PCR reaction requires 2~3 h including time for
identification of the PCR products by gel electro-
phoresis. Moreover, the one-step multiplex RT-PCR
takes RNA as templates, which not only saved the
time of cDNA synthesis required in the conventional
sRT-PCR, but also decreased the possibility that losing
the yield of RNA during the cDNA synthesis process.
This is because that in multiplex RT-PCR, the cDNA
synthesis and amplification are achieved at the same
time in a single tube. The merits of this method
suggested that it is suitable molecular method for the
rapid, accurate and reliable detection of PRRSV in
clinical samples. In particular, when distinguishing of
swine viruses (CSFV, SVDV and VESV) is required.
Considering the prevalence and economic impact of
PRRSV, a simple, cost effective, sensitive and rapid
diagnostic technique is urgently required. Although
the multiplex RT-PCR method described in this study
showed some advantages over the conventional
sRT-PCR, further evaluation of this assay on a larger
scale is strongly recommended.
3 Materials and Methods
3.1 Reference strains and samples
CH
-
1a (GenBank access number: AY032626) live
vaccine virus and other reference strains (Table 4) used
in this study was obtained from the virology
department of Lanzhou Veterinary Research Institute,
Table 4 10 reference strains used in this study
Genotype
Strain
GenBank accession No.
CH-1a
AY032626
JX-3
EU200961
HUB1
EF075945
HuN
EF517962
GD
EU109503
HN2007
EU880437
GD2007
EU880433
JX0612
EF488048
North America
JX143
EU708726
European
Lelystad
M96262
Gansu of P.R. China. Six true-negative samples were
collected from healthy animals. Twenty-five clinical
samples from 25 pigs, including lungs, livers, serums
and other organs, were obtained from the pigs that
were suspected with the infection of PRRSV and
stored at −70
℃
until total RNA extraction. Ten blood
samples from three experimental PRRSV-infected
swine and one healthy control pig were also included
in the study.
The samples were processed as described elsewhere
(Scortti et al., 2006), and used to inoculate monolayers
of Marc
-
145 cells in duplicate. These cells were
incubated for 90 min at 37
℃
to facilitate adsorption.
They were washed twice with DMEM (Dulbecco's
Modified Eagle Medium) and we then added fresh
DMEM supplemented with 10% fetal bovine serum
(FBS) ( HyClone, USA) and incubated the cells for 6
days at 37
℃
, in a humidified atmosphere containing
5% CO
2
. With Cells were examined for cytopathic
effect (CPE) on days 4~6. HuN strain cell virus were
be used as positive control. Virus-free DMEM or FBS
were used as negative controls. If CPE was observed,
RT-PCR was carried out to confirm the presence of
PRRSV.
3.3 Extraction of RNA
The viral RNA of all clinical samples were extracted
using QIA amp Viral RNeasy Mini Kit (Qiagen,
Germany), following manufacturer’s instructions. In
brief, after lysis of the specimens, the mixture was
applied to a spin column as described by the
manufacturer’s protocol. The extracted RNA was
eluted in a total volume of 60 μL of elution buffer and
was stored at −70
℃
until used.
3.4 Primers design
Based on the conserved nucleotide sequence of the
PRRSV, three pairs of PRRSV specific primers were
designed using DNAStar software (DNAstar 7.0
green.rar) and the sequence of the primers, their
position on the PRRSV genome and the expected
product size are shown in Table 5.
3.5 RT-PCR reaction
Conventional sRT-PCR was performed using a
commercially available two-step assay purchased
from Guangdong Biological Technology Co. Ltd
(China) following the manufacturer's instructions. The
one-step multiplex RT-PCR