Page 5 - Genomics and Applied Biology

Genomics and Applied Biology, 2010, Vol.1 No.4
http://gab.sophiapublisher.com
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samples. A conventional single RT-PCR (sRT-PCR)
assay for PRRSV is commercially available in China
and this technique has provided a simple and rapid
technique to detect clinically suspicious samples.
However, this method is unable to detect virus in
serum and blood in which the amount of PRRSV is
very low. To obtain a better detection method for
PRRSV, in this study three sets of primers were
designed and used in a multiplex RT-PCR format to
detect in a number of tissues and organs, including
serum, blood, heart, brain, lung and kidney. Meanwhile, as
a RNA virus, one-step multi-RT-PCR was developed will
detect suspected samples, which has certain
instructive significance to preventive treatment of
PRRSV in production practice extensively.
1 Results and Analysis
1.1 The sensitivity of CH-1a RNA detection by the
multiplex RT-PCR and the conventional RT-PCR
The sensitivity of multiplex RT-PCR reaction was
compared with RT-PCR reaction for detecting
PRRSV using 10-fold serial dilutions Marc-145
cell-adapted cultures infected by CH-1a live virus. All
amplified DNA products were electrophoresed on
1.5% agarose gel stained with ethidium bromide.
sRT-PCR reaction had a detection limit of 0.1
TCID50 while multiplex RT-PCR reaction had a
detection limit of 0.01 TCID50 (Figure 1). Therefore,
the sensitivity of multiplex RT-PCR reaction for
detecting PRRSV is about 10 times higher than that of
sRT-PCR reaction. As shown in Figure 1, the
multiplex RT-PCR could detect the presence of the
virus up to the 10−7 dilution whereas the conven-
tional sRT-PCR was only able to detect up to the 10−6
dilution. Therefore, the detection limit or sensitivity of
the multiplex RT-PCR was at least 10 fold higher than
that of the conventional sRT-PCR.
1.2 The specificity test for the multiplex RT-PCR
The multiplex RT-PCR was facilitated to amplify the
reference virus strain of one SVDV sample (HK/70),
two CSFV samples (C-strain and Shimen virus) and
two VESV isolates (serotypes B51 and H54). None of
the CSFV, SVDV and VESV isolates was positive by
the multiplex RT-PCR (Figure 2). This results showed
Figure 1 Sensitivity of CH- 1a RNA detection by the multiplex
RT-PCR and the conventional RT-PCR
Note: M: DL2000 DNA Molecular Marker; 1~5: The multiplex
RT-PCR products: 490 bp, 345 bp and 228 bp; Reaction carried
out using several concentrations of CH-1a virus solution 10-3,
10-4, 10-5, 10-6, 10-7, respectively; 6~10: The conventional
RT-PCR products 660 bp: Reaction carried out using several
concentrations of CH -1a virus solution 10-4, 10-5, 10-6, 10-7,
10-8, respectively; All the products were electrophoresed on a
1.5% agarose gels
Figure 2 The result of the specificity test for the multiplex RT-PCR
Note: M1: CH- 1a; 1: CSFV C strain; 2: CSFV Shimen strain; 3:
SVDV HK/70 strain; 4: VESV serotypes B51; 5: VESV serotypes
H54; 6: A water control; M2: DL2000 DNA Molecular Marker
that the multiplex RT-PCR is specific for PRRSV.
Three related viruses (CSFV, SVDV, VESV) were
assayed, and all of them were negative.
1.3 The accuracy evaluation of the multiplex RT-
PCR assay
The accuracy of the multiplex RT-PCR assay was
evaluated by detection of 25 field clinical samples
(collected from Guangxi, Gansu and Ningxia province
of China), 10 reference strains and 6 true-negative
samples. The coincidence of the multiplex RT-PCR
and the conventional sRT-PCR was 100% for 10