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Genomics and Applied Biology, 2010, Vol.1 No.4
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Notably, all sample detected positive by the conven-
tional RT-PCR were positive by the multiplex
RT-PCR. Meanwhile, sequence analysis and virus
isolation were verified this results. This indicated that
the sensi- tivity of the multiplex RT-PCR for PRRSV
was higher than the conven- tional sRT-PCR.
1.4 The feasibility analysis of the multiplex RT-PCR
during the course of PRRSV infection
In order to assess the performance of the multiplex
RT-PCR during the course of PRRSV infection, three
pigs were experimentally infected with PRRSV HuN
with 1.5 mil the titers of 10
5.8
TCID50 by dropped
nose (denoted No.1, No.2 and No.3). Blood samples
were collected over a 20 day period and tested using
the multiplex RT-PCR and the conventional RT-PCR.
One uninfected control pig was also included as a
negative control. All of the samples were positive
from the sixth to the fourteenth day after challenge by
both PCR methods. All of the samples were positive
from sixth to fourteenth day after challenge by both
PCR methods. However, one sample on the fourth day
and two samples on the sixteenth day were only
detected positive by the multiplex RT-PCR, indicating
the ability of the multiplex RT-PCR for detection of
low concentration virus. One negative control from,
tissue of a healthy swine was tested negatively during
the test process (Table 3).
Table 3
Comprison between the multiplex RT-PCR (M) and the conventional RT-PCR (C) for detection of PRRSV blood samples
from swine artificial infected
Days post infection
Item
Sample No.
2
th
4
th
6
th
8
th
10
th
12
th
14
th
16
th
18
th
20
th
No.1
-
+
+
+
+
+
+
-
-
-
No.2
-
-
+
+
+
+
+
+
-
-
No.3
-
-
+
+
+
+
+
+
-
-
M
Control
-
-
-
-
-
-
-
-
-
-
No.1
-
-
-
+
+
+
+
-
-
-
No.2
-
-
-
+
+
+
+
-
-
-
No.3
-
-
+
+
+
+
+
-
-
-
C
Control
-
-
-
-
-
-
-
-
-
-
Note: +: Positive;
-
: Negative
2 Discussion
Multiplex PCR is gaining popularity because of its
experimental simplicity and
greater
effectiveness as
well as decreased effort and shorter time required
(Chamblain et al., 1998). Meanwhile, this method
were be used to detect FMDV were confirmed 100
times more than sRT-PCR (Bao et al.,2008). However,
the care still needs to be taken to overcome the often
problems such as a number of primers used in a same
reaction tube might interact with each other, leading to
the block of the reaction (Elnifro et al., 2000;
Henegariu et al., 1997; Markoulatos et al., 2002) and
close size of PCR products or cross reactions, leading
to complex for distinguish the origin of the PCR
products. Therefore, much care has been taken to
select and design the multiplex RT-PCR primers in
this study so that the obtained PCR products could be
very specific and easily identified by separating the
products onto agarose gel by electrophoresis.
We showed that the multiplex RT-PCR method was
approximately 10 times more sensitive than the
standard conventional sRT-PCR. This makes the
multiplex RT-PCR a better choice over the
conventional sRT-PCR for the diagnosis of PRRSV in
case examination, especially when the lower concen-
tration of virus. Therefore, the method has a potential
advantage for detection of the virus from serum, blood,
spleen, asymptomatic carriers and co-infection cases
with other virus. Because of using of one-step
RT-PCR method in this multiplex RT-PCR, more time
was saved in this assay. A standard two-step PCR
takes at least 3~4 h whereas the one-step multiplex