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Genomics and Applied Biology, 2010, Vol.1 No.4
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Research Article Open Access
The Development of a Multiplex Reverse Transcription Polymerase Chain Reaction
for Detection of Porcine Reproductive and Respiratory Syndrome Virus
Jinyan Wu Hong Tian Yan Chen Youjun Shang Shuanghui Yin Na Zhao Ye Jin
Xiangtao Liu
Qingge Xie
Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiologic Biology, Lanzhou Veterinary Research Institute,
Chinese Academy of Agricultural Sciences, Gansu, 730046
Corresponding author, hnxiangtao@163.com;
Authors
Genomics and Applied Biology 2010, Vol 1 No 4 doi: 10.5376/gab.2010.01.0004
Received: 18 Nov., 2010
Accepted: 18 Nov., 2010
Published: 24 Dec., 2010
This is an Open Access article published under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:
Wu et al., 2010, The Development of a Multiplex Reverse Transcription Polymerase Chain Reaction for Detection of Porcine Reproductive and Respiratory
Syndrome Virus, Genomics and Applied Biology, 2010, Vol. 1, No. 4 (DOI: 10.5376/gab.2010.01.0004)
Abstract
A multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) was developed for the detection of
porcine reproductive and respiratory syndrome virus (PRRSV). A set of three pairs of primer were designed based on the sequence of
high conservative region of PRRSV. The diagnostic accuracy of the multiplex RT-PCR assay was evaluated using 25 field clinical
samples, 10 reference strains and 6 true-negative samples. Simultaneously, the specificity and sensitivity of this method were
evaluated. The result indicated that this assay could reliably differentiate between PRRSV and other swine viral disease, such as
classical swine fever virus (CSFV), swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV).
Meanwhile, this assay was shown to be 10-fold more sensitive than the conventional single RT-PCR (conventional sRT-PCR) method.
The results indicated that the multiplex RT-PCR developed in this study has high sensitivity, strong specificity and easy operation, it
is not only suitable for diagnosis of samples of insignifiance content, but also it has important applied value in getting rid of animals
with recessive virus and veterinary quarantine. The method may provide a new avenue to the rapid detection of this important
pathogen in one reaction.
Keywords
Porcine reproductive and respiratory syndrome virus (PRRSV); Multiplex RT-PCR; Conventional single RT-PCR
Background
Porcine reproductive and respiratory syndrome virus
(PRRSV) is a member of the family Arteriviridae
(Cavanagh, 1997). It is characterized by respiratory
disease in young pigs and severe reproductive failure
in sows, including abortion, stillbirths and weak
piglets (Hill, 1990). PRRSV has caused immense
economic losses in the pig industry and is considered
to be one of the most important infectious diseases of pigs
in the world (Polson et al., 1992; Li et al., 2007), and it
is comprised of two viral genotypes, the North American
type and the European type (NA-type and EU-type)
(Nelsen et al., 1999). An et al (2007) found that all of
the Chinese isolates examined belong to the North
American (NA) type and the viruses were
geographically restricted to regions in southeast China.
The virus was first confirmed in China in 1996, since
then, the virus has spread widely in China (Gao et al.,
2004; Chen et al., 2006). In June of 2006, outbreaks of
highly pathogenic (acute, atypical) PRRS in most
areas of China have been suggested to be caused by
highly virulent Chinese-type PRRSV (H-PRRSV)
strains. From January to July 2007, 39 455 morbid pigs
died among 143 221 infected pigs according to the
government numerical data. Rapid spread and their
persistence in some environments have made the
control of outbreaks difficult and, at times, even
impossible. Therefore, development of a rapid,
sensitive and specific diagnostic method is extremely
important to avoid or prevent the occurrence and
spread of this disease.
Due to the highly variability of PRRSV, three sets of
primers in different region of PRRSV genome were
designed would improve the detection sensitivity of
PRRSV. In the test, if one or more fragment was
amplified, but negative control didn't amplify any
fragment, which indicated that PRRSV existed in the