Detection of porcine reproductive respiratory syndrome virus by the polymerase chain reaction

doi:10.1016/0166-0934(94)90024-8

Journal of Virological Methods

Volume 47, Issue 3, May 1994, Pages 273-278

https://doi.org/10.1016/0166-0934(94)90024-8 Get rights and content

Abstract

A polymerase chain reaction (PCR) is described for the detection of the porcine reproductive respiratory syndrome virus (PRRSV). Using the PCR we were able to detect about 30 infectious units of PRRSV resolved in tissue culture medium or sperm. For the detection of PRRSV in sperm the PCR is 10-times more sensitive than culturing in alveolar macrophages. For the detection of PRRSV, PCR provides a good alternative to cell culture methods.

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Rapid and sensitive detection of type II porcine reproductive and respiratory syndrome virus by reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip
2014, Journal of Virological Methods
Citation Excerpt :
In our study, the reaction tubes were incubated at 61 °C for 40 min and then directly analyzed by using the VF visualization strip cassette. Importantly, the RT-LAMP-VF assay exhibits high specificity and sensitivity within 50 min, which is much rapid than RT-PCR used for PRRSV detection (Van Woensel et al., 1994; Chittick et al., 2011; Wernike et al., 2012) that takes at least 2 h. To the best of our knowledge, this is the first study to describe the RT-LAMP assay in combination with a sealed lateral flow strip to avoid any contamination during the detection of type II PRRSV. Moreover, the requirement for only limited equipment is another advantage of this method, making it applicable for rapid veterinary diagnosis and for use in rural areas.
Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was combined with a vertical flow (VF) nucleic acid detection strip to develop a universal assay for the detection of type II porcine reproductive and respiratory syndrome virus (PRRSV). The loop primers were labeled separately with biotin and fluorescein isothiocyanate (FITC) in this assay. Using optimized parameters, the whole reaction could be completed in <50 min in a completely enclosed environment. The detection limit of this assay was found to be 1 pg RNA, 30 tissue culture infective dose 50 (TCID₅₀) virus, or 230 copies of recombinant plasmid DNA, which is relatively higher than that of RT-LAMP analyzed by agarose gel, RT-LAMP visualized by calcein, and the conventional RT-polymerase chain reaction (PCR). No false-positive results were obtained in the specificity assay. The efficiency of the RT-LAMP method was tested by analyzing 43 clinical samples, and the results were compared with those obtained by RT-PCR analysis, with the respective positive rates of 32.56% and 27.91%. This result confirmed that the method described is a rapid, accurate, and sensitive method for universal type II PRRSV detection. Also, this method can be used for the rapid detection of type II PRRSV during the early phase of an outbreak, especially for rapid veterinary diagnosis on the spot and in rural areas.
Multiplex PCR for the simultaneous detection of porcine reproductive and respiratory syndrome virus, classical swine fever virus, and porcine circovirus inpigs
2013, Molecular and Cellular Probes
Citation Excerpt :
The PCR is an alternative rapid virus detection method and several single PCR-based methods have been reported for a number of porcine pathogens. Individual PCR or reverse transcriptase (RT)-PCR assays have been developed for detection and identification of PRRSV, CSFV, and PCV-2 [14–16]. However, using conventional PCR technology to detect several viruses individually is labour intensive and expensive.
A multiplex polymerase chain reaction (PCR) was designed for the simultaneous detection of three viruses involved in reproductive and respiratory failure in pigs: porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV) and porcine circovirus type 2 (PCV-2). Each target produced a specific amplicon with a size of 718 bp (PRRSV), 288 bp (CSFV), or 466 bp (PCV-2). The sensitivity of the multiplex PCR using purified plasmid constructs containing the specific viral target fragments was 2.0 × 10⁴, 2.5 × 10³, and 6.0 × 10² copies for PRRSV, CSFV, and PCV-2, respectively. Non-specific reactions were not observed when other viruses, bacteria, and PK-15/Marc-145 cells were used to assess the multiplex PCR. Among 82 clinical samples from Fujian province, co-infection by PRRSV and CSFV was 12.19%, co-infection by PRRSV and PCV-2 was 21.95%, CSFV and PCV-2 was 13.41%, and co-infection by the three viruses was 3.66%. In conclusion, the multiplex PCR should be useful for routine molecular diagnosis and epidemiology. The multiplex PCR was effective in detecting various combinations of one or more of these viruses in pig specimens.
Simultaneous detection of porcine circovirus type 2, classical swine fever virus, porcine parvovirus and porcine reproductive and respiratory syndrome virus in pigs by multiplex polymerase chain reaction
2010, Veterinary Journal
A multiplex polymerase chain reaction (PCR) was designed for the simultaneous detection of four viruses involved in reproductive and respiratory failure in pigs: porcine circovirus type 2 (PCV-2), porcine parvovirus (PPV), classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV). Each of the four pairs of oligonucleotide primers exclusively amplified the targeted fragment of the specific viruses. The sensitivity of the multiplex PCR using purified plasmid constructs containing the specific viral target fragments was 2.58 × 10⁷, 2.64 × 10⁵, 2.66 × 10⁷ and 2.73 × 10⁵ copies for PRRSV, PCV-2, CSFV and PPV, respectively. Using the multiplex PCR, co-infections with these four viruses were identified in 26/76 (34.2%) piglets born from sows with reproductive failure in China. This method is a rapid, sensitive and cost-effective diagnostic tool for the routine surveillance of viral infections in pigs.
Rapid detection of porcine reproductive and respiratory syndrome virus by reverse transcription loop-mediated isothermal amplification assay
2009, Journal of Virological Methods
A rapid detection assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been developed for detecting porcine reproductive and respiratory syndrome virus (PRRSV). The RT-LAMP assay utilized a set of six primers to amplify the open reading frame 6 (ORF6) of the PRRSV. The amplified products were analyzed by agarose gel electrophoresis or visualized by colorimetric method. The results demonstrated that the RT-LAMP assay detected all 22 different PRRSV isolates, had no cross-reaction with four other swine viruses (i.e., PCV2, SIV, CSFV, and PEDV), and obtained a 91.3% sensitivity in 23 positive clinical samples in reference to the permissive cells-based virus isolation procedure. Therefore, the RT-LAMP assay provides a specific and sensitive means for detecting PRRSV in a simple, fast, and cost-effective manner. Furthermore, the RT-LAMP assay can be performed in less well-equipped laboratories as well as fields.
Reverse transcription loop-mediated isothermal amplification for the detection of highly pathogenic porcine reproductive and respiratory syndrome virus
2008, Journal of Virological Methods
A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the open reading frames 1a of highly pathogenic porcine reproductive and respiratory syndrome virus genome was developed. The 10 reference strains, 1 clinical isolation strain and 122 positive samples were tested. Positive reactions were confirmed for all strains and specimens by reverse transcription loop-mediated isothermal amplification and nested reverse transcription polymerase chain reaction (RT-PCR). The results showed this detection technique is more reliable and convenient for rapid and sensitive diagnosis of highly pathogenic porcine reproductive and respiratory syndrome virus infection.

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Detection of porcine reproductive respiratory syndrome virus by the polymerase chain reaction

Abstract

Virology

Virology

Characterization of swine infertility and respiratory syndrome virus (isolate ATCC VR-2332)

J. Vet Diagn. Invest.

Rapid and simple method for purification of nucleic acids

J. Clin. Microbiol.