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. 2017:2017:8403642.
doi: 10.1155/2017/8403642. Epub 2017 Mar 23.

Development of Isothermal Recombinase Polymerase Amplification Assay for Rapid Detection of Porcine Circovirus Type 2

Affiliations

Development of Isothermal Recombinase Polymerase Amplification Assay for Rapid Detection of Porcine Circovirus Type 2

Yang Yang et al. Biomed Res Int. 2017.

Abstract

Porcine circovirus virus type II (PCV2) is the etiology of postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis, nephropathy syndrome (PDNS), and necrotizing pneumonia. Rapid diagnosis tool for detection of PCV2 plays an important role in the disease control and eradication program. Recombinase polymerase amplification (RPA) assays using a real-time fluorescent detection (PCV2 real-time RPA assay) and RPA combined with lateral flow dipstick (PCV2 RPA LFD assay) were developed targeting the PCV2 ORF2 gene. The results showed that the sensitivity of the PCV2 real-time RPA assay was 102 copies per reaction within 20 min at 37°C and the PCV2 RPA LFD assay had a detection limit of 102 copies per reaction in less than 20 min at 37°C. Both assays were highly specific for PCV2, with no cross-reactions with porcine circovirus virus type 1, foot-and-mouth disease virus, pseudorabies virus, porcine parvovirus, porcine reproductive and respiratory syndrome virus, and classical swine fever virus. Therefore, the RPA assays provide a novel alternative for simple, sensitive, and specific identification of PCV2.

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Figures

Figure 1
Figure 1
Optimal primers and probe combinations of PCV2 real-time RPA assay. The amplification results of nine different combinations of primers with the probe PCV2 RPA Pe are shown.
Figure 2
Figure 2
The sensitivity of PCV2 real-time RPA assay. (a) Fluorescence performance via real-time test using a dilution range of pPCV2/RPA. (b) Reproducibility of the PCV2 real-time RPA assay according to eight test runs. (c) Probit regression analysis was done on data from the eight runs of PCV2 real-time RPA assay.
Figure 3
Figure 3
Optimal detection conditions of PCV2 RPA LFD assay. (a)The assay works in a broad range of temperatures. (b) After 10 min of amplification, the test line is visible on the lateral flow dipstick.
Figure 4
Figure 4
Evaluation of the sensitivity of PCV2 RPA LFD assay. (a) In the lateral flow format (PCV2 RPA LFD) the sensitivity was 102 copies of the standard DNA. (b) Positive PCV2 RPA LFD reaction products (146 bp) could be detected on a stained agarose gel (3%).
Figure 5
Figure 5
(a) Specificity test results of PCV2 RPA LFD assay using total DNA extracted from PCV2 virus and other virus. (b) Positive PCV2 RPA LFD reaction products (146 bp) could be detected on a stained agarose gel (3%).

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