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

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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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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.
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fig2: 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.

Mentions: After running the PCV2 real-time RPA assay with different primer and probe combinations, PCV2 RPA F3/PCV2 RPA R1 combines with PCV2 RPA Pe which were used as they yielded the highest amplification efficiency (Table 1, Figure 1). The PCV2 real-time RPA assay was sufficiently sensitive for detecting 102 copies per reaction within 20 min at 37°C (Figures 2(a) and 2(b)). The limit of PCV2 real-time RPA assay detection in 95% was 102 copies per reaction of pPCV2/RPA (Figure 2(c)). In evaluating the specificity of PCV2 real-time RPA assay, no cross-reactions were observed with PCV1/JL, PRRSV/CH-1R, CSFV/c-strain, PRV/Fa, PPV/AV30, and FMDV/O/CHA and positive signal was only observed with PCV2/NX and PCV2/CQ strain (Table 2).


Development of Isothermal Recombinase Polymerase Amplification Assay for Rapid Detection of Porcine Circovirus Type 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.
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fig2: 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.
Mentions: After running the PCV2 real-time RPA assay with different primer and probe combinations, PCV2 RPA F3/PCV2 RPA R1 combines with PCV2 RPA Pe which were used as they yielded the highest amplification efficiency (Table 1, Figure 1). The PCV2 real-time RPA assay was sufficiently sensitive for detecting 102 copies per reaction within 20 min at 37°C (Figures 2(a) and 2(b)). The limit of PCV2 real-time RPA assay detection in 95% was 102 copies per reaction of pPCV2/RPA (Figure 2(c)). In evaluating the specificity of PCV2 real-time RPA assay, no cross-reactions were observed with PCV1/JL, PRRSV/CH-1R, CSFV/c-strain, PRV/Fa, PPV/AV30, and FMDV/O/CHA and positive signal was only observed with PCV2/NX and PCV2/CQ strain (Table 2).

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus