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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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(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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fig5: (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%).

Mentions: The sensitivity results showed that the detection limit of PCV2 RPA LFD assay was 102 copies per reaction of the recombinant plasmid pPCV2/RPA DNA (Figure 4(a)), and the amplicon in the PCV2 RPA LFD assay (146 bp) was also tested by subsequent visualization with 3% agarose gel electrophoresis (Figure 4(b)). In evaluating the specificity of PCV2 RPA LFD assay, no cross-reactions were observed with other important viruses of pigs as described above (Figure 5(a)), which were also confirmed by the agarose gel electrophoresis (Figure 5(b)).


Development of Isothermal Recombinase Polymerase Amplification Assay for Rapid Detection of Porcine Circovirus Type 2
(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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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5382309&req=5

fig5: (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%).
Mentions: The sensitivity results showed that the detection limit of PCV2 RPA LFD assay was 102 copies per reaction of the recombinant plasmid pPCV2/RPA DNA (Figure 4(a)), and the amplicon in the PCV2 RPA LFD assay (146 bp) was also tested by subsequent visualization with 3% agarose gel electrophoresis (Figure 4(b)). In evaluating the specificity of PCV2 RPA LFD assay, no cross-reactions were observed with other important viruses of pigs as described above (Figure 5(a)), which were also confirmed by the agarose gel electrophoresis (Figure 5(b)).

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