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Comparative Study
. 2009 Feb;33(2):139-47.
doi: 10.1007/s11259-008-9080-8. Epub 2008 Jul 24.

A DNA miniarray system for simultaneous visual detection of porcine circovirus type 1 (PCV1) and 2 (PCV2) in pigs

D J An  1 , D S Song, J Y Park, B K Park
Affiliations
Comparative Study

A DNA miniarray system for simultaneous visual detection of porcine circovirus type 1 (PCV1) and 2 (PCV2) in pigs

D J An et al. Vet Res Commun. 2009 Feb.

Abstract

A miniarray system was developed for the simultaneous detection of porcine circovirus type 1 (PCV1) and type 2 (PCV2) in pigs. The system consists of a polymerase chain reaction (PCR) step to amplify target viral DNA, followed by detection of the amplified DNA using a membrane-anchored probe array and an avidin-alkaline phosphatase (Av-AP) indicator system. The lower limit of detection of PCV using the miniarray was 10(1.9) tissue culture infectious dose 50 (TCID(50))/ml and 10(2.08)TCID(50)/ml for PCV1 and PCV2, respectively, and 100 viral copies/microl for both PCV1 and PCV2. We validated the miniarray system using 141 lymph node specimens from pigs with suspected postweaning multisystemic wasting syndrome or porcine dermatitis and nephropathy syndrome. Of the 141 samples evaluated, 55 were identified as positive for PCV by the miniarray. Relative to in situ hybridization, the sensitivity and specificity of the miniarray was 100% and 98.9%, respectively. In contrast to other microarray systems, the miniarray does not require a DNA chip reader, since the results can be determined by visual inspection of colorized spots on a nylon membrane. This system represents an effective alternative method for the differential detection of PCV1 and PCV2 in pigs, as well as the maintenance of PCV-free cell lines and pre-screening of commercial vaccines for possible contamination.

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Figures

Fig. 1
Fig. 1
Alignment of the PCV ORF1 gene sequences that were used to design the miniarray probes. The open reading frame (ORF) 1 regions of 7 PCV1 and 10 PCV2 isolates from various countries were used to determine the sequence variation in the probe region. GenBank accession numbers and countries of origin are shown (CA, Canada; CN, China; DE, Germany; AT, Austria; HU, Hungary; JP, Japan; NL, Netherlands; TW, Taiwan; UK, United Kingdom; US, United States of America)
Fig. 2
Fig. 2
PCR using biotin-16-dUTP and hybridization of PCV1 and PCV2 target sequences. (A) Lane M, 1 kb DNA ladder; Lane 1, mock; Lane 2, PCV1; Lane 3, PCV2. (B) Miniarrays were subjected to hybridization with amplified PCV1 and PCV2 target sequences. Probes were arranged as follows: PCV1/2, upper corner; PCV1 left corner; PCV2, right corner
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