Detection of PCV2 Nucleic Acids
There are several PCR assays for the detection of PCV2-specific nucleic acids described in the literature (Choi et al., 2000; Grierson et al., 2004; Hamel et al., 2000; Morozov et al., 1998; Shibata et al., 2003). Variants of the regular PCR assay include the following:
|Multiplex PCR for the detection of
PCV1 and PCV2
Multiplex PCR. More than one target sequence is detected in a single PCR step. The following multiplex PCR assays have been described in the literature: PCV2/PCV1 (Pogranichnyy et al. 2000; Ouardani et al., 1999), PCV2/PPV (Kim et al., 2001a), and PCV2/pseudorabies virus/PPV (Huang et al., 2004).
Nested PCR. To increase the ability to detect very small amounts of the target sequence the PCR assay is done in two steps. The first step generates a product that serves as template for the second step (Kim and Chae, 2001; Kim and Chae, 2004, Larochelle et al., 2000).
Multiplex-nested PCR. This assay combines multiplex and nested PCR and is designed to detect very small amounts of several target sequences. Assays have been described for the simultaneous detection of PCV1/PCV2/PPV (Kim et al., 2003; Kim and Chae, 2003) and PCV1/PCV2 (Kim et al., 2001b).
Quantitative real time PCR. Quantitative real-time PCR-assays have been developed and these assays allow for determination of the amount of PCV2 genomic copy numbers in the serum or tissues. PCR reaction and detection are combined in one step which decreases the turn-around time (Brunborg et al., 2004; Chung et al., 2005, Ladekjær-Mikkelsen, 2002; Liu et al., 2000; Olvera et al., 2004; Opriessnig et al., 2003; Rovira et al., 2002)
Reverse transcriptase (RT-) PCR. This is used to detect PCV2 RNA which is only present if PCV2 replicates (Yu et al., 2005). Reverse transcription of RNA is required to make a complementary DNA for further amplification.
Most pigs in the field (healthy or diseased pigs) are infected with PCV2 at some point in their lives. Therefore the use of PCR, which theoretically can detect one genomic copy, is considered by many to be too sensitive for most applications. One notable exception may be the detection of PCV2-nucleic acids in semen. PCR assays to detect PCV2 in semen have been described (Larochelle et al., 2000; Kim et al., 2003; Kim et al., 2001b); however, the infection dose required to transmit PCV2 infection via semen is unknown.
The amount of PCV2 nucleic acids in serum and tissues has been demonstrated to be predictive of the clinical outcome which might be an application for quantitative real-time PCR on individual pigs (Brunborg et al., 2004; Olvera et al., 2004). Those pigs with 107 or greater PCV2 genomic copies per milliliter of serum likely have PCV2-associated lymphoid lesions and disease (Brunborg et al., 2004).
|Kidney: PCV2 -In situ hybrization (black staining is PCV2 antigen)|
ISH for PCV2 uses a labeled DNA probe that corresponds to a specific portion of the PCV2-genome (Kim and Chae, 2003; McNeilly et al., 1999; Rosell et al., 1999; Sirinarumitr et al., 2000). Hybridization is usually done over-night and followed by a color reaction. This method allows localization of PCV2 but the method in general is time consuming (over-night hybridization step) and the reagents are expensive. Several ISH assays that detect multiple viruses within the same tissue section have been described: PCV1/PCV2 (Kim and Chae, 2001; Nawagitgul et al., 2000) PCV2/PRRSV (Choi and Chae, 2001; Sirinarumitr et al., 2001), and PCV2/PPV (Choi and Chae, 2000; Kim and Chae, 2002).
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