Development of strain-specific real-time PCR and RT-PCR assays for quantitation of chicken anemia virus
Introduction
Chicken anemia virus (CAV) is a small, nonenveloped, icosahedral virus belonging to the Circoviridae family (Pringle, 1999) and is the causative agent of chicken infectious anemia. Due to the highly stable and resistant nature of the virus particles, CAV is present ubiquitously in poultry farms worldwide. Although there is only one defined serotype to date since CAV was first isolated in Japan (Yuasa et al., 1979), different strains have been isolated with minor sequence differences. The 2.3-kb genome is a single stranded, covalently closed, negative sense DNA coding for three partially overlapping open reading frames, ORF1, 2 and 3. One polycistronic mRNA is transcribed from the genome from which the three corresponding viral proteins, VP1, 2 and 3 are translated (Noteborn et al., 1992; Phenix et al., 1994).
The disease affects primarily young birds and is characterized by aplastic anemia accompanied by a general immunosuppression due to lymphoid atrophy. Recent studies suggest that subclinical infection in older birds can result in immunosuppressive effects such as impaired cytotoxic T lymphocyte development (Markowski-Grimsrud and Schat, manuscript in preparation), which may have important implications for cell-mediated immunity to a multitude of pathogens.
Important questions regarding the pathogenesis of CAV have been raised recently. In the spring of 1997 it was discovered that the departmental specific-pathogen-free (SPF) poultry flocks at Cornell University had become seropositive for CAV. A new virus strain, SH-1, was isolated from the flocks (Cardona et al., 2000a, Cardona et al., 2000b). To determine the location of the virus in these birds a nested PCR assay was developed for detection of viral DNA from various organ tissues. An unexpected finding was that viral DNA could be detected in gonadal tissues independently of the long-term presence of antibodies (Cardona et al., 2000a). It is not yet known if the viral genome is maintained as episomal DNA, virus particles, or perhaps as integrated insertions in cellular DNA in the gonadal tissues. The development of quantitative assays to determine the number of copies present in the gonadal tissues and to measure mRNA levels will be important to address these questions.
Although extremely sensitive, the nested PCR assay does not provide a quantitative measure of copy numbers of the DNA in the template. Techniques for semi-quantitative, competitive PCR for detection of CAV-specific DNA have been reported (Yamaguchi et al., 2000; Miles et al., 2001). These methods, however, are not as accurate as real-time PCR and are more time consuming since for each sample at least four PCR reactions must be run simultaneously, each containing different concentrations of a competitive target fragment. The fluorogenic probes employed in real-time PCR allow for more rapid and sensitive standardization and quantitation. In addition, endogenous controls can easily be included in a multiplex PCR reaction.
In this study, a comparison was undertaken between nested PCR and real-time quantitative PCR using TaqMan technology. In addition, specific primers were shown to discriminate between different strains of CAV. Finally, a TaqMan-based real-time RT-PCR method was developed and used to estimate relative CAV-specific transcript levels.
Section snippets
Chickens
All experimental chicken samples were obtained from the N2a line from the SPF departmental flocks at Cornell University, which had become exposed inadvertently to CAV (Cardona et al., 2000a, Cardona et al., 2000b). This inbred line was originally developed based on resistance to Marek's disease and has a defined MHC haplotype, B21B21 (Weinstock and Schat, 1987).
Virus strains, cell lines and virus propagation
The Cux-1 (Bülow et al., 1983) and CIA-1 (Lucio et al., 1990) strains of CAV were propagated in MDCC-CU147 (CU147), a Marek's disease
Sensitivity and accuracy of quantitative real-time PCR using a CAV-containing plasmid
The assay was first optimized using a plasmid containing the CAV Cux-1 strain genome, pCuxAB, and the Cux-1 specific primer/probe set (Table 2). Fig. 1a depicts the amplification plot of a 10-fold dilution series of pCuxAB representing 1–108 copy numbers. The standard curve generated from this plot (Fig. 1b) exemplifies the high sensitivity and accuracy of amplification over a large concentration range (R2>0.99). Similar standard curves were generated for each new assay run with highly
Discussion
The advent of TaqMan technology has introduced a new era for the quantitation of pathogens. In this paper it is demonstrated that CAV real-time PCR was at least as sensitive as a two-round nested PCR assay. The lack of detection of one of the nested PCR samples in the real-time PCR may be explained by the fact that 2 μg of genomic DNA was used in the nested PCR versus only 1 μg in the real-time PCR. In addition, the CAV real-time PCR method allowed for the determination of viral load with a
Acknowledgements
The authors would like to thank Ole M. Grimsrud for assistance with the figures and proofreading the manuscript. Carrie Markowski-Grimsrud is the recipient of a graduate research fellowship from the American Association of University Women. This manuscript is based upon work supported in part by the Cooperative State Research, Education, and Extension Service, US Department of Agriculture, under Agreements no. 96-38420-3061 and no. 98-35204-6425 and a gift from Charles River Laboratories.
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