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In this study, we developed a real-time quantitative polymerase chain reaction (qPCR) assay based on a dual-labeled hydrolysis probe to simultaneously detect both duck circovirus (DuCV) 1 and DuCV-2. The reproducibility, sensitivity and specificity of the primer set and probe were evaluated using other duck pathogens. The detection limit was 20 copies per µL. The intra-assay coefficients of variation (CVs) were ≤ 0.73% and the inter-assay CVs were ≤ 1.89%. No cross-reaction occurred with other duck pathogens. In addition, the qPCR assay was successfully applied to the simultaneous detection of DuCV-1 and DuCV-2 in clinical field samples. Therefore, this assay will be useful for laboratory diagnosis and epidemiological field studies of DuCV., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Porcine circovirus 3 (PCV-3) is a newly emerging virus that poses a potential threat to the swine industry. We developed a sensitive assay utilizing droplet digital PCR (ddPCR) to detect PCV-3. Specificity of the assay was confirmed by the failure of amplification of DNA of other relevant viruses. The detection limit for ddPCR was 1 copy/μL, 10 times greater sensitivity than TaqMan real-time PCR (rtPCR). Both methods showed a high degree of linearity, although TaqMan rtPCR showed less sensitivity than ddPCR for clinical detection. Our findings indicate that ddPCR might offer faster and improved analytical sensitivity for PCV-3 detection.

Porcine viral diarrhea, mainly caused by porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine group A rotaviruses (RVA), porcine group C rotaviruses (RVC) and porcine circovirus 2 (PCV2), is a serious global problem, resulting in substantial economic losses to the swine industry. For fast and reliable diagnosis of the causative agent associated with viral diarrhea in pigs, an inexpensive and easy to perform gel-based multiplex PCR assay was developed in this study to detect and differentiate the different viruses by amplicon size. The assay was able to distinguish between all five viral agents without cross-reacting with other non-target pig viruses. The detection limits of the assay per reaction were 5 copies for PEDV, TGEV, RVC and PCV2 and 50 copies for RVA for the singleplex assays and 50 copies when all five viruses were multiplexed. Sixty-nine field samples were used to validate the developed multiplex assay. The overall prevalence of positive samples was 44.9% (31/69). PCV2 was detected in 37.7% of the samples, PEDV and RVC each in 4.3%, TGEV in 2.9%, and RVA was detected in 1.4% of the samples tested. A total of 5.8% of the samples were co-infected by two or more viruses, and the results of the multiplex assay were in agreement to those obtained by single PCR assays. These findings suggest that the developed cost-effective multiplex assay is specific, sensitive, and will serve as a valuable diagnostic tool for the rapid differential detection of these five viruses and for molecular epidemiological studies and diarrhea disease management., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The objective of this study was to develop a real-time recombinase polymerase amplification (rt-RPA) assay for the rapid detection of porcine circovirus 3 (PCV3). Specific RPA primers and exo probes were designed for the cap gene of PCV3 within the conserved region of viral genome. The amplification was performed at 38 °C for 20 min. The rt-RPA was specific for PCV3, as there was no cross-reaction with other pathogens tested. Using the recombinant plasmid pUC57-PCV3 as template, the analytical sensitivity was 23 copies. Of the 186 clinical samples, PCV3 DNA was identified in the 51 samples by the rt-RPA, and the positive rate was 27.4% (51/186). The diagnostic agreement between the rt-RPA and real-time PCR was 96.2%. The R 2 value of rt-RPA and real-time PCR was 0.919 by linear regression analysis. The developed rt-RPA assay shows promise for rapid and sensitive detection of PCV3 in diagnostic laboratories and at point-of-need, thus facilitating the prevention and control of PCV3., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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 10 2 copies per reaction within 20 min at 37°C and the PCV2 RPA LFD assay had a detection limit of 10 2 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.

Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility.

Porcine circovirus 2 (PCV2) is a common virus in pig population and is associated with the postweaning multisystemic wasting disease (PMWS). In this study, it was developed and evaluated the single-tube nested PCR (STNPCR) method for the detection of PCV2 DNA. PCV2 reference controls and swine tissue samples were used, and primers were selected for targeting specific regions of the viral genome. In comparison of the methods, STNPCR was 10 times more sensitive than conventional PCR and showed the same sensitivity to nested PCR (NPCR), but with reduction in the risk of cross-contamination. In clinical application, 55 tissue samples were analysed by conventional PCR and resulted in 67% (37/55) of positive reactions, while the NPCR and STNPCR were able to identify the presence of viral DNA in 100% (55/55) of the samples. The high sensitivity combined with the elimination of cross-contamination makes the STNPCR method suitable for the epidemiological studies of PCV2 and can aid in the diagnosis of PMWS., (© 2012 Blackwell Verlag GmbH.)

Superficial inguinal lymph nodes from 72 wild boars examined in a previous immunohistochemical (IHC) study on porcine circovirus type 2 (PCV2) were selected for a PCV2 polymerase chain reaction (PCR) analysis. Four of these lymph nodes were PCV2-IHC strongly positive with PMWS histological lesions (outcome 1), 6 weak to mild PCV2-IHC positive without PMWS histological lesions (outcome 2) and 62 PCV2-IHC negative. Considering IHC the gold standard for diagnosis, the aims of the study were to evaluate the suitability of the PCV2-DNA extraction from formalin-fixed and paraffin-embedded (FFPE) tissue and the sensitivity and specificity of PCR under two IHC interpretations criteria: (A) the sample was considered positive if the result was outcome 1; (B) the sample was considered positive if the result was outcome 1 or 2. Under (A) criteria, sensitivity and specificity of PCR were 100% and 89.7%, respectively; the Cohen's Kappa coefficient was 0.49. Under (B) criteria, sensitivity and specificity of PCR were 80.0% and 95.2%, respectively; the Cohen's Kappa coefficient was 0.72. The high Cohen's Kappa coefficient under the (B) interpretative criteria indicates good agreement between the two methods. In conclusion, 1) DNA extracted from FFPE specimens of wild boar is suitable for PCR and further represents a screening test for PCV2/PCVD (PCV2 Diseases) investigations in wild boar as well; 2) routine histological sampling can also be useful for PCV2 virological studies in wild boar.

Safety and quality are important issues for vaccines. Whereas reversion to virulence poses a safety risk with live attenuated vaccines, the potential for the presence of adventitious agents is also an issue of vaccine quality. The recent detection or porcine circovirus type 1 (PCV1) in human vaccines has further highlighted the importance of quality control in vaccine production. The purpose of this study was to use a novel conventional PCR to detect PCV1, and subsequently screen materials used in the manufacture of vaccines at Bharat Biotech International Limited, India. The genome or gene fragments of PCV1 were not detected in any of the vaccines and materials tested, including the live attenuated rotavirus vaccine candidate ROTAVAC(®). Further, the identity of the cells and the viruses used as starting materials in the manufacture of these vaccines was confirmed by species-specific PCR or virus-specific RT-PCR, and no cross-contamination was detected in any case. The methods can be applied for regular in-house quality control screening of raw materials and seeds/banks, as well as formulated vaccines., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Porcine circovirus 2 (PCV2) is believed to be a necessary but not sufficient underlying cause of porcine circovirus associated disease (PCVAD) in swine (Opriessnig et al., 2007). Since the potential threat of PCVAD is dependent on the prevalence of PCV2 in swine populations, accurate diagnostic tests are important for epidemiologic surveillance. Therefore, we evaluated the diagnostic sensitivity (Se) and specificity (Sp) of a new indirect ELISA and two quantitative PCR tests for PCV2 in a series of latent class models that used Bayesian estimation procedures. A total of 4140 samples from finisher pigs were tested for evidence of PCV2 by the ELISA and a TaqMan (TM) quantitative PCR, 995 by the ELISA and a SYBR Green (SG) dye-binding PCR, 998 by both PCRs and 993 by all three tests. Overall, the median (95% probability interval) ELISA Se and Sp was 0.85 (0.83-0.87) and 0.74 (0.68-0.79), respectively, when all three tests were analyzed together at an ELISA absorbance (optical density or OD) cutoff of ≥0.3. The TM PCR Se and Sp was 0.86 (0.84-0.88) and 0.94 (0.87-0.97), respectively, and the SG PCR Se and Sp was 0.83 (0.81-0.85) and 0.98 (0.94-1.00), respectively when all three tests were analyzed together at an ELISA OD cutoff of ≥0.3. Sensitivity analysis revealed that Sp estimates in general had less stability than Se estimates, but the SG PCR(Sp) was the most stable. Limited conditional dependence between the two PCR tests was detected. We conclude that the ELISA had the highest diagnostic Se at an absorbance cutoff of ≥0.3, while the SG PCR had the highest diagnostic Sp. The prevalence levels for exposure to PCV2 in finishing swine populations across all analyses ranged from 58 to 100%., (Published by Elsevier B.V.)

Classical postweaning multisystemic wasting syndrome (PMWS) diagnosis is based on postmortem findings (histopathology plus viral detection in lymphoid tissues). Because one of the major differences between PMWS-affected and nonaffected pigs is Porcine circovirus-2 (PCV-2) load in serum and tissues, real-time quantitative polymerase chain reaction (qPCR) has been suggested as a potential diagnostic technique for the disease. The objective of the present study was to assess the applicability of qPCR to quantify PCV-2 loads in pooled serum samples as an easy-to-use PMWS diagnostic tool at the herd level. The experimental design included two simulation studies with several serum pool sizes from pigs already screened for PMWS (by histopathology and detection of PCV-2 by qPCR). Several qPCR thresholds were defined and validated with experimental pools created in the laboratory. Quantitative PCR on pooled serum samples did not result in a sufficiently reliable alternate method to the classical PMWS diagnosis method based on individual clinical, histopathological, and PCV-2 detection criteria. However, serum pools seemed to be an alternative at a low economic cost for the quantification of PCV-2 loads in suspicious herds. A targeted (including only clinically diseased animals) sampling approach did not give better estimates compared with a random sampling approach.

Porcine circovirus type 2 (PCV2) and the associated disease postweaning multisystemic wasting syndrome (PMWS) have caused heavy losses in global agriculture in recent decades. Rapid detection of PCV2 is very important for the effective prophylaxis and treatment of PMWS. To establish a sensitive, specific assay for the detection and quantitation of PCV2, we designed and synthesized specific primers and a probe in the open reading frame 2. The assay had a wide dynamic range with excellent linearity and reliable reproducibility, and detected between 102 and 1010 copies of the genomic DNA per reaction. The coefficient of variation for Ct values varied from 0.59% to 1.05% in the same assay and from 1.9% to 4.2% in 10 different assays. The assay did not cross-react with porcine circovirus type 1, porcine reproductive and respiratory, porcine epidemic diarrhea, transmissible gastroenteritis of pigs and rotavirus. The limits of detection and quantitation were 10 and 100 copies, respectively. Using the established real-time PCR system, 39 of the 40 samples we tested were detected as positive.

Porcine circoviruses (PCV) are widespread in domestic pigs worldwide and there is growing information about the presence of PCV in other suid species. Based on serological studies with sera of wild boars, it was established that PCV1 was present in these animals and antibodies specific to PCV2 were also detected in wild boars living in captivity or in sylvatic areas, both with or without clinical signs of PMWS. Studies including PCV2 genome or antigen detection confirmed the previous findings. This is the first report about the presence of PCV in Transylvanian wild boar populations. Four hundred and sixty-nine samples were collected and grouped according to geographic origin, tested for the presence of PCV DNA using a real-time quantitative polymerase chain reaction assay, and 13.52% of the animals proved to be positive for one or in three cases both of the PCV genotypes. PCV2 was detected in all of the PCV-positive samples.

Light Upon eXtension real-time PCR (LUX real-time PCR) assay was developed for the detection of porcine circovirus type 2 (PCV2). The primers flanking a 114 bp fragment were selected from ORF1. The optimized assay could detect 20 viral copies of pBluescript SK+ plasmid containing inserted PCV2 DNA. The dynamic range of quantitative analysis covered a 7-order interval ranging from 20 to 2 x 10(8) genome equivalents per assay with the best results in the range from 2 x 10(2) to 2 x 10(7) viral copies. The LUX real-time PCR assay had a high specificity since it detected PCV2 but not PCV1, CSFV, PRRSV or negative samples. There was good agreement between the LUX real-time PCR and the conventional PCR when lymph nodes from PCV2 infected animals were tested. A comparison of the LUX real-time PCR with the TaqMan PCR and SYBR Green PCR indicated that the amount of viral copies determined using linear calibration curve differed from assay to assay but not more than an order. LUX real-time PCR, similar to the TaqMan PCR, was more specific for generation of fluorogenic signal than SYBR Green PCR., (Copyright 2010 Elsevier B.V. All rights reserved.)

Background/purpose: The postweaning multisystemic wasting syndrome, caused by the porcine circovirus type 2 (PCV-2), is a major disease that poses a significant threat to the global swine industry. The purpose of this study was to establish a real-time polymerase chain reaction (PCR) method for the quantification of PCV-2 and to enable the rapid differentiation of porcine circoviruses type 1 and 2 (PCV-1 and PCV-2). Such a method would significantly speed up the process of clinical diagnosis, and could also be used to study the pathogenic mechanisms of diseases associated with PCV-2., Methods: Multiplex real-time PCR, together with LightCycler PCR data analysis software, was used for the quantification of PCV-2, and for the rapid differentiation of PCV-1 and PCV-2. A 263-bp DNA fragment was amplified from the 3' end of the open reading frame-2 of PCV-2 by nested PCR, and its DNA sequence was verified as having 100% identity with a PCV-2 standard (NCBI accession number: AF055394). The 263-bp DNA fragment was cloned into the pGEM-T easy vector, and the recombinant plasmid was serially diluted and quantified using real-time PCR. A standard curve was then constructed for quantification of the PCV-2 levels in field samples. The differentiation of PCV-1 and PCV-2 was carried out by analyzing the melting temperatures of the genotype-specific PCR products., Results: To quantify the PCV-2 levels in field samples, a standard curve (1 x 10(2) -1 x 10(9) copies/microL) was constructed. PCV-2 concentrations as low as 1 x 10(2) copies/microL could be detected in specimens taken from the lymph nodes or infected tissues in samples of PCV-2-infected pigs. The diagnosis of PCV-1 and PCV-2 infections and the quantification of the viral load in the field samples could be completed within 45 minutes after extracting the viral DNA using a commercial extraction kit., Conclusion: This study demonstrate that real-time PCR is a clinically feasible method for the accurate quantification of PCV-2, and for the rapid differentiation of PCV-1 and PCV-2., (Copyright 2010 Taiwan Society of Microbiology. Published by Elsevier B.V. All rights reserved.)

A multiplex polymerase chain reaction (PCR) was designed for the simultaneous detection of four viruses involved in reproductive and respiratory failure in pigs: porcine circovirus type 2 (PCV-2), porcine parvovirus (PPV), classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV). Each of the four pairs of oligonucleotide primers exclusively amplified the targeted fragment of the specific viruses. The sensitivity of the multiplex PCR using purified plasmid constructs containing the specific viral target fragments was 2.58x10(7), 2.64x10(5), 2.66x10(7) and 2.73x10(5) copies for PRRSV, PCV-2, CSFV and PPV, respectively. Using the multiplex PCR, co-infections with these four viruses were identified in 26/76 (34.2%) piglets born from sows with reproductive failure in China. This method is a rapid, sensitive and cost-effective diagnostic tool for the routine surveillance of viral infections in pigs., (Copyright (c) 2008 Elsevier Ltd. All rights reserved.)

Taking advantage of the high sensitivity of polymerase chain reaction (PCR) and the cell-localizing ability of in situ hybridization (ISH), an indirect in situ PCR (ISPCR) method was developed for detecting the distribution of porcine circovirus type 2 (PCV2) in formalin-fixed and paraffin-embedded inguinal lymph nodes obtained from clinically healthy PCV2-carrier pigs and postweaning multisystemic wasting syndrome (PMWS)-affected pigs. Comparisons of the relative sensitivity of indirect ISPCR with other routinely used diagnostic methods for PCV2 indicated that nested PCR was the most sensitive method followed by indirect ISPCR, conventional PCR, ISH, and immunohistochemical (IHC) staining. Although indirect ISPCR, ISH, and IHC staining all revealed a similar signal distribution pattern of PCV2, using indirect ISPCR allowed specific amplification and detection of previously uneasily detected PCV2 signal than by routine ISH or IHC staining, particularly in those cells within the germinal center in clinically healthy PCV2-carrier pigs. Furthermore, six different PCV2 signal expression patterns in conjunction with the correlated lymphoid lesion stages were classified to describe the tissue morphological changes and viral infection. The result indicates that indirect ISPCR is a more effective, cell-based diagnostic tool with good specificity to detect limited PCV2 infection in formalin-fixed and paraffin-embedded tissue specimens and it would be a useful tool for further exploring the pathogenesis of PCV2 infection.

A real-time PCR assay, based on Primer-Probe Energy Transfer (PriProET), was developed to improve the detection and quantification of porcine circovirus type 2 (PVC2). PCV2 is recognised as the essential infectious agent in post-weaning multisystemic wasting syndrome (PMWS) and has been associated with other disease syndromes such as porcine dermatitis and nephropathy syndrome (PDNS) and porcine respiratory disease complex (PRDC). Since circoviruses commonly occur in the pig populations and there is a correlation between the severity of the disease and the viral load in the organs and blood, it is important not only to detect PCV2 but also to determine the quantitative aspects of viral load. The PriProET real-time PCR assay described in this study was tested on various virus strains and clinical forms of PMWS in order to investigate any correlation between the clinical signs and viral loads in different organs. The data obtained in this study correlate with those described earlier; namely, the viral load in 1 ml plasma and in 500 ng tissue DNA exceeds 10(7) copies in the case of PMWS. The results indicate that the new assay provides a specific, sensitive and robust tool for the improved detection and quantification of PCV2.

Diagnostic assays that use polymerase chain reaction (PCR) methods are increasingly available to veterinarians. Psittacine beak and feather disease virus (family Circoviridae, genus Circovirus) is a pathogen of clinical importance for which PCR assays have been developed. Several laboratories offer this diagnostic assay in the United States; however, there is little information on the sensitivity, specificity, or accuracy of these assays. In this study, known positive (n = 10) and negative (n = 10) samples were sent to 5 commercial laboratories. Accuracy was 100% for 2 laboratories, 95% for 2 laboratories, and 71% for 1 laboratory; the accuracy of the latter laboratory was affected because of a specificity of 20%. These results suggest that although the results from most laboratories are highly accurate, both false-positive and false-negative results are occasionally reported by at least 2 laboratories. These results also suggest that at least 1 laboratory may be generating large numbers of false-positive results.

A multiplex PCR (mPCR) assay was developed and evaluated for its ability to simultaneously detect multiple viral infections of swine. Specific primers were designed for each of the following four DNA or RNA viruses: porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), pseudorabies virus (PRV), and porcine reproductive and respiratory syndrome virus (PRRSV). Each target produced a specific amplicon with a size of 353 bp (PCV2), 271 bp (PPV), 194 bp (PRV), or 434 bp (PRRSV). The assay was sensitive and specific in detecting each target agent in composite cell cultures and clinical specimens. Results from mPCR were confirmed by PCR for individual viruses and by virus isolation. In conclusion, the mPCR has the potential to be useful for routine molecular diagnosis and epidemiology.

The emergence of multiple genotypes of PCV2, as demonstrated by phylogenetic analysis of whole genome or capsid sequences, makes it necessary to have quantitative diagnostic assays that perform equally well on all strains. The objectives of this study were to develop and validate a novel real-time polymerase chain reaction (PCR) assay targeting the highly conserved rep gene (ORF1) and investigate the effects of diagnostic specimen choice on its performance. The assay was tested in naturally infected conventional pigs, experimentally infected gnotobiotic pigs, and plasmid-spiked negative serum, lung tissue, and feces and found to have a linear detection range of 2.2x10(3) to 2.2x10(10) copies of PCV2 per mL. The assay successfully detected and quantified PCV2 DNA in serum, buffy coat, feces, and multiple lymphoid (bronchial, mesenteric, and superficial inguinal lymph nodes; thymus; tonsil; ileal Peyer's patches; and spleen), and non-lymphoid (myocardium; lung; kidney; liver; and gluteal muscle) tissues from naturally infected pigs. Across all tissues and sera of naturally infected pigs, the mean PCV2 concentration was 3.0logs higher in wasting versus non-wasting pigs. PCV2 concentration measured by tissue culture and immunohistochemical staining in homogenized liver samples of experimentally infected gnotobiotic pigs were compared to the concentrations estimated by quantitative PCR. Similar trends were noted with increasing PCV2 concentration detected in subclinically infected to severely PMWS-affected pigs across all assays. Our diagnostic assay was developed with a conserved target sequence, and performed efficiently in quantification of PCV2 in a variety of tissues from naturally and experimentally infected pigs.

Several real-time PCR assays for quantification of PCV2 DNA (qPCR) have been described in the literature, and different in-house assays are being used by laboratories around the world. A general threshold of 10(7) copies of PCV2 per millilitre serum for postweaning multisystemic wasting syndrome (PMWS) diagnosis has been suggested. However, neither inter-laboratory nor inter-assay comparisons have been published so far. In the present study, two different qPCR probe assays used routinely in two laboratories were compared on DNA extracted from serum, nasal and rectal swabs. Results showed a significant linear association between the assays (p<0.0001), and a systematic difference of 1.4 log10 copies of PCV2 per millilitre of sample (p<0.0001). This difference indicated that the assay from laboratory 1 yielded a higher output than the one from laboratory 2. Results also showed that there was no linear association between the amount of PCV2 DNA and the amount of total DNA, neither in nasal (p=0.86) nor in rectal (p=0.78) swabs, suggesting that normalizing of PCV2 DNA load in swab samples to total DNA concentration is not suitable. The present exploratory study highlights the need for the performance of ring trials on qPCV2 protocols between laboratories. Meanwhile, the proposed thresholds for PMWS diagnosis should only be considered reliable for each particular laboratory and each particular assay.

This study examined if pigs in a Porcine circovirus disease (PCVD)-affected herd (n = 100) had shed more Porcine circovirus-2 (PCV-2) in their feces than pigs in a PCVD-nonaffected herd (n = 101), and if differences in shedding among production stages within and between the herds existed. The PCV-2 shedding was quantified by real-time polymerase chain reaction. The highest median PCV-2 shedding was found in the nursery of the PCVD-affected herd and in the grower of the PCVD-nonaffected herd. The PCV-2 shedding was significantly higher in earlier stages (newly weaned, nursery, and pregrower) in the PCVD-affected herd (Wilcoxon rank sum; P < 0.001) compared with the PCVD-nonaffected herd. Porcine circovirus-2 DNA was not detected in a significant proportion of lactating sows (parity > or = 3) in the PCVD-nonaffected herd (Fisher's exact test; P = 0.001). The results of this study suggest there may be an association between the presence of PCV-2 in the feces of lactating sows and increased PCV-2 shedding in younger pigs.

A duplex real-time quantitative PCR (qPCR) method for the simultaneous detection of porcine circovirus type 2 (PCV2) and an exogenous internal positive control (IPC) in porcine semen samples was developed. The IPC was included to monitor DNA extraction and PCR inhibition and consisted of a mutated PCV2 plasmid clone which differed from the target PCV2 in the probe binding region and thus was detected by the use of a second probe with different end-labeling. The sensitivity, specificity and repeatability of the assay were validated by testing semen samples from 12 boars inoculated experimentally with PCV2, 10 boars infected naturally with PCV2, and 3 PCV2 negative control boars. The duplex qPCR assay was found to be more sensitive, specific, rapid, and repeatable than nested PCR (nPCR) methods for the detection of PCV2 DNA in semen. Analysis of separated semen fractions by the duplex qPCR assay showed PCV2 DNA to be present mainly in the cell fraction as opposed to the seminal plasma fraction which is in contrast to previous reports. The duplex qPCR assay was found to be a valuable tool for accurate and quantitative detection of PCV2 DNA in boar semen.

Infections of young racing pigeons with pigeon herpesvirus (PiHV), fowl adenovirus (FAdV) and pigeon circovirus (PiCV) are reported frequently. The role of these viruses in the pathogenesis of a disease complex called young pigeon disease syndrome (YPDS) is generally accepted. All of these viruses cause inclusion bodies in the liver so liver samples are particularly useful for the detection of infection. Consequently a multiplex polymerase chain reaction (PCR) was developed for the detection of PiHV, FAdV and PiCV in liver samples from racing pigeons. The detection limits were 10(1) genome equivalents for the detection of PiHV and PiCV and 10(3) genome equivalents for FAdV. The absence of PCR inhibitors was shown by the detection of cytochrome B gene as an internal control. No PCR products were amplified from related herpes and circoviruses or negative controls, demonstrating the specificity of the multiplex PCR. The addition of cellular DNA from liver samples or Q-solution to the reaction mix had no influence on its sensitivity. The usefulness of the multiplex PCR was demonstrated by re-investigation of liver samples from young racing pigeons previously tested positive by uniplex PCRs.

A novel hot-start multiplex PCR (mPCR) assay was developed and subsequently evaluated for its effectiveness in simultaneously detecting multiple viral infections of swine. Specific primers for each of five virus genomes, namely classical swine fever virus (CSFV), African swine fever virus (ASFV), porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine parvovirus (PPV) were used. Combined nucleic acid purification was carried out using a commercial RNA/DNA extraction kit. The mPCR consisted of a two-step procedure which included reverse transcription and PCR amplification. This mPCR and the corresponding separate assays were evaluated comparatively on serial ten-fold dilutions of each virus. Analysis of the sensitivity in comparison to the corresponding single PCR (sPCR) for the detection of each of the five targets was identical for CSFV, PCV2 and PPV, 1 log lower for PRRSV and 2 logs lower for ASFV. No spurious PCR amplification reactions among all five pathogens were noticed with various amounts of DNA and RNA mixtures. All the uninfected controls were scored negative. The relative efficiency of the mPCR developed in this study compared to performing sPCR for each virus, suggests its potential application for routine molecular diagnostic purposes.

Circoviruses are the causative agents of acute and chronic diseases in several animal species. Clinical symptoms of circovirus infections range from depression and diarrhoea to immunosuppression and feather disorders in birds. Eleven different members of the genus Circovirus are known so far, which infect pigs and birds in a species-specific manner. Here, a nested PCR was developed for the detection of a broad range of different circoviruses in clinical samples. Using this assay, a novel circovirus was detected in mute swans (Cygnus olor) found dead in Germany in 2006. Sequence analysis of the swan circovirus (SwCV) genome, amplified by multiply primed rolling-circle amplification and PCR, indicates that SwCV is a distinct virus most closely related to the goose circovirus (73.2% genome sequence similarity). Sequence variations between SwCV genomes derived from two different individuals were high (15.5% divergence) and mainly confined to the capsid protein-encoding region. By PCR testing of 32 samples derived from swans found dead in two different regions of Germany, detection rates of 20.0 and 77.3% were determined, thus indicating a high incidence of SwCV infection. The clinical significance of SwCV infection, however, needs to be investigated further.

The collection of biological material (e.g., blood) directly onto filter paper for subsequent use in laboratory assays such as polymerase chain reaction (PCR), has become a common practice. Dried cells or fluid on the paper can be readily rehydrated and retrieved into a standard volume of an appropriate elution buffer but introduces a dilution factor to the sample. The use of a common cutting instrument for excising a standard-sized piece of paper that contains the material also introduces the potential for transferring biological material from one sample to subsequent samples, causing false-positive results by PCR. In the present study, filter-paper-collected blood that contained beak and feather disease virus was used to determine if viral DNA could be transferred between samples by a hole punch used to excise sequential filter papers. It was determined that false-positive results could be obtained at least 13 times after a positive sample. Subsequently, the efficacy of 4 methods of hole punch disinfection, flaming, VirkonS, bleach, and a bleach-ethanol combination, was assessed. The only effective and practical method to destroy DNA was a method where the hole punch was agitated in commercial bleach, rinsed in water, the water was displaced with 100% ethanol and air-dried. This method was simple, cheap, and relatively rapid, and allowed for the use of a single hole punch for a series of samples, without carryover contamination and consequent false-positive results.

Rapid and reliable detection of viral pathogens is critical for the management of the diseases threatening the economic competitiveness of the swine farming industry worldwide. Molecular beacon assays are one type of real-time polymerase chain reaction (PCR) technology capable of fast, specific, sensitive, and reliable viral detection. In this paper, the development of molecular beacon assays as novel tools for the rapid detection of Aujeszky's disease virus, African swine fever virus, porcine circovirus type 2 and porcine parvovirus is described. The assays are capable of rapidly detecting 2 x 10(1) copies of target and are linear between 2 x 10(9) and 2 x 10(2) copies. They can detect virus specifically in clinical samples such as whole blood, serum and tissue. In comparison to conventional PCR they are either as sensitive or more sensitive. As such these molecular beacon assays represent a powerful tool for the detection of these viruses in swine.

The use of porcine organs is being developed as a means to alleviate the shortage of human organs for transplantation. Recommendations have been published for the microbiological specifications of organ-source pigs to reduce the possibility of a microorganism from pigs being inadvertently transferred to the recipient of the xenograft. The pseudorabies virus (PRV), porcine cytomegalovirus (PCMV), and porcine circovirus (PCV) are infectious agents in pigs that are considered to be of significance for the microbiological safety of xenotransplantation. A multiplex polymerase chain reaction (mPCR) was developed to detect and differentiate among PRV, PCMV, and PCV. The sensitivities of the multiplex PCR were 10(2.5) TCID(50)/ml for PRV, 10(1.8) TCID(50)/ml for PCMV, and 10(1.8) TCID(50)/ml for PCV. The lowest viral concentrations detected by single PCR were 10(1.5) TCID(50)/ml for PRV, 10(1.0) TCID(50)/ml for PCMV, and 10(1.4) TCID(50)/ml for PCV2. Non-specific reactions were not observed when other viruses, bacteria, and Vero cells were used to assess the multiplex PCR. The multiplex PCR was effective in detecting various combinations of one or more of these viruses in pig specimens collected for xenotransplantation.

A nested polymerase chain reaction (nPCR) protocol was applied to porcine semen to demonstrate the porcine circovirus type 2 (PCV2) shedding patterns and duration in naturally infected boars. Sperm morphology analysis was performed on a subset of samples to determine if the presence of PCV2 DNA in semen was associated with reduced semen quality. Semen was collected serially from 43 boars representing 6 breeds, aged 33.9 to 149.3 weeks. Of the 903 semen samples collected, 30 samples (3.3%) were positive for PCV2 DNA by nPCR from 13 boars. Boars shedding PCV2 DNA in semen ranged between 35.9 and 71.0 weeks of age, and shedding occurred during a period of up to 27.3 weeks. A semen nPCR test was 2.6 times more likely to be positive when collected from pigs that were < or =52 weeks of age, and 3.0 times more likely to be positive when collected from pigs that were < or =26 weeks from time of entry into the stud main unit (generalized estimating equations: P = 0.02; 95% confidence interval [CI] of the odds ratio 1.2 to 5.5, and P = 0.01; 95% CI of the odds ratio 1.3 to 6.9, respectively). These results demonstrate a sporadic and long-term shedding pattern of PCV2 DNA in semen from naturally infected boars. PCV2 DNA in semen does not appear to have detrimental effects on sperm morphology; however, boar age and, possibly, breed may contribute to the persistence of PCV2-shedding in semen.

The absence of extraneous viruses is a requirement in the quality control of vaccines for veterinary use in the European Pharmacopoeia. A polymerase chain reaction (PCR) assay for the detection of porcine circovirus type 1 (PCV1) and type 2 (PCV2) was evaluated in 18 commercial porcine vaccines. Since vaccine components may contain PCR enhancers or inhibitors, 13 of the studied vaccines (used as diluents) were subsequently spiked with different dilutions of PCV2 and tested by PCR. Although PCV2 DNA was not detected in any of the vaccines tested, PCV1 was detected in 2/18 vaccines (11%). Eleven out of 13 PCV2 spiked vaccines showed a positive PCR result. The lack of amplification observed in two spiked vaccines suggested that use of the PCR assay to detect PCV2 could depend on vaccine composition. The results of this exploratory study have demonstrated that PCR is a rapid and fairly sensitive method for the detection of porcine circoviruses as extraneous agents in vaccine products and can be used in the quality control of pig vaccines. The study has also indicated the need for optimising the sensitivity of PCR methods for PCV genome detection in vaccine products.

This article reports the complete nucleotide sequences of four duck circovirus (DuCV) isolates from sick ducks in Taiwan and development of a polymerase chain reaction (PCR) for detection and differentiation of goose circovirus (GoCV) and DuCV. Sequence comparison showed that Taiwanese DuCV isolates had 82.5%-83.8% nucleotide sequence identity to the German and North American DuCV isolates. This is the first report on the presence of DuCV and its associated diseases outside Germany. A PCR test was developed using a universal primer pair based on conserved sequences present in the genomes of GoCV and DuCV. This PCR test could detect and differentiate between GoCV and DuCV by the size of PCR product each virus produced (256 bp for GoCV and 228 bp for DuCV). Application of this PCR test to samples of bursa of Fabricius from sick birds in the field showed that 9 of 26 goose samples contained GoCV, while 13 of 34 duck samples contained DuCV. This PCR test could serve as a fast and sensitive method for detection and differentiation of DuCV and GoCV.

Multiplex PCR was established to detect porcine circovirus type 2 (PCV-2), porcine parvovirus (PPV) and porcine pseudorabies virus (PRV) and applied to samples from 137 piglets exhibiting clinical signs of postweaning multisystemic wasting syndrome (PMWS). PCV-2 DNA was detected from all samples. Moreover, 43 samples were positive for PPV but negative for PRV; 11 samples were positive for PRV but negative for PPV; and 35 samples were positive both for PPV and PRV. These results suggests that PCV-2 co-infection with PRV and PPV may play an important role in PMWS. Also, multiplex PCR is an appropriate candidate method for diagnosis of PCV-2, PRV and PPV simultaneously in field cases.

Real-time PCR assays were developed for quantitative detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2). The established real-time PCR for the quantitation of PRRSV cDNA and PCV2 DNA were found to be in the 9-log(10) linear dynamic range with excellent linearity and reliable reproducibility. Using these techniques, the distribution and quantitation of PRRSV and PCV2 in naturally infected and challenged pigs were investigated. The viral concentrations were expressed as the mean log(10) viral DNA or cDNA copy numbers per mg or ml of tested samples. For pigs infected naturally with both viruses, the lung, spleen, tonsil and lymphoid organs had the highest viral burdens with ranges from 5.73 to 8.38 and 5.65 to 6.91 for PRRSV and PCV2, respectively. The injection of formalin-inactivated Salmonella choleraesuis emulsified in complete Freund's adjuvant 1 week before and after the inoculation of both viruses resulted in PRRSV replication enhancement 2 weeks post-challenge. However, this facilitated the clearance of PRRSV 4 weeks post-challenge. Results from this study show that the established quantitative PCR could be a useful tool when applied to vaccine development and pathogenesis studies in the future.

Two common viral diseases in psittacine birds including budgerigar fledgling disease (BFD), generally called avian polyomavirus (APV) infection, and psittacine beak and feather disease (PBFD) have similar clinical manifestations characterized by feather disorders. A duplex shuttle PCR was developed for detection of APV and PBFD virus (PBFDV). Two pairs of oligonucleotide primers were designed to amplify a 298-bp fragment of the t/T antigen region of APV genome and a 495-bp fragment of the capsid protein region encoded by open reading frame (ORF) C1 of PBFDV genome, respectively. In the present study, APV and PBFDV were detected simultaneously in one tube by duplex shuttle PCR using these two pairs of primers. The detection limits were 2 viral copies of APV and 3 viral copies of PBFDV. In the clinical application, we detected 16 APV-positive, 15 PBFDV-positive, and 3 mixed infected samples in 39 samples examined. Sequences of the amplified products were read. The t/T antigen region was conserved in the APV-positive samples as expected. ORF C1 of PBFDV genome showed diversity. Phylogenic analysis indicated that PBFDV ORF C1 consisted of 6 clusters which were related to subfamilies of psittacine birds. Our duplex shuttle PCR could be a useful method for differential diagnosis and molecular epidemiology of BFD and PBFD.

A multiplex PCR (mPCR) assay was developed and subsequently evaluated for its effectiveness as a means to simultaneously detect multiple viral infections of swine. Specific primers for each of four common DNA viruses, namely, pseudorabies virus (PRV), porcine circovirus type I (PCV1), porcine circovirus type II (PCV2), and porcine parvovirus (PPV), were used for testing procedure. The assay was shown to be highly sensitive in that as little as 10(-4) ng of each of the respective amplicons (approximately equal to 10,000 molecules) was detected when a composite of all four viruses (including both field and gene-deleted permutations of PRV) was tested as a single sample. It was also effective for detecting one or more of these same viruses in various combinations in specimens including lymph nodes, lungs, spleens, and tonsils collected from clinically ill pigs, and in specimens in spleen collected from aborted fetuses. The relative efficiency (compared to performing separate assays for each virus) and apparent sensitivity of mPCR suggest its potential application for routine molecular diagnostic purposes.

To investigate sequence diversity of psittacine beak and feather disease virus, samples collected from 31 psittacine species with or without clinical signs were tested for the presence of the viral genome. A real-time polymerase chain reaction was developed amplifying a 202 base pair fragment of the region encoding the capsid protein C1 and detecting 100 to 1000 genome equivalents. The nucleotide sequences of the polymerase chain reaction products showed 84.1 to 100% identity with no consistent pattern with regard to the infected bird species. Amino acid exchanges were concentrated mainly in five of the 42 deduced positions. Sequences obtained from an outbreak of acute beak and feather disease in lories clustered in a separate branch of a phylogenetic tree. Sequences in samples from African grey parrots with feather disorders grouped together, whereas those from the same species with immunosuppression clustered in other branches. These results indicate the possible existence of beak and feather disease virus genotypes.

A polymerase chain reaction (PCR) and dot blot hybridization (DBH) test have been developed for the diagnosis of infection by a novel circovirus of geese (GoCV). These tests were applied to samples of bursae of Fabricius from sick and dead birds from commercial goose farms in Hungary. In this second report of the occurrence of circovirus infection in diseased geese, 103 of 214 (48.1%) and 37 of 150 (24.6%) birds, and 49 of 76 (64.5%) and 18 of 76 (23.7%) flocks were positive by PCR and DBH respectively. The sensitivity of the PCR test was such that 0.10 fg of virus DNA was detectable. The DBH test was less sensitive, only detecting larger amounts (40 pg) of DNA, but was used as a semi-quantitative method for detecting the presence of virus. The incidence of infection was affected by factors such as the age of the birds and rearing methods.

A multiplex seminested polymerase chain reaction (PCR) was developed for the simultaneous detection and differentiation among porcine circovirus 1 (PCV1), PCV2, and porcine parvovirus (PPV) from boar semen. Primers of PCV1, PCV2 and PPV were specific and did not react with other viruses respectively. Twenty (20.4%) and 42 (42.9%) out of 98 whole semen samples were found to be positive for PCV and PPV using multiplex conventional and seminested PCR, respectively. When the separated fractions of PCV or PPV-contaminated semen were analyzed using multiplex seminested PCR, PCV and PPV DNA were found to be present mainly in the seminal fluid and nonsperm cell fractions. This multiplex seminested PCR assay was sensitive, rapid and a good alternative method for the detection and differentiation of these viruses in boar semen.

Multiplex nested polymerase chain reactions (PCRs) were developed for the simultaneous detection and differentiation of genomic material of porcine circovirus 1 (PCV1), porcine circovirus 2 (PCV2), and porcine parvovirus (PPV) in formalin-fixed, paraffin-embedded tissues. Multiplex conventional and nested PCR and in situ hybridization were compared for their ability to detect the 3 viruses in such tissues. Xylene deparaffinization followed by proteinase K digestion yielded DNA of sufficient quality for reliable and consistent PCR analyses. The DNA from PCV1, PCV2, and PPV was detected by both multiplex nested PCR and in situ hybridization in lymph-node tissue from 12 pigs experimentally co-infected with the 3 viruses, as well as in formalin-fixed, paraffin-embedded lymph-node tissue from 30 pigs with naturally occurring postweaning multisystemic wasting syndrome; the agreement rates for the 2 methods were 100% in both groups of pigs. Thus, multiplex nested PCR could be applied successfully to formalin-fixed, paraffin-embedded tissues for simultaneous detection of these 3 porcine viruses.

Polymerase chain reaction (PCR) and dot blot hybridisation (DBH) tests for detecting pigeon circovirus (PiCV) DNA were developed and evaluated using tissue samples obtained from diseased and clinically normal pigeons, which originated in Belgium and Northern Ireland. When PCR product was visually detected, the limit of detection of the PCR test was 31 fg, while that of the DBH was 1.6p g. For evaluation purposes, the results of the PCR and DBH tests, performed with DNAs extracted from samples of bursa of Fabricius (BF), were compared with those of in situ hybridisation (ISH) and histology. In 32 samples tested by all four tests, 27 (84%) were positive by PCR, 24 (75%) were positive by ISH, 20 (63%) were positive by DBH, and 13 (41%) were positive by histology. Additional PCR testing showed that in some disease-affected birds, PiCV DNA could be detected in a range of tissues including thymus, spleen, liver, kidney and brain. The PCR detection of PiCV DNA in BF samples from clinically normal birds indicated that PCR can detect infections in the absence of disease, a finding that mitigates against its use as a disease diagnostic. In addition, nucleotide sequence determinations indicated that PCR test performance was adversely affected by the sequence diversity exhibited by selected PiCVs. The application of the DBH test to dilutions of test samples indicated that the BF from some diseased pigeons contained very large amounts of virus DNA, as much as 10(13)genome copies/g tissue, and suggested that this test may be a convenient method of providing a semi-quantitative estimate of virus load.

A retrospective study for the detection of porcine circovirus 2 (PCV-2) DNA was conducted by nested PCR method in 16 cases of swine post-weaning multisystemic wasting syndrome (PMWS) in Thailand. Histopathology showed characteristic lesions of PMWS and intracytoplasmic viral inclusion bodies in macrophages infiltrating in lymphoid tissues. PCV-2 DNA was detected from formalin-fixed and/or formalin-fixed paraffin-embedded tissues from all pigs with PMWS. The amplified products were digested with Hae III.

Three oligonucleotide primers for semi-nested polymerase chain reaction (PCR) were designed according to already published sequences of porcine circovirus types 1 (PCV-1) and 2 (PCV-2) isolates. These primers were used to detect PCV-2 DNA. A positive amplification reaction was visualized from a DNA suspension containing as few as 10 copies of virus DNA. In total. 77 samples of inguinal lymph nodes and nasal swabs from pigs in the Czech Republic were used to detect the virus. Thirty-seven of them were positive for PCV-2 DNA. In order to confirm specificity of the PCR reaction, seven DNA fragments were sequenced. Czech PCV sequences were found to have a 92-97% homology with other known PCV-2 strains and only 80-83% homology with PCV-1 strains.

An experimental study was conducted to evaluate the potential presence of porcine circovirus type 2 (PCV2) in the semen of infected boars. Four mature boars were inoculated intranasally with PCV2 isolate LHVA-V53 propagated on PK15 cells. Two boars inoculated with the supernatant of noninfected PK15 cells were kept as controls. Serum samples were collected from all boars at 4, 7, 11, 13, 18, 21, 25, 28, 35, and 55 days postinoculation (dpi) and from the four PCV2-infected boars at 90 dpi. Samples were tested for the presence of antibodies to PCV2 by an indirect immunofluorescence assay and for the presence of PCV2 DNA by PCR and nested PCR. Semen samples were collected from all six boars at 5, 8, 11, 13, 18, 21, 25, 28, 33, and 47 dpi and tested for the presence of PCV2 DNA by a nested PCR assay. Antibodies to PCV2 could be detected as early as 11 dpi in one boar, and all four infected boars were found positive for PCV2 antibodies by 18 dpi. Thereafter all infected boars remained positive for antibodies to PCV2 until 90 dpi. Analysis of serum samples by nested PCR demonstrated the presence of PCV2 DNA as early as 4 dpi in three of four infected boars. Serum samples from all infected boars were positive for PCV2 DNA from 11 dpi until 35 dpi but were negative at 90 dpi. PCV2 DNA was detected as soon as 5 dpi in the semen of two infected boars and intermittently thereafter in the semen of all four infected boars. The semen of two infected boars was positive for PCV2 DNA at 47 dpi. Following infection, PCV2 DNA can be detected in semen concurrently with the presence of PCV2 DNA and antibodies in the serum. The present study suggests that PCV2 may be shed intermittently in the semen of infected boars.

A competitive PCR (cPCR) assay was developed for monitoring porcine circovirus (PCV) DNA in serum samples from piglets. The cPCR was based on competitive coamplification of a 502- or 506-bp region of the PCV type 1 (PCV1) or PCV2 ORF2, respectively, with a known concentration of competitor DNA, which produced a 761- or 765-bp fragment, respectively. The cPCR was validated by quantification of a known amount of PCV wild-type plasmids. We also used this technique to determine PCV genome copy numbers in infected cells. Furthermore, we measured PCV DNA loads in clinical samples. More than 50% of clinically healthy piglets could harbor both types of PCV. While PCV1 was detected in only 3 of 16 pigs with postweaning multisystemic wasting syndrome (PMWS), all the sick piglets contained PCV2. A comparison of the PCV2 DNA loads of healthy and sick animals revealed a significant difference, indicating that the development of PMWS may require a certain amount of PCV2.

Sets of oligonucleotide primers were designed according to the sequences of the open reading frames (ORFs) ORF1 and ORF2 of the prototype nonpathogenic PK-15 strain of porcine circovirus (PCV) type 1 (PCV-1). By the PCR performed with the various primer sets, genomic DNA or RNA from other bacterial or viral pathogens of the respiratory tracts of pigs could not be amplified. A positive amplification reaction could be visualized with DNA extracted from a viral suspension containing as few as 10 viral particles per ml. No DNA fragment could be amplified from lysates of continuous porcine cell lines (PT, ST, and PFT cells) known to be negative for PCV. When tested with clinical samples from pigs, the results of the single PCR method showed nearly 93% (13 of 14 samples) correlation with histopathological and immunohistochemical findings. Interestingly, subclinical PCV infections could be detected by single PCR with clinical samples that have been submitted from animals with irrelevant cases of respiratory and/or enteric problems. On the basis of the nucleotide sequences of PCV strains (PCV-2) recently associated with outbreaks of postweaning multisystemic wasting syndrome (PWMS) in Quebec, Canada, pig farms, other primers were designed from the PCV-1 genome, and these primers failed to amplify genomic fragments specific to the ORF1 or ORF2 genes of clinical isolates associated with PWMS but amplified DNA from the PCV-1 strain. Two rapid multiplex PCR (mPCR) methods have been developed to distinguish between both genotypes of PCV. By those two mPCR methods, (i) species-specific primer pairs were used to amplify a DNA fragment of 488 bp specific for the ORF2 genes of both genotypes, whereas a 375-bp fragment was amplified from the ORF1 gene of the PCV-1 strain only, or (ii) species-specific primer pairs were used to amplify a DNA fragment of 646 bp specific for the ORF1 genes of both genotypes, whereas a 425-bp fragment was amplified from the ORF2 gene of the PCV-1 strain only. By both mPCR methods, a PCV-2 infection was demonstrated in tissues of 94.2% (33 of 35) of the sick pigs tested, in agreement with previous findings showing the close association of this new genotype of PCV with outbreaks of PMWS in Europe and North America. On the other hand, a PCV-1 infection was confirmed in only 5.7% (2 of 35) of the pigs, and confirmation of a mixed infection with PCV-2 was obtained by a single PCR with PCV-2-specific primers.

A universal PCR assay was designed that consistently detected psittacine beak and feather disease virus (BFDV) in psittacine birds affected with psittacine beak and feather disease (PBFD) from different geographic regions across Australia. Primers within open reading frame 1 (ORF1) of the BFDV genome consistently amplified a 717 bp product from blood and/or feathers of 32 birds with PBFD lesions. The PCR did not amplify a product from the feathers or blood from 7 clinically normal psittacine birds. Primers based on regions outside of ORF1 did not consistently produce a PCR product, suggesting there was some genomic variation outside ORF1. The amplified ORF1 PCR products of 10 BFDV isolates, from different psittacine species and from various regions around Australia, were cloned and comparative DNA sequence analysis demonstrated 88-99% of the ORF1 fragments. The derived amino acid sequences of the amplified ORF1 fragments demonstrated similar identity between all 10 isolates. Within ORF1, there was complete conservation of the putative nucleotide binding site and marked conservation of 2 other motifs previously identified as essential components of the replication-associated proteins of other circoviruses and geminiviruses.