The reagents and instruments
The common PCR was performed with commercial PCR kit (Promega, USA). The fluorescence PCR was performed with GoTaq® Probe qPCR Master Mix, 1000 rxn (A6102, Promega, USA) and monitored with the 7500 fluorescence PCR instrument (Applied Biosystems, Thermo Fisher Scientific, USA).
Plasmid construction
The nucleotide sequence of PCV3 capsid protein was synthesized based on the reference sequence retrieved from GenBank (PCV3-US/MO2015, KX778720.1). The sequence was linked to T3 vector and transferred into E. coli DH5α. The plasmid genome was extracted with Plasmid MiniPrep Kit (Qiagen, German) and quantified with Nanodrop 2000 (Thermo Fisher Scientific, USA). The copy number of purified capsid was measured and calculated according to the following formula: y copies / μL = [x (ng/μL) × 10− 9/ (DNA length× 660)] × 6.02 × 1023. The capsid was diluted in a concentration of 107 copies/μL and stored at − 80 °C until use.
The real-time PCR assay
Primers and probes: The genome sequences of existing 58 PCV3 isolates were retrieved from the GenBank and aligned with Mega software version 6.0. The conserved region of PCV3 sequence was identified as the sequence of capsid, which was selected for designing the primers and probe. The candidate primers and probes were designed with the Primer Premier 5.0 and then synthesized (Invitrogen, Shanghai, China).
Reaction system: a total volume of 20 μL, including 10 μL of GoTaq® Probe qPCR Master Mix (2X); 1.2 μL of primers pair; 0.6 μL of probe; 2 μL of template, 6.2 μL of dH2O. The amplification procedure was as follows: 95 °C for 2 min; 40 cycles of 95 °C for 15 s and 60 °C for 1 min.
Characterization
Sensitivity: The constructed capsid plasmid (with initial concentration of 107 copies/μL) was diluted to 106,105,104,103, 102 and 101 copies/μl. The diluted plasmid was applied for sensitivity assay. The sensitivity test was repeated in triplicate.
Specificity: Common swine viruses and bacterium were tested for specificity, including porcine circovirus type 2 (PCV2, commercial vaccine), pseudorabies virus (PRV, bartha K61, GenBank: JF797217), porcine reproductive and respiratory syndrome virus European strain (PRRSV-EU, Lelystad virus strain, GenBank: M96262) and American strain (PRRSV-NA, JXA1 strain, GenBank: EF112445), classical swine fever (CSFV, C strain, GenBank: Z46258), porcine parvovirus (PPV, NADL-2, ATCC VR-742, GenBank: KF913351.1), Escherichia coli (Migula) Castellani and Chalmers (E.coli., ATCC@25922™); Actinobacillus pleuropneumoniae (Shope) Pohl et al. (App., ATCC@27088™). The negative control was SPF negative porcine serum obtained from VRMD. The positive control was PCV3 capsid plasmid. All these viruses were stored, cultured and identified by the National Veterinary Diagnosis Center of Chinese Animal Disease Control Center. The specificity test was repeated in triplicate.
Combining the results of sensitivity and specificity, the sequence of primers pair and probe after optimization was provided (Table 1, marked as “This study”).
Clinical samples
One hundred ten tissue samples were collected from diseased pigs in domestic swine farms, because of dermatitis, respiratory and reproductive disorders. These samples were archived in our institution at − 40 °C for less than 1 year. The whole DNA of the tissue homogenate was extracted with 5*MAGMAX-96 Viral 1 Kit (AM1836–5, ABI, USA) for following assays. The collection of samples involved in our study complied with national guidelines. The study proposal was reviewed and approved from Institutional Review Board of Veterinary Diagnosis Centre, China Animal Disease Control Center. The Written Informed Consent was obtained from the owners of all the involved farms before the samples were applied.
Detection and comparison with other methods
The DNA of clinical samples after extraction was detected with both the established method in this study and two previously reported methods: the common PCR (Palinski, 2016) and real-time fluorescence PCR methods (Wang, 2017). The common PCR was performed according to the reports of Palinski and determined with 2% agarose gel electrophoresis. The sample with amplification stripe was considered positive. Both real-time fluorescence PCR methods were performed with GoTaq® Probe qPCR Master Mix, 1000rxn (A6102, Promega, USA) and monitored with the 7500 fluorescence PCR instrument. The sample presenting typical amplification curve was interpreted as positive.