Advanced Detection Method for PCV3 and PCV4 Using Duplex Real-Time RAA

The accurate detection of Porcine circovirus 3 (PCV3) and Porcine circovirus 4 (PCV4) is crucial for managing viral diseases in pork herds. Recent advancements in molecular diagnostic techniques have led to the development of a highly sensitive and specific duplex real-time recombinase amplification assay (RAA) for simultaneous detection of these viruses.

Standard Plasmid Construction

To create standard plasmids for the duplex real-time RAA assay, the complete genome sequences of PCV3 (2,000 bp) and PCV4 (1,770 bp) were synthesized and cloned into the pUC57 vector by Sangon Biotech Co., Ltd. The resultant plasmids, designated as pUC57-PCV3 and pUC57-PCV4, were quantified using NanoDrop and Qubit 2.0 fluorometer. The DNA copy number was calculated using the formula: DNA copy number/μL = [plasmid concentration (ng/μL) × 10−9 × 6.02 × 1023] / [DNA length (nt) × 660]. Sequencing was performed to ensure accuracy, and the plasmids were stored at -20°C.

Primer and Probe Design

The design of effective primers and probes is essential for the success of real-time RAA. The genomic sequences of 30 PCV3 strains and 29 PCV4 strains from the GenBank were aligned using Lasergene version 7.1 software to identify conserved regions. Primer Premier 5.0 software was utilized to create the primers and probes according to TwistAmpTM amplification guidelines. These designs were then validated using NCBI Primer-BLAST web tool. The primers and probes were synthesized by Sangon Biotech Co., Ltd.

The duplex real-time RAA assay for PCV3 and PCV4 utilized two primer sets and two RAA probes targeting the ORF2 gene of PCV3 (Fig. 1). For PCV4, three primer sets and two RAA probes were designed to target the ORF1 or ORF2 gene (Fig. 2 and Fig. 3). The details of the primers and probes are summarized in Table 1.

Positions of the duplex real-time RAA primers and probes in the ORF1 (Rep gene) sequences of different PCV4 strains in the GenBank database. Dots represent nucleotide residues that match the majority. The forward primers are marked with red bars, the reverse primers are marked with blue bars, and probes are marked with green bars

Positions of the duplex real-time RAA primers and probes in the ORF2 (Cap gene) sequences of different PCV4 strains in the GenBank database. Dots represent nucleotide residues that match the majority. The forward primers are marked with red bars, the reverse primers are marked with blue bars, and probes are marked with green bars

Duplex Real-Time RAA Assay Setup

The duplex real-time RAA assay is performed using a commercial probe-based RAA kit. The reaction mix includes Buffer A, forward and reverse primers, probes, template DNA, nuclease-free water, and Buffer B. The reaction mixture is prepared in a 50 μL volume and incubated at 39°C for approximately 20 minutes. Real-time fluorescence monitoring determines the positivity of the samples, with positive samples showing an exponential amplification curve above the negative control threshold.

Optimization of Reaction System

To optimize the duplex real-time RAA assay, several primer-probe combinations were tested using standard plasmids pUC57-PCV3 (20.0 ng/μL) and pUC57-PCV4 (20.0 ng/μL) as templates. The best performing combination was further optimized by adjusting the concentrations of primes and probes. The primer concentration ranged from 200 to 600 nM, while the probe concentrations varied from 60 to 180 nM (Table 2).

Nucleic Acid Extraction

Nucleic acids were extracted from both clinical and artificially spiked samples using the MagNA Pure LC Total Nucleic Acid Isolation Kit. The extracted nucleic acids were eluted in nuclease-free water and stored at -80°C. For standard plasmids, the Plasmid Mini Kit was used for extraction following the manufacturer’s instructions.

Analytical Specificity

The specificity of the duplex real-time RAA assay for detecting PCV3 and PCV4 was evaluated using nucleic acids and cDNA from other swine pathogens such as Foot and Mouth Disease Virus (FMDV), Classical Swine Fever Virus (CSFV), Pseudorabies Virus (PRV), Porcine Parovirus (PPV), Porcine Circovirus 2 (PCV2), Porcine Epidemic Diarrhea Virus (PEDV), Transmissible Gastroenteritis Virus (TGEV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and Porcine Deltacoronavirus (PDCoV).

Analytical Sensitivity

The sensitivity of the duplex real-time RAA assay was assessed by using serially diluted standard plasmids (ranging from 107 to 100 copies per 2.5 μL). The lower limit of detection (LOD) was calculated using probit regression analysis of eight independent runs, providing a reliable measure of the assay’s sensitivity.

Repeatability and Reproducibility

To evaluate the repeatability and reproducibility of the assay, three different concentrations (104, 103, and 102 copies per 2.5 μL) of standard plasmids were analyzed. Each dilution was tested in triplicate in one run or over three independent runs on separate days. The coefficients of variation (CVs) were calculated from the cycle threshold (CT) values.

Detection of Clinical Samples

The duplex real-time RAA assay was applied to 60 samples, including 55 clinical samples and 5 artificially spiked samples. These samples were initially screened using qPCR assays specific for PCV3 and PCV4. The artificially spiked samples were then evaluated by the duplex RAA assay to assess its clinical performance. The results from the RAA assay were compared to those from qPCR to assess the assay’s reliability.

Statistical Analysis

Kappa statistics were used to compare the coincidence rates between the duplex real-time RAA assay and qPCR assays. All statistical analyses and data plotting were conducted using SPSS 24.0 software and GraphPad Prism.

Conclusion

The development of a duplex real-time RAA assay for the detection of PCV3 and PCV4 represents a significant advancement in molecular diagnostics. This method facilitates simultaneous detection of both viruses with high specificity and sensitivity. The assay’s reliability, as demonstrated through various optimization and testing steps, underscores its potential for widespread application in healthcare settings.

Additionally, the ability to accurately distinguish between these two closely related viruses is crucial for effective disease management and prevention in pork production. Future research could focus on the implementation of this assay in routine diagnostic practices and its adaptation for use in field conditions.

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