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Development of a triplex TaqMan probe-based real-time PCR assay for simultaneous detection of swine influenza virus, porcine reproductive and respiratory syndrome virus, and porcine circovirus type 2

BMC Veterinary Research volume 21, Article number: 297 (2025) Cite this article

Abstract

Background

Porcine respiratory disease complex poses a substantial economic challenge for the swine farms due to its prevalence in pig farming environments. The disease is primarily caused by viral pathogens such as swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine circovirus type 2 (PCV2). Given the high incidence and morbidity associated with these viruses, effective control strategies rely on rapid and accurate diagnosis.

Results

To this end, we developed and validated a triplex TaqMan probe-based real-time PCR assay for the simultaneous detection and differentiation of SIV, PRRSV, and PCV2. This assay demonstrated high specificity, with no observed cross-reactivity between the target viruses. The assay’s sensitivity was determined to be 100 copies/µL for SIV and 10 copies/µL for PRRSV and PCV2. Comparison with a national standard detection method using 110 clinical samples revealed 100% agreement, confirming the reliability of the newly developed assay for clinical diagnostic applications.

Conclusions

Given the zoonotic potential of SIV, this assay not only provides a valuable diagnostic tool for veterinary medicine but also contributes to enhanced public health surveillance efforts.

Peer Review reports

Background

The intensification of livestock farming in recent years has led to the widespread occurrence of porcine respiratory disease complex (PRDC), posing a significant challenging to its control. PRDC is primarily driven by viral pathogens that damage respiratory epithelial cells and lung tissues, predisposing animals to secondary bacterial pathogens [1]. SIV, PRRSV, and PCV2 are recognized as the main viral pathogens associated with PRDC [2]. Co-infections with these viral pathogens can negatively impact growth performance and reduce the efficacy of vaccines [3, 4].

SIV is globally distributed and represents a substantial threat to the swine industry due to its detrimental impact on animal health and productivity [5]. The zoonotic potential of SIV, including the transmission of swine-origin H1N1 influenza subtypes to humans, has resulted in human fatalities [6]. Subtypes such as H1N1, H1N2, and H3N2 are prevalent in swine populations worldwide, often leading to high morbidity (up to 100%) but relatively low mortality in pigs [7]. The conserved nature of matrix protein (M) gene makes it a common target for diagnostic primer and probe design [7].

PRRSV, a positive single-stranded RNA virus belonging to Arteriviridae family, can cause immunosuppression and significant economic losses in the swine industry [8, 9]. The high mutation rate and recombination frequency of PRRSV strains make it a persistent and challenging pathogen in global animal husbandry [10]. The matrix (M) and nucleocapsid (N) genes of PRRSV are relatively conserved compared to other genomic regions.

PCV2 is a small, non-enveloped icosahedral virus belonging to the genus Circovirus within the family Circoviridae [11]. The capsid (Cap) protein, the sole structural protein and principal immunogen of PCV2, is crucial for vaccine development and diagnostic assays [12, 13]. PCV2 infections can lead to immunosuppression and a spectrum of subclinical symptoms, increasing susceptibility to secondary infections and further compromising animal health [1, 14].

While single real-time fluorescent quantitative PCR (qPCR) assays exist for detecting SIV, PRRSV, and PCV2 [15,16,17], single-plex assays require separate reaction, limiting efficiency in terms of time and resources. Furthermore, a readily available, single quantitative assay for the direct simultaneous detection of these three pathogens is lacking. In this study, we designed specific primers and probes targeting M gene of SIV, the M and N genes of PRRSV, and the Cap gene of PCV2 to develop a triplex qPCR assay. This novel assay offers a robust and efficient approach for the diagnosis and surveillance of these important swine pathogens.

Materials and methods

Primers and probes design

Based on the relatively conserved regions of the M gene of SIV, the M and N genes of PRRSV, and the Cap gene of PCV2, gene sequences of these pathogens were retrieved from NCBI and aligned using MegAlign software to facilitate primers and probes design (Figure S1). Primers were designed using Premier 5 and Primer Express 3.0.1 software, targeting amplification lengths between 100 and 200 bp and incorporating distinct fluorophore wavelengths (Table 1). To confirm specificity, the designed primers and probes were validated using NCBI Primer-BLAST before synthesis.

Table 1 The primers/probes for detection in this study
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Viral nucleic acid extraction and reverse transcription

Viral DNA or RNA was extracted from clinical samples exhibiting respiratory symptoms using the TIANGEN Virus DNA/RNA Kit (Beijing, China). Briefly, nasal swabs and tissue samples were resuspended in 2 mL PBS, homogenized, and centrifuged at 5,000 rpm for 10 min. The resulting supernatant was used for viral nucleic acid extraction. Complementary DNA (cDNA) was then synthesized utilizing the 5×Prime Script RT Master Mix (for Real Time) from Takara (Beijing, China). Our laboratory’s nucleic acid collection, including classical swine fever virus (CSFV), pseudorabies virus (PRV), SIV, PRRSV, PCV2, Streptococcus suis (SS), and Haemophilus parasuis (HPS) was also used in this study. All nucleic acid samples were stored at -80 °C until use.

Standard plasmids construction

The M gene of SIV, the M and N genes of PRRSV, and Rep and Cap gene fragment of PCV2 were amplified using the primers listed in Table 2 and subsequently cloned into the pMD18-T Vector. The resulting plasmids were sequenced to verify the correct insertion of the target fragments. The concentrations of the plasmids were then quantified using a Thermo Scientific NanoDrop2000 spectrophotometers and calculated using the following formula: Plasmid copies/µL= (A260 ng/µL×10− 9 × 6.02 × 1023) /(plasmid length ×660). Three standard plasmids, serially diluted tenfold from 107copies/µL to 102 copies/µL, were used to generate standard curves.

Table 2 The primes for standard plasmids construction and phylogenetic analysis
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Optimization of the triplex qPCR

The triplex qPCR was conducted on the Applied Biosystems QuantStudio™ 5 instrumentation (Thermo Fisher Scientific) utilizing Premix Ex Taq™ enzyme (Takara Biotechnology, Dalian, China). The reaction mixture contained 10 uL of enzyme, 0.2 uL of ROX reference dye for normalization, and 1 uL of mixed plasmid templates containing the target genes. To optimize the assay conditions, we evaluated primer concentrations ranging from 0.1 to 0.4 µM and probe concentrations ranging from 0.2 µM to 0.6 µM. The cycling conditions consisted of an initial denaturation step at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at a temperature between 52 °C and 62 °C for 30 s. The optimal reaction conditions were determined based on a combination of achieving the lowest Ct values (cycle threshold) and the highest fluorescence intensity, while also considering cost-effectiveness. Standard plasmids with a known concentration of 1 × 10^7 copies/µL were used to optimize the reaction conditions.

Specificity, sensitivity, and repeatability assessment

To assess the specificity of the primers and probes used in this experiment, negative controls were selected to include viruses such as CSFV and PRV, as well as bacteria like HPS and SS, which are known to cause PRDC. Additionally, the genome of porcine alveolar macrophage (PAM) was used as a negative control, given that the nucleic acids extracted from clinical samples were derived from the cells of pigs exhibiting respiratory symptoms.

The sensitivity of the triplex qPCR was assessed under optimized reaction conditions by performing 10-fold serial dilutions of a standard plasmid, spanning a concentration range from 108 copies/µL to 1 copy/µL. To ascertain the limit of detection (LOD) for this assay, we conducted 23 replicate experiments using three specific dilutions of the standard plasmid: 100 copies/µL, 10 copies/µL, and 1 copy/µL. The LOD was defined as the concentration at which the standard plasmid consistently yielded a positive detection rate of 95%.

To evaluate the repeatability of the method, both intra-assay and inter-assay variability were evaluated by conducting experiments with four different concentrations of the standard plasmid: 106, 105, 104, and 103 copies/µL. Each concentration was tested in triplicate, and the entire assay was repeated three times using plasmids from different batches. The coefficient of variation (CV) was calculated for both in-batch and out-of-batch cycle threshold (Ct) values across all concentrations to quantitatively assess the reproducibility of the assay.

Clinical sample detection and phylogenetic analysis

A total of 110 clinical samples showing signs of respiratory tract disease were collected from the southwestern region of China. The nucleic acids extracted from these samples were tested using the triplex qPCR assay developed in this study. Subsequently, the extracted viral nucleic acids were also tested for various pathogens using the National Standard Detection Methods, as recommended. The primers and probes used in these assays are listed in Table S1.

The clinical samples consisted of both diseased tissues and nasal swabs. One portion of the samples was used for viral nucleic acid extraction and analysis via the triplex qPCR assay, while the other portion was sent for testing according to standard diagnostic methods. Since the experimental animals were either deceased or euthanized for other research purposes, no additional interventions or injuries were inflicted upon them. According to the Regulations on the Administration of Laboratory Animals in China, this study did not require review or approval by an ethics committee.

Some of the positive samples identified by the triplex qPCR assay were further analyzed by sequencing after amplification with a single sequencing primer. Representative strain sequences were retrieved from NCBI and used to construct a phylogenetic tree using MEGA6.4 software. The tree was generated using the neighbor-joining method and bootstrap sampling with 1000 replicates. To enhance the clarity of the phylogenetic tree, iTOL software was used for sequence visualization and analysis.

Result

Optimization of the triplex qPCR reaction system and conditions

To enhance the efficiency and cost-effectiveness of the triplex qPCR assay, we focused on optimizing key parameters, including primer and probe concentrations and the annealing temperature. Our primary objective was to minimize non-specific amplification while achieving lower Ct values. Among comparable conditions, those that exhibited higher fluorescence intensity and required lower amounts of primers and probes were preferred.

After conducting a series of experiments involving varying temperature gradients, an optimal annealing temperature of 60 °C was determined. To further refine the assay conditions, a matrix cross-test was performed, varying the primer concentrations between 0.1 and 0.4 µM and the probe concentrations between 0.2 and 0.6 µM. The optimized reaction system is summarized in Table 3.

Table 3 Triple fluorescence quantitative PCR reaction system
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Validation of the specificity of primers and probes used in the triplex qPCR assay

To evaluate the specificity of the primers and probes selected for the triplex qPCR assay, templates were extracted from positive controls, including PRRSV, PCV2, and SIV, as well as from negative controls, such as PRV, CSFV, HPS, SS, and PAM. The results demonstrated that the designed primers and probes effectively recognized and amplified the target gene fragments of the corresponding viruses. No amplification or fluorescent signal was observed for any unrelated pathogens (Table 4). These findings confirm that each primer-probe pair exhibits high specificity, making them well-suited for use in the triplex qPCR assay (Fig. 1).

Table 4 Specificity of the multiplex real-time PCR assay
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Fig. 1
figure 1

Specificity test of the triple qPCR assay. Amplification curves represent samples positive for PRRSV, SIV and PCV2 detected by the triple qPCR assay. Negative samples included CSFV, PRV, HPS, SS, PAM

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Establishment of standard curves for the triplex qPCR assay

Using the optimized reaction conditions, we established standard curves by selecting six different concentrations of plasmids, ranging from 107 to 102 copies/µL, diluted in 10-fold increments. The results showed the following coefficients of determination (R2) and amplification efficiencies (Eff%) for each pathogen: SIV (R2 = 0.997, Eff% = 91.562), PRRSV (R2 = 0.999, Eff% = 100.530), and PCV2 (R2 = 0.999, Eff% = 108.767). With R2 values close to 1 and amplification efficiencies within the optimal range of 90-110%, these results indicated a strong linear correlation between the template plasmid concentrations and the corresponding Ct values (Fig. 2). This demonstrates the reliability of the assay for quantitative analysis of nucleic acids in amplified samples.

Fig. 2
figure 2

Standard curve test of the triple triplex qPCR assay. A standard curve test of SIV; B standard curve test of PRRSV; C standard curve test of PCV2

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Evaluation of the triplex qPCR sensitivity

The sensitivity of the triplex qPCR experiments was evaluated using an optimized reaction system with varying concentrations of standard plasmids. The results of 23 replicates conducted at low concentrations are summarized in Table 5. At concentration of 100 copies/µL, the positive detection rate of the three pathogens was 100%, the assay achieved a positive detection rate of 100% for all three pathogens. When the concentration was reduced to 10 copies/µL, the positive detection rates were 52% for SIV, 96% for PRRSV, and 100% for PCV2. At the lowest concentration tested, 1 copy/µL, none of the pathogens were detected. The LOD for this experiment was defined as the lowest concentration at which the positive detection rate exceeded 95%. Accordingly, the sensitivity thresholds for the triple qPCR were determined to be 100 copies/µL for SIV, 10 copies/µL for both PRRSV and PCV2 (Fig. 3). At LOD, the Ct values ranged from approximately 34 to 36. A cutoff value of 36 was established, which indicates that samples with a Ct value of 36 or less are considered positive, while those with a Ct value above 36 are classified as negative.

Fig. 3
figure 3

Sensitivity test of the triple fluorescence quantitative PCR assay. A sensitivity test of SIV; B sensitivity test of PRRSV; C sensitivity test of PCV2

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Table 5 Sensitivity of triplex qPCR assay
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Assessment of repeatability in triplex qPCR assay

A series of repetitive tests were conducted using standard plasmids diluted from 106 to 103 copies/µL to assess the repeatability of the triplex qPCR assay. The evaluation was performed by calculating the mean values and standard deviations of the results. As shown in Table 6, the coefficient of variation (CV) for intra assay repeatability was ≤ 1%, while the inter CV was ≤ 5%. These results indicate that the triplex qPCR detection method demonstrates high stability and reliability.

Table 6 Repeatability test of the multiplex real-time PCR assay
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Assessment of co-infection in triplex qPCR assay

In co-infection analysis, standard plasmids were used to simulate mixed infections commonly observed in clinical samples (Fig. 4). The results demonstrated that the triplex qPCR was capable of detecting two or three pathogens simultaneously.

Fig. 4
figure 4

Co-infection simulation experiments with two or three pathogens. A PCV2 + SIV; B PCV2 + PRRSV; C PRRSV + SIV; D PCV2 + PRRSSV + SIV with the same concentration

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Detection of clinical samples

To evaluate the clinical applicability of the established method, we tested 110 clinical samples. The results revealed that 2.7% of the samples were positive for SIV, 10.9% for PRRSV, and 7.2% for PCV2 (Table 7). To confirm the accuracy of our detection method, all samples were subsequently analyzed using national standard detection method of China. The results showed a 100% concordance with our method, indicating that the assay is reliable for detecting pathogens in clinical samples.

Table 7 The detection of clinical samples
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Phylogenetic analysis

Some of the clinically positive samples tested were subjected to genetic analysis using primers of Table 2 to assess their evolutionary relationships. Using MEGA6.4, phylogenetic trees for PRRSV and PCV2 were constructed employing the neighbor-joining method and bootstrap analysis with 1000 repetitions. For PRRSV, a phylogenetic tree was generated that included the ORF5 sequences of 12 positive strains from this study, alongside 28 reference strains obtained from the GenBank database. The result showed that the sequences of the 12 positive samples were classified into two major lineages: lineage 1 (NADC30/34-like PRRSV) and lineage 8 (highly-pathogenic PRRSV). Notably, four out of seven samples in lineage 1 were closely related to sublinage 1.5 (NADC30-like PRRSV), and three clustered into sublinage 1.8 (NADC34-like PRRSV). For PCV2, a genetic evolution tree was constructed based on full-length sequences, incorporating 23 reference sequences from the GenBank database along with seven positive sequences identified in this study. The analysis indicated that the positive sequences belonged to subtypes PCV2b, PCV2c, PCV2d, and PCV2e. Among these, subtype PCV2d was predominant across all tested samples, suggesting that it may be the most prevalent subtype in this context (Fig. 5).

Fig. 5
figure 5

Phylogenetic analysis of PRRSV and PCV2. The GenBank accession number of the reference sequence are shown on each branch of the phylogenetic tree. Strains detected in this study are labeled with). (A) Phylogenetic tree of PRRSV strains. (B) Phylogenetic tree of PCV2 strains

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Discussion

Co-infection is a prevalent challenge in livestock farming, particularly affecting pig production through the interaction of bacterial and viral pathogens. A notable example of this phenomenon is PRDC, which is significantly influenced by viral pathogens, including SIV, PRRSV, and PCV2 [18]. Studies have shown higher detection rates of PRRSV and PCV2 in clinical samples related to PRDC, indicating their crucial roles in disease manifestation [1, 19]. Moreover, co-infections involving PRRSV and SIV can impair the efficacy of PRRSV vaccines, while the combination of PCV2 and SIV exacerbates clinical symptoms [20, 21]. Consequently, establishing accurate and efficient detection methods is imperative for the timely identification and control of these pathogens.

Current detection strategies are primarily categorized into pathogen-based and serology-based methods. Among pathogen-based approaches, nucleic acid detection technologies, such as PCR, are commonly employed to amplify pathogen-specific nucleic acid sequences. Multiplex fluorescent qPCR offers notable advantages over single PCR methods, including reduced reaction time and resource consumption by enabling the simultaneous detection of multiple pathogens in a single assay. This approach minimizes sample handling steps and lowers the risk of cross-contamination. For instance, a multiplex PCR and microarray system have been developed to differentiate four viruses and four bacteria associated with PRDC [22].While this method demonstrates high specificity, its sensitivity is comparatively lower than the method introduced in this study. In addition to multiplex techniques, single and dual PCR methods are also utilized for detecting pathogens linked to porcine respiratory diseases [23, 24].Dual nested PCR for simultaneous detection of both PCV2 and PRRSV pathogens have been developed [25]. However, this method increases the risk of cross-contamination due to the need to perform two PCR reactions, and the double-stranded nested PCR involves more steps and requires the design of two pairs of primers compared to a single PCR. Although the Point-of-Care and Label-Free assays applied to the field detection of PRRSV and SIV viruses show good sensitivity and specificity, they are limited to two respiratory pathogens [26]. Additionally, existing detection methods require updates to address the rapid emergence of new subtypes, such as NADC30-like and NADC34-like PRRSV strains, PCV2f subtype, and G4EA H1N1 subtype [27, 28]. Thus, this study aimed to develop a method capable of detecting three respiratory disease-related pathogens while also accommodating recently prevalent strains. Primers and probes were designed to target conservative regions of new subtypes, shown in supplementary material. The reaction conditions, including temperature and concentrations of primers and probes, were optimized to enhance amplification efficiency and reduce fluorescence interference. Notably, the sensitivity of SIV detection was lower within the optimized system, likely due to interference from other fluorescence signals. Sensitivity testing established minimum detection limits of 100 copies/µL for SIV, and 10 copies/µL for both PRRSV and PCV2, demonstrating good sensitivity compared to established methods [29]. For sensitivity test, the selected analogue items are constructed standard plasmids, viral nucleic acids extracted directly from the virus and the addition of standard plasmids and viruses to negative material for sensitivity test. However, all of these have certain drawbacks, nasal swabs or even more tissue homogenates are more complex than standard plasmids and viral nucleic acids; the reference standard of negative patient material and the actual clinical negative and positive samples have certain differences, and can not completely simulate the actual clinical samples. Therefore, sensitivity test results may differ slightly from the sensitivity of clinical samples.Specificity testing confirmed that this triplex real-time PCR assay accurately detects SIV, PRRSV, and PCV2 without cross-reactivity to other pathogens in the analyzed clinical samples. Additionally, intra- and inter-batch stability tests yielded coefficients of variation below 5%, indicating the method’s reliability for clinical application [30].

The triplex qPCR detection method was evaluated using clinical samples collected from pig farms in China in 2023, where signs of respiratory tract disease were present. The results indicated positivity rates of 10.9% for PRRSV, 7.2% for PCV2, and 2.7% for SIV, with co-infections observed in 2.7% of cases involving PRRSV and PCV2, and 1.8% with all three pathogens. The high prevalence of PRRSV aligns with previous findings [19]. In contrast, a study of Italian pig farms reported significantly higher positivity rates for PCV2 [31]. The variation in pathogen prevalence affecting swine respiratory diseases necessitates continuous monitoring and timely intervention strategies. Furthermore, clinical samples were verified against national standards, achieving a compliance rate of 100%, affirming the applicability of triplex qPCR for clinical diagnostics. To further characterize positive samples, a genetic evolution analysis was performed, revealing that PRRSV positive samples in this study were classified as PRRSV-2 type 1 and 8 lineages. This aligns with the prevailing PRRSV-2 genotype in China, particularly lineages 1, 3, 5, and 8 [32]. The phylogenetic analysis of PCV2 identified subtypes PCV2b, PCV2c, PCV2d, and PCV2e, highlighting the virus’s genetic variability and propensity for recombination [31, 33]. The detection of multiple virus subtypes underscores the potential risks to pig productivity, emphasizing the necessity for timely and accurate testing to mitigate such threats.

Conclusions

In summary, our study developed a method to detect respiratory diseases in pigs early. The triplex qPCR assay, developed in this study, can detect three main pathogens including PRRSV, PCV2 and SIV without interference from other pathogens. This method is quick, flexible, and sensitive, making it a reliable tool for early diseases detection. Using this test helps us catch these diseases early, which helps manage them better. Moreover, it keeps pork safe by ensuring it’s free from these pathogens, which is good for consumers and the pork industry.

Data availability

Data is provided within supplementary information files.

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Acknowledgements

Not applicable.

Funding

This study was supported by Grants from the Central Government-guided Local Science and Technology Development Project (ZY04JD03), National Center of Technology Innovation for Pigs (NCTIP-XD/C09), Central Public-interest Scientific Institution Basal Research Fund (1610302022018).

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Authors and Affiliations

  1. State Key Laboratory for Animal Disease Control and Prevention, National Poultry Laboratory Animal Resource Center, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China

    MengJie Wang, Yu Pan, Wengjie Ma, Xingrong Wang, Changyou Xia & Yue Wang

  2. College of Veterinary Medicine, Southwest University, Chongqing, 400715, China

    MengJie Wang, Xingrong Wang & Yue Wang

Authors
  1. MengJie Wang
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  2. Yu Pan
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  3. Wengjie Ma
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  4. Xingrong Wang
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  5. Changyou Xia
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  6. Yue Wang
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Contributions

MW: Investigations, data curation, data analysis, original draft preparation. YP: Investigations, data curation, review and editing. WM: Data curation, review and editing. XW: Data curation, review and editing. HC: Supervision and funds acquisition. CX: Conceptualization, review and editing, funds acquisition. YW: Conceptualization, supervision, review and editing, funds acquisition.

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Correspondence to Changyou Xia or Yue Wang.

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Wang, M., Pan, Y., Ma, W. et al. Development of a triplex TaqMan probe-based real-time PCR assay for simultaneous detection of swine influenza virus, porcine reproductive and respiratory syndrome virus, and porcine circovirus type 2. BMC Vet Res 21, 297 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12917-025-04688-x

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Keywords

BMC Veterinary Research

ISSN: 1746-6148

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