Development of a droplet digital PCR method for detection of porcine circovirus 4
BMC Veterinary Research volume 19, Article number: 129 (2023)
Porcine circovirus 4 (PCV4), a newly emerging virus that was first discovered in 2019, may pose a potential threat to the pig industry. Droplet digital PCR (ddPCR) is an absolute quantitative method that has high sensitivity and accuracy. In this study, we developed a novel ddPCR assay to detect PCV4. Furthermore, we evaluated the detection limit, sensitivity, specificity and reproducibility of the ddPCR and TaqMan real-time quantitative PCR (qPCR) and tested 160 clinical samples to compare the detection rate of the two methods.
The detection limit for ddPCR was 0.54 copies/µL, 10.6 times greater sensitivity than qPCR. Both ddPCR and qPCR assays exhibited good linearity and repeatability, and the established ddPCR method was highly specific for PCV4. The results of clinical sample testing showed that the positivity rate of ddPCR (5.6%) was higher than that of qPCR (4.4%).
This study successfully developed a sensitive, specific and repeatable ddPCR assay for PCV4 detection, which can be widely used in clinical diagnosis of PCV4 infections.
Porcine circoviruses (PCVs) are non-enveloped, circular single-stranded DNA viruses, which belong to the family Circoviridae, genus Circovirus. Until 2019, only three types of PCVs have been characterized, named PCV1, PCV2, and PCV3. PCV1 is a non-pathogenic virus derived from the porcine kidney cell line PK-15 . PCV2 is confirmed to be the primary causative agent of porcine circovirus-associated diseases (PCVAD), resulting in huge economic losses to the global pig industry . PCV3 was first identified in sows with porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs in 2015 in the United States and then in different countries around the world .
In 2019, a distinct novel PCV, designated PCV4, was discovered in the Hunan province of China in pigs with PDNS, respiratory and enteric signs . Subsequently, PCV4 infections have been reported in Jiangsu, Anhui, Henan, Shanxi, Inner Mongolia and other provinces of China [5,6,7,8], as well as in South Korea , which indicate that PCV4 has a widespread epidemic trend and may be a potential threat to the global pig industry. To better investigate the epidemiology of PCV4, several diagnostic methods, including conventional PCR (PCR) , loop-mediated isothermal amplification (LAMP) , multienzyme isothermal rapid amplification (MIRA) , real-time quantitative PCR (qPCR) [6, 11] and enzyme-linked immunosorbent assay (ELISA) , have been developed for the detection of PCV4 infection. However, these methods may lack specificity and sensitivity, or do not allow direct quantification of viral DNA, thus rendering them unsuitable for routine diagnosis in the early stages of viral infection. Therefore, the development of a rapid, simple, and reliable diagnostic method is imperative for managing PCV4.
Droplet digital PCR (ddPCR) is an innovative third-generation PCR technology for absolute quantification of nucleic acids without the requirement of a standard curve . The ddPCR uses the same target-specific primers and fluorescent probe as TaqMan-based qPCR. In ddPCR, the reaction mixture is partitioned into tens of thousands to millions water-in-oil droplets prior to massive parallel PCR amplification. At end point, each droplet is classified as positive or negative based on the recorded fluorescence signal, and the positive fraction of counted droplets is employed to calculate the target copy number using Poisson algorithms [13, 14]. The ddPCR method has been demonstrated to have higher sensitivity and specificity than qPCR, especially when the quantity of the target is very low [15,16,17]. At present, ddPCR has been used widely in the detection and quantification of a range of microorganisms, including other circoviruses, such as porcine circovirus type 2 (PCV2) , porcine circovirus type 3 (PCV3)  and pigeon circovirus (PiCV) . However, no ddPCR assay is currently available for PCV4. In this study, a ddPCR assay was developed for detection and quantification of PCV4 in serum samples of pig. Furthermore, the sensitivity, specificity and repeatability of the PCV4 ddPCR assay was compared with qPCR.
Materials and methods
Plasmids, viruses and field samples
To prepare the standard positive control, the whole genome of PCV4-LY2020 (Accession no. MW759026) was synthesized and cloned into pBluescript II SK (+) vector. The recombinant plasmid named pSK-PCV4 was transformed into in E. coli DH5α and subsequently purified with a Plasmid MiniPrep Kit (OMEGA Biotech, Shanghai, China). The DNA concentration of the plasmid construct pSK-PCV4 was quantified using the NanoDrop 2000 spectrophotometer (Thermo Fisher, Waltham, MA, USA). The estimated copy number of the pSK-PCV4 plasmid in solution was calculated using methods described previously .
Classical swine fever live vaccine (CSFV, strain CVCC AV1412), and porcine reproductive and respiratory syndrome live vaccine (PRRSV, JXA1-R strain) were purchased from Wuhan Keqian Biology Co., Ltd. and stored in our lab. Porcine epidemic diarrhoea virus (PEDV) LYL strain (Accession no. ON960076), porcine rotavirus (PoRV) CC0812 strain (Accession no. JF835112), PCV2 SH strain (Accession no. HM038027), and porcine circovirus type 3 (PCV3; Accession no. MZ449239) culture media were stored in our laboratory. One hundred and sixty samples (105 blood samples, 55 tissue samples) were collected from 24 pig herds located in five cities (Nanyang, Zhengzhou, Pingdingshan, Xinyang and Luoyang) of Henan province from September 2019 to July 2022. Informed consent from the herd´s owners have been obtained to collect the samples used in this study.
Primers and probe for PCV4 droplet digital PCR
According to the genomic sequence of the PCV4 strains listed in GenBank, the conserved sequences of PCV4 ORF2 gene was analyzed using MEGA 6.0 software. One probe and a pair of specific primers were prepared subsequently. The primer and probe sequences were as follows: PCV4-F (5’- CGTTCCAAGAGGGCGTG − 3’), PCV4-R (5’-GCCAGTAGGCGGAGATACC-3’), and PCV4-P (FAM-5’- ACCTCCC.
TCATGAAGCGCGCA-3’-BHQ1). All primers and probes were synthesized by Sangon Biotech (Shanghai, China).
Nucleic acid extraction and reverse transcription
Viral RNA from PEDV, PoRV, PRRSV and CSFV were extracted using the RNA Viral Genome Extraction Kit (Solarbio Biotech Co., Ltd., Beijing, China), following the instructions of the manufacturer. Each viral RNA was employed for the synthesis of the first strand cDNA in a 20 µL reverse transcription (RT) reaction mixture containing 1 µg of total RNA, 4 µL of 5 × AMV buffer, 2 µL of dNTPs (2.5 mmol/L), 0.5 µL of RNase Inhibitor (40 U/µL), 1 µL of random primer, 1 µL of AMV reverse transcriptase (5 U/µL), and RNase-freeH2O and then incubated at 42 °C for 60 min and 95 °C for 5 min (Sigma-Aldrich, St. Louis, MO, USA). Viral DNA was extracted from PCV2 and PCV3 and using a DNA Viral Genome Extraction Kit (Solarbio, Beijing, China).
Droplet digital PCR (ddPCR) assay
The ddPCR assay for PCV4 was performed in a TD-1 Droplet Digital PCR system (TargetingOne, Beijing, China) following manufacturer’s instructions. The reaction volume was 20 µL, containing 10 µL of 2 × ddPCR Supermix (TargetingOne, 23,003), 800 nM of each primer PCV4-F/R, 250 nM of PCV4-P probe, and 2 µL of the template. The reaction mixture and 180 µL oil were placed in a droplet generator, followed by heat-sealing for PCR. In order to optimize the annealing temperature, the amplification reaction protocol was as follows: 95 °C for 10 min, 40 cycles at 94 °C for 30 s and a temperature gradient from 55 to 61 °C for 1 min; the temperature ramp rate was set to 1.5 °C/s on a T100 thermal cycler (TargetingOne, Beijing, China). Finally, the droplets were analyzed using a chip reader (TargetingOne, Beijing, China). Then, ddPCR was optimized for primer and probe concentrations (300:200 nm, 800:250 nm, 500:300 nm and 400:400 nm). The ddPCR was performed in triplicate.
The qPCR assay for PCV4 was performed with the same primers and probe as ddPCR. The PCR was performed in 20 µL volume, including 10 µL of 2 × TaqMan™ Fast Advanced Master Mix (Thermo Scientific, Waltham, MA, USA), 1.6 µL of each primer (10 µM), 0.5 µL of probe (10 µM), 2 µL of the template and 4.3 µL of ddH2O. The PCR was conducted as follows: 50 °C for 2 min, 95 °C for 2 min, 40 cycles at 95 °C for 20 s and 57 °C for 20 s.
Analytical sensitivity and repeatability
Dilutions of pSK-PCV4 plasmid ranging from calculated 2.0 × 105 to 2.0 × 101 copies per µL were used in analytical sensitivity determination of the ddPCR and qPCR assays. Two microliter of each plasmid dilution was used as template to ascertain the detection limit (LoD), which represents as the highest dilution detected by each PCR assay. To ensure the assay result accuracy, inter-assay and intra-assay repeatability tests were performed three times independently.
To investigate specificity, assays including PCV4, PCV3, PCV2, CSFV, PEDV, PoRV, and PRRSV as templates were evaluated. Nuclease-free water was used in place of samples as no template control.
Clinical sample detection by qPCR and ddPCR assays
To evaluate the applicability of the method, a total of 160 clinical samples from the pigs without any symptoms were assayed using the above ddPCR and qPCR procedure in parallel. In qPCR, any sample that has a Ct value more than 40 was considered as negative. Samples giving inconsistent PCR results were further verified by sequencing.
Development of a PCV4 ddPCR assay
For ddPCR, annealing temperature gradients from 55 to 61 °C were performed to optimize the separation between positive and negative partitions. The results indicated that 59 °C provided the greatest difference in the fluorescence signal between the positive and negative droplet populations (Fig. 1), thus it was chosen as the optimal annealing temperature. To further determine if the ddPCR system for PCV4 could be improved, the primer-to-probe concentration was optimized. The results suggested that the optimal concentration ratio was 300:200 nM because this ratio of reagents resulted in optimal separation between positive and negative droplet populations (Fig. 2).
Analytical sensitivity and reproducibility
Assays with serially diluted pSK-PCV4 plasmid solution exhibited good linearity in both qPCR and ddPCR. In qPCR, the standard curve exhibited a good linear correlation (Y = − 3.52X + 48.71) with R2 value of 0.9935 (Fig. 3A), the detection limit was 5.71 copies/µL (Table 1). In contrast, the standard curve of the ddPCR assay was Y = 1.01x − 1.56 with R2 value of 0.9996 (Fig. 3B), the LoD was 0.54 copies/µL (Table 1). The results revealed that the LoD of ddPCR was ~ 10.6-fold lower than that of qPCR, which indicated that ddPCR was significantly more sensitive for PCV4 detection. In the repeatability tests, the intra-assay coefficient of variation ranged from 1.22 to 3.70%, and the coefficient of variation of the inter-assay ranged from 2.79 to 7.57% (Table 2). These results showed that the developed PCV4 ddPCR has a good reproducibility.
Analytical specificity of the ddPCR assay
For the specificity analysis, nucleic acid templates from different pathogens were assayed, including PCV4, PCV3, PCV2, PEDV, PoRV, CSFV and PRRSV. As shown in Fig. 4, only the PCV4 test was positive, while other pathogen tests were negative. The results indicated that this method exhibits specificity for the detection of PCV4.
Clinical samples testing
To further determine if ddPCR can be performed in a routine involving real world subjects, 160 clinical samples collected from 24 pig farms in Henan Province were evaluated using ddPCR and qPCR. As shown in Table 3, PCV4 was detected with a positive rate of 4.4% (7 of 160) by ddPCR and 5.6% (9 of 160) by qPCR. Two samples detected as negative by qPCR were positive by ddPCR. The amplicons from samples giving conflicting positive results were further sequenced by Sangon Biotech Co. Ltd. (Beijing, China), and the sequencing results confirmed that the two samples were positive for PCV4. According to these data, ddPCR was found to be more sensitive than qPCR for PCV4 detection in clinical samples.
Since its discovery in 2019, PCV4 has been detected in pigs of all ages and in both clinically healthy and on diseased pigs [21,22,23]. A latest study showed that PCV4 was pathogenic to piglets after challenge with the virus generated from infectious clones , indicating that PCV4 may pose a potential threat to the pig industry. To date, PCV4 has not been isolated from clinical samples, which severely hinders the in-depth research of the epidemiology and pathogenic mechanism of the virus infection. To monitor PCV4 continuously and effectively, several etiological and serological methods have been developed and played an important role in the diagnosis of PCV4 infection. However, these methods are time-consuming, complex to operate, and unsuitable for samples with low virus load . Therefore, it is urgently needed to develop a rapid, simple and sensitive detection method for PCV4.
The ddPCR is emerging as an attractive platform that enables absolute quantification of nucleic acid targets without relying on the establishment of a standard curve as required in qPCR. The ddPCR technology does not depend on a standard curve and the reaction is efficient and highly sensitive, thus it has been used to detect a variety of diseases and is especially useful for low viral load samples [16, 17, 26]. In addition, ddPCR is highly tolerant to many PCR inhibitors, making it more suitable for the detection of complex clinical samples such as blood and faeces . Because of these features of high sensitivity, absolute quantification and high reproducibility, ddPCR has been widely used for viral load quantification [28, 29], mutant genes detection , target verification following genome editing , copy number variations analysis , etc.
In this study, a sensitive and specific ddPCR method for detection and quantification of PCV4 was successfully established. Meanwhile, a qPCR assay, which used the same primers and probe as ddPCR was also developed to cross-validate both assays. To evaluate linearity, sensitivity, and repeatability of ddPCR and qPCR, serially diluted pSK-PCV4 plasmid solution were prepared in triplicate, and then used to conduct parallel tests. The results indicated that both ddPCR and qPCR exhibited good linearity, with R2 values of 0.9996 and 0.9935, respectively. The LoD of ddPCR and qPCR were 0.54 copies/µL and 5.71 copies/µL, respectively. Indicating that the sensitivity of the ddPCR assay was 10.6 times higher than that of qPCR, which is consistent with the findings from PCV2 and PCV3 ddPCR assay [18, 19]. Plasmid standard dilutions with different copy numbers were also used to evaluate the robustness and reproducibility of the ddPCR assay. The results showed that the intra- and inter-assay coefficients of variation (CV) for concentration (copies/µL) were 1.22 to 3.70% and 2.79 to 7.57%, respectively, which indicated that the robustness and repeatability of the ddPCR reaction system was high. Additionally, the ddPCR assay exhibited high specificity, presenting no cross-reactivity signals with other common swine pathogens such as PCV3, PCV2, PEDV, PRRSV, CSFV and PoRV. These advantages make the PCV4 ddPCR assay more suitable for the early detection of PCV4 infection.
Subsequently, the method was used for the detection of PCV4 in clinical samples to evaluate the practicability of ddPCR and qPCR. The qPCR-positive detection rate was 4.4%, while PCV4 ddPCR exhibited a greater positive (5.6%). Different results were obtained from ddPCR and qPCR, indicating that the sensitivity of the ddPCR method was higher than that of qPCR. In addition, all the positive samples were collected from the pigs without any symptoms, indicating that PCV4 could cause subclinical infection and cofactors may be essential for the virulence of PCV4. Thus, ddPCR is a specific, sensitive and rapid-detection method of high clinical significance for the early infection diagnosis and a potential epidemic tracking tool for PCV4 in pig farms.
Despite the advantages described above, the utility of the ddPCR assay is limited by several factors. First, the ddPCR technology is not widely available in veterinary clinical diagnosis due to its high cost. Second, the ddPCR method started to lose linearity when the initial concentration of the nucleic acid templates was higher than 1 × 105 copies/µL, thereby presenting a relatively narrow linear dynamic range compared with the qPCR assay [33, 34]. Third, the controls used in the ddPCR assay might be not precise representative of the target template, leading to the underestimation of the true sample concentration. In conclusion, further efforts are necessary to develop more accurate and standardized approaches for improving the ddPCR assays.
We first established and evaluated a droplet digital PCR assay for rapid and accurate detection of PCV4. PCV4 ddPCR exhibits higher sensitivity compared with qPCR, and it was analytically specific and reproducible, making it a reliable tool for the diagnosis and epidemiological investigation of PCV4.
The data presented in this manuscript are available through the corresponding author upon reasonable request. The datasets generated and/or analysed during the current study are available in the GenBank repository, accession numbers: PEDV strain LYL: ON960076; PoRV strain CC0812: JF835112; PCV2 strain SH: HM038027; PCV3: MZ449239.
Porcine circovirus 4
Droplet digital PCR
Real-time quantitative PCR
Porcine circovirus-associated disease
Porcine dermatitis and nephropathy syndrome
Porcine circovirus type 2
Porcine circovirus type 3
Porcine epidemic diarrhoea virus
Classical swine fever virus
Porcine reproductive and respiratory syndrome virus
Tischer I, Gelderblom H, Vettermann W, Koch MA. A very small porcine virus with circular single-stranded DNA. Nature. 1982;295:64–6.
Opriessnig T, Meng XJ, Halbur PG. Porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, intervention strategies. J Vet Diagn Invest. 2007;19:591–615.
Palinski R, Piñeyro P, Shang P, Yuan F, Guo R, Fang Y, et al. A novel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure. J Virol. 2017;91:e01879–16.
Zhang HH, Hu WQ, Li JY, Liu TN, Zhou JY, Opriessnig T, et al. Novel circovirus species identified in farmed pigs designated as porcine circovirus 4, Hunan province, China. Transbound Emerg Dis. 2020;67:1057–61.
Lian Z, Liu J, Liu P, Zhu Z, Yao X, Yuan L, et al. Development and application of an indirect ELISA for the detection of antibody to porcine circovirus 4 in pigs. Transbound Emerg Dis. 2021;68(6):2975–9.
Zhang D, Bai C, Ge K, Li Y, Gao W, Jiang S, et al. Establishment of an SYBR Green-based real-time PCR assay for porcine circovirus type 4 detection. J Virol Methods. 2020;285:113963.
Ha Z, Yu C, Xie C, Wang G, Zhang Y, Hao P, et al. Retrospective surveillance of porcine circovirus 4 in pigs in Inner Mongolia, China, from 2016 to 2018. Arch Virol. 2021;166:1951–9.
Tian RB, Zhao Y, Cui JT, Zheng HH, Xu T, Hou CY, et al. Molecular detection and phylogenetic analysis of porcine circovirus 4 in Henan and Shanxi provinces of China. Transbound Emerg Dis. 2021;68:276–82.
Kim DY, Kim HR, Park JH, Kwon NY, Kim JM, Kim JK, et al. Detection of a novel porcine circovirus 4 in korean pig herds using a loop-mediated isothermal amplification assay. J Virol Methods. 2022;299:114350.
Li Y, Zhao Y, Li C, Yang K, Li Z, Shang W, et al. Rapid detection of porcine circovirus type 4 via multienzyme isothermal rapid amplification. Front Vet Sci. 2022;9:949172.
Chen W, Jiang D, Xiao L, Zhang P, Luo Y, Yang Z, et al. Development of a real-time TaqMan PCR assay for the detection of porcine circovirus 4. J Vet Res. 2022;66(1):29–33.
Zhang Y, Qu S, Xu L. Progress in the study of virus detection methods: the possibility of alternative methods to validate virus inactivation. Biotechnol Bioeng. 2019;116:2095–102.
Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Hindson BJ, et al. Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods. 2013;10(10):1003–5.
Hudecova I. Digital PCR analysis of circulating nucleic acids. Clin Biochem. 2015;48(15):948–56.
Verhaegen B, De Reu K, De Zutter L, Verstraete K, Heyndrickx M, Van Coillie E. Comparison of droplet digital PCR and qPCR for the quantification of Shiga toxin-producing Escherichia coli in bovine feces. Toxins (Basel). 2016;8(5):157.
Taylor SC, Laperriere G, Germain H. Droplet digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Sci Rep. 2017;7(1):2409.
Suo T, Liu X, Feng J, Guo M, Hu W, Guo D, et al. ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load specimens. Emerg Microbes Infect. 2020;9(1):1259–68.
Zhao S, Lin H, Chen S, Yang M, Yan Q, Wen C, et al. Sensitive detection of porcine circovirus-2 by droplet digital polymerase chain reaction. J Vet Diagn Invest. 2015;27(6):784–8.
Liu Y, Meng H, Shi L, Li L. Sensitive detection of porcine circovirus 3 by droplet digital PCR. J Vet Diagn Invest. 2019;31(4):604–7.
Stenzel T, Dziewulska D, Tykałowski B, Koncicki A. The clinical infection with pigeon circovirus (PiCV) leads to lymphocyte B apoptosis but has no effect on lymphocyte T subpopulation. Pathogens. 2020;9(8):632.
Liu H, Shi K, Zhao J, Yin Y, Chen Y, Si H, Qu S, Long F, Lu W. Development of a one-step multiplex qRT-PCR assay for the detection of african swine fever virus, classical swine fever virus and atypical porcine pestivirus. BMC Vet Res. 2022;18(1):43.
Hou CY, Zhang LH, Zhang YH, Cui JT, Zhao L, Zheng LL, et al. Phylogenetic analysis of porcine circovirus 4 in Henan Province of China: a retrospective study from 2011 to 2021. Transbound Emerg Dis. 2022;69(4):1890–901.
Chen N, Xiao Y, Li X, Li S, Xie N, Yan X, et al. Development and application of a quadruplex real-time PCR assay for differential detection of porcine circoviruses (PCV1 to PCV4) in Jiangsu province of China from 2016 to 2020. Transbound Emerg Dis. 2021;68:1615–24.
Sun W, Du Q, Han Z, Bi J, Lan T, Wang W, et al. Detection and genetic characterization of porcine circovirus 4 (PCV4) in Guangxi, China. Gene. 2021;773:145384.
Niu G, Zhang X, Ji W, Chen S, Li X, Yang L, et al. Porcine circovirus 4 rescued from an infectious clone is replicable and pathogenic in vivo. Transbound Emerg Dis. 2022;69(5):e1632–41.
Frías M, Rivero-Juárez A, Téllez F, Palacios R, Jiménez-Arranz Á, Pineda JA, et al. Evaluation of hepatitis C viral RNA persistence in HIV-infected patients with long-term sustained virological response by droplet digital PCR. Sci Rep. 2019;9(1):12507.
Dingle TC, Sedlak RH, Cook L, Jerome KR. Tolerance of Droplet-digital PCR vs real-time quantitative PCR to inhibitory substances. Clin Chem. 2013;59(11):1670–2.
Ruelle J, Yfantis V, Duquenne A, Goubau P. Validation of an ultrasensitive digital droplet PCR assay for HIV-2 plasma RNA quantification. J Int AIDS Soc. 2014;17(4 Suppl 3):19675.
Sanders R, Huggett JF, Bushell CA, Cowen S, Scott DJ, Foy CA. Evaluation of digital PCR for absolute DNA quantification. Anal Chcm. 2011;83:6417–84.
Pinheiro LB, Coleman VA, Hindson CM, Herrmann J, Hindson BJ, Bhat S, et al. Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem. 2012;84:1003–11.
Ling L, Michael S, Rachel C, Damien B, John W, Howard S. A powerful new approach to measuring engraftment using copy number variations and ddPCR, exemplified in a complex allogeneic bone marrow transplantation case. Pathology. 2015;47:87.
Zhang Y, Tang ET, Du Z. Detection of MET gene copy number in cancer samples using the ddPCR method. PLoS ONE. 2016;11(1):e0146784.
Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem. 2011;83(22):8604–10.
Zhang Z, Zhang Y, Lin X, Chen Z, Wu S. Development of a novel reverse transcription droplet digital PCR assay for the sensitive detection of Senecavirus A. Transbound Emerg Dis. 2019;66(1):517–25.
The authors gratefully acknowledge the staff at the TargetingOne Corporation for their help in providing technical support.
This work was financially supported by the National Natural Science Foundation of China (No. 32002302 and 31870917), the fund of Scientific and Technological research project of Henan province (No. 202102110097) and the Foundation of Nanyang Normal University (No. 2017ZX012). The funding bodies played no role in the design of the study, the collection, analysis, and interpretation of data and in writing the manuscript.
Ethics approval and consent to participate
The samples used in this study were collected in different pig farms. The written consents for the use of the samples before participation in the study were obtained from the farmers. The study was carried out in compliance with the ARRIVE guidelines. All methods were carried out in accordance with Chinese Law for the Care and Use of Animals. The research protocol was approved by the Animal Welfare and Ethics Committee of Nanyang Normal University (approval no. No 21015, year: 2021).
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Cq values (qPCR) and target copy number (ddPCR) of PCV4 positive clinical samples.
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Liu, Y., Zhang, X., Han, X. et al. Development of a droplet digital PCR method for detection of porcine circovirus 4. BMC Vet Res 19, 129 (2023). https://doi.org/10.1186/s12917-023-03690-5
- Porcine circovirus 4
- Droplet digital PCR
- Real-time quantitative PCR