Porcine Epidemic Diarrhea Virus (PEDV), Porcine Parvovirus (PPV), Porcine Pseudorabies Virus (PRV), Transmissible Gastroenteritis Virus (TGEV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), PCV2 strains were isolated and preserved by the National Pig Breeding Swine Disease Control Laboratory of the National Pig Breeding Industry Engineering Technology Research Center; the positive nucleic acid of PCV3 was derived from the lung samples of pigs in Kaiping, Guangdong. From June to August 2021, clinical samples (stool, gut, and other materials) were collected from 138 cases of diarrhea pigs from 20 different farms in Guangdong, Jiangxi, and Hainan provinces. The detailed information of clinical samples is shown in Table 3.

The samples were homogenized with sterilized PBS. The supernatant was taken after centrifugation; the RaPure Viral RNA/DNA Kit (Magen, Shanghai, China) was used to extract viral nucleic acid. Using the PrimeScript™ reagent Kit with gDNA Eraser (Takera, China), the viral RNA (TGEV, PRRSV, and PEDV) was reverse transcribed for cDNA. The experimental procedures are strictly conducted in accordance with the product instructions.

The nucleotide sequences of PEDV (GenBank: MK458329.1), PPV (GenBank: AF257079), TGEV (GenBank: U26210.1), PRV (GenBank: AF257079), PRRSV (GenBank: EU926974.1), PCV2 (GenBank: HM038024.1), and PCV3 (GenBank: MH491016.1) were downloaded from GenBank. Primers are designed on the Primer3website and Synthesized by Sangon company (Shanghai, China). Porcine circovirus-like virus (PCLV) ORF4 sequences with fewer mutations were selected for primer design by comparing published PCLV genes (Figure 7). The Basic Local Alignment search tool was used to validate all primers. Primer sequence as shown in Table 1.

As a template, nucleic acids isolated from PCLV positive clinical samples were employed. PCLV-ORF4-F1 (EcoRI) and PCLV-ORF4-R1 (KpnI) primers with restriction sites were used for PCR amplification according to the extracted template, and the length of PCLV ORF4 with 597 bps was obtained and cloned into PCMV-HA (N) vector.

The qPCR detection system for PCLV was 2× Taq PCR Master Mix II (Tiangen, China) 12.5 μl, ddH₂O 8.5 μl, PCLV-F primer 1 μl (10 mM), PCLV-R primer 1 μl (10 mM), and template 2 μl. PCR was performed under the following conditions: after treatment at 95°C for 5 min, 35 cycles of 95°C for 15 s, 56°C for 30 s, 72°C for 60 s, then 72°C for 10 min, 12°C for 5 min, 1% agarose gel electrophoresis was used to validate PCR products.

Eastep® qPCR Master Mix (2×; promega, USA) was used to perform the real-time PCR assay and conducted in the Applied BiosystemsStepOnePlus Real-Time PCR System (Life Technologies). The assay system includes 10 μl of Eastep® qPCR Master Mix (2×), 2 μl of the template, 4 μl of 10-mM PCLV-F2 primer and PCLV-R2 primer, 7 μl of nuclease-free water, 2 μl of CXR 100 x^*, 95°C for 120 s, 40 cycles of 95°C for 15 s; finally, 60°C for 40–60 s. In the range of 65–95°C. The melting curve was analyzed by collecting a SYBR green fluorescent signal.

The standard curves for qPCR were established using 1.7 × 10⁸-1.7 × 10² copies/μl PCMV-HA(N)-PCLV-ORF4 at different concentrations. Each group of samples was repeated three times, and the corresponding Ct value was measured by collecting the fluorescence signal, and then, consequently, the amplification curve was obtained. The standard curve was drawn with the Ct value of each plasmid concentration as the ordinate and the logarithm of standard plasmid copy number as the abscissa.

The sensitivity of this assay was compared with that of the conventional PCR method. The constructed PCMV-HA (N)-PCLV-ORF4 plasmid and the positive sample nucleic acid were diluted 10 times for detection. The detection limit of real-time quantitative PCR was defined as the minimum plasmid concentration and minimum nucleic acid dilution concentration of the positive sample that can be quantified and amplified by this method and maintained within the linear range of the standard curve. The limit of detection (LoD) of conventional PCR was the lowest plasmid concentration observed in the band detected by agarose gel electrophoresis. The lowest detection concentration of the two methods was compared.

In order to explore the specificity of this method, the ddH₂O was used as the negative control, the DNA of PPV, PCV2, PCV3, and PRV, and the cDNA of TGEV, PRRSV, and PEDV were used as templates for amplification.

Using plasmid PCMV-HA(N)-PCLV-ORF4 as a template, the reproducibility of SYBR Green real-time fluorescence quantitative PCR was evaluated with three concentration gradients (1.7 × 10⁸ copies/μl, 1.7 × 10⁶ copies/μl, and 1.7 × 10⁴ copies/μl). The assay was performed in three periods with five repeats per sample. By analyzing the variation of Ct values in different replicates, the coefficient of variation (CV) was calculated by dividing the standard deviation (SD) by the mean.

The accuracy of PCLV detection in clinical samples was based on this method. We used the RaPure virus RNA/DNA kit (Magen, China) to extract viral nucleic acid for detection. A Ct value <35 is considered positive for PCLV. The conventional PCR was used for further validation, and the results were compared. All positive specimens were sequenced.

The ORF1 sequences of the Chinese and American endemic strains were downloaded from GenBank. Bo-Circo-like virus, identified from a calf with severe hemorrhagic enteritis in China, its ORF1 sequences, was also downloaded from GenBank, and the serial numbers were displayed in the evolutionary tree. All permutations were further aligned with MegAlign (Lasergene) using ClustalW alignment. MEGA7 software was used to construct the phylogenetic tree with 1,000 bootstrap repeats using the maximum likelihood method.

The amplification curve showed good repeatability, and the fluorescence intensity changed regularly with the multiple dilution of plasmid concentration (Figure 1A). The coefficient of determination was 0.999, while the slope of the standard curve was −3.679, the Y-intercept was 39.229, and the amplification efficiency was estimated to be 86.994%. The linear relationship is well presented (Figure 1B). According to the dissolution curve, all positive samples were unimodal, and the melting temperature was 77.48 ±.5°C (Figure 1C). At the same time, no melting curve or dimer curve was collected in the negative control.

To detect the dilutions of PCMV-HA (N)-PCLV-ORF4 (1.7 × 10⁸ copies/μl−1.7 × 10² copies/μl) and the positive sample nucleic acid by using conventional PCR showed that the minimum plasmid concentration was 1.7 × 10³ copies/μl, and the minimum nucleic acid dilution concentration of positive samples was 10⁻³ (Figures 2A,C). Based on the analysis of the amplification curve and the standard curve, the LoD detected by real-time qPCR was 1.7 × 10¹ copies/μl plasmid concentration of 10⁻⁶ (Figures 2B,D). Therefore, real-time qPCR was 100 to 1,000 times more sensitive than that of conventional PCR.

Only the PCLV group collected a strong fluorescence signal (Figure 3A) and a specific melting peak as shown at 77.18 ± 0.5°C through specificity analysis (Figure 3B). No signal amplification was found in the nucleic acid of PCV2, PCV3, PPV, TGEV, PRV, PRRSV, and PEDV, which were extracted successfully (Figure 3C). Both Ct values were >35, and there was no distinct melting peak (Figures 3A,B). The method showed high specificity in clinical sample detection and repeatability detection.

Reproducibility analysis of this method and the intra-assay SD and CV were 0.04–0.14% and 0.37–0.78%, respectively. The SD and CV between assays were 0.053–0.143% and 0.042–0.139% (Table 2). These results indicated that the real-time PCR assay is highly reproducible.

According to the results of clinical samples from six positive pig farms, the positive detection rate using real-time qPCR and conventional PCR was 22.46% (31/138) and 17.39% (24/138), respectively. Sequencing was used to confirm all positive samples (Table 3). The geographic distribution maps of porcine circovirus-like viruses in all provinces of China and cities of Guangdong province were made based on the reported results and our findings. The color-coded area indicates the locations of farms where porcine circovirus-like virus has been identified (Figure 4). The results showed that positive samples were detected in China's Hainan and Jiangxi provinces, as well as Yunfu, Huizhou, and Shanwei cities in Guangdong Province, indicating that the virus has a tendency to spread.

Sequences uploaded to GenBank from Guangdong, Hainan, and Guangxi provinces of China and the United States were used to construct a phylogenetic tree based on the nucleotide sequence of PCL ORF1 (Figure 5). Previous studies have shown that PCL virus strains we detected had 89% homology with Bo-Circo-like virus from Bovine. In this study, four PCL virus strains showed a sequence similarity of 86.7%–87.8% for the rep gene and 92.2%–92.9% for the Rep protein with Bo-Circo-like virus (Figure 6), demonstrating further the two viruses from swine and bovine share high homology.