The nucleotide sequences for the target fragments of MPXV-I-D14L and MPXV-II-D18L were synthesized by Suzhou Hongxun Biotechnology Company Limited (Suzhou, China). Foot-and-mouth disease virus type A/O (FDMV type A RE-A/WH/09 strain and type O 0/MYA98/BY/2010 strain) vaccine was purchased from Zhongmu Industrial Compane Limited (Beijing, China). GTPV (CVCC AV41 strain) vaccine was obtained from Harbin Pharmaceutical Group Biological Vaccine Company Limited, China. Peste des petits ruminants virus (PPRV) vaccine strain, ORF virus (ORFV), and Mycoplasma pneumoniae (CCPP) were kept in the laboratory. We conducted a comparative analysis of the nucleotide sequences of D14L and D18L genes against other orthopoxviruses available from the GenBank. Specific primers and corresponding probes targeting the highly conserved regions of the D14L and D18L genes were designed using Beacon Designer 8.0 (Table 1). The locations of these primers and probes are shown in Figures 1A, B. Primers and probe for GTPV were selected based on literature reports (23).

Sheep tissues and nasal swabs were collected from sheep farms, slaughterhouses, and individual farmers in Sichuan, China between January 2022 and December 2023 after the first MPX case in humans was reported in China. Tissue samples from golden monkey including heart, lung, kidney, and tumor tissues, were kindly provided by Prof. Hou Rong of Northwestern University, China. The tissue samples were added to 500 μL of phosphate-buffered saline (PBS, pH 7.2) and homogenized by a frozen tissue grinder. The resulting homogenate was subjected to three cycles of freeze-thawing, followed by centrifugation at 12,000 rpm for 5 min. Nasal swabs were processed by adding 500 μL of PBS and vortexing for 2 min. Total nucleic acids from the supernatant (200 μL) and PPRV, A/O FMDV preparations were extracted using TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver. 5.0 (TaKaRa, Beijing, China) and GeneJET™ Viral DNA and RNA Purification Kit (Thermo Fisher Scientific, Beijing, China). RNA extracted from PPRV and A/O FMDV preparations were reverse transcribed into complementary DNA (cDNA) using ReverTra Ace qPCR RT Master Mix with gDNA Remover kit (Thermo Fisher Scientific, Beijing, China).

The multiplex real-time PCR reaction systems and procedures were optimized in accordance with the manufacturer's instructions for Taq Pro U+ Multiple Probe qPCR Mix (Novozymes, Nanjing, China). In this experiment, primers volume ranged from 0.2 to 2.0 μL (0.1–1 μM), probes concentration ranged from 0.1 to 0.5 μL (0.05–0.25 μM), and the range of the annealing temperature ranged from 56 to 61°C. The multiplex real-time PCR amplification was conducted using ArchimedX4 system, a Medical fluorescence quantitative PCR instrument from Kunpeng Gene Technology Company Limited (Beijing, China).

MPXV-I-D14L and MPXV-II-D18L plasmids were synthesized by Suzhou Hongxun Biotechnology Company Limited and subsequently cloned into the PUC 57 vector. Additionally, a fragment of GTPV RPO30 gene was amplified and cloned into a pMD 19T vector, resulting in the GTPV-RPO30 plasmid. To determine the copy number of the plasmid, the concentration was converted using the following formula:

The three standard plasmids (MPXV-I-D14L, MPXV-II-D18L, and GTPV-RPO30) were 10-fold serially diluted (ranging from 1.5 × 10⁷ to 1.5 × 10⁰ copies/μL) and equal volumes of each plasmid were mixed in a 1:1:1 ratio. This mixture served as the template for generating the standard curve of the multiplex real-time PCR assay.

For the specificity analysis of the developed multiplex real-time PCR, DNA from ORFV and CCPP as well as cDNA from PPRV and A/O FMDV RNA were used as templates, with ddH₂O included as a negative control. The clinical manifestations after infection by the aforementioned virus are similar to those of MPXV and GTPV. To evaluate sensitivity, three standard plasmids (MPXV-I-D14L, MPXV-II-D18L, and GTPV-RPO30) were subjected to a 10-fold serial dilution ranging from 1.5 × 10⁷ to 1.5 × 10⁻¹ copies/μL. The limit of detection (LOD) for the developed multiplex real-time PCR was determined using these 10-fold serially diluted standard plasmids.

The three standard plasmids (MPXV-I-D14L, MPXV-II-D18L, and GTPV-RPO30) were used at concentrations of 1.5 × 10⁴, 1.5 × 10³, and 1.5 × 10² copies/μL to assess the intra-assay and inter-assay variability. Three replicate experiments (three repetitions for each test) were conducted for each dilution and the coefficient of variation (CV%) of the Ct values was calculated.

The multiplex real-time PCR assay developed was validated using a variety of sample types, including sheep tissue, nasal swabs and monkey tissue samples as described above. To ensure a comprehensive comparison, the assay was also tested against assays published previously: the multiplex real-time PCR assay for MPXV clades I and II developed by Huo et al. (24), and multiplex real-time PCR assay for goat pox or sheep pox virus by Wang et al. (23). Due to lack of MPXV positive sample, 40 spiked samples with viral standard plasmids were prepared as follows: all of these samples used was detected and confirmed negative for MPXV clades I, II, and GTPV by multiplex real-time PCR assay published previously (24). Five samples were spiked with GTPV plasmids, 16 samples spiked with MPXV clade I and MPXV clade II plasmids, seven samples spiked with MPXV clades I and II plasmids, 10 samples spiked with either MPXV clade I and GTPV plasmids or MPXV clade II and GTPV plasmids, and two samples spiked with MPXV clades I, II and GTPV plasmids. The concentrations of viral standard plasmids blended into tissue samples and nasal swabs were 1.5 × 10⁶, 1.5 × 10⁴, and 1.5 × 10² copies/μL. This process generated a set of positive samples to evaluate the applicability and accuracy of the developed multiplex real-time PCR.

The TaqMan probes for MPXV-I-D14L, MPXV-II-D18L, and GTPV-RPO30 were labeled with Cy5, FAM, and HEX at the 5′ end, and with BHQ2, BHQ1, and BHQ1 at the 3′ end, respectively (Table 1). Primers and probes were synthesized by Sangon Biotech (Shanghai, China). The optimal reaction system was as follows: in a total volume of 20 μL, the reaction mixture included 10 μL of 2 × Taq Pro U+ Multiple Probe qPCR Mix, 0.4 μL of each prime (10 μM), 0.2 μL of each probe (10 μM), 3 μL of nucleic acid template, and 4 μL of enzyme-free water. The amplification conditions were set at 37°C for 2 min, followed by 95°C for 30 s, and then 45 cycles of 95°C for 10 s, and 60°C for 30 s. A CT value of <35 cycles was judged as positive.

The standard curves for the multiplex real-time PCR assay were generated using three standard plasmids ranging from 1.5 × 10⁷ to 1.5 × 10⁰ copies/μL. The amplification efficiencies, slopes and correlation coefficients (R²) for MPXV-I, MPXV-II and GTPV were found to be 101.99%, −3.275 and 0.997, 100.67%, −3.306 and 0.994, and 109.42%, −3.115 and 0.993, respectively (Figures 2B–D). The amplification efficiency of the developed multiplex real-time PCR assay fell within the acceptable range of 90–110% and the correlation coefficients complied with 0.99, indicating a strong linear relationship.

In specificity analysis, 1.5 × 10⁶ copies/μL of standard plasmids (MPXV-I-D14L, MPXV-II-D18L, and GTPV-RPO30), DNA from ORPV and CCPP and cDNA from PPRV and FMDV A/O were used. The results showed that MPXV-I-D14L, MPXV-II-D18L, and GTPV-RPO30 exhibited amplification curves while the other viruses exhibited no amplification curves. Importantly the fluorescent signals of MPXV-I, MPXV-II, and GTPV did not interfere with one another, indicating that the developed multiplex real-time PCR assay had good specificity (Figure 3).

For sensitivity testing, standard plasmids (MPXV-I-D14L, MPXV-II-D18L, and GTPV-RPO30) were 10-fold serially diluted, ranging from 1.5 × 10⁷ to 1.5 × 10⁻¹ copies/μL. The results showed that the LOD of the developed multiplex real-time PCR assay for detection of MPXV-I, MPXV-II, and GTPV was 1.5 × 10¹ copies/μL (Figure 2A). Assessing the sensitivity of each primers/probe set alone, the results showed that the LOD for detection of MPXV-I, MPXV-II, and GTPV was 1.5 × 10⁰ copies/μL, 1.5 × 10¹ copies/μL, and 1.5 × 10⁰ copies/μL, respectively (Supplementary Figure 1). The assay's accuracy was further evaluated with the standard plasmids at a concentration of 300–30 copies/reaction (Table 2). Analysis showed 208.72 copies/reaction (95% confidence interval 180.16–237.27 copies/reaction), 207.83 copies/reaction (95% confidence interval 148.26–267.39 copies/reaction) and 252.07 copies/reaction (95% confidence interval 188.97–315.17 copies/reaction) for GTPV-ORF30, MPXV-I-D14L, and MPXV-II-D18L, respectively.

To evaluate the repeatability of the developed multiplex real-time PCR assay, three different concentrations of standard plasmids (1.5 × 10⁴, 1.5 × 10³, and 1.5 × 10² copies/μL) were tested. The results showed that the coefficients of variation (CV%) for intra- and inter assay variation ranged from 0.17 to 0.89% and 0.58 to 1.09%, respectively (Table 3).

A total of 249 sheep tissue samples, nasal swab and monkey tissue samples, along with 40 spiked samples were tested using the developed multiplex real-time PCR assay and the assays published previously [Huo et al. for MPXV clades I and II (24) and Wang et al. for GTPV (23)]. Real-time PCR for GAPDH gene were used to validate negative results (Supplementary Figure 2). As shown in Table 4, the developed multiplex real-time PCR assay showed that 248 tissue samples and nasal swabs were negative (no amplification curve/Ct > 35), while 40 spiked samples and one sheep nasal swab sample were positive for GTPV (Ct <35). Sanger sequencing results confirmed that the nasal swab sample was tested positive for GTPV. The published assays revealed that 249 tissue samples and nasal swabs and 14 spiked samples were negative (no amplification curve, Ct > 35) and 26 spiked samples were positive (Ct <35). The detection results of MPXV clades I and II in the 14 spiked samples were all negative. The samples were spiked with the standard plasmid at a concentration of 1.5 × 10² copies/μL. Based on the sample testing data and the results from the spiked samples (Table 4), the detection sensitivity of the developed triple qPCR assay significantly surpassed that of the published method. The developed multiplex real-time PCR assay demonstrated a clinical sensitivity and specificity of 100%, while the published assay had a relative sensitivity of 63.42% and a clinical specificity of 100%. In the performance comparison between the developed multiplex real-time PCR and the published real-time PCR detection approaches for detecting MPXV I and II as well as GPTV in clinical and spiked samples, the relative sensitivity of the developed multiplex real-time PCR method amounted to 157.69%, while the relative specificity reached 100% (Table 5).