N-GPV used in this study was isolated from morbid ducks liver in Liaocheng city, Shandong province, China (named SDLC strain). GPV (HN strain), duck plague virus (ZB strain), duck tembusu virus (GL strain), duck hepatitis virus (LC strain), duck reovirus (HB strain), Muscovy duck parvovirus (GX strain), and duck-origin H9N2-AIV (GT strain) were preserved by the Research Institute of Poultry Disease of Shandong Agricultural University. Sixty clinical liver samples and 60 cloacal swabs were collected from dead ducks and clinical healthy ducks respectively in Shandong, Jiangsu, and Henan province of China in different large-scale duck farms.

The liver samples were homogenized in dulbecco’s modified eagle medium (DMEM), frozen and thawed three times, then centrifuged at 5000 ×g for 15 min. The supernatant were filtered through a 0.22 μm filter, and the filtrate was inoculated into duck embryo fibroblasts (DEF) cells. The culture supernatant was harvested after 5 days post-inoculated for three passages, and detected by PCR.

DNA was extracted by TIANamp Genomic DNA Kit (TIANGEN biotech, Beijing, China). RNA was extracted by MiniBEST Universal RNA Extraction Kit (TaKaRa, Dalian, China).

VP3 gene of N-GPV, GPV, and MDPV were aligned by the MegAlign software, and phylogenetic tree was constructed with MEGA version 7.0 using the neighbor-joining method (Figure 1). The reference waterfowl parvovirus isolates are listed in Table 1.

A set of four primers and PCR primers were designed based on the VP3 gene of N-GPV using an online program (PrimerExplorer V4, http://primerexplorer.jp/elamp4.0.0/index.html) and Primer 5.0 software respectively. Primers including two inner (FIP and BIP), two outer (F3 and B3), and PCR primers are shown in Table 2.

Outer primers were used to amplify target DNA, and the amplified PCR products were purified with a gel extraction kit (Takara, Dalian, China), cloned into the pMD18-T vector (Takara, Dalian, China) and transformed into E. coli DH5α competent cells. Clones were sequenced by BGI Tech (BGI Tech, Shenzhen, China), and analyzed results. High fidelity clone was cultured and extracted plasmid with Pureplasmid Mini Kit (CWBIO, Beijing, China). Meanwhile, 1.0 × 10⁷ copies/μL–1.0 × 10² copies/μL substances were used for constructing standard curve.

The qLAMP assay was conducted in a 25 μL reaction system, which containing 1 μL FIP and BIP primers (80 μM of each), 1 μL F3 and B3 primers (10 μM of each), 2.5 μL 10 × Isothemal amplification buffer, 1.5 μL 100 mM MgSO₄, 3.5 μL 10 mM dNTP Mix, 1.25 μL 20 × EvaGreen^® dye, 1 μL Bst DNA polymerase, 1 μL target DNA, 11.5 μL nuclease-free water, and performed using 7300 Real time PCR System (Applied Biosystems, Foster City, CA, United States). Mixtures were incubated at 65°C in water bath for 60 min, and terminated by heating at 80°C for 20 min (followed by NEB typical LAMP protocol), or carried out on 7300 Real time PCR system followed by procedure: 60 cycles of 64°C 10 s and 65°C 50 s, fluorescence signals were collected at the end of 65°C step, and the cutoff point for Tt (time threshold, a cycle indicates 1 min) value was determined as 50 by the previous reported method(Tang et al., 2016). Data was analyzed by SDS software program (version 1.4). The qLAMP results were also analyzed by gel electrophoresis with 2% agarose gel, and observed under UV light.

The PCR assay was conducted in 20 μL reaction system, containing 10 μL 2 × Ex Taq Mix, 6 μL ddH₂O, 1 μL forward primer, 1 μL forward primer, 1 μL reverse primer, and 2 μL DNA, and performed using PCR System (Applied Biosystems, Foster City, CA, United States). PCR conditions were as follows: 95°C for 5 min, 30 cycles of denaturation (95°C for 30 s), annealing (55°C for 30 s), and extension (72°C for 30 s), followed by a final extension at 72°C for 10 min.

Ten-fold series dilutions (10⁷–10⁰ copies/μL) of standard substance were used as templates for qLAMP amplification to confirm its sensitivity.

Quantitative loop-mediated isothermal amplification assay was carried out using different viruses including GPV, duck plague virus, duck tembusu virus, duck hepatitis virus, duck reovirus, Muscovy duck parvovirus, and H9N2-AIV to validate specificity of this method.

Sensitivity test was performed to compare PCR and LAMP assay, and plasmid with different dilutions was used as templates for PCR detection. F3, B3 Primers for PCR amplification were shown in Table 2. PCR results were electrophoresed in 2% TAE agarose gel and observed in the UV transilluminator.

To confirm that the qLAMP detection method has good reproducibility to detect N-GPV, tenfold series dilutions of plasmid substances, from 1.0 × 10⁷ copies/μL to 1.0 × 10⁰ copies/μL were used. For the intra-assay test, triplicate from each dilution were tested in the same run. For the inter-assay test, samples from each dilution were tested in three independent runs. The results were evaluated by coefficient of variation.

Sixty clinical liver samples and 60 cloacal swabs from dead ducks and clinical healthy ducks respectively were collected to test reliability of qLAMP, PCR assay, and virus isolation assay.

To determine genetic relationship between SDLC and other GPV and MDPV strains, phylogenetic tree of VP3 genes was constructed. Phylogenetic trees based on VP3 genes showed that the SDLC strains were in the same branch with European GPV and vaccine isolates, and shared 93.4–98.9% identity with GPV isolates, while only shared 80.4–88.7% with MDPV, which indicated that evolutionary relationship of SDLC strain is closer to GPV stains, and N-GPV is a novel variant of GPV.

Concentration of positive plasmid was 253.50 ng/μL, determined by DS-11 Spectrophotometer (Denovix, America), and the copy number was 7.91 × 10¹⁰ copies/μL. The standard curve was generated by Tt values obtained from this study, and a good linear relationship was established between the log of the plasmid copy numbers (copies/μL) and the Tt values (R²= 0.997), with a regression line revealing an average intercept of 51.14 and an average slope of –5.2 (Figure 2).

Performed by ten-fold series dilutions (1.0 × 10⁷–1.0 × 10⁰ copies/μL) of standard substance, the qLAMP method for N-GPV detection has the lowest limit of 10² copies, and no amplification signals were observed in negative control (NC) (Figure 3A). In our strategy, the results of qLAMP assay could not only be evaluated by naked eyes at the endpoint with EvaGreen^® fluorescent dye in the UV light (Figure 3B) and agarose gel electrophoresis (Figure 3C), it could also be quantitatively monitored using the real-time PCR system during the reaction.

Specificity test of qLAMP method was carried out by seven duck-origin viruses and a goose-origin virus (mentioned in virus and samples). The results showed that our assay could only detect N-GPV and GPV, while no amplification signals were detected in other viruses (Figure 4A). The specificity of qLAMP assay could also be confirmed by green fluorescence in the UV light (Figure 4B), and typical ladder pattern seen in the agarose gel electrophoresis (Figure 4C). In addition, no positive results were obtained in negative control (NC), which indicates that this qLAMP assay has a good specificity for N-GPV detection.

In the intra-assay test, the coefficient of variation of the Tt values varied from 0.74 to 1.81%; In the inter-assay test, the coefficient of variation of the Tt values varied from 0.82 to 2.25% (Table 3). These results revealed that the qLAMP method has a high reproducibility and excellent stability in detecting N-GPV.

Different dilution plasmids (1.0 × 10⁷–1.0 × 10⁰ copies/μL) were used as templates for LAMP and PCR amplification. Results showed that the detection limit of qLAMP assay was 10² copies, while PCR assay was 10⁴ copies, which indicated the sensitivity of LAMP method was 100 times higher than PCR method.

Loop-mediated isothermal amplification, PCR, and virus isolation assay were performed on clinical samples and cloacal swabs of ducks from different regions of China. The results were shown by statistical analysis in Table 4. The results showed that the consistency of these three methods in every sample. The sensitivity of qLAMP assay was the highest, and there was no positive amplification in negative control in qLAMP assay, PCR, and virus isolation assay.