Detection and plasmid construction of ASFV

The qPCR method based on ASFV p72 gene was established for accurate testing of environmental swabs, and the primers used are shown in Table 1. The automatic nucleic acid extractor used for DNA extraction was purchased from Luoyang Ascend Biotechnology Co., Ltd (Henan, China). The TIANamp Soil DNA Kit was purchased from Tiangen Biotech CO., LTD (Beijing, China). The qPCR was performed under the conditions of 1 cycle at 25 ℃ for 10 min for uracil-N-glycosylase activation, 1 cycle at 95 ℃ for 20 s for predegeneration, followed by 45 cycles at 95 ℃ for 1 s for degeneration and 58 ℃ for annealing/extension.

Table 1 Primers used for qPCR in the study

The reference sequence of p72 gene, the detected samples in our lab, was used to design the primers, and then the target sequence (the sequence of 103,682 to 103,931) was cloned into the pEASY-blunt to generate a pEASY-blunt-p72 recombinant plasmid. The precise viral load in the positive samples was determined using the solutions of the pEASY-blunt-p72 plasmid diluted in the range of 101 to 1010. The qPCR runs of the experimental samples involved three replicates.

Different types of sample processing methods

For qPCR detection of environmental samples, 500 µL ASFV eluant and sterile cotton swabs were prepared, followed by using the cotton swabs dipped with the ASFV eluant to wipe the sampling area repeatedly, squeezing the cotton swabs along the tube wall and centrifugation for 3 min at 14,000 g to collect the supernatant. Finally, the DNA was extracted from the supernatant for qPCR analysis.

For qPCR detection of soil samples, 2 steel balls and 500 µL ASFV eluant are mixed into the tissue in a tube, followed by oscillating the tube for 10 min and centrifugation for 3 min at 14,000 g to to collect the supernatant for DNA extraction and qPCR detection.

For qPCR detection of blood swab samples, the blood swabs were dipped in 500 µL ASFV eluant for 10 min, followed by squeezing the swabs along the tube wall to collect solution for DNA extraction and qPCR detection. The ASFV eluant was used to desorb ASFV from swabs and protect ASFV from degradation.

Ingredients of the ASFV eluant

The ASFV eluant, a mixed solution of glycine, SDS, pure water and D-trehalose dehydrate, can desorb ASFV from environmental samples. Similarly, these ingredients embodied in the patent certificate.

Firstly, reagents that may aid in the collection and elution of ASFV from environmental samples were screened: glycine-sodium hydroxide solution, SDS, BSA, and FBS.

Then several reagents with virus enrichment effects were combined in order to discover synergistic effects and further enhance the ability to enrich and elute ASFV from the environment and environmental samples. Due to difficulties in dissolving FBS with other reagents, FBS was excluded from the collection solution formulation. The other reagents were combined into three different combinations: glycine-sodium hydroxide solution with 10% SDS, glycine-sodium hydroxide solution with 10% BSA, and glycine-sodium hydroxide solution with 10% SDS and 10% BSA. These 3 combinations were used to prepare the collection solution for collecting simulated environmental samples. After nucleic acid extraction using an automated nucleic acid extractor, qPCR was performed for detection.

A preliminary environmental sample collection solution was prepared based on a 7.5 mg/ml glycine-sodium hydroxide solution. It was combined with a protein-lipid soluble substance solvent of 4 mg/ml SDS and a bioprotectant of 5 mg/ml trehalose (trehalose acts to protect viral nucleic acid from degradation and does not affect the elution efficiency of the established collection solution, data not shown). To optimize the proportions of the components, 6 different concentrations of collection solutions were prepared as follows:

(1)

1.875 mg/mL glycine-sodium hydroxide solution, 1 mg/mL SDS, 1.25 mg/mL trehalose.

(2)

3.75 mg/mL glycine-sodium hydroxide solution, 2 mg/mL SDS, 2.5 mg/mL trehalose.

(3)

7.5 mg/mL glycine-sodium hydroxide solution, 4 mg/mL SDS, 5 mg/mL trehalose.

(4)

15 mg/mL glycine-sodium hydroxide solution, 8 mg/mL SDS, 10 mg/mL trehalose.

(5)

22.5 mg/mL glycine-sodium hydroxide solution, 12 mg/mL SDS, 15 mg/mL trehalose.

(6)

30 mg/mL glycine-sodium hydroxide solution, 16 mg/mL SDS, 20 mg/mL trehalose.

(The concentrations of the reagents in the preliminary environmental sample collection solution were diluted 1 and 2 times, and 2, 3, 4 times were added.)

Desorption effect of virus from environmental samples

Briefly, a cement floor was partitioned into different zones with an area of 1 × 1 cm2, followed by dropping the positive virus blood samples (100 µL each with 5 to 55-fold dilution) as simulated environmental samples. 24 h later, the positive environmental areas were eluted separately by swabs dipped with the eluant and 0.85% saline solution to collect the environmental samples, followed by qPCR analysis of the virus concentration differences between the ASFV eluant and 0.85% saline solution. The qPCR analysis was repeated 3 times for each experimental sample, and the average value was used for data analysis. Before dropping the positive virus blood samples, the cement floor was detected negative, and each zone was used only once in the test of environmental samples.

Desorption of virus from soil samples

Soil samples were prepared by dropping 100 µL positive blood samples into 0.2 g soil and incubation for 24 h, followed by treating the soil samples with the ASFV eluant and the TIANamp Soil DNA Kit via mixing 500 µL ASFV eluant and 2 steel balls with the soil in a tube. Then, the tube was oscillated for 10 min and centrifuged for 3 min at 14,000 g to obtain the supernatant for DNA extraction and qPCR detection. The qPCR was performed for each experimental sample with 5 replicates, and the average value was used for data analysis.

Desorption and protection of virus in blood swabs

In the blood swab experiment, cotton swabs were dipped in a positive blood sample and put in a tube, followed by treating the simulated blood swabs at 37 ℃ for 24, 48 and 72 h just like the case of sending samples to a test station from a pig farm. The experimental samples were divided into 2 groups by treating the simulated blood swabs separately with the eluant and saline solution, where the blood swabs were squeezed along the wall of each tube to collect the solution for DNA extraction and qPCR detection to verify the protective efficacy of the ASFV eluant.

Comparison of the ASFV eluant and saline solution in detection of clinical samples

A comprehensive collection of 102 environmental samples was conducted at a slaughterhouse in Hubei, China, utilizing swabs immersed in the eluant and 0.85% saline solution. These samples encompassed various elements such as transport vehicles, production equipment, and human bodies, enabling a comparative analysis of the test outcomes between the 2 treatment methods.

Clinical application of the ASFV eluant

Environmental samples were collected by swabs dipped in the ASFV eluant to test the effect of the eluant in clinical practice. A total of 175 environmental swabs were collected from a large-scale pig farm in Hubei, China and detected by the ASFV eluant. There were 17 categories of environment samples collected by the ASFV eluant after the pigs developed symptoms of ASFV and samples of these categories were collected again after 3 thorough disinfection. Furthermore, the trestles of farrowing bed, the most contaminated area, were tracked and detected to reveal the disinfection effect.