Development and application of a droplet digital polymerase chain reaction (ddPCR) for detection and investigation of African swine fever virus

Abstract

The aim of this study was to develop a droplet digital polymerase chain reaction (ddPCR) method to detect African swine fever virus (ASFV). The methods of ASFV real-time PCR and ddPCR were established and optimal reaction conditions were confirmed. Each method was evaluated for linearity, limit of detection, and specificity. The results indicated that ASFV ddPCR had a high degree of linearity (R² ≥ 0.998) and specificity. The detection limit was 10 copies/reaction, which was approximately a 10-fold greater sensitivity than real-time PCR. This sensitive method could be used as an efficient molecular biology tool to diagnose ASFV, which is very important for preventing the spread of diseases across borders.

African swine fever (ASF) is a severe porcine disease caused by the African swine fever virus (ASFV), which can cause high mortality in swine and lead to huge economic losses worldwide. The virus infects domestic pigs, warthogs, and bush pigs, as well as soft ticks (Ornithodoros genus), which likely act as a vector (1). The clinical symptoms of ASF are very similar to classical swine fever, and the 2 diseases normally have to be confirmed by laboratory diagnosis (2–4). Therefore, development of a novel, sensitive, and rapid method to detect ASFV is important to prevent and monitor the spread of this disease.

Droplet digital polymerase chain reaction (ddPCR) is a novel molecular technology that enables the quantification of nucleic acids without using the standard samples (5). Since absolute quantification and enhanced detection sensitivity can be achieved, ddPCR has been used in various research applications, such as pathogen diagnosis (6), mutation detection (7), and transgenic research (8). In our research, a specific and sensitive ASFV ddPCR detection method was developed, which is very important for preventing the spread of diseases across borders.

The DNA of ASFV was obtained from commercial kits and provided by the Animal Quarantine Laboratory, Sichuan Agricultural University. As negative controls, classic swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus (JEV), porcine circovirus type 2 (PCV–2), pseudorabies virus (PRV), and foot-and-mouth disease virus (FMDV) were provided by the Animal Quarantine Laboratory, Sichuan Agricultural University. A total of 163 porcine samples including the excrement of wild boar, serum from sentinel animals from the Chinese border, clinical samples (visceral tissues, abortus, semen, and blood) from domestic pigs, and serum from imported pigs from Canada were tested. The samples were provided by the Institute of Military Veterinary Medicine, Academy of Military Medical Science, Sichuan Entry-Exit Inspection and Quarantine Bureau, China, and Sichuan Agricultural University. All samples were extracted using a kit (Magnetic Viral DNA/RNA Kit; TIANGEN, Biotech Company, Beijing, China) and were stored at −20°C. Animal welfare and experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals, and were approved by the animal ethics committee of Sichuan Agricultural University.

A pair of specific primers and one TaqMan probe were designed in a highly conserved region of the ASFV K205R gene and were evaluated using computer software (Primer–Blast, National Center for Biotechnology Information, Bethesda, Maryland, USA and Oligo, Molecular Biology Insights, Cascade, Colorado, USA). The primers and probe sequences were as follows: F1 (5′-CAGGCAAAACAAGTGAAACA-3′); F2 (5′-GCAAACTGCTCATCCAATATC-3′); and probe (FAM-5′-TGTTCTTCACGCGTAGCGAATGGGC-3′-BHQ). The primers and probe were synthesized (Invitrogen; Shanghai, China), the plasmid including the K205R gene of ASFV was constructed in previous studies (9).

The ddPCR assay was done in 20 μL of solution containing 10 μL of 2 × ddPCR super mix (Bio-Rad, Hercules, California, USA), 1 μL of template, and the primers and probe at final concentrations of 900 nM and 250 nM, respectively. The mixtures were then emulsified automatically with droplet generator oil (QX-100 droplet generator; Bio-Rad), which partitioned each sample into 15 000 to 20 000 water-in-oil, nanoliter-sized droplets. The droplets were then transferred to a 96-well reaction plate and heat-sealed with pierceable sealing foil sheets. The cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 94°C for 30 s and 60°C for 60 s, 1 cycle of 98°C for 10 min, and ending at 12°C. Finally, the plate was placed into a droplet reader (QX-100 droplet reader; Bio-Rad). Each well was queried for fluorescence to determine the quantity of positive events (droplets), and data were analyzed (Figure 1). The real-time quantitative PCR (qPCR) was done using 10 μL of 2 × Premix Ex Taq, 1 μL of template, and the primer and probe at final concentrations of 250 nM and 200 nM, respectively. Amplification programs were as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 30 s.

Figure 1.

The assay was optimized with different annealing temperatures and the fluorescence amplitude of amplification determined the optimal reaction condition. The annealing temperature gradient (lanes 1–7) were 60°C, 59.2°C, 58°C, 56.1°C, 53.8°C, 51.0°C, and 50°C, respectively. Lane 8 — negative control.

In order to evaluate the linearity, sensitivity, and efficiency of quantification of ASFV real-time PCR and ddPCR, the concentration of ASFV plasmid standard was determined by using ultramicro spectrophotometer (NanoDrop 2000; Thermo Fisher Scientific, Delaware, USA), and the copy number of the plasmid was calculated (10). Serial 10-fold dilutions of templates from 1 × 10⁵ copies/μL to 1 × 10⁰ copies/μL were used to perform and evaluate the linearity and detection limits of ASFV real-time PCR and ddPCR. The standard curve of real-time reverse transcriptase (RT)-PCR was automatically generated after amplification and the results showed good linearity (Figure 2A); efficiency (E) was 95.1%, the R² value was 0.999. The results of the trend line of ddPCR were determined using computer software (Excel; Microsoft, Redmond, Washington, USA) and the curve formula, and showed a high degree of linearity (R² ≥ 0.998). The detection limit of qPCR and ddPCR were determined as the last serial dilution that gave a positive result. The results showed both methods had a high detection limit; the detection limit of qPCR was ~10² copies/reaction. The minimum detection limit of the ddPCR assay was 0.36 copies, and approximately equal to ~10 copies/reaction (Figure 2B). However, the established ASFV ddPCR had an approximately 10-fold greater detection limit than qPCR.

Figure 2.

Standard curves of real-time polymerase chain reaction (RT-PCR) were constructed (A) and linear quantitative range of droplet digita l PCR (ddPCR) assay was constructed using serial dilutions of templates from 1 × 10⁵ copies/μL to 1 × 10⁰ copies/μL (B). A — A quantification correlation was obtained by plotting the quantification cycle value against the log starting concentration. B — The concentration was determined by ddPCR, the detection limit was approximately 10 copies/reaction.

To determine the specificity and cross-reactivity of ASFV ddPCR assay, the DNA/cDNA of ASFV, CSFV, PRRSV, JEV, PCV–2, PRV, and FMDV were tested. The specificity of the amplicons was confirmed by DNA sequencing. The results showed that ASFV could be detected without cross-reactivity (Figure 3A), the absolute number of ASFV nucleic acid molecules could be calculated directly from the ratio of positive events to total partitions, and other pathogens showed as no positive events happened (Figure 3B). The ASFV amplicons were sequenced, confirming that the reaction produced specific amplifications, indicating that this method had a high degree of specificity for detection of ASFV.

Figure 3.

Specificity and cross-reactivity assay of African swine fever virus (ASFV) droplet digital polymerase chain reaction (ddPCR) was tested. The fluorescence amplitude of ASFV amplification was observed without cross–reactivity (A) and the ratio of positive events to total partitions (B). The assay was done using classic swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus (JEV), porcine circovirus type 2 (PCV–2), pseudorabies virus (PRV), and foot-and-mouth disease virus (FMDV), ASFV, and nuclease-free water, respectively (lanes 1 to 8).

To analyze repeatability (intra-assay) and reproducibility (inter-assay) of the ddPCR method, each assay was tested in triplicate. The standard deviation (SD) and coefficient of variation (CV) were calculated based on the determined concentration. The assay showed intra- and inter-assay coefficient of variations of 2.84% to 5.16% and 3.68% to 5.14% (Table I), respectively.

Table I.

Repeatability and reproducibility of African swine fever virus (ASFV) droplet digital polymerase chain reaction (ddPCR).

Initial concentration (copies/μL)	Repeatability (intra-assay variation)			Reproducibility (inter-assay variation)
	Mean (copies/reaction)	SD	CV (%)	Mean (copies/reaction)	SD	CV (%)
104	10 680	448.56	4.20	9880	363.58	3.68
103	1164	33.06	2.84	1006	51.71	5.14
102	81	4.18	5.16	72	3.63	5.04
101	9.9	0.41	4.14	8.4	0.36	4.29

SD — standard deviation; CV — coefficient of variation.

To investigate ASFV in China and prevent the spread of diseases across borders, the 163 porcine samples included the excrement from wild boar, serum from sentinel pigs from the Chinese border, clinical samples and serum from imported pigs from Canada were tested by qPCR and ddPCR. In addition, the serums were re-tested using a commercial ASFV enzyme-linked immunosorbent assay (ELISA) kit (Innovative Diagnostics Veterinary, Montpellier, France). The results showed all samples were negative in qPCR, ddPCR, and ELISA, and the results of the 3 methods were consistent. Development of a sensitive and accurate method of ASFV ddPCR is very important for preventing the spread of the ASFV disease.

The ddPCR is the third-generation implementation of conventional PCR that facilitates the quantitation of nucleic acid targets without the need for calibration curves (11). In ddPCR, the reaction mixture is partitioned into 10 000 to 20 000 water-in-oil droplets, such that each droplet in the emulsion is an independent nano-PCR containing zero, 1, or more copies of the target nucleic acid, assorted in a random fashion (12). The ddPCR is highly sensitive as the fluorescence of each droplet is individually measured after PCR amplification. The absolute number of target nucleic acid molecules could be calculated directly from the ratio of positive events to total partitions, using binomial Poisson statistics. The initial quantity of template should be present at < 1 × 10⁵ copies because high concentration template may lead to nonlinear results (13). From a cost-benefit perspective, ddPCR seems to be more expensive than RT-PCR, although it has a higher detection limit. However, as development of ddPCR continues, its costs are also expected to decrease. Therefore, ddPCR is a novel, accurate, affordable, and sensitive molecular biology technology with great potential.

In our study, an accurate and sensitive detection method of ASFV ddPCR was established, which helped to diagnose the disease, especially when the samples contained low concentrations of the virus. This method had a high degree of linearity and specificity, the detection limit of ASFV ddPCR was higher sensitivity than qPCR. The specific, sensitive, and accurate ASFV ddPCR method can be applied as a novel diagnostic tool for ASFV infection.