Introduction
African swine fever is a severe disease of domestic and wild pigs that originated from Africa, initially in several sub-Saharan countries. The disease became endemic in some European regions with the 2007, 2014, and 2018–2019 European and Asian outbreaks.1 Being an extremely contagious and fatal viral disease, ASF in just less than 1 month since the first outbreak in China in August 2018 spread nationwide in 25 provinces, expanding to Mongolia, Vietnam, Cambodia, North Korea, Laos, Myanmar, Philippines, South Korea, Timor-Leste, and Indonesia despite governmental efforts to control the disease.2 In China, a total of 149 ASF outbreaks have been detected in 32 of 34 provincial-level administrative divisions and > 1,160,000 pigs have been culled. By the end of 2019, Vietnam had ASF outbreaks in all 63 provinces and a total of 6,000,000 pigs had been culled.2
Although deemed finally well controlled in the early months of 2020 in the aforementioned Asian nations, ASF has caused serious consequences such as socioeconomic losses and an enormous decrease in domestic and wild pig populations and diversity, particularly among endemic native breeds.3 Clinical signs of ASF are typically similar to those of CSF and some other swine diseases,4 making diagnosis challenging. Some strains of ASFV, which is a double-stranded DNA virus, are easily spread and highly virulent, associated with an almost 100% mortality rate. In total, 24 genotypes of ASFV have described on the basis of sequencing of the p72 ASFV capsid protein gene.5 To the authors' knowledge, no effective treatment or vaccine exists for ASF, making control of the disease imperative.
Control of ASF will only be successful through timely and accurate identification of disease outbreaks, herd culling, and concerted biosecurity efforts in noninfected regions.6,7 Because of the similarities in clinical and postmortem signs of ASF with other swine diseases, ASF can only be accurately diagnosed by a combination of clinical and postmortem examination and laboratory or onsite diagnostic tests. A rapid and reliable diagnostic test would therefore be an essential tool in a national strategy to prevent and control an ASF epidemic.
The most accurate protocol for laboratory diagnosis of ASF is virus isolation, in combination with other virological tests such as the hemadsorption test and viral genome detection by conventional or real-time PCR assay, as recommended by OIE.8 Unfortunately, this protocol requires the use of highly skilled and experienced technicians and a well-equipped laboratory and is time-consuming. Although useful to confirm the positive results obtained by other tests, this protocol is impractical for monitoring and controlling disease spread.4 Another approach recommended by OIE is the use of serologic tests such as ELISAs, immunoblotting techniques,9 and indirect fluorescent antibody tests.8,10 Compared with virus isolation, such serologic tests are quite rapid and easily conducted, even on-site, for detection of ASFV antigens without the need for highly skilled technicians.4 However, such tests are low in sensitivity, particularly when used for pigs with subacute or at early-stage ASFV infection.4,11
Consequently, real-time PCR assay has been considered the method most moderately time-consuming and reliable for ASFV detection because of its accuracy, diagnostic and analytic sensitivity, specificity, and acceptable performance time, making it a routine tool for ASFV diagnosis in national and reference laboratories.1,7,10 Nevertheless, this type of assay would be unsuitable for on-site ASFV detection in the field (eg, on farms or small-scale pig-rearing operations) because of the need for thermal cycling instruments and standard laboratory conditions.11,12,13 Isothermal amplification techniques, such as recombinase polymerase amplification, loop-mediated isothermal amplification, and cross-priming amplification, have been also developed for rapid detection of ASFV,4 especially in combination with immune-chromatographic strips for on-site detection of ASFV.14,15 Such tests are rapid and require no expensive thermal instrument or skilled technician; however, their diagnostic sensitivity and accuracy are limited because of primer and probe specificity.11,16 The need remains for a rapid test that can be used in the field with a manageable amount of equipment yet sufficient accuracy to provide accurate screening for ASFV.
The iiPCR assay consists of a modified PCR technique based on Rayleigh-Bénard convection. This assay involves the typical PCR steps of denaturation, annealing, and extension. The difference is that the PCR reaction is carried out in a capillary vessel which is heated through the bottom end and fluid containing target DNA or cDNA, and all amplification materials are cycled through different temperature levels inside the tube to complete denaturation, annealing, and extension steps of the PCR assay continuously and simultaneously,17,18 rather than changing the temperature of the whole reaction tube. With an easily transported iiPCR system, nucleic acid (DNA and RNA) amplification and detection is automated, making the detection of pathogens easy, convenient, and rapid. Moreover, with the automated nucleic acid extraction, iiPCR assay, and integrated magnetic bead-based nucleic acid extraction and purification provided by this system, the time from sample collection to pathogen detection is as short as 1 to 1.5 hours. Simplified reporting of assay results as positive or negative readouts also renders the iiPCR system more convenient and user-friendly than alternative methods, which may allow operators to perform the assay without substantial training and experience.17 Overall, iiPCR assays with iiPCR instruments or systems have been used for the detection of many human and animal pathogens, such as FMDV, malarial parasites, Plasmodium spp, Salmonella spp, influenza A virus, Middle East respiratory syndrome virus, Zika virus, avian influenza, bovine torovirus, severe acute respiratory syndrome coronavirus 2, Dengue virus, and yellow fever virus.19,20,21,22,23,24,25,26,27,28,29
Vietnam, among other countries, has been a hot spot for ASF, and rapid, reliable on-site detection of ASFV is important for early prevention and control of disease. The purpose of the study reported here was to evaluate the ability of an integrated automated nucleic acid extraction system and iiPCR kit (ie, iiPCR system) to detect ASFV in biological samples from pigs in Northern and Southern Vietnam with and without ASF, with real-time PCR assay used as a reference method.
Materials and Methods
Ethics statement
The present study was conducted in accordance with principles for ethics in biological research and rules for biosafety assurance as set forth by the Institute of Biotechnology, Vietnam Academy of Science and Technology. Collection and provision of samples by the RAHO-6 and NCVD were officially in compliance with the national standard for pathogenic anatomy and sampling approved by the Vietnam Ministry of Agriculture and Rural Development (regulation No. TCVN 8402:2010).
Sample collection
Samples for this study were provided independently from 2 institutions: RAHO-6 (Southern Vietnam) and NCVD (Northern Vietnam). For analytical sensitivity testing, 2 samples identified as ASFV positive by means of real-time PCR assay with Ct values of 19.7 (swine tissue homogenate) and 14.0 (swine blood) were provided by the RAHO-6 and the NCVD, respectively. Dilutions were made by adding sufficient volume of fresh PBS solution to samples before automated DNA extraction for the iiPCR assay and by adding deionized water to DNA samples after extraction for the real-time PCR assay.
For analytical specificity testing, 2 samples each of virus-infected cell culture medium predetermined to be ASFV negative and CSFV, PRRSV, or FMDV positive (total of 6 samples) were provided by the RAHO-6. Two samples each of virus-infected cell culture medium positive for CSFV and homogenized tissue solution positive for PRRSV or PCV2, all of which were predetermined to be ASFV negative (total of 6 samples), were provided by the NCVD.
For evaluation of diagnostic performance, various swine biological samples predetermined to be ASFV positive or ASFV negative or that remained untested were provided by the NCVD and RAHO-6. Specifically, from the RAHO-6, a total of 216 samples were provided, including predetermined 31 ASFV-positive and 29 ASFV-negative lymph node tissue samples, 30 predetermined ASFV-positive and 30 ASFV-negative whole blood samples, 30 predetermined ASFV-positive and 30 ASFV-negative serum samples, and 30 predetermined ASFV-positive and 6 ASFV-negative spleen samples. From the NCVD, a total of 150 samples were provided, including 37 predetermined ASFV-positive and 23 ASFV-negative tissue homogenate (mostly lymph nodes) samples, 31 predetermined ASFV-positive and 29 ASFV-negative serum samples, 24 untested tonsil samples, and 6 untested spleen samples. All samples except those of whole blood were frozen after collection at −20 °C until use.
Sample preparation
Prior to extraction of nucleic acid from tissue samples, 0.1 g of each tissue sample was well grinded in 1 mL of PBS solution by use of a small disposable tissue grinder, then centrifuged at 5,000 × g for 3 minutes. Then, 200 µL of supernatant of each sample was submitted for DNA extraction. The same volume (200 µL) of each whole blood and serum sample was immediately submitted for nucleic acid extraction without any pretreatment.
Nucleic acid extraction for real-time PCR assay
At the RAHO-6, DNA extraction was performed by use of a purification systema and reagents in accordance with the manufacturer's instructions, resulting in 200 µL of extracted nucleic acid solution. At the NCVD, nucleic acid extraction was performed with an automated nucleic acid extraction systemb and DNA-RNA extraction kitc in accordance with the manufacturer's instructions. Briefly, 200 µL of each sample was pipetted into the first well of the extraction plate prior to the process of automated extraction, resulting in 200 µL of nucleic acid solution. Each DNA sample was stored separately at −80 °C until further use.
For analytical sensitivity tests, 2 and 3 extractions were performed per sample from the RAHO-6 and NCVD, respectively. For analytical specificity and diagnostic performance evaluation, only 1 extraction was performed per sample.
Real-time PCR assay
At the RAHO-6, the real-time PCR assay was carried out with a commercial real-time PCR master mix for real-time DNA amplification.d primers and probes for ASFV detection primers per standard recommendations of the OIE,8,30 and a real-time thermocycler.e Amplification conditions included an initial denaturation step at 94 °C for 120 seconds, followed by 45 cycles at 94 °C for 30 seconds, 58 °C for 60 seconds, and 72 °C for 45 seconds and an elongation step at 72 °C for 420 seconds before an indefinite holding period at 4 °C. At the NCVD, the real-time PCR assay was performed with a different PCR master mixf and thermocyclerg and similar amplification conditions to those used at the RAHO-6. Signals were analyzed by the built-in software of real-time thermocyclers. A sample was considered to have yielded a positive result for ASFV when the measured Ct value was < 40.
ASFV detection by the iiPCR system
The iiPCR systemh consisted of a combined automated DNA extraction process and iiPCR assay involving magnetic bead–based nucleic acid purification, robotic arm liquid handling, and iiPCR technologies in a fully automated molecular detection platform. For nucleic acid extraction and purification, a cartridge seti containing preloaded magnetic bead–based reagents and chemicals for nucleic acid extraction,j liquid transfer.k For iiPCR amplification of ASFV DNA, a reagent containing a mix (lyophilized pellet) of dNTPs, ASFV-specific primers, probe, and enzymesl was used.
Briefly, 200 µL of supernatant from the homogenate sample was transferred into the first well of the extraction cassette containing lysis buffer and 95% ethanol. The system automatically carried out DNA extraction and iiPCR steps focused on the nucleocapsid vp72 gene (amplification size, 88 bp) that were designed for all 24 ASFV genotypes, providing test results in approximately 85 minutes. The device automatically determined the fluorescent signal of each sample and control before and after iiPCR assay to generate S/Ns and, by a built-in algorithm, converted these ratios into the following simplified results for display: positive (+); negative (−); inconclusive, suggesting retesting (?); warning (!); or abnormal, with signals outside detection limits (*).
Experimental design
Analytical sensitivity was determined by comparing the ability of the iiPCR assay and reference real-time PCR assay to detect 10-fold serial dilutions (101 to 1010) of 1 ASFV-positive sample, with 2 and 3 repeats performed for each dilution at the RAHO-6 and NCVD, respectively. A predetermined ASFV-positive sample (positive control) and negative control (deionized water) were also assayed.
Analytical specificity of the iiPCR assay was determined for the 4 other swine viral pathogens (CSFV, FMDV, PCV2, or PRRSV) independently at the RAHO-6 and NCVD (2 positive samples/pathogen; 3 pathogens/institution), compared with results for real-time PCR assay. A predetermined ASFV-positive sample and negative control (deionized water) were also assayed.
Diagnostic performance of the iiPCR assay was determined independently at the RAHO-6 and NCVD with swine biological samples from each institution. Each sample was tested once with both the iiPCR assay and real-time PCR assay. A predetermined ASFV-positive sample and a negative control (deionized water) were also assayed. When results of the 2 as-says disagreed or results of the real-time PCR assay disagreed with results for a predetermined ASFV-positive sample, the sample was again tested in triplicate with the 2 assays as a discrepancy test.
Statistical analysis
The real-time PCR assay was considered the reference method against which the iiPCR assay was compared. The number of true- or false-positive and negative results of the iiPCR assay was determined. Sensitivity was computed as the number of true-positive results/ (number of true-positive results + number of false-negative results). Specificity was computed as the number of true-negative results/(number of true-negative results + number of false-positive results). Cohen κ values were calculated to indicate the degree of agreement between the iiPCR and real-time PCR assay. Statistical analysis was performed with the aid of spreadsheet software.m Data for S/N and Ct values are reported as mean ± SD.
Results
Analytical sensitivity and specificity
The maximum dilution at which ASVF could be detected in swine tissue homogenate or blood was 106 with the iiPCR system at both institutions, whereas with the real-time PCR assay, the maximum dilution was 106 at the RAHO-6 and only 105 at the NCVD (Table 1).
Detection rates (DRs)* and analytical sensitivity† of an iiPCR system and real-time PCR assay for detection of ASFV in swine tissue homogenate or blood.
| Dilution | RAHO-6 | NCVD | ||||||
|---|---|---|---|---|---|---|---|---|
| DR | Mean ± SD S/N | DR | Mean ± SD Ct | DR | Mean ± SD S/N | DR | Mean ± SD Ct | |
| 101 | 2/2 | 3.99 ± 0.25 | 2/2 | 22.09 ± 0.25 | 3/3 | 3.86 ± 0.29 | 3/3 | 18.87 ± 0.26 |
| 102 | 2/2 | 3.91 ± 0.02 | 2/2 | 22.02 ± 0.20 | 3/3 | 3.54 ± 0.09 | 3/3 | 22.05 ± 0.11 |
| 103 | 2/2 | 3.95 ± 0.08 | 2/2 | 24.82 ± 0.07 | 3/3 | 2.81 ± 0.60 | 3/3 | 28.74 ± 0.24 |
| 104 | 2/2 | 3.56 ± 0.15 | 2/2 | 29.07 ± 0.44 | 3/3 | 3.12 ± 0.64 | 3/3 | 28.18 ± 1.28 |
| 105 | 2/2 | 3.63 ± 0.27 | 2/2 | 33.72 ± 0.45 | 3/3 | 3.74 ± 0.31 | 3/3 | 33.14 ± 1.33 |
| 106 | 2/2 | 2.46 ± 0.11 | 2/2 | 37.19 ± 0.64 | 3/3 | 3.18 ± 0.63 | 0/3 | N/A |
| 107 | 0/2 | 1.03 ± 0.01 | 0/2 | N/A | 0/3 | 1.00 ± 0.02 | 0/3 | N/A |
| 108 | 0/2 | 1.03 ± 0.00 | 0/2 | N/A | 0/3 | 1.00 ± 0.00 | 0/3 | N/A |
| 109 | 0/2 | 1.02 ± 0.01 | 0/2 | N/A | 0/3 | 1.00 ± 0.00 | 0/3 | N/A |
| 1010 | 0/2 | 1.03 ± 0.01 | 0/2 | N/A | 0/3 | 1.00 ± 0.00 | 0/3 | N/A |
| Positive control | 1/1 | 3.62 | 1/1 | 33.04 | 1/1 | 4.10 | 1/1 | 27.27 |
| Negative control | 0/1 | 1.00 | 0/1 | N/A | 0/1 | 1.03 | 0/1 | N/A |
Bold values represent results for the maximum dilution at which ASFV was detectable.
The DR represents the total number of assays performed in which ASFV was detected.
Analytical sensitivity was calculated as the number of positive results divided by the total number of assays.
N/A = Not applicable.
The iiPCR system detected ASFV without cross-reactivity with the 4 other common swine pathogens that produce similar clinical signs (Table 2).
Analytical specificity (detection rates) of the iiPCR system and real-time PCR (rt-PCR) assay for detection of ASFV but not other common swine pathogens that produce similar clinical signs.
| Pathogen | RAHO-6 | NCVD | ||
|---|---|---|---|---|
| iiPCR | rt-PCR assay | iiPCR | rt-PCR assay | |
| CSFV | 0/2 | 0/2 | 0/2 | 0/2 |
| PPRSV | 0/2 | 0/2 | 0/2 | 0/2 |
| FMDV | 0/2 | 0/2 | N/A | N/A |
| PCV2 | N/A | N/A | 0/2 | 0/2 |
| ASVF (positive control) | 1/1 | 1/1 | 1/1 | 1/1 |
| Negative control | 0/1 | 0/1 | 0/1 | 0/1 |
See Table 1 for remainder of key.
Diagnostic performance
At the RAHO-6, complete agreement was found between results of the iiPCR system and the reference method for the total of 121 predetermined ASFV-positive and 95 predetermined ASFV-negative lymph node, whole blood, serum, and spleen samples (Supplementary Table S1). Overall sensitivity and specificity of the iiPCR system were therefore 100% (95% CI, 97.2% to 100.0%) and 100% (95% CI, 97.8% to 100.0%). Accuracy was also 100% (95% CI, 98.8% to 100.0%), and the Cohen κ value was 1.00.
At the NCVD, 4 discrepancies in results were observed for the total of 68 predetermined ASFV-positive samples, 52 predetermined ASFV-negative samples, 24 nonpretested tonsil samples, and 6 nonpretested spleen samples (Supplementary Table S1). These discrepant results involved 3 false-positive samples and 1 false-negative sample overall. Overall sensitivity and specificity were therefore 98.9% (95% CI, 95.5% to 100.0%) and 94.8% (95% CI, 87.9% to 100.0%), accuracy was 97.3% (95% CI, 94.3% to 100.0%), and the Cohen κ value was 0.94. Results of discrepancy testing for the 4 samples that yielded different results between the 2 test methods were summarized (Table 3), showing final false-positive results for 2 samples (tissue homogenate and spleen) with the iiPCR system, with correct, negative results obtained with the real-time PCR assay, the Ct values for which were below threshold.
Results of discrepancy testing for the 4 samples at the NCVD with results that differed between the iiPCR system and rtPCR assay.
| Sample | Discrepancy | iiPCR | rt-PCR assay | Final conclusion | ||
|---|---|---|---|---|---|---|
| S/N | Result | Ct | Result | |||
| 19S146 (tissue) | iiPCR+ vs rtPCR− | 1.57 ± 0.62 | + | BT | − | False positive |
| D13963 (tonsil) | iiPCR+ vs rtPCR− | 1.02 ± 0.03 | − | BT | − | False negative |
| D13957 (tonsil) | iiPCR− vs rtPCR+ | 1.03 ± 0.00 | − | BT | − | True negative |
| S13271 (spleen) | iiPCR+ vs rtPCR− | 1.88 ± 0.56 | + | BT | − | False positive |
Each S/N value represents the mean ± SD of 3 repeated assays of each sample retested because of an observed discrepancy.
+ = Positive. − = Negative. BT = Below threshold.
Discussion
Control of ASF is challenging but important. Because of the high virulence and fatality rate of ASFV infection, domestic pigs in infected herds are typically culled. Infected wild pigs are typically detected by passive surveillance (animals found dead), allowing the disease to rapidly spread in susceptible populations and then fade out.31 However, the risk of infection can linger because of prolonged persistence of the virus in contaminated carcasses, which may remain for weeks in fields or forests where the infected wild pigs died.31,32 As omnivores, pigs—particularly wild pigs—can become infected through ingestion, sniffing, or rooting around infected carcasses.33 The virus can also be spread among pigs by vectors such as soft ticks of the genus Ornithodoros.34 Furthermore, ASFV is extremely stable in the environment and efficiently transmitted via blood and meat of infected animals. It can persist at 4 °C for > 1 year in blood, several months in boned meat, and several years in frozen carcasses35,36 and survives putrefaction.37
The less accurate the diagnosis of ASF is and the longer it takes to achieve a definitive diagnosis, the greater the opportunity for spreading the disease becomes. Thus, in the present study, the usefulness of an iiPCR system for rapid and accurate detection of ASFV in swine biological samples was evaluated. Except for the stage involving grinding of tissue samples in PBS solution prior to DNA extraction, samples were processed automatically by the iiPCR system through to the results stage, which could prevent user error and sample contamination. The detection limit of the iiPCR system for ASFV was found to be low and comparable to that of real-time PCR assay, the reference method. The iiPCR system was able to detect ASFV in sample dilutions of 101 to 106, demonstrating greater analytical sensitivity than the real-time PCR assay, which, at the NCVD, was able to detect the virus in dilutions of up to 105 only. Similar to the results for the real-time PCR assay, the iiPCR system had excellent analytical specificity for detection of ASFV, with no cross-reaction observed with other swine viral pathogens, particularly CSFV—the virus that causes clinical signs indistinguishable from those of ASFV. These findings suggested that the iiPCR system was reliable for ASFV detection and comparable in analytical performance to the reference method. Therefore, we believe that the iiPCR system could serve as a substitute protocol for real-time PCR assay in the detection of ASFV, yielding faster results without the requirement for an expensive, real-time PCR thermocycler.
Diagnostic performance of the iiPCR system was evaluated with a large number of samples collected from ASF-epidemic regions by the RAHO-6 and NCVD, which are both authorized institutions for veterinary administration and animal disease control in Northern and Southern Vietnam. These samples represented a wide range of types, including lymph node, whole blood, serum, tissue homogenate, spleen, and tonsil. Sensitivity, specificity, and accuracy was very high at both institutions, with high Cohen κ values of 1.0 and 0.94 at the RAHO-6 and NCVD, respectively. For samples with predetermined ASFV status, only 1 false-positive result was obtained at the NCVD, and this pertained to an ASFV-negative tissue homogenate sample for which the Ct yielded by the real-time PCR assay was below threshold. Discrepancy tests yielded 2 false-positive results in 3 repeated assays for the iiPCR system: one involving a spleen sample and the other a homogenized tissue sample. It was remarkable that at NCVD, the analytical sensitivity of the iiPCR system was higher than that of real-time PCR assay; consequently, the viral concentration of these discrepant samples might have been just within the detectable limit of the iiPCR system but below that of the real-time PCR assay.
For the nonpretested spleen and tonsil samples, diagnostic performance tests yielded 2 false-positive results and 1 false-negative result for the iiPCR system. Subsequent discrepancy testing revealed that the false-negative result was actually a true-negative result, suggesting that the sensitivity and accuracy of the iiPCR system at the NCVD was actually as high as 100% and 98.0%, respectively.
Timeliness of a national strategy for ASF epidemic control is currently limited by the time required to detect ASFV in collected samples, which is largely influenced by test performance time. The total time required for ASFV detection with the iiPCR system was approximately 2 hours, including a sample pretreatment time of 0.5 hour plus a DNA extraction and iiPCR assay time of 1.5 hour. This is approximately half the time required for the real-time PCR assay, which consumes at least 4 hours, including 0.5 hours for sample pretreatment, 1 hour for DNA extraction, and at least 2.5 hours for the PCR assay itself and does not include the time required to transfer samples from the field to the laboratory for testing. The iiPCR system, on the other hand, can be used on-site. The reagents for ASFV detection with this system are in lyophilized format and could be stored at 2 to 8 °C for at least 2 years after manufacture, with long open-bag stability: 2 to 8 °C for 2 months or −20 °C for 6 months.
We found that the iiPCR system for ASFV detection could be successfully applied for a wide range of sample types with high sensitivity, specificity, and accuracy. The evaluated sample types were those that could be collected by farmers or veterinarians in combination with information about clinical signs. Furthermore, the iiPCR system was user-friendly, yielding results that were easily interpreted. These and our other findings suggested that the iiPCR system for ASFV detection has great potential for use in the field for disease surveillance and quarantine in national strategies for early ASFV control.
Supplementary Materials
Supplementary materials are available online at: avmajournals.avma.org/doi/suppl/10.2460/javma.259.6.662.
Acknowledgments
No third-party funding or support was received in connection with this study or the writing or publication of the manuscript.
The authors declare that there were no conflicts of interest.
Footnotes
KingFisher Flex purification system, Thermo Fisher Scientific, Waltham, Mass.
Taco nucleic acid automatic extraction system, GeneReach Biotechnology Corp, Taichung City, Taiwan.
Taco DNA/RNA extraction kit, GeneReach Biotech, Taiwan.
Platinum quantitative PCR SuperMix-UDG, Thermo Fisher Scientific, Waltham, Mass.
QuantStudio 5, Thermo Fisher Scientific, Waltham, Mass.
qScript XLT 1-step RT-qPCR ToughMix, Quantabio, Beverly, Mass.
ABI 7500, Applied Biosystems, Waltham, Mass.
POCKIT Central nucleic acid analyzer, GeneReach Biotechnology Corp, Taichung City, Taiwan.
POCKIT Central cartridge set (24 tests/set), GeneReach Bio-technology Corp, Taichung City, Taiwan.
POCKIT extraction cartridge, GeneReach Biotechnology Corp, Taichung City, Taiwan.
POCKIT transfer cartridge, GeneReach Biotechnology Corp, Taichung City, Taiwan.
POCKIT Central ASFV premix reagent (24 tests/set), Gene-Reach Biotechnology Corp, Taichung City, Taiwan.
Excel professional edition 2016, Microsoft Corp, Redmond, Calif.
Abbreviations
| ASF | African swine fever |
| ASFV | African swine fever virus |
| CSF | Classical swine fever |
| CSFV | Classical swine fever virus |
| Ct | Cycle threshold |
| FMDV | Foot-and-mouth disease virus |
| iiPCR | Insulated isothermal PCR |
| NCVD | National Center for Veterinary Diagnostics, Vietnam |
| OIE | World Organization for Animal Health |
| PCV2 | Porcine circovirus type 2 |
| PRRSV | Porcine reproductive and respiratory syndrome virus |
| RAHO-6 | Regional Animal Health Office 6, Vietnam |
| S/N | Signal-to-noise ratio |
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