MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCESEVALUATING ABILITY OF DETECTION OF AFRICAN SWINE FEVER VIRUS ASFV IN SWINE OF THE TRIA
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African Swine Fever (ASF) is a highly infectious and deadly disease caused by the African Swine Fever virus, with mortality rates reaching up to 100% in pigs First identified in Kenya in the 1920s, ASF spread to Europe in 1957 and the Caucasus region and southern Russia in 2007 The disease made its way to East Asia when China reported its first outbreak in August 2018, leading to rapid transmission across most provinces and significant disruptions to the swine industry By the end of 2018, ASF cases were also reported in several other Asian countries.
In February 2019, the African Swine Fever Virus was first identified in Vietnam, leading to a rapid and widespread outbreak within five months that significantly impacted the country's pig production industry.
The African Swine Fever (ASF) epidemic emerged in Vietnam in 2020 and persisted through 2021 and 2022, significantly affecting the pig industry, which constitutes 60% of the nation's total livestock output This outbreak has resulted in substantial economic losses for farmers and negatively impacted the overall pig production sector, contributing to a decline in the country's gross domestic product (GDP).
Early detection of this serious virus is crucial, prompting the development of real-time PCR reaction kits to fulfill market needs effectively.
The TraceDetectTM qPCR ASFV Kit is designed to streamline the detection and quantification of African Swine Fever Virus (ASFV) through real-time PCR technology With numerous real-time PCR kits available on the market, it is crucial to rigorously test and evaluate these kits during the research phase to ensure their sensitivity and specificity Consequently, a study titled "Evaluating ability of detection of African Swine Fever Virus (ASFV) on the trial kit TraceDetectTM qPCR ASFV" was conducted to assess the kit's performance.
Evaluating ability of detection of African Swine Fever virus (ASFV) on the trial kit TraceDetectTM qPCR ASFV.
Content 1: Evaluate the parameters (specificity and limit of detection) of TraceDetectTM qPCR ASFV kit.
Content 2: Successfully comparison and evaluation the ability to detect ASFV on different field samples with a trial kit.
LITERATURE REYVIEYW - L1 L2 12 1 1 1 1H 1111111 111 re 3 2.1 Introduction of African Swine Fever virus (ASTFV) 2c 2 S2ccc+ssceeecseecexeee2
Diseases and associated syndromes . 5 2+ + +2 +21 *2E 221 2121 1E se rrke 5
African Swine Fever Virus (ASFV) initially enters the body through the tonsils or dorsal pharyngeal mucosa, subsequently spreading to the mandibular or retropharyngeal lymph nodes and disseminating systemically via viremia The virus can be detected in nearly all tissues of infected pigs, with the clinical progression of African Swine Fever in domestic pigs categorized as peracute, acute, subacute, or chronic (Greig, 1972).
The clinical signs and pathology of African Swine Fever Virus (ASFV) vary based on several factors, as noted by Schulz et al (2017) In the peracute form of ASFV, key indicators include a body temperature exceeding 41°C, significant loss of appetite, and pronounced depression in affected swine.
Cutaneous hyperemia in swine can lead to death within 1 to 4 days after the onset of clinical signs The peracute form of the disease is typically observed in regions where African Swine Fever Virus (ASFV) is absent, with the presentation of clinical symptoms varying based on several influencing factors.
Acute African Swine Fever (ASF) is caused by highly or moderately virulent strains of the virus and is characterized by a rapid clinical progression Key symptoms include a high fever ranging from 40 to 42°C, lethargy, anorexia, inactivity, respiratory failure, and severe pulmonary edema Affected animals may also exhibit widespread necrosis and hemorrhage, particularly in lymphatic tissues, skin (notably on the ears and flanks), and splenomegaly, leading to high mortality rates Additional clinical signs can include nasal discharge, vomiting, and diarrhea, with the latter sometimes resulting in black stains around the perianal region.
2015) Pregnant sows may be miscarry (Wu ef al/., 2021).
Subacute African Swine Fever (ASF) presents symptoms similar to those of acute ASF, albeit generally less severe Infected pigs typically succumb to the disease 7 to 20 days post-infection, with mortality rates ranging from 30% to 70% These pigs often exhibit moderate to high fever, and vascular changes such as hemorrhages and edema can be more pronounced in the subacute form compared to the acute form (Gomez-Villamandos et al., 2003) Post-mortem examinations reveal hydropericardium, ascites, and multifocal edema, particularly in the gall bladder wall and perirenal fat (Sanchez-Vizcaino et al., 2015) Additional clinical signs may include hemorrhagic splenomegaly or partial splenomegaly with affected patches, as well as multifocal hemorrhagic lymphadenitis (Gómez-Villamandos et al., 1995).
The clinical manifestations of chronic ASF were skin multifocal necrosis,arthritis, growth retardation, weight loss, dyspnea, and miscarriage.
Table 2.1 Main lesions observed in the different forms of ASF (Sanchez ef al., 2015)
Peracute form Acute form Subacute form — Chronic form Virulence High/moderate Moderate Low x High tan Eien fever, See acute form Respiratory
Clinical appetite loss, appetite loss, :
: but less signs, signs lethargy, lethargy, gastro- tegraynaisl —— hypernode intestinal signs P
- esin hemorrhages in necrotic skin, Pathology Erythema szszergiẽ ue — several organs, pneumomia, kumxane cs l hemorrhagic pericarditis,
Làn Wan ° lymph nodes, abortion abortionMortality Variable Low
Acute African Swine Fever (ASF) is characterized by severe hemorrhagic splenomegaly, as seen during post-mortem examinations, where the abdominal cavity reveals a large, dark-colored spleen and a congested liver Additionally, multiple areas of partial hemorrhagic splenomegaly can be observed in the spleen of subacute ASF cases Notable findings include multifocal hemorrhages in lymph nodes, particularly a marbled appearance in acute ASF, and severe hemorrhagic lymphadenopathy in various lymph nodes, including the gastrohepatic, renal, and ileocecal lymph nodes Moderate hemorrhagic lymphadenopathy is also present in the mesenteric lymph node, highlighting the severe impact of acute ASF on the lymphatic system (Salguero, 2020).
African Swine Fever (ASF) has been reported in twenty-five countries across sub-Saharan Africa, exhibiting various epidemiological patterns The disease was recognized in the Caucasus region following an outbreak in Georgia in June 2007, subsequently spreading to Armenia and Azerbaijan Since 2011, ASF has expanded into the northwest, affecting new areas in the Russian Federation and Ukraine In August 2018, ASF was identified for the first time in China, quickly spreading to multiple provinces and municipalities ASF remains endemic in Africa, primarily transmitted between wild pigs and soft ticks, with species such as O moubata in Africa and O erraticus in the Iberian Peninsula identified as vectors of the ASF virus.
African Swine Fever Virus (ASFV) is primarily transmitted through direct contact between infected and healthy animals, utilizing oral and nasal routes, as well as through tick bites and various injection methods (Guinat et al., 2016; Plowright et al., 1969) The incubation period for ASFV ranges from 4 to 19 days, influenced by factors such as the virus strain, exposure route, and virulence During this incubation phase, infected domestic pigs can transmit the virus despite showing no clinical symptoms Once clinical signs appear, ASFV is present in high concentrations across all bodily secretions, including nasal secretions, saliva, feces, urine, conjunctival secretions, and genital secretions.
African Swine Fever Virus (ASFV) can survive in contaminated environments for extended periods, remaining viable for over three days in barns and several weeks in pig manure Remarkably, ASFV can be isolated at room temperature after 18 months and from rotting blood even after 15 weeks In frozen or uncooked meat, the virus can persist for months, but processed products like Parma ham show no infectious virus after 300 days of curing Cooking or canning hams at 70°C (158°F) eliminates ASFV, while infectious virus is undetectable in chilled deboned meat, bone-in meat, or ground pork after 110 days, and in smoked deboned meat after 30 days.
ASFV was inactivated by organic solvents, detergents, oxidizing agents including hypochlorite and phenol, and commercial disinfectants For example, ASF V was inactivated in 30 minutes by exposure to 2.3% chlorine, 3% ortho- phenylphenol,
9 or iodine-containing compounds Other effective virucidal treatments include formalin, sodium hydroxide, beta-propiolactone, glyceraldehyde’s, or acetyl-ethyleneimine (Sanchez-Vizcainoa ef ạ., 2010).
Real-time PCR, or Quantitative PCR (qPCR), integrates traditional PCR principles with fluorescence detection of amplified products after each cycle, eliminating the need for post-amplification electrophoresis on agarose gel This method is widely utilized in research and diagnostics for both qualitative analysis—determining the presence of target genes—and quantitative analysis, which measures the gene quantity in a sample The ability to quantify genes is a significant advantage of Real-time PCR, offering two levels of quantification: relative and absolute.
Fluorescence plays a crucial role in Real-time PCR reactions, enabling computers to detect signals after each cycle When target DNA is amplified, the tubes emit specific fluorescence wavelengths upon excitation Currently, the most widely used fluorescent agents are those incorporated into double-stranded DNA and probes (Monis et al., 2006).
Real-time fluorescent PCR chemistries play a crucial role in real-time PCR reactions, facilitating DNA analysis through two primary methods These methods include the use of dsDNA binding dyes for both specific and non-specific detection of amplified products, as well as the detection of specific PCR products using fluorophore-linked oligonucleotides, such as primer-probes and nucleic acid analogues (Navarro et al., 2015).
DNA binding dyes function by absorbing excitation light and fluorescing upon binding with double-stranded DNA (dsDNA) present in the reaction medium This category of luminescent agents encompasses various chemicals, including Ethidium.
10 bromide, YO-PRO-1, BEBO, LC Green, SYBR® Green I, SYTO9, SYTO-82 and SYTO-13, EvaGreen, SYBR® Gold.
Primer-probes are designed to incorporate fluorescent elements directly into primers, and they come in various structural forms These include hairpin primer-probes, which can be categorized into three types: scorpion primer-probes, AmplifluorTM primer-probes, and LUXTM (Light-Upon-eXtension) primer-probes Additionally, there are pseudo-cyclic forms known as "cyclicons" (Cyclicon primer-probes) and linear structures that work in conjunction with a DNA minor-groove binding dye, such as Angler® primer-probes (Navarro et al., 2015).
The probe is an oligonucleotide sequence with an attached donor and/or acceptor fluorophore, which included two groups, hydrolysis, and hybridization (Navarro et al.,
The TaqMan probe, a hydrolysis probe known for its high specificity and applicability, consists of a short-chain oligonucleotide designed to complement a specific region on the target sequence It features a fluorescence emitter (Reporter) at the 5' end and a fluorescent absorber (Quencher) at the 3' end Upon excitation by light, the fluorescence absorber transitions from a stable state to an excited state, releasing energy as it returns to its initial stable state within a brief period, typically one to ten nanoseconds This mechanism enhances the specificity of amplification, making TaqMan probes a valuable tool in molecular biology applications.
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MATERIALS AND METHOD s.sccccce nen n2 101101 12 G2 long nee 16 3.1 Location and duration na ố 16 3.2, Objectiand material S sang gu 060116605 50015800115G399G100GGN5E0IS88650NSNGbyGUAIBSBN RARE ERS EEE 16
Materials research
Clinical samples: 90 samples including 50 serum samples, 20 tissue samples and
Twenty stool samples were collected from pig farms across various provinces in Vietnam These samples were promptly shipped to the laboratory within 24 hours for analysis The DNA was extracted from the samples, while the untreated samples were preserved at a temperature of -20°C to ensure their integrity.
The trial kit's specificity was tested using unrelated DNA/RNA samples from various bacteria and viruses responsible for common pig diseases These included Escherichia coli, Salmonella spp., Actinobacillus suis, Staphylococcus aureus, Streptococcus suis, Porcine Circovirus type 2 (PCV2), and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), all provided by BIOMIN Vietnam Co Ltd.
Standard solutions containing plasmids carrying the target gene of ASFV with concentrations 10° copies/HL were provided by the TraceDetectTM qPCR ASFV Kit producer.
DNA extraction was conducted using the TracePureTM Viral DNA/RNA Extraction Kit and the TraceBeadTM Nucleic Acid Extraction Kit from KT BIOTECH Furthermore, a sterile TE buffer at pH 8.0, prepared through filtration from BIO BASIC, was utilized to dilute the standard plasmid.
Real-time PCR reactions were performed using the trial kit TraceDetectTM qPCR ASFV Kit for detection of ASFV.
This study utilized a range of essential equipment and tools, including the Linegene K Plus Real-time PCR from BIOER, a centrifuge from MRC Laboratory Instruments, and temperature-controlled storage solutions such as a 4°C refrigerator and deep freezers set at -20°C and -80°C Additional instruments included a vortex machine, a biosafety cabinet, a Thermo Scientific dry bath incubator, various micropipettes (10µl, 100µl, 1000µl), Eppendorf tubes (1.5 ml), and filter tips (10µl, 100µl, 1000µl), all crucial for conducting the research effectively.
Methodology 20157 17
To process whole blood samples for serum collection, the blood is allowed to clot and is then transferred into a 1.5 ml Eppendorf tube The samples are centrifuged at 14,000 rpm for 5 minutes, which separates the supernatant, resulting in the collection of serum.
Treatment of stool samples: Stool samples were processed into a 10% suspension with PBS solution and centrifuged at 14000 rpm for 5 minutes to collect the supernatant. 3.3.2 Extraction DNA
DNA extraction from raw samples is a crucial step for conducting experimental manipulations This process typically involves several key stages, including the disruption of cells to release DNA into solution, followed by the precipitation of DNA while eliminating contaminating biomolecules such as proteins, polysaccharides, lipids, phenols, and other secondary metabolites Modern techniques often utilize silica columns found in DNA extraction kits to enhance this process.
3.3.2.1 Extraction DNA from serum, whole blood, and saliva
In this study, the extraction of total nucleic acid from serum samples was performed using TracePureTM Viral DNA/RNA Extraction Kit with the following steps:
Step 1: Add 200 uL samples and 20 pL Proteinase K into 1.5 ml tubes.
Step 2: Add 200 uL Lysis Solution (Lysis Buffer mixed with Carrier RNA based on the manufacturer’s instructions) and mix through by vortex for 20 seconds Incubate at 56°C for 15 minutes, vortex 2 to 3 times during incubation.
Step 3: Add 300 uL Ethanol (96 - 100%) to the samples and mix by vortex and transfer the solution into the filter column, incubate for 3 minutes Centrifuge at 8000 xg for 1 minute.
Step 4: Add 700 uL Wash Buffer 1 Centrifuge at 8000 xg for 1 minute.
Step 5: Add 500 hL Wash Buffer 2 Centrifuge at 16000 xg for 1 minute (repeat). Centrifuge the empty column at 16000 xg for 1 minute.
Step 6: Transfer the spin column to a new 1.5 mL tube Add 50 to 100 pL Elution Buffer (Elution Buffer heated to 56°C) and incubate for 3 minutes Centrifuge at 8000 xg for 1 minute.
Step 7: Remove the column and collect the 1.5 mL centrifuge tube The extracted DNA cuold be used immediately or stored at -20°C.
For tissue samples, the process of extracting total DNA was performed using TraceBeadTM Nucleic Acid Extraction Kit with the following steps:
Step 1: Cut 10 to 25 mg tissue samples into 1.5 ml tubes Add 180 nL Digestion Solution + 20 pL Proteinase K Mix through by vortex Incubate for 1 to 3 hours until the sample was completely digested Vortex several times during incubation Let the sample cool to room temperature and then dd 10 pL RNase A Incubate at room temperature for 10 minutes.
Step 2: Add 200 pL Lysis Solution Vortex for 30 seconds.
Step 3: Add 300 uL Ethanol (96 - 100%) to the samples and mix by vortex and transfer the solution into the spin column, incubate for 3 minutes at room temperature. Centrifuge at 16000 xg for 1 minute.
Step 4: Add 500 hL Wash Buffer 1 Centrifuge at 16000 xg for 1 minute Discard the liquid in the collection tube and reattach the collection tube to the filter column.
Step 5: Add 500 hL Wash Buffer 2 Centrifuge at 16000 xg for 3 minutes Discard the liquid in the collection tube and reattach the collection tube to the filter column Step 6: Repeat step 5 and centrifuge empty column at 16000 xg for 1 minute to dry the spin column.
Step 6: Add 50 to 100 pL Elution Buffer (Elution Buffer heated to 56°C) and incubate at 3 minutes Centrifuge at 8000 xg for 1 minute Discard flow-through and collection tube.
Step 7: DNA cuold be used immediately or stored at -20°C.
The TraceDetectTM qPCR ASFV Kit was utilized for real-time PCR reactions following the manufacturer's instructions Positive results were detected using a Taqman probe in the FAM channel, which operates with an excitation wavelength of 495 nm and an emission wavelength of 520 nm Each 20 µL reaction consisted of 10 µL of 2X Master mix, 3 µL of Primer/Probe mix, 2 µL of nuclease-free water, and 5 µL of DNA template, including both positive and negative controls The thermal cycling conditions included an initial denaturation at 95°C for 2 minutes, followed by 45 cycles of denaturation at 95°C for 15 seconds and annealing at 60°C for 60 seconds.
The detection limits of the TraceDetectTM qPCR ASFV Kit were assessed by conducting qPCR reactions using a standard sample containing ASFV DNA plasmid The plasmid concentrations ranged from 10° copies/µL, with further dilutions down to concentrations of 10°, 104, 103, and 107 copies/µL.
101, 10° copies/uL The Ct value corresponding to each concentration was used to find the lowest detectable DNA concentration value by the trial kit The experiment repeated
The standard curve was generated by analyzing the reaction results from ten-fold serial plasmid dilutions, ranging from 10° to 10° copies/µL, using Excel 2016 This analysis provided key metrics, including the correlation coefficient (R²), the slope of the standard curve, and the y-intercept Additionally, the efficiency value (E) of the PCR reaction can be calculated using the formula outlined by Bustin et al (2009).
The trial kit was evaluated for analytical specificity through real-time PCR reactions on seven DNA samples of common pathogenic bacteria found in pigs, including Escherichia coli, Salmonella spp., Staphylococcus aureus, Clostridium perfringens, Streptococcus, Porcine Circovirus type 2 (PCV2), and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) This experiment was conducted in triplicate to ensure accuracy and reliability of the results.
3.3.6 Determination of detection limits on field samples
The detection limit for field samples was established using 10 positive DNA samples from a reference kit during qPCR testing with the trial kit To determine the trial kit's detection limit within the field sample matrix, DNA samples exhibiting a positive Ct value of less than 35 were subjected to serial dilutions of 10, 100, 1,000, and 10,000-fold.
Ct values can quantify the number of copies per reaction based on a standard curve, with the experiment replicated three times The results were statistically analyzed using SPSS software.
3.3.7 Check accuracy in ASFV detection
The accuracy of African Swine Fever Virus (ASFV) detection was assessed using 50 serum samples from which DNA was extracted These samples were randomly assigned identifiers from W1 to WS0 A Real-time PCR reaction was conducted with a trial kit, and the negative, positive, and Ct values of the positive samples were recorded, with each sample tested three times The evaluation of the results was based on comparisons with a commercially available reference kit, allowing for the determination of true negative (TN), true positive (TP), false negative (FN), and false positive (FP) outcomes as outlined in Table 3.1.
Table 3.1 Evaluate the negative and positive results of the trial kit
The Reference kit The trial kit
The analysis of 50 field serum samples enabled the calculation of key diagnostic metrics for the trial kit, including diagnostic specificity, diagnostic sensitivity, positive predictive value, and negative predictive value.
True Negative Diagnostic specificity (Sp) =8 P ow False Negative + True Negative
True Positive Diagnostic sensitivity (Se) = — -
True Positive Positive predictive value(PPV) = — —
True Negative Negative predictive value (NPV) = - :
3.3.8 Checking the trial kit's ability to detect ASFV in different sample platforms
The study evaluated the trial kit's effectiveness in detecting African Swine Fever Virus (ASFV) across various sample types, utilizing a total of 40 samples consisting of 20 tissue samples and 20 stool samples Each experiment was conducted three times, and the results were compared against those obtained from a commercially available reference kit.
3.3.9 Evaluation of the reliability of the trial kit for the detection of ASFV
Data processing na
Microsoft Office Excel 2016 software was used for statistics, calculating the coefficient of variation (CV%), plot graphs, build standard curves, LineGene4080 software is used to perform Real-time PCR reaction.
RESULTS AND DISCUSSION 2-2-2
The limit of detection for the trial Real-time PCR kit targeting the ASFV agent was evaluated using a standard sample of ASFV DNA plasmid, which was diluted from 10° copies/uL to concentrations of 10°, 10, 103, 107, 101, and 10° copies/uL The experiment, conducted in triplicate, demonstrated the kit's capability to detect ASFV DNA across a ten-order magnitude range, from 10° to 10° copies/uL, with the lowest detectable concentration being 10° Results indicated strong linearity throughout this range, confirming the limit of detection at | copy/uL.
Table 4.1 Results of three replicates for serial 10-fold dilutions of ASFV DNA
Concentration of plasmid ASFV (copies/reaction)
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Figure 4.1 Determination LOD of the ASFV TracdetectTM qPCR ASFV Kit
The calibration curve should show a strong linear correlation, slopes betwee -3.3 and -3.6 giving reaction efficiencies between 90 and 105% are typically acceptable, with
The study achieved R² values between 0.98 and 1.00, indicating high precision in the calibration curve influenced by operational accuracy, instrument quality, and chemical reagents (Green and Sambrook, 2018) Using the average Ct values from three replicates and ASFV DNA concentrations, a standard curve was created in Microsoft Office Excel 2016, resulting in a linear equation of y = -3.5854x + 33.722 This curve exhibited a slope coefficient of -3.5854, a correlation coefficient of R² = 0.9998, and an amplification efficiency (E%) of 90.06% Since these parameters fall within acceptable ranges, the standard curve equation is reliable for quantifying the original ASFV DNA copies in the reaction.
Figure 4.2 The standard curve of ASFV plasmid DNA.
The specificity of the test kit was evaluated using DNA/RNA samples from various bacteria and viruses associated with common diseases in pigs, including Escherichia coli, Salmonella spp., Staphylococcus aureus, Clostridium perfringens, and Streptococcus.
The study focused on the detection of Porcine Circovirus type 2 (PCV2) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) The amplification results indicated that the positive control and ASFV-positive serum DNA samples produced fluorescent signals in the kit's FAM color channel, while unrelated DNA/RNA did not show amplification Therefore, the trial kit was confirmed to be specific for the detection of African Swine Fever Virus (ASFV).
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Figure 4 3 Result Real-time PCR to testing analytical.
4.1.3 Determination of detection limits on field samples
To assess the limit of detection for field samples, ten positive DNA samples were analyzed using a reference kit and a trial kit via qPCR DNA samples exhibiting a positive Ct value of less than 35 were subsequently diluted in factors of 10, 100, 1,000, and 10,000 to evaluate the trial kit's detection limit within the field sample matrix The Ct values obtained from the Real-time PCR reactions were subjected to ANOVA analysis, revealing a significant difference in Ct values across concentrations with a significance level (Sig.) of less than 0.05 after three repetitions These findings facilitate the quantitative assessment of DNA copy numbers in the samples.
The quantification of DNA copy number in the original sample is determined using a standard curve represented by the equation y = -3.5854x + 37.307, based on the obtained Ct value This process allows for accurate measurement of DNA concentration, identifying the minimum number of copies present in the test sample.
24 kit could be detected for ASFV is 1 copy/uL In conclusion, the detection limit of the kit in the field sample was lcopy/uL.
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Figure 4.4 The results of real-time PCR of 10 samples after 4 times 10-fold.
4.1.4 Comparative values between the trial kit and Reference kit
4.1.4.1 Check accuracy in ASFV detection
To evaluate the effectiveness of the trial kit in detecting African Swine Fever Virus (ASFV), a total of 50 serum samples were collected from pig farms across various provinces in Vietnam, consisting of 21 positive and 29 negative samples as confirmed by a reference kit The DNA from these samples was then extracted and analyzed for ASFV presence using the Realtime PCR method, with the findings summarized in Table 4.2.
Table 4.2 ASFV detection results on field samples of 2 Real-time PCR kits
Out of 50 field samples collected, there were 29/50 (58%) samples negative and 21/50 (42%) samples positive for detection of ASFV in 3 replicates The results of
The detection of ASFV in 50 samples demonstrated comparable results between the trial kit and the reference kit As detailed in Table 4.2, both kits achieved 100% true positive and true negative results, with no instances of false negatives or false positives Therefore, it can be concluded that the trial kit is equally effective in detecting ASFV as the reference kit.
The analysis of 50 serum samples revealed that the trial kit achieved a diagnostic specificity, diagnostic sensitivity, negative predictive value, and positive predictive value of 100%.
4.1.4.2 Checking the trial kit's ability to detect ASF'V in different sample platforms
To check the trial kit’s ability to detect ASF V in different sample platforms, this study used 40 samples included 20 tissue samples and 20 stool samples The results of
In a study involving three replicates, 40 samples were tested for African Swine Fever Virus (ASFV), revealing that 80% (32/40) were negative and 20% (8/40) were positive The findings indicated no significant difference in detection rates between the trial and reference kits Consequently, the results confirmed that the Real-time PCR Test kit is effective in identifying ASFV in both tissue and stool samples.
Table 4.3 The trial kit's ability to detect ASFV in 2 samples platforms
Type of Number of Trial kit sample samples (+/+) (-/-) (-/+) (+/-)
Tissue Sfoolsample Serum Tissue Stoolsample Serum sample sample sample sample
The trial kit The Reference kit m=Positive mNegative
Figure 4.5 The result of detection ASFV of both kits in tissue and stool samples.
The comparison of ASFV detection accuracy between two kits was conducted using 90 samples, including 50 serum samples, 20 tissue samples, and 20 stool samples, as illustrated in Figure 4.5 The results indicated no significant difference in the detection of positive and negative samples between the two kits Consequently, the trial kit demonstrated its effectiveness in detecting ASFV across various sample types.
4.1.4.3 Evaluation of the reliability of the test kit for the detection of ASFV
The evaluation of the trial kit's reliability for detecting ASFV involved twenty-nine positive samples tested three times, with Ct values documented in Appendix 3 Figure 4.4 illustrates that the coefficient of variation (CV%) ranged from a low of 0.43% in samples W8 and W20 to a high of 7.39% in sample W1, remaining below 15% These results indicate that the Real-time PCR kit demonstrated stability in detecting ASFV in field samples.
Figure 4.6 The stability of the trial kit of 29 positives with ASFV samples.
It has been almost a hundred years since ASF was first described in Kenya since
Since 1921, African Swine Fever (ASF) has increasingly threatened global pig populations Today, various methods exist to identify ASF virus-infected animals, with real-time PCR being the preferred diagnostic assay in many laboratories due to its speed and quantitative capabilities A previous study demonstrated that real-time PCR, utilizing SYBR Green and primers targeting the A137R gene, successfully detected 85.29% of positive samples for ASFV.
In 2013, researchers utilized a TaqMan probe and primer targeting the VP72 gene to effectively detect African Swine Fever Virus (ASFV) The experiment demonstrated a remarkable detection rate of 100% for positive samples, successfully identifying all 94 out of 94 cases, with an enhanced detection limit for field samples.
In 2022, Hwang HJ conducted a study on the development and validation of a rapid quantitative real-time PCR assay for detecting the African swine fever virus, utilizing nucleic acid sequences of the major capsid protein p72 The assay demonstrated a turnaround time of just 50 minutes, achieving 100% agreement for both positive and negative samples Additionally, the limit of detection (LoD) values for various genotypes ranged from 10 to 20 copies per reaction (Hwang et al., 2022).