The topic research "Evaluation for specificity and sensitivity of TopSPEC®PEDV RI-qPCR KIT for detection the presence of PEDV in pigs” was conducted totest the effectiveness detection of
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The thesis was performed from March 2023 to July 2023 at Gene Technology Laboratory - BIO313, Faculty of Biological Sciences, Nong Lam University Ho Chi Minh City
The positive control for PEDV detection is provided inside each corresponding the trial kit.
The detection kit's specificity was established using the DNA of various bacteria and viruses responsible for common pig diseases, including Escherichia coli, Salmonella, Actinobacillus pleuropneumoniae, Staphylococcus aureus, Clostridium perfringens, Streptococcus suis, Porcine circovirus type 2 (PCV2), Porcine Reproductive and Respiratory Syndrome virus (PRRSV), and African swine fever virus (ASFV) These samples were provided by the Gene Technology Lab - Bio 313 at the Faculty of Biological Sciences, Nong Lam University, Ho Chi Minh City.
The TopSPEC® kit's manufacturer supplied the DNA control necessary for creating a standard curve, which consists of a solution containing plasmid DNA that targets the PEDV gene, with a concentration of 10° copies/µL.
Eighty-two clinical samples, including swabs, stool, and tissue, were collected from pig farms across several provinces in Southern Vietnam for RNA extraction The detection of Porcine Epidemic Diarrhea Virus (PEDV) was performed using the TopSPEC® PEDV RT-qPCR Kit, and the results were compared to those obtained from another commercial kit.
Chemicals: the the trial kit TopSPEC® PEDV RT-qPCR KIT for the qPCR reaction for PEDV, AccuRive pDNA/ RNA Prep Kit (CatEX-DRA05.1A, KT BIOTECH).
Equipment: Linegene K Plus Real-time PCR instrumentation (Cat#FQD-48A, BIOER), centrifuge (Cat#ECEN209-24, MRC Laboratory Instruments), 4°C refrigerator; deep freezers (-20°C, -80°C); vortex machine; biosafety cabinet; dry bath incubator (Thermo Scientific), etc.
Tools: Micropipettes, eppendorf, filter tips, etc.
RNA extraction by AccuRive pDNA/RNA Prep Kit |
Perform the Real-time RT-PCR reaction
Determine the specificity and the limit of detection (LOD) of the the trial kit TopSPEC® PEDV RT-gPCR KIT
[ Evaluate the kit's ability to detect PEDV on field samples
Samples collected for analysis included swab, stool, and tissue samples stored in 1.5 mL Eppendorf tubes Viral RNA was extracted using the AccuRive pDNA/RNA Prep Kit (Cat#EX-DRA0S.1A, KT BIOTECH), following the manufacturer's guidelines.
Step 1: Add 200 uL of the sample and 900 pL of KTS1 into a 1.5 uL tube, then mix by vortex and anneal at room temperature for 10 minutes.
Step 2: Add 200 pL of KTS2 to the mixture and centrifuge at 13000 rpm for 10 minutes.
In Step 3, transfer 600 mL of the supernatant from the centrifuged mixture into a new tube containing 600 mL of KTS3 Vortex the mixture at room temperature for 10 minutes, followed by centrifugation at 13,000 rpm for another 10 minutes Finally, discard the supernatant while retaining the pellet.
Step 4: Add 900 uL of KTS4 in the tube in step 3 then continue centrifugation at
13000 rpm for 10 minutes Discard the supernatant, retain the pellet, and dry at 60°C for 10 minutes.
Step 5: Add 50 uL of KTSŠ to resuspend the RNA and store the RNA at -20°C.
3.4.2 Reverse transcription Real-time PCR
RT-qPCR reactions were conducted using trial kits with a total volume of 25 µL, which included 20 µL of PEDV RT-qPCR Mix and 5 µL of DNA/RNA extraction, following the manufacturer's guidelines The thermal cycling protocol consisted of a preconditioning phase at 45°C for 15 minutes, an initial denaturation at 95°C for 3 minutes, followed by 45 cycles of denaturation at 95°C for 15 seconds, and annealing and elongation at 60°C for 30 seconds.
The results were evaluated using 2 color channels: FAM color channels (representing the signal record of the target PEDV) and HEX (representing the signal
IC record of the sample in the kit) Two the trial kits PEDV were evaluated as positive, negative, or inconclusive as shown in Table 3.1.
Table 3.1 Evaluating of results qPCR to detect PEDV
Result PEDV (FAM) IC (HEX)
3.4.3 Evaluation of the specificity and sensitivity of TopSPEC® PEDV RT-qPCR kit
3.4.3.1 Evaluation of the specificity of the trial kit
The specificity of the TopSPEC® PEDV RT-qPCR Kit was tested using DNA samples from six bacterial species, including Escherichia coli, Salmonella, Actinobacillus pleuropneumoniae, Staphylococcus aureus, Clostridium perfringens, and Streptococcus suis, as well as four viruses: PCV2, PRRSV, ASFV, and PEDV, which are commonly found and pathogenic in pigs To ensure the accuracy of the assay, the experiment was conducted three times.
3.4.3.2 Determine the limit of detection (LOD)
The sensitivity of the trial kits was assessed using the lower limit of detection (LOD), which was established through a serial 10-fold dilution of the Porcine Epidemic Diarrhea Virus (PEDV).
RNA plasmid standards were prepared at concentrations ranging from 10° copies/µL to 10° copies/µL Multiple RT-qPCR reactions were conducted with these standard samples, and the corresponding Ct values for each concentration were recorded To ensure accuracy, the experiment was repeated three times, and a standard curve was generated using the charting tool.
In Microsoft Excel, you can analyze the concentration of standards in a reaction by plotting them against the average Ct value from three repetitions This process allows you to determine the slope, correlation coefficient (R²), and amplification efficiency (E%).
Furthermore, the formula could be used to calculate the efficiency value (E) of a PCR reaction (Bustin ef al., 2009).
E = (10 C1) _1) in which: E: the efficiency of a PCR reaction (%)
Slope: Value of the slope of the standard curve
3.4.4 Evaluating the detectability of PEDV in field samples
3.4.4.1 Evaluating the ability of detecting PEDV by the the trial kit and commercial control kit
To assess the detection capability of PEDV, experiments were conducted using a commercial control kit on 82 samples, including tissue, swab, and stool samples from various swine farms in Southern Vietnam Following the extraction with the AccuRive pDNA/RNA Prep Kit, real-time PCR was executed in triplicate according to the manufacturer's guidelines The results facilitated the calculation of True Positive (TP), False Negative (FN), True Negative (TN), and False Positive (FP) percentages for the trial kit, as presented in Table 3.2.
Table 3.2 Determination of TP, TN, FP, and FN of the trial kit
Commercial kit The trial kit Conclusion
+ + True positive (TP) + - False negative (FN)
& + False positive (FP) (+) Positive sample, (-) Negative sample
3.4.4.2 Evaluating the stability of the the trial kit for detecting PEDV
Following an investigation into the presence of Porcine Epidemic Diarrhea Virus (PEDV) in field samples, the positive samples were analyzed for their Ct values in both the FAM and HEX channels The coefficient of variation (CV%) was calculated after three repetitions to evaluate the stability of the trial kit This assessment utilizes a specific formula to compute the CV%, ensuring accurate measurement of the results.
3.4.4.3 Sensitivity, specificity, positive and negative predict value
Sensitivity, specificity, and accuracy are essential statistics for evaluating the reliability of diagnostic tests Sensitivity (Se) measures the test's ability to correctly identify positive cases, while specificity (Sp) assesses the percentage of samples that test negative for the disease In 2015, Safari et al highlighted the importance of positive and negative predictive values (PPV and NPV) in screening performance The positive predictive value reflects the proportion of true positive diagnoses among all positive test results, whereas the negative predictive value indicates the proportion of true negative diagnoses among all negative test results (Baratloo et al., 2015; Saah and Hoover, 1997; Zhu et al., 2010).
In 2015, Safari et al demonstrated the screening performance characteristics, focusing on positive predictive value (PPV) and negative predictive value (NPV) The PPV represents the proportion of correctly diagnosed positive samples among all positive test results, while the NPV indicates the ratio of accurately diagnosed negative samples to all negative test results Additionally, they successfully calculated diagnostic sensitivity and specificity using established formulas.
The number of True Positive The number of True Positive + The number of False Negative
The number of True Negative The number of False Positive + The number of True Negative
The number of True Positive The niimber of True Positive +The number of False Positive
The number of True Negative The number of False Negative + The number of True Negative
Statistical results, graphing, standard curve construction using Microsoft Excel
4.1.1 Evaluating of the specificity of the trial kit
The TopSPEC® PEDV RT-qPCR KIT demonstrated specificity by effectively distinguishing PEDV from other pathogens, including Escherichia coli, Salmonella, and others Results indicated that the positive control sample exhibited a fluorescent signal in the FAM channel at Ct = 32, while the negative control and unrelated DNA/RNA showed no signal Internal controls consistently amplified in the HEX channel with Ct values ranging from 29 to 32, confirming the proper functioning of both the chemicals and equipment during the qPCR process These findings validate the trial kit's specificity for PEDV without cross-reactivity to the tested pathogens.
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Figure 4.1 Specific evaluation of the qPCR test for PEDV detection A) The fluorescence signal in the FAM channel; B) The fluorescence signal in the HEX channel (internal control).
4.1.2 Determination for the limit of detection (LOD)
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To determine the limit of detection (LOD) for the TopSPEC® PEDV RT-qPCR KIT, a standard plasmid with a concentration of 10° copies/µL was utilized, focusing on the specificity of the PCR reaction The experiment, conducted in triplicate, measured Ct values across seven different template concentrations ranging from 10° to 10° copies/µL Results indicated that the FAM signal positively detected pathogens at a concentration of 5*10! copies/µL after three repetitions, while all concentrations yielded a HEX signal (internal control) Consequently, the LOD for detecting PEDV was established at 5*10! copies/reaction, as detailed in Table 4.1 and illustrated in Figure 4.2.
Table 4.1 Ct values of diluted PEDV RNA plasmid concentrations of 3 repetitions
Concentration of plasmid PEDV (copies/reaction)
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3 129.28 ey (62 4 6 8 1012 14 16 8 20 đệ 28 28 30 32 34 36 36 40 42 44 46 Figure 4.2 Determine the PEDV RT-qPCR kit's LOD 4) The fluorescence signal in the FAM channel; B) The fluorescence signal in the HEX channel (internal control).
A standard curve was established by plotting the threshold cycle numbers (Ct) from real-time PCR against the known copy counts of recombinant plasmid (Balboni et al., 2015) Using Microsoft Office Excel 365, the Ct values were represented on the vertical axis (x) and the logarithm of plasmid concentration on the horizontal axis (y) after three repetitions The resulting linear standard curve was expressed as y = -3.6223x + 36.455, demonstrating a strong linear correlation with an R² value of 0.9947, a slope of -3.6223, and an efficiency (E%) of 89%, as illustrated in Figure 4.3.
Figure 4.3 Standard curve of PEDV RT-qPCR kit at 7 point concentrations.
4.1.3 Evaluating the ability detection PEDV in field samples
4.1.3.1 Evaluating the ability of detection PEDV by the trial kit and commercial kit
After confirming the specificity and limit of detection of qPCR, the kit was utilized to evaluate the detection of PEDV in 82 field samples including 18 positive samples and
64 negative samples with repeating three times The commercial kit was PEDV of PrimerDesign PEDV was identified in the examined samples, according to the results.
An experiment was conducted to evaluate the accuracy of a trial Real-time PCR kit in identifying Porcine Epidemic Diarrhea Virus (PEDV) using 82 field samples collected from farms The results indicated that both the commercial and trial kits detected 64% of the samples as negative and 18% as positive for PEDV, with consistent findings across three repeats Consequently, the trial kit demonstrated the same level of accuracy in detecting PEDV RNA in field samples as the commercial kit.
Commercial kit |, Rep.l Rep.2 Rep.3,
#Positive samples #Negative samples Figure 4.4 The result to detect PEDV in field samples of two PEDV
4.1.3.2 Evaluate the stability of the the trial kit in detecting PEDV
The stability of the PEDV RT-qPCR kit was assessed by analyzing the coefficient of variation (CV%) of Ct values from 18 positive samples, as illustrated in Figure 4.5 In the FAM channel, sample P1 exhibited the lowest CV% at 0%, while sample P11 showed the highest CV% at 13% Similarly, in the HEX channel, sample P5 recorded a CV% of 0%, and sample P16 had the highest CV% at 14%.
The coefficient of variation indicates the correlation of the threshold cycle (Ct) in detecting PEDV using the TopSPEC® PEDV RT-qPCR KIT The survey results demonstrate that this kit effectively identifies the presence of PEDV across various field sample platforms utilized in the study.
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Figure 4.5 Result of PEDV detection test on negative samples of PEDV RT-qPCR kit 4) The fluorescence signal in the FAM channel;
B) The fluorescence signal in the HEX channel.
4.1.3.3 Diagnostic sensitivity and specificity, negative and positive predictive values
The analysis in section 4.1.1.3.1 revealed no discrepancies in positive and negative PEDV results between the trial kit and the commercial kit, with 82 field samples showing no false negatives or false positives The experiment yielded 18 true negative samples and 64 true positive samples, resulting in a diagnostic specificity, sensitivity, positive predictive value, and negative predictive value of 100%.
The E value should reach 100%, signifying that the template doubles with each thermal cycle during exponential amplification Ideal slope values for PCR efficiency are around -3.32, although various factors can influence this efficiency (Bustin et al., 2009) For optimal probe-based assays, efficiency values typically range from 90% to 110%, corresponding to slope values between -3.10 and -3.58 Additionally, an acceptable R² value should range from 0.98 to 1 (Pfaffl, 2004).
The R² value of the prepared line significantly impacts the accuracy of operations, equipment, and chemicals used In this study, the parameters of the standard curve—including the y-intercept, slope, efficiency, and R²—were found to be within the acceptable range for general data quality.
Tran Duc Hoan's research identified 26 positive samples for Porcine Epidemic Diarrhea Virus (PEDV), with phylogenetic analysis revealing 98-99% similarity among PEDV isolates from Korea, China, Vietnam, and Thailand, distinguishing them from European strains Insufficient maternal milk nutrition for piglets increases vulnerability to external microorganisms, reducing their resistance to diseases The prevalence of PEDV peaks in spring, likely due to temperature fluctuations, increased rainfall, and high humidity, which favor virus replication Trong Thang's findings indicated clinical symptoms of PEDV in ecological regions, with 76.67% of intestinal and mesenteric samples and 51.67% of stool samples testing positive Clinical manifestations are monitored through camera observations in the cages.
Diarrhea in pigs is characterized by yellow or gray liquid stools, lethargy, and a tendency to pile up or lie prone (100%) A significant number of affected pigs are very thin (82.60%), exhibit excessive thirst (60.86%), and have a reduced body temperature (54.34%) Vomiting is also observed in some cases (54.34%), alongside systemic diseases, particularly in emaciated pigs that display symptoms such as wrinkled skin, yellow feces stuck in the anus (100%), and a bloated abdomen with undigested milk Additionally, affected pigs may show signs of yellowing and excessive foam, along with a thin intestinal wall (100%) Internal organ issues include hemorrhagic glomerulonephritis (73.91%), swollen liver (58.69%), enlarged gallbladder (65.21%), lung hematomas (71.74%), and spleen swelling and congestion (63.04%).
In a 2014 study by Zhao PanDeng et al., intestinal sample analysis revealed a remarkable 92.8% positivity rate, exceeding the conventional RT-PCR results from 42 clinical samples Both TaqMan and RT-PCR methods demonstrated a higher positivity rate for variant Porcine Epidemic Diarrhea Virus (PEDV) compared to classic PEDV, highlighting the significant prevalence of variant PEDV over the past three years Additionally, it's important to note that both PEDV-positive and negative samples may harbor other pathogens linked to diarrhea, such as porcine group A rotavirus (GARV) and transmissible gastroenteritis virus (TGEV), as indicated in previous research (Zhang et al., 2013) Among the 42 intestinal samples from pigs suffering severe diarrhea, 36 tested positive for the variant strain.
Recent analyses identified both PEDV variant and classic samples, revealing viral loads ranging from 10? to 10° copies/uL and 10° to 10° copies/uL, respectively The detection limit for both variants is established at 5 x 10? DNA copies.
In the study by Wang et al (2014), standard curves were established using 10-fold serial dilutions of pCR 2.1-OH851 and pCR 2.1-OH1715 for virulent and variant strains of PEDV, achieving a detection limit of 1 copy/reaction for both strains A strong linear correlation (R² > 0.99) was observed between Ct values and plasmid copy numbers for both PEDV strains, with standard curve slopes of -3.40 and -3.31, respectively The duplex real-time RT-PCR assay effectively identified the virulent strain using a Cy5 probe and the variant strain using a FAM probe, without cross-reactivity with other pig viruses, including PRRSV and TGEV Among 295 positive samples retested, 45 were positive for variant PEDV, while 250 were positive for virulent PEDV, with results confirmed by sequencing using the P160-P161 primer combination This study demonstrates the development of a reliable duplex real-time RT-PCR assay for distinguishing between virulent and variant strains of PEDV.
In a study by Han et al (2019), a duplex quantitative real-time PCR method using SYBR Green I was developed for the detection of Porcine Epidemic Diarrhea Virus (PEDV) The researchers created standard plasmids for PEDV, which were serially diluted, resulting in a standard curve with a linear correlation (R²) of 0.99 and an efficiency of 94.58% The assay demonstrated a detection limit of 3.46 × 10^1 copies/μL and showed specificity for PEDV Among 66 field samples from piglets with diarrhea, 29 tested positive for PEDV, indicating a singular infection rate of 43.94% and an overall detection rate of 71.21% These results confirm that duplex qPCR is a highly sensitive and specific method for the differential diagnosis of PEDV in field samples, supporting its use as an effective surveillance tool for the virus.
A study by Su et al (2018) introduced a novel duplex TaqMan probe-based real-time RT-qPCR for the detection and differentiation of classical and variant Porcine Epidemic Diarrhea Virus (PEDV) in China This method utilized two probes per response to ensure specificity, successfully amplifying DNA without interference from seven other swine viruses Standard curves were created from 10-fold serial dilutions of recombinant plasmid DNA, achieving identical regression coefficients of -3.213 and -3.119, and a correlation coefficient (R²) of 0.999 for both PEDV subtypes Over the past five years, 79 clinical samples from pigs with acute diarrhea in Central China were analyzed, with 51 confirmed positive for PEDV by conventional RT-PCR The duplex TaqMan RT-PCR detected 67 positive samples (84.81%), including 63 variant PEDV, 3 co-infections, and 1 classical PEDV, with viral loads ranging from 10² to 10⁸ copies/reaction.
29 time RT-PCR can be used as a useful clinical diagnostic tool for detecting and differentiating classical and variant PEDV.
CONCLUSION AND RECOMMENDA TION 29 5a, CUOI G LUIS OT esse ores ear reinetaemeae a oe oa menses Soerarsie ees E eae 29 5.22 RECOMMMEHIST ON sesiisiissisiiitiL1680460356840636036430.0663664030354156368086GG08405s 8381453188 8389830.28.88 29
In this study, the TopSPEC® PEDV RT-qPCR KIT was utilized to assess the specificity and limit of detection for specific pathogens The findings confirmed that both trial kits demonstrated high specificity without cross-reacting with unrelated pathogens The limit of detection for the kits was established at 5 x 10^1 copies per reaction, and analysis of eighty-two field samples revealed no presence of the targeted pathogens.
Continue to evaluate the PEDV RT-qPCR kit with a larger number of samples and to combine testing on more sample platforms for kit.
Adams G., A beginner’s guide to RT-PCR, qPCR and RT-qPCR The Biochemist, (2020),vol 42,pp 48-53.
Annamalai T., Saif, L J., Lu, Z., andJung, K., Age-dependent variation in innate immune responses to porcine epidemic diarrhea virus infection in suckling versus weaned pigs Veterinary Immunology and Immunopathology, (2015),vol 168,pp. 193-202.
Anon U., to require reports of PED J Am Vet Med Assoc, (2014),vol 244,pp. 1234.
Arya M., Shergill, I S., Williamson, M., Gommersall, L., Arya, N., andPatel, H. R.H., Basic principles of real-time quantitative PCR Expert Review of Molecular Diagnostics, (2005),vol 5,pp 209-219.
Balboni et al (2015) developed a SYBR Green real-time PCR assay that incorporates melting curve analysis, enabling the simultaneous detection and differentiation of canine adenovirus types 1 and 2 This innovative method, published in the Journal of Virological Methods, enhances diagnostic capabilities in veterinary virology, providing a reliable tool for identifying these pathogens efficiently.
In their 2015 study, Baratloo et al provide a straightforward definition and calculation methods for accuracy, sensitivity, and specificity, essential metrics in diagnostic testing Additionally, Bustin's 2005 article in the Expert Review of Molecular Diagnostics discusses the current procedures and preferences in real-time, fluorescence-based quantitative PCR, highlighting its significance in molecular diagnostics.
Bustin S A., Benes, V., Garson, J A., Hellemans, J., Huggett, J., Kubista, M., et al., The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments Clinical Chemistry, (2009),vol 55,pp 611-622.
In the study by Cankar et al (2006), critical factors influencing DNA quantification through real-time PCR were analyzed, focusing on the impact of different DNA extraction methods and sample matrices on the quantification of genetically modified organisms The findings emphasize the importance of selecting appropriate extraction techniques to ensure accurate measurements in biotechnology applications.
10.Coussement W., Ducatelle, R., Debouck, P., andHoorens, J., Pathology of Experimental CV777 Coronavirus Enteritis in Piglets I Histological and Histochemical Study Veterinary Pathology, (1982),vol 19,pp 46-56.
11.Chang S H., Bae, J L., Kang, T J., Km, J., Chung, G H., Lim, C W., et al., Identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus Mol Cells, (2002),vol 14,pp 295- 299.
12 Chen J., Liu, X., Shi, D., Shi, H., Zhang, X., Li, C., et al., Detection and molecular diversity of spike gene of porcine epidemic diarrhea virus in China Viruses, (2013),vol 5,pp 2601-2613.
A study by Chen et al (2016) published in the Journal of General Virology examines the pathogenesis of the United States porcine epidemic diarrhea virus (PEDV) prototype compared to S-INDEL-variant strains in conventional neonatal piglets The research highlights significant differences in disease progression and severity between these viral strains, providing insights into their impact on swine health Understanding these variations is crucial for developing effective prevention and control strategies against PEDV in pig populations.
14 Debouck P., Pensaert, M., andCoussement, W., The pathogenesis of an enteric infection in pigs, experimentally induced by the coronavirus-like agent, CV 777 Vet Microbiol, (1981),vol 6,pp 157-165.
A study by Dee et al (2014) published in BMC Veterinary Research evaluated the role of contaminated complete feed in transmitting porcine epidemic diarrhea virus (PEDv) to naive pigs The research demonstrated that natural feeding behavior could facilitate infection, highlighting the significance of feed as a potential vector for PEDv spread.
16.Fraga D., Meulia, T., andFenster, S (2014) Current Protocols Essential Laboratory Techniques In (Vol 8, pp 10.13.11-10.13.40).
17.Gibson U E., Heid, C A., andWilliams, P M., A novel method for real time quantitative RT-PCR Genome Res, (1996),vol 6,pp 995-1001.
In 2019, Han et al developed a SYBR green I-based duplex real-time fluorescence quantitative PCR assay, enabling the simultaneous detection of porcine epidemic diarrhea virus and porcine circovirus 3 This innovative method, published in Molecular and Cellular Probes, enhances diagnostic capabilities in veterinary medicine, facilitating more efficient monitoring of these significant swine pathogens The study is detailed in volume 44, pages 44-50.
19 Jung K., Ahn, K., andChae, C., Decreased activity of brush border membrane- bound digestive enzymes in small intestines from pigs experimentally infected with porcine epidemic diarrhea virus Res Vet Sci, (2006),vol 8/,pp 310-315.
20 Jung K., Wang, Q., Scheuer, K A., Lu, Z., Zhang, Y., andSaif, L J., Pathology of US porcine epidemic diarrhea virus strain PC21A in gnotobiotic pigs Emerg Infect Dis, (2014),vol 20,pp 662-665.
21.Kocherhans R., Bridgen, A., Ackermann, M., andTobler, K., Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence Virus Genes, (2001),vol 23,pp 137-144.
22 Lee C., Porcine epidemic diarrhea virus: An emerging and re-emerging epizootic swine virus Virology Journal, (2015),vol 12,pp 193.
23.Lee M., Leslie, D., andSquirrell, D., Internal and External Controls for Reagent Validation Edited by Edwards K, Logan J, Saunders N, (2004),vol.,pp.
24 Li W., van Kuppeveld, F J M., He, Q., Rottier, P J M., andBosch, B.-J., Cellular entry of the porcine epidemic diarrhea virus Virus Research, (2016),vol 226,pp. 117-127.
25.Lohse L., Krog, J S., Strandbygaard, B., Rasmussen, T B., Kjaer, J., Belsham,
G J., et al., Experimental Infection of Young Pigs with an Early European Strain of Porcine Epidemic Diarrhoea Virus and a Recent US Strain Transbound Emerg Dis, (2017),vol 64,pp 1380-1386.
26 Lowe J., Gauger, P., Harmon, K., Zhang, J., Connor, J., Yeske, P., et al., Role of transportation in spread of porcine epidemic diarrhea virus infection, United States Emerg Infect Dis, (2014),vol 20,pp 872-874.
27.Mackay I M., Real-time PCR in the microbiology laboratory Clin Microbiol Infect, (2004),vol 70,pp 190-212.
28 Madson D M., Magstadt, D R., Arruda, P H., Hoang, H., Sun, D., Bower, L P., et al., Pathogenesis of porcine epidemic diarrhea virus isolate (US/Iowa/18984/2013) in 3-week-old weaned pigs Vet Microbiol, (2014),vol. 174,pp 60-68.
29.Martelli P., Lavazza, A., Nigrelli, A D., Merialdi, G., Alborali, L G., andPensaert, M B., Epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in Italy Veterinary Record, (2008),vol 162,pp 307-310.
30.Moldovan E., andMoldovan, V., Controls in Real-Time Polymerase Chain Reaction Based Techniques Acta Marisiensis - Seria Medica, (2020),vol 66,pp. 79-82.
31.Mouritzen P., Noerholm, M., Nielsen, P S., Jacobsen, N., Lomholt, C., Pfundheller, H M., et al (2005) ProbeLibrary: a new method for faster design and execution of quantitative real-time PCR In: Nature Publishing Group US New York.
32 Nath K., Sarosy, J., Hahn, J., andDi Como, C., Effects of ethidium bromide and SYBR® Green I on different polymerase chain reaction systems Journal of Biochemical and Biophysical Methods, (2000),vol 42,pp 15-29.
33.Navarro E., Serrano-Heras, G., Castaủo, M J., andSolera, J., Real-time PCR detection chemistry Clin Chim Acta, (2015),vol 439,pp 231-250.
A study by Niederwerder et al (2016) published in the Journal of Veterinary Diagnostic Investigation examined the tissue localization, shedding, virus carriage, antibody response, and aerosol transmission of Porcine Epidemic Diarrhea Virus (PEDv) in 4-week-old feeder pigs The findings provide crucial insights into the dynamics of PEDv infection, highlighting the virus's behavior in young pigs and its implications for disease management in swine populations.
A study by Pasick et al (2014) investigated the potential role of contaminated feed in the initial outbreaks of porcine epidemic diarrhea (PED) in Canada The research highlighted the significance of feed as a transmission pathway for this viral disease, emphasizing the need for stringent biosecurity measures in the swine industry to prevent future outbreaks The findings contribute to understanding how feed contamination can impact animal health and disease spread, underscoring the importance of monitoring and controlling feed sources.
Ramyasoma, H P B K D (2014) conducted a doctoral dissertation at the University of Colombo focused on developing a real-time PCR-based assay This assay aims to quantify BCR-ABL fusion gene transcripts and assess minimal residual disease in leukemic patients in Sri Lanka.
37 Reguera J., Santiago, C., Mudgal, G., Ordofio, D., Enjuanes, L., andCasasnovas,
J M., Structural Bases of Coronavirus Attachment to Host Aminopeptidase N and Its Inhibition by Neutralizing Antibodies PLOS Pathogens, (2012),vol 8,pp. e1002859.
38.Saah A J., andHoover, D R., "Sensitivity" and "specificity" reconsidered: the meaning of these terms in analytical and diagnostic settings Ann Intern Med,(1997),vol 126,pp 91-94.
39 Scott A., McCluskey, B., Brown-Reid, M., Grear, D., Pitcher, P., Ramos, G., et al., Porcine epidemic diarrhea virus introduction into the United States: Root cause investigation Prev Vet Med, (2016),vol 123,pp 192-201.
40 Seraj Z L, Elias, S M., Haque, T., Jewel, N A., andSunfi, T R., Combination of DNA markers and eQTL information for introgression of multiple salt- tolerance traits in rice Advancement in Crop Improvement Techniques, (2020),vol.,pp.
In 2013, Stevenson et al published a study in the Journal of Veterinary Diagnostic Investigation detailing the emergence of Porcine Epidemic Diarrhea Virus (PEDv) in the United States The research highlighted the clinical signs and lesions associated with the virus, along with the analysis of viral genomic sequences This significant finding contributes to the understanding of PEDv's impact on swine health and the broader implications for the livestock industry.
A novel duplex TaqMan probe-based real-time RT-qPCR assay has been developed for the detection and differentiation of classical and variant strains of porcine epidemic diarrhea viruses, as reported by Su et al in their 2018 study published in Molecular Cell Probes This innovative method enhances the accuracy and efficiency of diagnosing these viral infections in swine, contributing to better disease management and control strategies in the pig farming industry.
43.Tichopad A., Dilger, M., Schwarz, G., andPfaffl, M W., Standardized determination of real-time PCR efficiency from a single reaction set-up Nucleic Acids Res, (2003),vol 37,pp e122.
In a case report published in BMC Veterinary Research, Trujillo-Ortega et al (2016) detail the isolation and characterization of the porcine epidemic diarrhea virus linked to the significant outbreak in Mexico in 2014 This research highlights the virus's impact on swine health and provides valuable insights for veterinary medicine and disease management strategies.
A study by Van Diep et al (2020) published in "Transboundary and Emerging Diseases" explores new tropisms of porcine epidemic diarrhoea virus (PEDV) in pigs that are naturally coinfected with variants exhibiting significant deletions in the spike (S) protein alongside PEDVs that have an intact S protein The research highlights the implications of these findings for understanding PEDV's behavior and spread among swine populations.
46.Van Reeth K., andPensaert, M., Prevalence of infections with enzootic respiratory and enteric viruses in feeder pigs entering fattening herds Ver Rec, (1994),vol 735,pp 594-597.
47 Wang L., Byrum, B., andZhang, Y., New variant of porcine epidemic diarrhea virus, United States, 2014 Emerg Infect Dis, (2014),vol 20,pp 917-919.
48.Wicht O., Li, W., Willems, L., Meuleman, T J., Wubbolts, R W., van Kuppeveld, F J., et al., Proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture J Virol, (2014),vol. 88,pp 7952-7961.
In their 2023 study, Zhang et al conducted a comprehensive investigation into porcine epidemic diarrhea (PED) cases, assessing various immunization strategies within large-scale swine farming systems The findings, published in Porcine Health Management, highlight the importance of effective vaccination approaches to mitigate the impact of PED on swine health and farm productivity This research underscores the need for tailored immunization strategies to enhance disease management in the swine industry.