• Journal of Veterinary Science
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• v.23 no.4
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• pp.51.1-51.10
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• 2022

Background: Due to the unavailability of an effective vaccine or antiviral drug against the African swine fever virus (ASFV), rapid diagnosis methods are needed to prevent highly contagious African swine fever. Objectives: The objective of this study was to establish the ladder-shape melting temperature isothermal amplification (LMTIA) assay for the detection of ASFV. Methods: LMTIA primers were designed with the p72 gene of ASFV as the target, and plasmid pUC57 was used to clone the gene. The LMTIA reaction system was optimized with the plasmid as the positive control, and the performance of the LMTIA assay was compared with that of the commercial real-time polymerase chain reaction (PCR) kit in terms of sensitivity and detection rate using 200 serum samples. Results: Our results showed that the LMTIA assay could detect the 10⁴ dilution of DNA extracted from the positive reference serum sample, which was the same as that of the commercial real-time PCR kit. The coincidence rate between the two assays was 100%. Conclusions: The LMTIA assay had high sensitivity, good detection, and simple operation. Thus, it is suitable for facilitating preliminary and cost-effective surveillance for the prevention and control of ASFV.

• Korean Journal of Clinical Laboratory Science
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• v.51 no.3
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• pp.309-315
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• 2019

Cervical cancer is the fourth most common malignant neoplasm in women worldwide. Most cases of cervical cancer are caused by an infection by the human papillomavirus. Molecular diagnostic methods have emerged to detect the HPV for sensitivity, specificity, and objectivity. In particular, real-time PCR has been introduced to acquire a more sensitive target DNA or RNA. RNA extraction and complementary DNA synthesis are proceeded before performing real-time PCR targeting RNA. To identify an adequate and sensitive cDNA synthesis kit, this study evaluated the two commonly used kits for cDNA synthesis. The results show that the $R^2$ and efficiency (%) of the two cDNA synthesis kits were similar in the cervical cancer cell lines. On the other hand, the Takara kit compared to Invitrogen kit showed P<0.001 in the $10^2$ and $10^3$ SiHa cell count. The Takara kit compared to the Invitrogen kit showed P<0.001 in the $10^1$ and $10^2$ HeLa cell count. Furthermore, 8, 4, 2, 1, and 0.5 ml of forty exfoliated cell samples were used to compare the cDNA synthesis kits. The Takara kit compared to the Invitrogen kit showed P<0.01 in 8, 4, and 1 ml and P<0.05 in 0.5 mL. The study was performed to identify the most appropriate cDNA synthesis kit and suggests that a cDNA synthesis kit could affect the real-time PCR results.

• Korean Journal of Microbiology
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• v.41 no.3
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• pp.168-176
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• 2005

Vibrio vulnificus, one of the marine bacterial pathogens causing septicemia, was detected using molecular methods, namely, PCR and/or Southern hybridization, and real-time PCR. Extracted and purified total DNAs by using commercial kits were used as templates for PCR. Multiplex-PCR was conducted by employing three sets of primers for the genes, hemolysin (vvhA), phosphomannomutase (pmm), and metalloprotease (vvpE), for V vulnificus virulence. The presence of DMSO ($5\%$) and BSA ($0.1\%$) in PCR reaction mixture improved a detection efficiency by higher PCR band intensities. TaqMan real-time PCR was carried out by using gene segment of vvhA as a target. Detection limit of PCR/Southern hybridization without enrichments was to be around $10^2\;cells\;g^{-1}$ of sample. However, those three methods using the enrichment at $35^{\circ}C$ in APW showed high sensitivity ($2\~10\;cells\;g^{-1}$ of sediments). Highly sensitive detection of V vulnificus by real-time PCR was achieved within $5\~6$ hr, whereas the detection by PCR/Southern hybridization required about 36 hr. Thus, it was evident that real-time PCR is the most rapid and efficient method for detecting V vulnificus in tidal flat sediments.

• Journal of Veterinary Clinics
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• v.36 no.5
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• pp.245-247
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• 2019

Taylorella equigenitalis (T. equigenitalis), Klebsiella pneumoniae (K. pneumoniae), and Pseudomonas aeruginosa (P. aeruginosa) are sexually transmittable bacteria known to cause venereal diseases (VD) in horses. T. equigenitalis causes contagious equine metritis (CEM), which is a considerable concern for equine breeding industry. K. pneumoniae and P. aeruginosa may cause endometritis and infertility in susceptible mares. The purpose of this study was to investigate the prevalence of these bacteria among breeding Thoroughbreds in South Korea. External genital swabs were collected from 178 breeding Thoroughbreds, including 11 stallions and 167 mares. The samples were tested using a commercial multiplex real-time PCR kit. T. equigenitalis, P. aeruginosa, and K. pneumoniae were present in 5.6%, 7.3%, and 5.6% of tested Thoroughbreds, respectively. The results highlight the need for regular testing of South Korean Thoroughbreds, particularly those used for breeding, for these bacteria. The regular pre-breeding test for these bacteria will prevent health complications for the horse and financial losses for the owner as a result of VD.

• Korean Journal of Veterinary Research
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• v.54 no.1
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• pp.55-57
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• 2014

Paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (MAP) has extended latent periods of infection. Due to this property, difficulties in the detection of fecal shedder have been raised. A newly designed method for DNA extraction from fecal specimens, mGITC/SC was evaluated in terms of diagnostic efficiency. The detection limit of IS900 real-time PCR was about 50 MAP (1.5 cfu) in 250 mg of feces (6 cfu per g). Also, this DNA extraction method was faster and cheaper than that using commercial kit or other methods. Consequently, the mGITC/SC is an economical DNA extraction method that could be a useful tool for detecting MAP from fecal specimens.

• Biomedical Science Letters
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• v.16 no.3
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• pp.193-196
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• 2010

Multiplex PCR-based short tandem repeat (STR) analysis is considered as a good tool for monitoring bone marrow engraftment after sex-mismatched allogeneic transplantation and provides a sensitive and accurate assessment of the contribution of both donor and/or recipient cells in post-transplantation specimens. Forensic STR analysis and quantitative real time PCR are used to determine the proportion of donor versus recipient each contained within the total DNA. The STR markers were co-amplified in a single reaction by using commercial $PowerPlex^{(R)}$ 16 system and $AmpFISTR^{(R)}$ $Identifiler^{(R)}$ / $Yfiler^{(R)}$ PCR amplification kits. Separation of the PCR products and fluorescence detection were performed by ABI $PRIS^{(R)}$ 3100 Genetic Analyzer with capillary electrophoresis. The $GeneMapper^{TM}$ ID software were used for size calling and analysis of STR profiles. Extracted DNA was quantified by the $Quantifiler^{TM}$ Human DNA / Y Human Male DNA Quantification Kit The intent of this study was to analyze the ratio of donor versus recipient cells in the post-transplant peripheral blood, spleen, lung and kidney specimens. Specimens were taken from the traffic accident male victim who had been engrafted from bone marrow female donor. Blood and spleen specimens displayed female donor DNA profile. Kidney specimen showed male recipient DNA profile. Interestingly, lung tissue showed mixed profiles. The findings of this study indicate that the forensic STR analysis using fluorescence labeling PCR combined with capillary electrophoresis is quick and reliable enough to assess the ratio of donor versus recipient cells and to monitor the mixed chimeric patterns.

• Korean Journal of Veterinary Service
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• v.43 no.2
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• pp.107-112
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• 2020

Foot-and-mouth disease (FMD) and avian influenza (AI) are highly pathogenic viral disease which affects the livestock industry worldwide. Outbreak of these viruses causes great impact in the livestock industry; thus, disease infected animals were immediately disposed. Burial is the commonly used disposal method for deceased animals. However, there is potential for secondary environmental contamination, as well as the risk that infectious agents persisting in the environment due to the limited environmental controls in livestock burial sites during the decomposition of the carcasses. Therefore, this study aimed to investigate the detection of FMD and AI viruses from animal carcass disposal sites using real-time reverse transcription PCR. Soil samples of more than three years post-burial from livestock carcass disposal sites were collected and processed RNA isolation using a commercial extraction kit. The isolated RNA of the samples was used for the detection of FMDV and AIV using qRT-PCR. Based on the qPCR assay result, no viral particle was detected in the soil samples collected from the animal disposal sites. This indicates that 3 years of burial and their carcass disposal method is efficient for the control or at least reduction of spread infections in the surrounding environment.

• Korean Journal of Soil Science and Fertilizer
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• v.44 no.6
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• pp.1181-1184
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• 2011

Total aerobic bacteria, Esherichia coli O157:H7, and Salmonella spp. were examined in commercial liquid pig manures. Commercial liquid pig manures (n=33) were collected from muck joint resource recovery plant at April, June, August, October 2009, Korea. Total aerobic bacteria were incubated at $37^{\circ}C$ for 24-48 hrs, and quantified as a colony-forming unit (CFU) $mL^{-1}$. Analysis of Esherichia coli O157:H7 and Salmonella spp. were followed by Korean Food Standards Codex method. Colony of Salmonella spp. was confirmed by API kit and real time polymerase chain reaction (PCR). Total aerobic bacteria isolated from fermented commercial liquid pig manures ranged from 2.8 to $24.3{\times}10^4\;CFU\;mL^{-1}$. Esherichia coli O157:H7 was not detected, and Salmonella spp. showed the low detection frequency at only 1 sample. This study suggests that continuous monitoring in commercial liquid pig manures is required to improve the agricultural food through management of agricultural land contaminated with liquid pig manures.