• Restorative Dentistry and Endodontics
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• v.30 no.5
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• pp.372-384
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• 2005

The purpose of this study was to monitor the secretion of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) by human periodontal ligament (PDL) fibroblasts stimulated with Prevotella nigrescens lipopolysaccharide (LPS), and to examine the effect of calcium hydroxide treatment on P. nigrescens LPS. LPS was extracted and purified from anaerobically cultured P. nigrescens. PDL fibroblasts were stimulated by the LPS (0, 0.1, 1, 10 ${\mu}g/ml$) or LPS (10 ${\mu}g/ml$) pretreated with 12.5 mg/ml of $Ca(OH)_2$ for 3 days, for various periods of time (12, 24, 48 h). Immunoprecipitation were performed for protein level analysis of MMP-1 MMP-2 and TIMP-1. Total RNA was isolated and real-time quantitative polymerase chain reaction (PCR) was performed for quantification of MMP-1 mRNA. According to this study, the results were as follows: 1. The p개duction of MMP-1 by stimulation with P. nigrescens LPS increased in time-dependent manner, and showed maximum value at 48 h in both protein and mRNA level. But there was no dose-dependent increas. 2. MMP-2 production time-dependently increased when stimulated with 1 and 10 ${\mu}g/ml$LPS, but there was no dose-dependent increase. 3. TIMP-1 p개duction increased to 24 h, but decreased at 48 h. It increased when stimulated with 0.1 and 1${\mu}g/ml$, but suppressed at 10 ${\mu}g/ml$ .4. P. nigrescens LPS pretreated with $Ca(OH)_2$ markedly downregulated MMP-1 gene expression.

• Journal of Food Hygiene and Safety
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• v.33 no.1
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• pp.50-57
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• 2018

Salmonella continue to be a major cause of food poisoning worldwide. The rapid detection method of food-borne Salmonella is an important food safety tool. A real-time polymerase chain reaction (PCR) has been used as a rapid method for the detection of pathogens. It has been recently reported that NBS LabChip real-time PCR is a novel, ultrafast, and chip-type-convenient real-time PCR system. We developed the assay method based on NBS LabChip real-time PCR for the rapid detection of Salmonella, which its reaction time was within 20 minutes. Two target genes (invA and stn) were selected to design target specific primers and probes. The new method was validated by checking specificity and sensitivity (limit of detection). This study included forty-two target and twenty-one non-target strains to assess the specificity. This assay was able to identify the 42 Salmonella strains correctly. The limit of detection (LOD) was $10^1copies/{\mu}L$ in Salmonella genomes DNA, while LOD incubated for 4 hr in the inoculated sausage sample ranged from $10^1CFU/g$ to $10^2CFU/g$ as an inoculated cell count. The assay developed in this study could be applied for the investigation of food poisoning pathogens.

• Korean Journal of Microbiology
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• v.55 no.1
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• pp.25-32
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• 2019

Influenza A (H1N1) virus caused a worldwide pandemic in 2009-2010 and still remains in seasonal circulation. Continuous surveillance activities are encouraged in the post pandemic phase to watch over the trend of occurrence every year, this is better to be done by a rapid and sensitive method for its detection. This study was conducted to detect proportions of occurrence of influenza A virus (H1N1) in patients with influenza-like illness. Samples from 500 patients with influenza or influenza-like clinical presentation were tested by real-time reverse transcription polymerase chain reaction (RT-PCR) and virus tissue culture. Among the total 500 participants, 193 (38.6%) were females and 307 (61.4%) males. Seventy-one patients (14.2%) were positive for H1N1 virus infection with real-time RT-PCR while 52 (10.4%) were positive by tissue culture. Non-statistically significant relation was found between age and gender with the positivity of H1N1. Sensitivity and specificity of real-time RT-PCR was 98.08% and 95.54%, respectively, in comparison to virus isolation with accuracy 95.8%. This study showed that H1N1 virus was responsible for a good proportion of influenza during the post-pandemic period. Real-time RT-PCR provides rapidity and sensitivity for the detection of influenza A virus (H1N1) compared with virus isolation and thus it is recommended as a diagnostic tool.

• Journal of IKEEE
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• v.23 no.1
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• pp.295-301
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• 2019

A heater in a portable real-time polymerase chain reaction(PCR) system is one of the important factors for controlling the PCR thermocycle precisely. Since heaters are integrated on a small-sized PCR chip for rapid heating and fabricated by semiconductor processes, the cost of producing PCR chips is high. Here, we propose to use chip resistors as an inexpensive and accurate temperature control method. The temperature distribution was simulated using one or two chip resistors on a real-time PCR chip and the PCR chip with uniform temperature distribution was fabricated. The temperature rise and fall rates were $18^{\circ}C/s$ and $3^{\circ}C/s$, respectively.

• Korean Journal of Veterinary Service
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• v.44 no.3
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• pp.163-168
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• 2021

The rapid and reliable detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a key role in isolating infected patients and preventing further viral transmission. In this study, we evaluated the clinical diagnostic performances of a commercial real-time reverse transcription loop-mediated isothermal amplification (RRT-LAMP) assay (Isopollo^® COVID-2 assay, M-monitor, Daegu, Korea) using eighty COVID-19 suspected clinical samples and compared these with the results of a commercial real-time reverse transcription polymerase chain reaction (RT-qPCR) assay (Allplex^TM 2019-nCoV rRT-QPCR Assay, SeeGene, Seoul, Korea). The results of the RRT-LAMP assay targeting the N or RdRp gene of SARS-CoV-2 showed perfect agreement with the RT-qPCR assay results in terms of detection. Furthermore, the RRT-LAMP assay was completed in just within a 20-min reaction time, which is significantly faster than about the 2 h currently required for the RT-qPCR assay, thus enabling prompt decision making regarding the isolation of infected patients. The RRT-LAMP assay will be a valuable tool for rapid, sensitive, and specific detection of SARS-CoV-2 in human or unexpected animal clinical cases.

• Biomedical Science Letters
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• v.29 no.4
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• pp.363-370
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• 2023

Human respiratory viral infections such as COVID-19 are highly contagious, so continuous management of airborne viruses is essential. In particular, indoor air monitoring is necessary because the risk of infection increases in poorly ventilated indoors. However, the current method of detecting airborne viruses requires a lot of time from sample collection to confirmation of results. In this study, we proposed a system that can monitor airborne viruses in real time to solve the deficiency of the present method. Air samples were collected in liquid form through a bio sampler, in which case the virus is present in low concentrations. To detect viruses from low-concentration samples, viral RNA was concentrated and extracted using silica-magnetic beads. RNA binds to silica under certain conditions, and by repeating this binding reaction, bulk samples collected from the air can be concentrated. After concentration and extraction, viral RNA is specifically detected through real-time qPCR (quantitative polymerase chain reaction). In addition, based on liquid handling technology, we have developed an automatic machine that automatically performs the entire testing process and can be easily used even by non-experts. To evaluate the system, we performed air sample collection and automated testing using bacteriophage MS2 as a model virus. As a result, the air-collected samples concentrated by 45 times then initial volume, and the detection sensitivity of PCR also confirmed a corresponding improvement.

• Toxicological Research
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• v.32 no.1
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• pp.81-87
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• 2016

Aflatoxin B1 (AFB1) is produced by Aspergillus flavus growing in feedstuffs. Early detection of maize contamination by aflatoxigenic fungi is advantageous since aflatoxins exert adverse health effects. In this study, we report the development of an optimized conventional PCR for AFB1 detection and a rapid, sensitive and simple screening Real-time PCR (qPCR) with SYBR Green and two pairs of primers targeting the aflR genes which involved aflatoxin biosynthesis. AFB1 contaminated maize samples were divided into three groups by the toxin concentration. Genomic DNA was extracted from those samples. The target genes for A. flavus were tested by conventional PCR and the PCR products were analyzed by electrophoresis. A conventional PCR was carried out as nested PCR to verify the gene amplicon sizes. PCR-RFLP patterns, obtained with Hinc II and Pvu II enzyme analysis showed the differences to distinguish aflatoxin-producing fungi. However, they are not quantitative and need a separation of the products on gel and their visualization under UV light. On the other hand, qPCR facilitates the monitoring of the reaction as it progresses. It does not require post-PCR handling, which reduces the risk of cross-contamination and handling errors. It results in a much faster throughout. We found that the optimal primer annealing temperature was $65^{\circ}C$. The optimized template and primer concentration were $1.5{\mu}L\;(50ng/{\mu}L)$ and $3{\mu}L\;(10{\mu}M/{\mu}L)$ respectively. SYBR Green qPCR of four genes demonstrated amplification curves and melting peaks for tub1, afIM, afIR, and afID genes are at $88.0^{\circ}C$, $87.5^{\circ}C$, $83.5^{\circ}C$, and $89.5^{\circ}C$ respectively. Consequently, it was found that the four primers had elevated annealing temperatures, nevertheless it is desirable since it enhances the DNA binding specificity of the dye. New qPCR protocol could be employed for the determination of aflatoxin content in feedstuff samples.

• 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 Veterinary Service
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• v.46 no.2
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• pp.123-135
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• 2023

Feline calicivirus (FCV) is considered the main viral pathogen of feline upper respiratory tract disease (URTD). The frequent mutations of field FCV strains result in the poor diagnostic sensitivity of previously developed molecular diagnostic assays. In this study, a more sensitive real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for broad detection of currently circulating FCVs and comparatively evaluated the diagnostic performance with previously developed qRT-PCR assay using clinical samples collected from Korean cat populations. The developed qRT-PCR assay specifically amplified the FCV p30 gene with a detection limit of below 10 copies/reaction. The assay showed high repeatability and reproducibility, with coefficients of intra-assay and inter-assay variation of less than 2%. Based on the clinical evaluation using 94 clinical samples obtained from URTD-suspected cats, the detection rate of FCV by the developed qRT-PCR assay was 47.9%, which was higher than that of the previous qRT-PCR assay (43.6%). The prevalence of FCV determined by the new qRT-PCR assay in this study was much higher than those of previous Korean studies determined by conventional RT-PCR assays. Due to the high sensitivity, specificity, and accuracy, the new qRT-PCR assay developed in this study will serve as a promising tool for etiological and epidemiological studies of FCV circulating in Korea. Furthermore, the prevalence data obtained in this study will contribute to expanding knowledge about the epidemiology of FCV in Korea.

• Korean Journal of Veterinary Service
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• v.35 no.4
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• pp.255-261
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• 2012

Foot and mouth disease (FMD) is highly infectious disease of cloven-hoofed animals, particularly cattle, sheep, pigs and goats. Last outbreak reported in November, 2010 induced the enormous social and economical impacts. Culling of infected animals, movement control, and vaccination are the major control measures of FMD. The aim of this study was to detection foot-and-mouth disease virus (FMDV) in the soil and leachate from modeling burial for pig carcass as measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR). FMDV and Coxsakievirus B1 (CVB1) were detected in soil by week 16 and Coxsakievirus B1 (CVB1) by weeks 12, respectively. FMDV and CVB1 also detected by weeks 8 in the leachate. Results from this study provides an evidence that FMDV could be inactivated for safe of pig carcasses infected with FMDV within 4 month in the carcass burial site.