• Journal of the Korea Institute of Military Science and Technology
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• v.22 no.4
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• pp.575-580
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• 2019

In biological warfare, it is important to identify biological agents for proper treatment. We focused on developing a real-time RT-PCR kit that can detect multiple species of biological agents. AccuPower(R) Biothreat Real-Time RT-PCR Kit(v3.0) could detect Bacillus anthracis, Yersinia pestis, Vibrio cholerae, Francisella tularensis, Salmonella typhi, Rickettsia prowazekii, Variola virus, Hantaan virus, Yellow fever virus, Brucella spp., Shigella dysenteriae in a single reaction. The results showed that the kit was verified to be able to detect at least 0.005 ng of nucleotide and 10,000 CFU/ml of bacteria. Therefore, the kit is expected to be used as a rapid and sensitive detection kit for 11 species of biological agents within 2 hours.

• Food Science and Preservation
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• v.30 no.2
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• pp.262-271
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• 2023

The objective of this study is to compare and assess the effectiveness of real-time polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), and the selective agar plate method for the detection of Salmonella spp. and Listeria monocytogenes in ready-to-eat (RTE) foods. In RTE foods, the detection performance of the three methods (RT-PCR [SureTect^TM kit and PowerChek^TM kit], LAMP [3M MDS], selective agar) were similar at 0-10, 10-50, 50-100, and 100- CFU/mL of Salmonella spp. and L. monocytogenes. We found that with RT-PCR, the Ct value of salad was significantly higher (p<0.05) than other RTE foods, indicating that fiber plays a critical role as an obstacle to the rapid detection of Salmonella spp. However, the Ct value displayed a mixed pattern according to the inoculation level of L. monocytogenes. The use of rapid detection kits and machines mostly depends on the user's choice, with accuracy, ease of use, and economy being the primary considerations. As an RT-PCR kit, SureTect^TM and PowerChek^TM showed high accuracy in detecting Salmonella spp. and L. monocytogenes in RTE foods, showing that they can replace the existing RT-PCR kits available. Additionally, LAMP also showed excellent detection performance, suggesting that it has the potential to be used as a food safety management tool.

• Journal of Microbiology and Biotechnology
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• v.20 no.10
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• pp.1450-1456
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• 2010

Accurate and rapid diagnosis of Pandemic Influenza A/H1N1 2009 virus (H1N1 2009) infection is important for the prevention and control of influenza epidemics and the timely initiation of antiviral treatment. This study was conducted to evaluate the performance of several diagnostic tools for the detection of H1N1 2009. Flocked nasopharyngeal swabs were collected from 254 outpatients of suspected H1N1 2009 during October 2009. This study analyzed the performances of the RealTime Ready Inf A/H1N1 Detection Set (Roche), Influenza A (H1N1) Real-Time Detection Kit (Bionote), Seeplex Influenza A/B OneStep Typing Set [Seeplex Reverse Transcriptase PCR (RT-PCR)], BinaxNow Influenza A & B Test Kit [Binax Rapid Antigen Test (RAT)], and SD BIOLINE Influenza Ag kit (SD RAT). Roche and Bionote real-time RT-PCR showed identical results for the H1N1 2009 hemagglutinin gene. Compared with real-time RT-PCR, the sensitivities and specificities were 83.7% and 100% for Seeplex RT-PCR, 64.5% and 94.7% for Binax RAT, and 69.5% and 100% for SD RAT. The sensitivities of Seeplex RT-PCR, Binax RAT, and SD RAT in patients aged over 21 years were 73.7%, 47.4%, and 57.9%, respectively. The sensitivities of Seeplex RT-PCR, Binax RAT, and SD RAT on the day of initial symptoms were mostly lower (68.8%, 56.3%, and 31.3%, respectively). In conclusion, multiplex RT-PCR and RAT for the detection of H1N1 2009 were significantly less sensitive than real-time RT-PCR. Moreover, a negative RAT may require more sensitive confirmatory assays, because it cannot be ruled out from influenza infection.

• Journal of Microbiology and Biotechnology
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• v.21 no.1
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• pp.100-108
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• 2011

A method for the rapid detection and quantification of Newcastle disease virus (NDV) produced in an animal cell culture-based production system was developed to enhance the speed of the NDV vaccine manufacturing process. A SYBR Green I-based real-time RT-PCR was designed with a conventional, inexpensive RT-PCR kit targeting the F gene of the NDV LaSota strain. The method developed in this study was validated for specificity, accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), and robustness. The validation results satisfied the predetermined acceptance criteria. The validated method was used to quantify virus samples produced in an animal cell culture-based production system. The method was able to quantify the NDV samples from mid- or late-production phases, but not effective on samples from the early-production phase. For comparison with other quantifiable methods, immunoblotting, plaque assay, and tissue culture infectious dose 50 ($TCID_{50}$) assay were also performed with the NDV samples. The results demonstrated that the real-time RT-PCR method is suitable for the rapid quantification of virus particles produced in an animal cell-culture-based production system irrespective of viral infectivity.

• Korean Journal of Environmental Agriculture
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• v.30 no.4
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• pp.367-376
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• 2011

BACKGROUND: In order to develop effective assessment method for Korean paddy soil microbial community structure, reliable genomic DNA extraction method from paddy soil and quantitative real-time PCR (qRT-PCR) method are needed to establish METHODS AND RESULTS: Out of six conventional soil genomic DNA extraction methods, anion exchange resin purification method was turn to be the most reliable. Various PCR primers for distinguishing five bacterial phylum (${\alpha}$-Proteobacteria, ${\beta}$-Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes), all bacteria, and all fungi were tested. Various qRT-PCR temperature conditions were also tested by repeating experiment. Finally, both genomic DNA extraction and qRT-PCR methods for paddy soil were well established. CONCLUSION: Quantitative real-time PCR (qRT-PCR) method to assess paddy soil microbial community was established.

• Microbiology and Biotechnology Letters
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• v.45 no.2
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• pp.87-92
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• 2017

The first Ebola hemorrhagic fever outbreak occurred in the Democratic Republic of Congo and Sudan in 1976 and then emerged in West Africa in 2014 with a total of 27,741 cases and 11,284 deaths. The fever is caused by the Ebola virus, which belongs to the Filoviridae family and contains a ssRNA genome. The known subtypes of the virus are Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, $Ta\ddot{i}$ Forest ebolavirus, and Zaire ebolavirus. The Ebola outbreak was historically originated majorly from the East and Central African tropical belt. The current outbreaks in West Africa caused numerous deaths and spread fear in global society. In the absence of effective treatment strategies and any vaccine, accurate diagnosis is the most important contributing factor in the management and control of the epidemic disease. WHO (World Health Organization) has announced emergency guidance for the selection and use of Ebola in in vitro diagnostic assays. Numerous companies and research institutions have studied the various diagnosis methods and identified four WHO procurement approved as diagnosis kits: RealStar Ebolavirus Screen RT-PCR kit 1.0 (Altona), Liferiver-Ebola Virus (EBOV) Real time RT-PCR kit, Xpert Ebola Assay, and ReEBOV Antigen Rapid Test Kit. The efficiency of novel diagnostic kits such as Rapid Diagnosis Test (RDT) is currently being evaluated.

• 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 Clinical Laboratory Science
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• v.41 no.4
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• pp.145-152
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• 2009

Novel influenza A virus, subtype H1N1 of swine-lineage, has been transmitted rapidly to many regions of the world. Rapid detection of the virus is essential to instigate appropriate patient care and public health management and for disease surveillance. The aim of this study is to determine the prevalence of novel influenza A (H1N1) virus in Korea using reverse-transcription real time polymerase chain reaction (rRT-PCR). Novel H1N1 virus was detected in a total of 8,948 nasopharyngeal samples from patients with influenza-like illness throughout Korea from August to September 2009. RNA was extracted from $300{\mu}l$of sample using an RNA extraction kit (Zymo Research, CA, USA). In the present study, Genekam kit (Genekam, Duisburg, Germany) was used to detect novel H1N1 virus. Novel H1N1 virus was found in 1,130 samples from a total of 8,948 samples (12.6%). The highest frequency was found in 10- to 19-year-olds (M: 29.3% vs. F: 16.4%), followed by 20- to 29-year-olds (M: 17.9% vs. F: 15.4%), 40- to 49-year-olds (M: 6.5% vs. F: 8.1%), 50- to 59-year-olds (M: 6.0% vs. F: 5.5%), and 30- to 39-year-olds (M: 4.6% vs. F: 3.8%). The mean positive rate was higher in men than in women (M: 14.7% vs. F: 7.4%). Novel H1N1 virus showed the lowest prevalence in patients over 60 years old. The positive rate increased daily and showed a significant high peak in mid-September 2009. In 19 provinces of Korea, Cheonan (41.1%), Busan (37.3%), Gangneung (33.3%), Jinju (32.1%), Ulsan (24.6%), Deajeon (23.7%) areas showed high frequencies and other provinces were found less than 10% of novel H1N1 virus. Since reverse-transcription real time PCR assay is rapid, accurate, and convenient, it may assist public health laboratories in detecting novel H1N1 virus. Moreover, these data could be useful for the management of patients with influenza-like illness.

• The Korean Journal of Nuclear Medicine Technology
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• v.23 no.1
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• pp.35-39
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• 2019

Purpose Hepatitis B virus (hepatitis B virus, HBV) infection is a worldwide major public health problem and it is known as a major cause of chronic hepatitis, liver cirrhosis and liver cancer. And serologic tests of hepatitis B virus is essential for diagnosing and treating these diseases. In addition, with the development of molecular diagnostics, the detection of HBV DNA in serum diagnoses HBV infection and is recognized as an important indicator for the antiviral agent treatment response assessment. We performed HBeAg assay using Immunoradiometric assay (IRMA) and Chemiluminescent Microparticle Immunoassay (CMIA) in hepatitis B patients treated with antiviral agents. The detection rate of HBV DNA in serum was measured and compared by RT-PCR (Real Time - Polymerase Chain Reaction) method Materials and Methods HBeAg serum examination and HBV DNA quantification test were conducted on 270 hepatitis B patients undergoing anti-virus treatment after diagnosis of hepatitis B virus infection. Two serologic tests (IRMA, CMIA) with different detection principles were applied for the HBeAg serum test. Serum HBV DNA was quantitatively measured by real-time polymerase chain reaction (RT-PCR) using the Abbott m2000 System. Results The detection rate of HBeAg was 24.1% (65/270) for IRMA and 82.2% (222/270) for CMIA. Detection rate of serum HBV DNA by real-time RT-PCR is 29.3% (79/270). The measured amount of serum HBV DNA concentration is $4.8{\times}10^7{\pm}1.9{\times}10^8IU/mL$($mean{\pm}SD$). The minimum value is 16IU/mL, the maximum value is $1.0{\times}10^9IU/mL$, and the reference value for quantitative detection limit is 15IU/mL. The detection rates and concentrations of HBV DNA by group according to the results of HBeAg serological (IRMA, CMIA)tests were as follows. 1) Group I (IRMA negative, CMIA positive, N = 169), HBV DNA detection rate of 17.7% (30/169), $6.8{\times}10^5{\pm}1.9{\times}10^6IU/mL$ 2) Group II (IRMA positive, CMIA positive, N = 53), HBV DNA detection rate 62.3% (33/53), $1.1{\times}10^8{\pm}2.8{\times}10^8IU/mL$ 3) Group III (IRMA negative, CMIA negative, N = 36), HBV DNA detection rate 36.1% (13/36), $3.0{\times}10^5{\pm}1.1{\times}10^6IU/mL$ 4) Group IV(IRMA positive, CMIA negative, N = 12), HBV DNA detection rate 25% (3/12), $1.3{\times}10^3{\pm}1.1{\times}10^3IU/mL$ Conclusion HBeAg detection rate according to the serological test showed a large difference. This difference is considered for a number of reasons such as characteristics of the Ab used for assay kit and epitope, HBV of genotype. Detection rate and the concentration of the group-specific HBV DNA classified serologic results confirmed the high detection rate and the concentration in Group II (IRMA-positive, CMIA positive, N = 53).

• Journal of Korean Dental Science
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• v.11 no.2
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• pp.62-70
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• 2018

Purpose: Bone marrow has long been a source of primary cells. This study was performed to evaluate the effects of age and sex on the cellular viability and expression of stem cell markers of mRNA and on the protein expression of bone marrow stem cells (BMSCs) derived from healthy donors. Materials and Methods: Stem cells were isolated from human bone marrow and plated in culture plates. The shape of the BMSCs was observed under inverted microscope. Quantitative cellular viability was evaluated using a Cell-Counting Kit-8 assay. The expression of stem cell surface markers was tested and a series of quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot was performed to evaluate the expression in each group. Result: The shapes of the cells at 20s, 30s, and 50s were similar to each other. No significant changes in cellular viability were noted among different age groups or sex groups. The BMSCs expressed CD44, CD73, and CD90 surface markers but did not express CD14 and CD34. There were no noticeable differences in CD surface markers among the different age groups. The expressions of CD surface markers were similar between men and women. No significant differences in the secretion of vascular endothelial growth factors (VEGFs) were noted at Day 3 between different age groups. qRT-PCR regarding the expression showed differences between the age groups. However, Western blot analysis showed a decrease in expression but did not reach statistical significance (P>0.05). Conclusion: This study clearly showed no significant differences in shape, cell viability, expression of stem cell surface markers, or secretion of human VEGF among different age groups. However, western blot analysis showed a tendency of age-related decrease which did not reach statistical significance. Collectively, autologous or allogeneic BMSCs should be meticulously applied to obtain optimal results regarding age and sex.