• Food Science of Animal Resources
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• v.30 no.3
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• pp.410-418
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• 2010

Staphylococcus aureus is one of the major pathogens that can cause staphylococcal infection and food poisoning. In this study, we compared conventional culture methods and real-time PCR for detection of S. aureus in artificially inoculated milk, sausage, raw pork, and vegetable salad. The performance of a coagulase test for confirming S. aureus was also compared with a colony PCR test. Bulk food samples (500 g each) were artificially inoculated with S. aureus and divided into 20 samples (25 g or mL each). All samples were added to tryptic soy broth (225 mL/sample) with 10% NaCl and incubated at $37^{\circ}C$ for 24 h. After the enrichment, broth cultures were streaked onto Baird-Parker (BP) agar with egg yolk tellulite, and incubated at $37^{\circ}C$ for 24 h. In addition, 1 mL of broth cultures was collected to perform real-time PCR. Two suspicious colonies from the BP agar were picked up and plated on nutrient agar and incubated at $37^{\circ}C$ for 24 h followed, by a coagulase confirmation test and a colony PCR analysis. There were no statistical differences between culture methods and realtime PCR in food samples with low background microflora, such as milk and sausage. However, a significant statistical difference was found between the culture methods and real-time PCR for raw pork and vegetable salad. Furthermore, the colony PCR test of the presumptive colonies on BP agar for confirming S. aureus is more accurate and efficient than the coagulase test for unprocessed foods.

• Genomics & Informatics
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• v.18 no.4
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• pp.40.1-40.8
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• 2020

Avian influenza (AIV) outbreaks can induce fatal human pulmonary infections in addition to economic losses to the poultry industry. In this study, we aimed to develop a rapid and sensitive point-of-care AIV test using loop-mediated isothermal amplification (LAMP) technology. We designed three sets of reverse transcription LAMP (RT-LAMP) primers targeting the matrix (M) and hemagglutinin (HA) genes of the H5 and H9 subtypes. RT-LAMP targeting the universal M gene was designed to screen for the presence of AIV and RT-LAMP assays targeting H5-HA and H9-HA were designed to discriminate between the H5 and H9 subtypes. All three RT-LAMP assays showed specific amplification results without nonspecific reactions. In terms of sensitivity, the detection limits of our RT-LAMP assays were 100 to 1,000 RNA copies per reaction, which were 10 times more sensitive than the detection limits of the reference reverse-transcription polymerase chain reaction (RT-PCR) (1,000 to 10,000 RNA copies per reaction). The reaction time of our RT-LAMP assays was less than 30 min, which was approximately four times quicker than that of conventional RT-PCR. Altogether, these assays successfully detected the existence of AIV and discriminated between the H5 or H9 subtypes with higher sensitivity and less time than the conventional RT-PCR assay.

• The Plant Pathology Journal
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• v.38 no.6
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• pp.665-672
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• 2022

Cymbidium mosaic virus (CymMV) is one of economically important viruses that cause significant losses of orchids in the world. In the present study, a reverse transcription recombinase polymerase amplification (RT-RPA) assay combined with a lateral flow immunostrip (LFI) assay was developed for the detection of CymMV in orchid plants. A pair of primers containing fluorescent probes at each terminus that amplifies highly specifically a part of the coat protein gene of CymMV was determined for RT-RPA assay. The RT-RPA assay involved incubation at an isothermal temperature (39℃) and could be performed rapidly within 30 min. In addition, no cross-reactivity was observed to occur with odontoglossum ringspot virus and cymbidium chlorotic mosaic virus. The RT-RPA with LFI assay (RT-RPA-LFI) for CymMV showed 100 times more sensitivity than conventional reverse transcription polymerase chain reaction (RT-PCR). Furthermore, the RT-PCR-LFI assay demonstrated the simplicity and the rapidity of CymMV detection since the assay did not require any equipment, by comparing results with those of conventional RT-PCR. On-site application of the RT-RPA-LFI assay was validated for the detection of CymMV in field-collected orchids, indicating a simple, rapid, sensitive, and reliable method for detecting CymMV in orchids.

• The Korean Journal of Nuclear Medicine
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• v.28 no.2
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• pp.220-226
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• 1994

This study was performed to evaluate the clinical applicability of the reverse transcription polymerase chain reaction (RT-PCR ) kit of HCV-RNA using biotinylated and radioiodinated primers. Study subjects were 118 patients with positive anti-HCV. HCV-RNA in patient's serum was extracted by guanidium thiocyanate method. After first amplification, the product was reamplified by primers labelled with biotin and I-125. The final amplification product was defected by counting the radioactivity after incubation in avidin coated tubes. In 51 samples, the test was repeated for evaluation of reproducibility. This new method was also compared with conventional RT-PCR methods in 34 samples from patients with chronic liver disease. The results were as follows ; 1) HCV-RNA was positive in 85(97%)of 88 patients with chronic liver disease, and in 23 (73%) of 30 patients with normal liver function. 2) In comparison with conventional method, HCV-RNA was detected in 32(94%) of 34 patients with new method, whereas in 27(79%) of the same group with conventional method. 3) Repeated test with new method in 52 samples demonstrated 82% of concordant result. In conclusion, new method with biotinylated and radioiodinated primers was more sensitive than conventional method. However, great care must be taken for quality control because there were considerable interassay variation and possiblity of false positivity and false negativity.

• Restorative Dentistry and Endodontics
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• v.33 no.6
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• pp.537-544
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• 2008

Polymerase chain reaction (PCR) can detect bacteria more rapidly than conventional plate counting. However DNA-based assays cannot distinguish between viable and dead cells due to persistence of DNA after cells have lost their vitality. Recently, propidium monoazide (PMA) treatment has been introduced. The purpose of this study is to evaluate the applicability of the PMA treatment and real-time PCR method for cell counting in comparison with plate counting and to evaluate the antibacterial efficacy of 2% CHX on E. faecalis using PMA treatment in combination with real-time PCR. Firstly, to elucidate the relationship between the proportion of viable cells and the real-time PCR signals after PMA treatment, mixtures with different ratios of viable and dead cells were used. Secondly, relative difference of viable cells using PMA treatment in combination with real-time PCR was compared with CFU by plate counting. Lastly, antibacterial efficacy of 2% CHX on E. faecalis was measured using PMA treatment in combination with real-time PCR. The results were as follows : 1. Ct value increased with decreasing proportion of viable E. faecalis. 2. There was correlation between viable cells measured by real-time PCR after PMA treatment and CFU by plate counting until Optical density (OD) value remains under 1.0. However, viable cells measured by real-time PCR after PMA treatment have decreased at 1.5 of OD value while CFU kept increasing. 3. Relative difference of viable E. faecalis decreased more after longer application of 2% CHX.

• Tuberculosis and Respiratory Diseases
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• v.39 no.6
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• pp.517-525
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• 1992

Background: Since its development by Saiki et al, polymerase chain reaction (PCR) has been very useful in various fields of molecular biology. PCR can be used for the detection of a very small amount of microbial agent, and is especially useful in those patients who are difficult to diagnose microbiologically or serologically. Mycobacterium tuberculosis is a very slowly growing organism and AFB staining frequently shows false negative results, and therefore PCR would be a very rapid, easy, and sensitive diagnostic method for the diagnosis of Mycobacterium tuberculosis. Method: To compare PCR with conventional methods in diagnosing Mycobacterium tuberculosis in sputum, we used sputa of patients who visited or were admitted to Seoul National University Hospital. The amplification targets were 383 base pair DNA, a part of 2520 base pair DNA encoding 65 kD Mycobacterium tuberculosis specific protein (the primers are TB-1, -2), and 123 base pair DNA, a part of IS6110 fragment, which multiple copies are known to exsist PCR one genome (the primers are Sal I-1, -2). We also requested AFB staing and culture to the lab of Seoul National University Hospital with the same sample and compared the results. Results: 1) Using TB-1, -2 primers, PCR was positive in 73.1% (19/26) of culture positive sputa, in 12.5% (1/8) of culture negative. but clinically diagnosed tuberculous sputa, and was negative in all sputa of patients who were clinically diagnosed as non-tuberculous etiology. 2) Using Sal I-I, -2 primers, PCR was positive in 94.1% (32/34) of culture positive sputa, in 23.1% (6/26) of culture negative, but clinically diagnosed tuberculous sputa, and was negative in 87.5% (14/16) of sputa from patients who were clinically diagnosed as non-tuberculous etiology. Conclusion: PCR could be a very rapid, sensitive and specific method for the diagnosis of Mycobacterium tuberculosis in sputa, and further studies should be followed for the development of easier method.

• Journal of Food Hygiene and Safety
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• v.30 no.2
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• pp.150-158
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• 2015

This article reports the development of an effective test procedure for detection of norovirus (NoV) in foods of diverse matrices. In this study, target foods included fermented milk, soybean paste, powders made from uncooked grains and vegetables, sesame leaves preserved in soy sauce, pickled mooli, and mooli. Viral recovery varied depending on the food matrices or elution buffers tested. Buffers were compared to determine effective elution buffers from artificially virus-contaminated foods. The conventional test procedure for concentrating viruses from food (elution-polyethylene glycol(PEG) precipitation-chloroform-PEG precipitation) was modified to save time by eliminating one PEG precipitation step. The modified procedure (elution-chloroform-PEG precipitation) was able to concentrate viruses more effectively than the conventional procedure. It also removed RT-PCR inhibitors effectively. The modified procedure was applied to target food for genogroup II NoV detection. NoV RNA was detected at the initial inoculum levels 3.125-12.5 RT-PCR units per 10-25 g tested food. The use of this newly established procedure should facilitate detection of low levels of norovirus in diverse foods.

• Biomedical Science Letters
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• v.22 no.1
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• pp.24-28
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• 2016

Tuberculosis (TB) remains an unsolved community health problem since identification of its causing microorganism called Mycobacterium tuberculosis (MTB) by Robert Koch in 1882. Annually, eight million TB cases are newly reported and 2~3 million patients die from TB. Pulmonary TB is highly infectious and untreated pulmonary TB patients are believed to infect >10 people in a year. The conventional methods for diagnosis of TB are chest X-ray and isolation of the causing microorganisms from patient specimens. Screening of TB is conducted with smeared sputum in slides, and TB is confirmed by identification of MTB in cultured specimens. One of the fatal pitfalls of screening detection for smeared sputum is that it is impossible to distinguish MTB and other acid-fast bacilli (AFB) because they are stained equally with Ziehl-Neelsen (ZN) stain. Culture of MTB is the most reliable method for diagnosis of TB but it takes 4~8 weeks. In this report, we suggest a fast and highly-reliable MTB detection method that distinguishes AFB in sputum samples. Purified DNA from the AFB stained slide samples offered by The Korean Institute of Tuberculosis were used to detect infected MTB in patients. PCR, real-time PCR and reverse blot hybridization assay (REBA) methods were applied to purified DNA. Conclusively, the real-time PCR method was confirmed to produce high sensitivity and we were able to further detect drug-resistant MTB with REBA.

• Korean Journal of Veterinary Service
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• v.38 no.2
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• pp.107-116
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• 2015

In this study, we developed a rapid, sensitive and specific reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for detection of swine influenza viruse (SIV) including major subtypes of swine influenza viruses H1N1, H1N2 and H3N2, and a novel subtype of influenza A virus that accidentally infected in pig population. The RT-LAMP was completed in 40 min at $58^{\circ}C$ and the sensitivity of the RT-LAMP ($1copy/{\mu}L$) was 10-fold higher than conventional reverse transcription-polymerase chain reaction (RT-PCR) ($10copy/{\mu}L$) and the same to real time RT-PCR ($1copy/{\mu}L$). Also, the result of the RT-LAMP can be confirmed without any detection system. Therefore, the RT-LAMP could be a alternative diagnostic method for SIV detection in national SIV monitoring system and clinical diagnostic laboratory in the future.

• Journal of Dairy Science and Biotechnology
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• v.33 no.3
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• pp.197-202
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• 2015

While various foodborne pathogenic bacteria can be detected more rapidly via polymerase chain reaction than via conventional plating methods, it is impossible to distinguish between viable and dead cells in DNA-based assays. Hence, propidium monoazide (PMA) treatment has been introduced to detect living cells. The purpose of this study is to evaluate the applicability of the PMA treatment and real-time qPCR method for the detection of Cronobacter sakazakii and to compare it to that of plate counting. Based on our positive results, we suggest the use of PMA treatment and real-time qPCR for the detection of viable Cronobacter sakazakii in various food sources and an update of the Korean Food Code.