• Journal of Food Hygiene and Safety
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• v.33 no.1
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• pp.71-76
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• 2018

Norovirus is a leading cause of sporadic pathogenic non-bacterial gastroenteritis worldwide. For the detection of norovirus, reverse transcription real-time PCR (RT qPCR) has quickly become a major tool due to its sensitivity and specificity. However, accurate viral RNA extraction methods are essential for RT qPCR analysis. TRIzol reagents are used to extract RNA from biological materials and are therefore widely used for norovirus RNA extraction. In this study, the yield of viral RNA extraction using TRIzol from genogroup II (GII) among the human norovirus genogroup I (GI) and GII, and murine norovirus (GV) depended on the pH of the virus sample solution. The yield of RNA extraction was higher at the alkaline pH than in the acidic region compared with the Ct (threshold cycle) value of the real-time PCR. From the results of this study, it was found that the pH condition is very important for the quantitative analysis of norovirus by extracting GII RNA using TRIzol.

• Journal of Microbiology
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• v.44 no.1
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• pp.106-112
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• 2006

In this study, the genetic diversities of multi-resistant Salmonella typhimurium (ST) isolates were analyzed via the application of both pulsed field gel electrophoresis (PFGE) and Polymerase chain reaction (PCR) analysis methods, using 6 kinds of primers (REP, ERIC, SERE, BOX, P-1254 and OPB-17). And their discriminative abilities (DA) were also compared in order to determine the most effective and reliable analysis method. 118 S. typhimurium isolates, cultured from diverse animals and human patients in Korea beginning in 1993, were analyzed and subjected to a comparison of Simpson's index of diversity (SID), using both PFGE and PCR methods. PFGE by XbaI enzyme digestion allowed for discrimination into 9 pulsotypes, with high SID values (0.991) on the genomic DNA level. This shows that PFGE is a very discriminative genotypic tool, and also that multiple clones of S. typhimurium isolates had existed in domestic animals and humans in Korea since 1993. However, we could ultimately not to trace the definitive sources or animal reservoirs of specific S. typhimurium isolates examined in this study. Depending on the SID values, the combined method (7 kinds of method) was found to be the most discriminative method, followed by (in order) SERE-PCR, REP-PCR, ERIC-PCR, PFGE & OPB-17 (RAPD), P-1254 (RAPD), and BOX-PCR at the $80\%$ clone cut-off value. This finding suggests that the REP-PCR method (which utilizes 4 primer types) may be an alternative tool to PFGE for the genotyping of S. typhimurium isolates, with comparable cost, time, and labor requirement. The establishment of a highly reliable and discriminatory method for epidemiologic analysis is considered necessary in order for researchers to trace the sources of specific pathogens and, consequently, to control and prevent the spread of epidemic S. typhimurium isolates to humans.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.12 no.8
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• pp.3517-3522
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• 2011

Contamination of public bath water by waterborne pathogens can cause disease outbreaks and contribute to background rates of disease. The aim of this study is to determine the prevalence of waterborne pathogens in public baths. A total of 30 water samples were collected from 30 different public baths in seoul, Korea. Pathogens in water samples were concentrated by 0.45 ${\mu}m$ nitrocellulose membrane filter, analyzed by both cultivation and polymerase chain reaction-reverse blot hybridization (PCR-REBA) of partial 16S rRNA gene. Various microorganisms including Escherichia coli and Shigella spp. were identified by microbiological cultivation. E. coli, Shigella spp., Salmonella spp., Pseudomonas spp. and Mycobacterium spp. were identified by PCR-REBA. Our results suggest that appropriate hygiene practice and continuous monitoring is needed for reducing health risk associated with public bath houses.

• Tuberculosis and Respiratory Diseases
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• v.68 no.6
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• pp.345-349
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• 2010

Background: The polymerase chain reaction (PCR) test is important for the confirmatory diagnosis of tuberculosis (TB) caused by Mycobacterium tuberculosis. The aim of this study was to analyze the yield of repeated PCR testing in patients with confirmed pulmonary TB. Methods: The medical records of 130 patients, who had more than two consecutive PCR tests and a M. tuberculosis-positive sputum culture from August, 2006 to December, 2007, were retrospectively reviewed for the purposes of this study. A positive TB-PCR test was defined as at least one positive test result. Results: The cumulative positive PCR test rate was 80% (104/130), with gradually increasing rates of positive findings upon the first, second and third TB-PCR tests with 52.3%, 68.5% and 75.4%, respectively. However, further testing did not increase the positive rate further. Conclusion: Repeated PCR testing at least three times for M. tuberculosis is helpful for diagnosis of pulmonary TB.

• Journal of Microbiology and Biotechnology
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• v.18 no.11
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• pp.1841-1847
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• 2008

Vibrio vulnificus is a causative agent of serious diseases in humans, resulting from the contact of wound with seawater or consumption of raw seafood. Several studies aimed at detecting V. vulnificus have targeted vvh as a representative virulence toxin gene belonging to the bacterium. In this study, we targeted the rpoS gene, a general stress regulator, to detect V. vulnificus. PCR specificity was identified by amplification of 8 V. vulnificus templates and by the loss of a PCR product with 36 non-V. vulnificus strains. The PCR assay had the 273-bp fragment and the sensitivity of 10 pg DNA from V. vulnificus. SYBR Green I-based real-time PCR assay targeting the rpoS gene showed a melting temperature of approximately $84^{\circ}C$ for the V. vulnificus strains. The minimum level of detection by real-time PCR was 2 pg of purified genomic DNA, or $10^3$ V. vulnificus cells from pure cultured broth and $10^3$ cells in 1 g of oyster tissue homogenates. These data indicate that real-time PCR is a sensitive, species-specific, and rapid method for detecting this bacterium, using the rpoS gene in pure cultures and in infected oyster tissues.

• Journal of Plant Biotechnology
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• v.49 no.3
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• pp.193-206
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• 2022

Astringent persimmon (Diospyros kaki Thunb.) is an important fruit crop in Korea; it possesses significant medicinal potential. However, knowledge regarding the pathogens affecting this crop, particularly, viruses and viroids, is limited. In the present study, reverse transcription-polymerase chain reaction (RT-PCR) and high-throughput transcriptome sequencing (HTS) were used to investigate the viruses and viroids infecting astringent persimmons cultivated in Korea. A one-step multiplex RT-PCR (mRT-PCR) method for the simultaneous detection of the pathogens was developed by designing species-specific primers and selecting the primer pairs via combination and detection limit testing. Seven of the sixteen cultivars tested were found to be infection-free. The RT-PCR and HTS analyses identified two viruses and one viroid in the infected samples (n = 51/100 samples collected from 16 cultivars). The incidence of single infections (n = 39/51) was higher than that of mixed infections (n = 12/51); the infection rate of the Persimmon cryptic virus was the highest (n = 31/39). Comparison of the monoplex and mRT-PCR results using randomly selected samples confirmed the efficiency of mRT-PCR for the identification of pathogens. Collectively, the present study provides useful resources for developing disease-free seedlings; further, the developed mRT-PCR method can be extended to investigate pathogens in other woody plants.

• The Journal of Korean Institute of Communications and Information Sciences
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• v.33 no.4B
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• pp.199-206
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• 2008

In this paper, we propose a novel Route_Reconf message as the RSVP message to maintain an RSVP session for hard handoff and consider path-comparing route (PCR) a PCR algorithm to find an intermediate node in the charge of downlink re-establishment. And, we consider information form each mode for the PCR algorithm, which also reduces the frequency and amount of exchanged RSVP message to minimize packet loss and delay between an intermediate node and a receiver. According to the proposed algorithm, a new support node (NSN) and an existing support node (ESN) along the RSVP path can be found; the former is a supporting RSVP session node newly searched and the latter is the last supporting node holding the previous session after handoff. On receiving the Route_Reconf message at the ESN, a new allocated route from a NSN to the MN waiting for the handoff via a new AR is configured by the ESN.

• International Journal of Oral Biology
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• v.38 no.1
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• pp.21-27
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• 2013

Anginosus group streptococci (AGS) were classified based on the nucleotide sequences of the 16S rRNA gene (16S rDNA) and comprised Streptococcus anginosus, Streptococcus intermedius, and Streptococcus constellatus. It is known that AGS is a causative factor of oral and systematic diseases. The purpose of this study was to discriminate the 56 clinical strains of AGS isolated from Korean oral cavities using phylogenetic analysis of 16S rDNA and species-specific PCR at the species-level. The 16S rDNA of clinical strains of AGS was sequenced using the dideoxy chain termination method and analyzed using MEGA version 5 software. PCR was performed to identify the clinical strains using species-specific primers described in previous studies and S. intermedius-specific PCR primers developed in our laboratory. The resulting phylogenetic data showed that the 16S rDNA sequences can delineate the S. anginosus, S. intermedius, and S. constellatus strains even though the 16S rDNA sequence similarity between S. intermedius and S. constellatus is above 98%. The PCR data showed that each species-specific PCR primer pair could discriminate between clinical strains at the species-level through phylogenetic analysis of 16S rDNA nucleotide sequences. These results suggest that phylogenetic analysis of 16S rDNA and PCR are useful tools for discriminating between AGS strains at the species-level.

• The Journal of Korean Society of Virology
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• v.30 no.3
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• pp.179-187
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• 2000

The etiology of rheumatic arthritis (RA) is associated with a number of genetic and environmental factors, but is not definitively elucidated. Recently, more attention has been paid to the possibility of microbial etiology in the pathogenesis of RA, because many different infectious agents have been reported to precede the onset or exacerbation of RA. Adenovirus (ADV) may be one cause of persistent or recurrent inflammatory arthritis. Varicella zoster virus (VZV) arthritis is detected frequently in RA patients treated with low dose methotrexate. The demonstration of simultaneous presence of both viral agents of specific viral nucleic acid in synovial fluids from synovitis patients would provide more direct evidence for arthritis etiological relationship, but there are no confirmed results. Therefore, we studied the ability of adenovirus and VZV to establish coinfection and persistent infection in synovial fluid from synovitis patients. The presence of viral agents in the synovial fluid demonstrated by isolation of cell culture, enzyme immunoassay and nested-PCR. The synovial fluids were also investgated for the presence of viral nucleic acid by nested-PCR using specific primer. ADV produced 220 bp and VZV produced 447 bp by each nested-PCR with specific primers. We detected 4/6 cases (66.7%) with persistent infection of ADV and 5/6 cases (83.3%) of VZV with 13 synovial fluids (between 7 to 52 day intervals) from synovitis patients by monoclonal ErA and nested-PCR. 21/28 cases (75%) with coinfection of adenovirus and VZV with synovial fluids from synovitis patients by nested-PCR. ADV and VZV coinfection and persistent infection of synovial fluids may provide a chronic antigenic stimuli to the immune system therefore provoking a continuing inflammatory response and caused the possibility of synovitis and arthritis.

• KSBB Journal
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• v.29 no.5
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• pp.361-371
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• 2014

Mycoplasma is well recognized as one of the most prevalent and serious microbial contaminants of biologic manufacturing processes. Conventional methods for mycoplasma testing, direct culture method and indirect indicator cell culture method, are lengthy, costly and less sensitive to noncultivable species. In this report, we describe a new TaqMan probe-based real-time PCR method for rapid and quantitative detection of mycoplasma contamination during manufacture of biologics. Universal mycoplasma primers were used for mycoplasma PCR and mycoplasma DNA was quantified by use of a specific TaqMan probe. Specificity, sensitivity, and robustness of the real-time PCR method was validated according to the European Pharmacopoeia. The validation results met required criteria to justify its use as a replacement for the culture method. The established real-time PCR assay was successfully applied to the detection of mycoplasma from human keratinocyte and mesenchymal stem cell as well as Vero cell lines artificially infected with mycoplasma. The overall results indicated that this rapid, specific, sensitive, and robust assay can be reliably used for quantitative detection of mycoplasma contamination during manufacture of biologics.