• Korean Journal of Microbiology
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• v.44 no.3
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• pp.228-236
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• 2008

Validation of viral safety is essential in ensuring the safety of mammalian cell culture-derived biopharmaceuticals, because numerous adventitious viruses have been contaminated during the manufacture of the products. Mammalian cells are highly susceptible to Reovirus type 3 (Reo-3), and there are several reports of Reo-3 contamination during the manufacture of biopharmaceuticals. In order to establish the validation system for the Reo-3 safety, a real-time RT-PCR method was developed for quantitative detection of Reo-3 in cell lines, raw materials, manufacturing processes, and final products as well as Reo-3 clearance validation. Specific primers for amplification of Reo-3 RNA was selected, and Reo-3 RNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be $3.2{\times}10^0\;TCID_{50}/ml$. The real-time RT-PCR method was proven to be reproducible and very specific to Reo-3. The established real-time RT-PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with Reo-3. Reo-3 RNA could be quantified in CHO cell as well as culture supernatant. When the real-time RT-PCR assay was applied to the validation of virus removal during a virus filtration process, the result was similar to that of virus infectivity assay. Therefore, it was concluded that this rapid, specific, sensitive, and robust assay could replace infectivity assay for detection and clearance validation of Reo-3.

• Korean Journal of Microbiology
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• v.41 no.3
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• pp.216-224
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• 2005

Chromatography has now been used successfully to provide the requisite purity for human plasma-derived biop-harmaceuticals such as coagulation factors and immunoglobulins. Recently, increasing attention has been focused on establishing efficient cleaning procedures to prevent potential contamination by microorganisms as well as carry-over contamination from batch to batch. The purpose of present study was to develop a cleaning validation system for the assurance of virus removal and/or inactivation during chromatography process. In order to establish an assay system for the validation of virus clearance during chromatography cleaning process, a quantitative real-time PCR method for porcine parvovirus(PPV) was developed, since PPV, a model virus for human parvovirus B19, has a high resistance to a range of physico-chemical treatment. Specific primers for amplification of PPV DNA was selected, and PPV DNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be 1.5 $TCID_{50}/ml$. The established real-time PCR assay was successfully applied to the validation of PPV removal and cleaning during SP-Sepharose cation chromatography for thrombin purification and Q-Sepharose anion chromatography for factor VIII purification. The comparative results obtained by real-time PCR assay and infectivity titrations suggested that the real-time PCR assay could be a useful method for chromatography cleaning validation and that it could have an additive effect on the interpretation and evaluation of virus clearance during the virus removal process.

• Korean Journal of Food Science and Technology
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• v.37 no.2
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• pp.279-286
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• 2005

Microbial contamination origin of Kimbab was determined using nine types of ready-to-use ingredients, three each from animal, seafood, and vegetable sources. Effect of radiation on microbiological safety was also investigated. Total aerobic bacteria were not detected in seasoned beef, ham, and seasoned burdock, whereas 3.50, 5.41, 8.83, and 5.07 log CFU/g were detected in surimi gel, seasoned and blanched spinach, dried laver, and cucumber, respectively. Total aerobic bacterial and mold numbers were 8.73 and 5.08 log CFU/g in prepared Kimbab. Gamma irradiation reduced level of contaminated aerobic bacteria and mold population in Kimbab and its ingredients, Salmonella mutagenicity assay (Ames test) showed Kimbub ingredients irradiated at 10 kGy did not show any mutagenicity. These results indicate ready-to-use kimbab ingredients were mostly responsible for total aerobic bacteria and mold population of Kimbab, and low dose irradiation and low temperature storage ($10^{\circ}C$) effectively ensured microbiological safety of Kimbab and ready-to-use ingredients.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.16 no.1
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• pp.25-30
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• 1983

The concentrations of dissolved vitamin $B_{12}$, thiamine and biotin in the water of Gyokpo coast, were determined by microbiological assay methods. Also the relations between the distribution of B group vitamin and other environmental factors were studied. Vitamin $B_{12}$ was assayed with Euglena gracilis strain Z, thiamine with Cryptococous albidus and biotin with Achromo bacter sp. yH-51. It was found that the concentration of B group vitamin in the water of Gyokpo coast were normal level : vitamin $B_{12}$; 1.36-3.95 ng/l, thiamine ; u-0.4 ng/l and biotin; 1.40-14.60 ng/l. The concentration of B group vitamin was high in summer than in winter. In the water of Gyokpo coast during summer, B group vitamin occurred slightly lower level than normal, the concentration suficiently neccessary for phytoplankton development. The concentration of biotin was positively correlated with abundance of phytoplankton, but not aerobic heterotrophic bacteria. It was suggested that the concentration of biotin in water might be much more influenced with the growth of phytoplankton and any environmental factors than bacteria and the other vitamin, especially.

• Korean Journal of Veterinary Research
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• v.38 no.2
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• pp.345-352
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• 1998

The diagnosis of brucellosis is currently based on serological and microbiological tests. However, the microbiological isolation and identification have several disadvantages such as time-consuming and laborious, and the serological methods have been reported to cross-react with antigens other than those from Brucella spp. To develop a sensitive and rapid diagnostic method for detection of Brucella species, the genus-specific primers were designed and synthesized from the sequence of gene encoding a 31kDa cell surface protein(BCSP) and a 36kDa outer membrane protein(OMPB) of B abortus. The amplified 711bp and 982bp DNA fragments were only visible in each species of Brucella by PCR method using the BCSP and OMPB primers, respectively. However, PCR product was not obtained with DNA from other Gram-negative bacteria. As little as 1pg of the B abortus genomic DNA could be detected by this PCR method. Using the PCR technique, semen samples from 185 bulls of Brucella-seronegative herds in Cheju island were examined for comparison of this PCR method with conventional methods in 1995. The semen samples from 5 bulls were positive by culture method and PCR, and one was positive and 5 were suspect by semen plasma agglutination test. However, the semen samples obtained from 177 bulls were negative by semen plasma agglutination, culture and PCR methods in 1996. The results of comparison tests suggested that PCR was a better test than agglutination test against semen of bulls. This study indicated that the PCR technique was a valuable for the diagnosis of bovine brucellosis, particulary in bull semens.

• Journal of Environmental Health Sciences
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• v.40 no.2
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• pp.88-97
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• 2014

Objectives: The aim of this study was to evaluate the occurrence of microbiological contamination of kitchen utensils and environments of food service operations at university located in Seoul, Korea. Methods: We collected swab samples from the surfaces of knives, chopping boards, floors, and drains, as well as drinking water and airborne bacteria samples from 20 food service operations. Three bacterial indicators and five food poisoning bacteria were measured quantitatively and qualitatively, respectively. We used selective culture media and the PCR assay targeting 16S rRNA gene for the microbiological analysis. Results: We detected bacterial indicators on knives or chopping boards in eight different food service operations and, three food service operations (I, M, and O) showed more than 3 log colony forming units $(CFU)/100cm^2$ on their knives, significantly higher than the others. The levels of bacterial indicators on the floors and drains in the cooking areas were much higher than those on the cooking utensils. S. aureus was detected on 10 floors and 8 drains. Culturable bacteria were identified in 5 drinking water samples, and food service operation B ($431.1CFU/m^3$) and C ($551.2CFU/m^3$) showed more than $400CFU/m^3$ of total airborne bacteria. Conclusions: These results suggest that some of food service operations in this study may require additional investigation to secure the microbial safety of cooking environments. In addition, further actions including hygiene education for employees and proper guidelines to maintain clean cooking environments should be prepared.

• Journal of Microbiology
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• v.41 no.3
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• pp.239-247
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• 2003

The LRV1-4 capsid protein possesses an endoribonuclease activity that is responsible for the single site-specific cleavage in the 5' untranslated region (UTR) of its own viral RNA genome and the formation of a conserved stem-loop structure (stem-loop IV) in the UTR is essential for the accurate RNA cleavage by the capsid protein. To delineate the nucleotide sequences, which are essential for the correct formation of the stem-loop structure for the accurate RNA cleavage by the viral capsid protein, a wildtype minimal RNA transcript (RNA 5' 249-342) and several synthetic RNA transcripts encoding point-mutations in the stem-loop region were generated in an in vitro transcription system, and used as substrates for the RNA cleavage assay and RNase mapping studies. When the RNA 5' 249-342 transcript was subjected to RNase T1 and A mapping studies, the results showed that the predicted RNA secondary structure in the stem-loop region using FOLD analysis only existed in the presence of Mg$\^$2＋/ ions, suggesting that the metal ion stabilizes the stem-loop structure of the substrate RNA in solution. When point-mutated RNA substrates were used in the RNA cleavage assay and RNase T1 mapping study, the specific nucleotide sequences in the stem-loop region were not required for the accurate RNA cleavage by the viral capsid protein, but the formation of a stem-loop like structure in a region (nucleotides from 267 to 287) stabilized by Mg$\^$2＋/ ions was critical for the accurate RNA cleavage. The RNase T1 mapping and EMSA studies revealed that the Ca$\^$2＋/ and Mn$\^$2＋/ ions, among the reagents tested, could change the mobility of the substrate RNA 5' 249-342 on a gel similarly to that of Mg$\^$2＋/ ions, but only Ca$\^$2＋/ ions identically showed the stabilizing effect of Mg$\^$2＋/ ions on the stem-loop structure, suggesting that binding of the metal ions (Mg$\^$2＋/ or Ca$\^$2＋/) onto the RNA substrate in solution causes change and stabilization of the RNA stem-loop structure, and only the substrate RNA with a rigid stem-loop structure in the essential region can be accurately cleaved by the LRV1-4 viral capsid protein.

• Korean Journal of Microbiology
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• v.51 no.3
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• pp.199-208
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• 2015

Biologics and medical devices manufactured with bovine-derived raw materials have the risk of viral contamination. Therefore, viral validation study is essential to ensure the safety of the products. Bovine adenovirus type-1 (BAdV-1) is one of the common bovine viral pathogens. For quantitative detection of BAdV-1 during the manufacture of biologics and medical devices, a TaqMan probe real-time PCR method was developed. Specific primers and TaqMan probe for amplifying and detecting BAdV-1 DNA were designed. Specificity, limit of detection (LOD), and robustness of the method was validated according to international guideline on the validation of nucleic acid amplification tests for the pathogen detection. The sensitivity of the assay was found to be $7.44{\times}10^1\;TCID_{50}/ml$. The real-time PCR method was reproducible, very specific to BAdV-1, and robust. Moreover, the method was successfully applied to the validation of Chinese Hamster Ovary (CHO)-K1 cells artificially infected with BAdV-1, a commercial CHO master bank, and bovine type 1 collagen. The overall results indicate that this rapid, specific, sensitive, and robust assay can be reliably used for quantitative detection of BAdV-1 contamination during the manufacture of biologics and medical devices using bovine-derived raw materials.

• Journal of Nutrition and Health
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• v.48 no.1
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• pp.94-104
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• 2015

Purpose: The purpose of this study was to estimate dietary folate intake and food sources of children and adolescents using a nutrient database revised based on measured folate in selected foods. Methods: Folate content in 51 foods known as folate sources was measured by microbiological assay after trienzyme extraction. Folate intake was estimated from a part of the data of 'Dietary Intake Survey of Infants, Children and Adolescents in 2007~2008' conducted by the Korea Food and Drug Administration (KFDA) and the Korea Health Industry Development Institute (KHIDI). The study subjects were 567 children aged 1~19 years living in the Chungcheong area who completed two 24-hour recalls. Results: Folate values were revised by replacing the values in the current database with the analyzed values except when the value in the current database was between the analytical values or was not different from the mean analytical value by more than 10%. Among the revised values of 40 food items, folate values of 36 foods were lower than the current values. Mean folate intakes estimated with the revised database were approximately 70~80% lower than those estimated with the current database. Mean folate intakes of males aged 12~14 and females aged 12~19 were lower than the 2010 Recommended Nutrient Intakes (RNIs). Chicken's eggs, Kimchi, rice, mandarin, and laver were found to be main food sources of folate. Conclusion: In this study, mean dietary folate intakes were lower than those estimated with the current database. Further analyses for folate content especially in cooked foods commonly consumed in Korea are needed using a reliable assay in order to accurately assess folate intake of the Korean population. In addition, nutrition education should be provided for adolescent females in order to increase consumption of folate-rich foods.

• Korean Journal of Microbiology
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• v.40 no.4
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• pp.334-341
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• 2004

Polysaccharides were prepared from Orostachys japonicus by extration with hot steam water (OJPl). The OIPl fraction was further purified by Sephadex G-50 gel filtration chromatography to produce FI (polysaccharides) and FII (oligosaccharides) fraction. The average molecular masses o fFI and FII fraction were determined to be 3050 kDa and 13 kDa, respectively. The antimicrobial activity of OIPl was tested against 8 strains of bacteria and one strain of yeast by the disc diffusion method, fluorescein diacetate (FDA) method and broth dilution method. The OIPl exhibited a very strong growth inhibition to Candida albicans. The OIPl remarkably sup­pressed the growth of Salmonella typhimurium and Staphylococcus aureus. The OIPl showed higher growth inhibition to Escherichia coli and Pseudomonas aeruginosa than propolis, positive control. When the anticancer activity of the OIPl, FI or FII was examined against human cancer cell lines and the Sarcoma 180 cells, these widely suppressed the proliferation of cell lines in the MTT assay and morphology study. Especially, they remarkably inhibited the growth of A549, HeLa and AGS cells. Also treatment of cancer cells with OJPl, FI or FII induced apoptotic cell death characterized by DNA fragmentation. The OJPl, FI or FII exhibiting various biological activities such as antimicrobial activity and anticancer activity is expected to be developed as new biohealth products.