Filtration과 Integrated Cell Culture/Real-Time Reverse Transcription PCR 기법을 이용한 채소류에서 Human Rotavirus 신속 검출

현지연,천정환,송광영,황인균,곽효선,이정수,김무상,이중복,서건호,Hyeon, Ji-Yeon,Chon, Jung-Whan,Song, Kwang-Young,Hwang, In-Gyun,Kwak, Hyo-Sun,Lee, Jung-Soo,Kim, Moo-Sang,Lee, Jung-Bok,Seo, Kun-Ho 한국미생물학회 2011 미생물학회지 Vol.47 No.2

본 연구는 human rotavirus (HRV)의 검출법을 최적화하기 위해 real-time RT-PCR과 세포 배양법을 이용하여 여러 가지 탈리 농축법을 비교 및 평가하는 것을 목적으로 하였다. 채소류 중 배추, 상추, 깻잎을 선정하여 바이러스 희석액을 접종하고 탈리액 비교를 위하여 buffer A (100 mM Tris-HCl, 50 mM glycine, 3% beef extract, pH 9.5)와 buffer B (250 mM Threonine, 300 mM NaCl, pH 9.5)를 이용하여 탈리하였고, 농축방법을 비교하기 위하여 PEG (polyethylene glycol) 침전법 또는 filtration [Nanoceram filter$^{(R)}$ (Argonide corporation)]을 이용하여 농축하였다. 또한 바이러스의 감염성 평가를 위하여 MA-104 cell을 배양하여 탈리, 농축 방법을 거쳐 회수된 HRV를 접종하고 1, 48, 72, 96, 120, 144, 168시간 후 세포를 수거하여 real-time RT-PCR을 시행하고 세포병변을 관찰하였다. 탈리 용액은 buffer A가 회수율 29.54%로 buffer B의 18.32%보다 더 뛰어난 탈리효과를 보였으며 농축방법을 비교했을 때 filtration 방법이 회수율 51.89%를 나타내며 PEG 침전법에 비해서 바이러스의 농축에 효과적이었으며 검출 소요시간이나 간단한 과정 면에서 효율적이었다. ICC/real-time RT-PCR을 시행하였을 때 세포병변 72시간 후부터 나타나기 시작했지만 Ct value는 48시간부터 감소하기 시작하여 더 빠른 시간 내에 감염성을 평가할 수 있었다. 따라서, filtration과 integrated/cell culture real-time RT-PCR을 이용하면 기존의 검출방법보다 빠른 시간 내에 바이러스 검출이 가능할 것으로 여겨진다. The purpose of this study was to evaluate and compare different elution and concentration methods for optimization of human rotavirus (HRV) detection method using real-time RT-PCR and cell culture techniques. The leafy vegetable samples (lettuce, Chinese cabbage) were artificially inoculated with HRV. Viruses were extracted from the vegetables by two different elution buffers, buffer A (100 mM Tris-HCl, 50 mM glycine, 3% beef extract, pH 9.5) and buffer B (250 mM Threonine, 300 mM NaCl, pH 9.5), and the extracted viruses were concentrated by filtration and PEG precipitation sequentially. To determine infectivity of the viruses, the viruses recovered from the samples were infected to the MA-104 cells, and integrated cell culture real-time RT-PCR was performed at 1, 48, 72, 96, 120, 144, 168 h post-infection (p.i.). The elution buffer A was more efficient in extracting the virus from the produce samples tested than the buffer B, 29.54% and 18.32% of recoveries, respectively. The sensitivity of real-time RT-PCR method was markedly improved when the virus was concentrated by the filtration method. When the viruses were eluted and concentrated by buffer A and filtration, respectively, the average recovery rate was approximately 51.89%. When the viruses recovered from samples were infected to MA-104 cell, infectious HRV was detected within 48 h p.i. by ICC/real-time RT-PCR, whereas cytopathic effects were not observed until 72 h p.i. The optimized detection method evaluated in this study could be useful for rapid and reliable detection of HRV in fresh produce products and applied for detection of other food-borne viruses.

기후대별 사무소 건물 PCW 창호 적용에 따른 에너지 성능 분석

현지연(Hyun, Ji-Yeon),박보랑(Park, Bo-Rang),조지현(Cho, Ji-Hyeon),조혜운(Cho, Hye-Un),문진우(Moon, Jin-Woo) 대한건축학회 2021 대한건축학회 학술발표대회 논문집 Vol.41 No.1

유제품 및 가공식품에서 Listeria monocytogenes 검출을 위한 배지법과 신속 검사키트의 유효성 검증

한소리,현지연,김희연,박종석,허석,신호철,서건호,Han, So-Ri,Hyeon, Ji-Yeon,Kim, Hee-Yun,Park, Jong-Seok,Heo, Seok,Shin, Ho-Chul,Seo, Kun-Ho 한국축산식품학회 2008 한국축산식품학회지 Vol.28 No.5

Listeria monocytogenes is a foodborne pathogen inducing listeriosis in human. We compared two different culture methods for detection of L. monocytogenes and validated two commercial kits, $VIDAS^{(R)}$ and $REVEAL^{(R)}$ for Listeria. L. monocytogenes was inoculated into various food samples to generate partial positive samples. The inoculated samples were enriched in half-Fraser broth for 48 hr at $30^{\circ}C$. The enriched samples were streaked onto Oxford agar at 24 and 48 hr postincubation followed by biochemical confirmation and concurrently analyzed by using the two commercial kits for comparison. When the enrichment period was extended from 24 to 48 hr, the numbers of positive samples were dramatically increased from 6 to 52 out of 80 samples tested using the culture method. With the commercial kits, the numbers of positive samples were also significantly increased from 10 to 18 and 1 to 18, respectively, when the enrichment period was extended from 48 to 72 hr. There was no statistical difference between the 24 hr culture method and $VIDAS^{(R)}$ or $Reveal^{(R)}$ with 48 hr enrichment. In conclusion, the 24 hr for the culture method was insufficient to detect L. monocytogenes in various foods. The commercial kits could be adequate means for presumptive screening of L. monocytogenes in food.

DSSC BIPV 창호 적용 업무시설 에너지 성능 분석

김남현(Nam Hyeon Kim),현지연(Ji Yeon Hyeon),최은지(Eun Ji Choi),조혜운(Hye Un Cho),문진우(Jin Woo Moon) 한국생태환경건축학회 2021 한국생태환경건축학회 학술발표대회 논문집 Vol.21 No.1

이미지 인식 기반 CLO 산출 모델 적용에 따른 열환경 제어 성능 평가

김남현(Nam Hyeon Kim),최은지(Eun Ji Choi),현지연(Ji Yeon Hyun),문진우(Jin Woo Moon) 대한설비공학회 2022 대한설비공학회 학술발표대회논문집 Vol.2022 No.6

본 연구의 목적은 실제 건물 환경에서 실시간 착의량 정보를 반영한 PMV 제어가 열환경 및 열쾌적에 미치는 성능을 확인하는 것이다. 이때, 실시간 착의량 정보는 이미지 처리 신경망 모델을 통해 실내 영상이미지를 기반으로 산출된다. PMV 기반 제어의 성능 평가를 위해 실제 건물 환경을 구현한 Test-bed 내에서 겨울철 난방기간 동안 실험을 수행하였다. 본 연구에서 제시하는 제어방법의 성능 분석을 위해 기존 제어방법인 setpoint 기반 제어방법, 고정착의량(1.0 clo)을 반영한 PMV 제어방식과 비교 분석 실험을 수행하였다. 그 결과, PMV 기반 제어방법은 기존 제어방식에 비해 쾌적한 열환경 조성이 가능함을 확인하였으며, 이러한 결과를 토대로 실제 건물 제어에 있어 재실자의 정보를 고려한 맞춤형 제어의 가능성을 보여준다.

시판 횟감어류에서의 Vibrio parahaemolyticus 분포 및 항생제 감수성에 관한 연구

성창현 ( Chang Hyeon Sung ),천정환 ( Jeong Hwan Cheon ),현지연 ( Ji Yeon Hyeon ),황인균 ( In Gyun Hwang ),곽효선 ( Hyo Sun Kwak ),윤상현 ( Sang Hyeon Yoon ),이정수 ( Jeong Soo Lee ),정윤희 ( Yun Hee Chung ),송광영 ( Kwang Young 한국수의공중보건학회 2010 예방수의학회지 Vol.34 No.3

Vibrio parahaemolyticus (V. parahaemolyticus) has been recognized as a significant food-borne pathogen around the world. In this study, we investigated the prevalence and antimicrobial resistance of V. parahaemolyticus isolated from raw fishes. A total of 64 samples of raw fishes purchased from a traditional seafood market in Seoul, Korea. were examined for the presence of V. parahaemolyticus using intestines, gills, and fins. Twenty five grams of all samples were enriched in 225ml of alkaline peptone water at 37℃ for 24h and then streaked onto thiosulfate citrate bile sucrose agar. Suspected colonies were inoculated into triple sugar iron agar for biochemical screening test and were finally confirmed with API 20NE strip. Antimicrobial resistance tests were performed with disc diffusion method in accordance with National Committee for Clinical Laboratory Standard. Thirty three V. parahaemolyticus strains were isolated from raw fishes among 33 out of 64 (51.6%). Among 33 isolates, 16 isolates (48.5%) were resistant to ampicillin, 7 isolates (21.2%) were resistant to amikacin, and all isolates were not resist to other antibiotics such as amoxicillin & clavulanic acid, sulfamethoxazole & trimethopenem, ciprofloxacin, cefotaxime and cefepime. Although the prevalence of V. parahaemolyticus was high in raw fishes compared to other studies, antimicrobial resistance rate of the isolates was relatively low. These results could be useful information for risk assessment of V. parahaemolyticus in raw fishes.

가공식품과 비가공식품에서의 황색포도상구균 검출을 위한 배지법과 Real-time PCR법의 비교

이재훈,송광영,현지연,황인균,곽효선,한정아,정윤희,서건호,Lee, Jae-Hoon,Song, Kwang-Young,Hyeon, Ji-Yeon,Hwang, In-Gyun,Kwak, Hyo-Sun,Han, Jeong-A,Chung, Yun-Hee,Seo, Kun-Ho 한국축산식품학회 2010 한국축산식품학회지 Vol.30 No.3

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.

다양한 식품에서 Campylobacter jejuni 검출을 위한 real-time PCR과 배지배양법의 비교검증

천정환(Jung-Whan Chon),현지연(Ji-Yeon Hyeon),황인균(In-Gyun Hwang),곽효선(Hyo-Sun Kwak),한정아(Jeong-A Han),김무상(Moo-Sang Kim),김종현(Jong-Hyun Kim),송광영(Kwang-Young Song),서건호(Kun-Ho Seo) 한국식품과학회 2011 한국식품과학회지 Vol.43 No.1

본 연구에서는 두 종류의 선택배지를 활용한 배지배양법과 realtime PCR의 C. jejuni 검출능력을 비교하였다. 소시지, 쇠고기 분쇄육, 무순에 C. jejuni를 접종하고 Hunt broth로 증균배양 하였으며, mCCD agar와 Preston agar에 배양액을 획선도말하여 미호기적으로 배양하였다. 동시에 증균배양액에서 1 ㎖을 채취하여 realtime PCR을 실시하였다. 실험결과, real-time PCR은 쇠고기 분쇄육과 소세지에서 두 가지 선택배지와 비교하여 동일한 검출력을 보였으나 무순에서는 훨씬 더 많은 양성을 검출하였다(p<0.05). 두 배지간의 비교에서는 Preston agar와 mCCD agar는 통계학적 유의차가 없는 민감도를 보였다(p>0.05). 결론적으로 real-time PCR 은 표준검출법인 배지배양법과 비교하여 동등하거나 우수한 민감도를 지닌 신속검출기법인 것으로 사료되며, 배지배양법에 앞서 선별검사로 사용할 경우 시간, 비용, 노동력 절감에 있어서 매우 유효한 방법이 될 것으로 판단된다. In this study, performances of culture methods using two selective media and real-time PCR were evaluated for detection of Campylobacter jejuni (C. jejuni) in various food samples. Sausage, ground beef, and radish sprouts inoculated with C. jejuni were enriched in Hunt broth and then streaked onto modified cefoperazone charcoal deoxycholate agar and Preston agar, followed by incubation under microaerobic conditions. The enriched Hunt broth (1 ㎖) was used in real-time PCR assay. No statistical differences were observed in sensitivity among the two selective media and real-time PCR for sausage and ground beef. However, the number of positives by real-time PCR in radish sprouts was much higher than the two selective media (p<0.05). It appears that real-time PCR could be used as an effective screening tool to detect C. jejuni, particularly in foods with a high number of background microflora such as fresh vegetables.

분말식품에서 Cronobacter spp. 검출을 위한 Real-Time PCR과 배지배양법의 비교검증

천정환,송광영,김선영,현지연,김윤경,황인균,곽효선,서건호,Chon, Jung-Whan,Song, Kwang-Young,Kim, Sun-Young,Hyeon, Ji-Yeon,Kim, Yun-Gyeong,Hwang, In-Gyun,Kwak, Hyo-Sun,Seo, Kun-Ho 한국미생물학회 2011 미생물학회지 Vol.47 No.1

본 연구에서는 분말 식품에서 real-time PCR과 배지배양법을 사용하여 Cronobacter spp.를 검출하는 방법이 비교검증 되었다. 조제분유, 이유식, 미숫가루에 Cronobacter를 인위적으로 접종시킨 후, 식품공전의 방법에 따라 멸균증류수와 Enterobacteriaceae enrichment (EE) broth에서 각각 1, 2차 증균배양 하였으며, Druggan-Forsythe-Iversen에 선택배양하여 Cronobacter를 검출하였다. Real-time PCR은 멸균증류수 및 EE broth에서 1 ml을 채취한 후 DNA를 추출하여 시행하였다. 실험결과 모든 식품에서 배지배양법과 real-time PCR간에는 통계학적 유의차가 존재하지 않았다(p>0.05). 한편 모든 실험회차에서 real-time PCR 수행 시, 1차 증균액인 멸균증류수에서의 양성검출율이 2차 증균액인 EE broth에서보다 높았는데, 이는 2차 증균액 내의 구성성분 중 일부분이 real-time PCR의 반응을 저해했기 때문으로 사료된다. 연구결과를 종합해 볼 때, 1차 증균 후, real-time PCR을 통해 Cronobacter를 검출하는 방법은 정확한 민감도를 보이면서도 시간과 노동력을 절감할 수 있는 효과적인 방법으로 사료된다. The aim of this study was to compare the performance of conventional culture and real-time PCR for detection of Cronobacter spp. in powdered foods. Infant formula, baby food and Misugaru inoculated with Cronobacter were enriched in distilled water as first enrichment step, followed by incubating in Enterobacteriaceae enrichment (EE) broth as second enrichment step. A loopful of enriched sample was streaked onto Druggan-Forsythe-Iversen agar, followed by incubating at $37^{\circ}C$ for 24 h. One milliliter of the enriched distilled water and EE broth were used in real-time PCR assay. No statistical differences were observed in the number of positive samples between culture method and real-time PCR (p>0.05) in all types of food samples. The number of positives of real-time PCR was higher in the first enrichment media (distilled water) than the second enrichment media (EE broth), though there was no significant difference (p>0.05). It appears that some components of the second enrichment broth, EE broth, inhibit the reaction of real-time PCR. These results show that real-time PCR using a single enrichment with distilled water could be useful as an effective screening method for detection of Cronobacter while saving much time and labor compared to conventional culture method.

시중에서 판매되는 닭고기의 저장온도에 따른 미생물학적 평가

천정환 ( Jung Whan Chon ),현지연 ( Ji Yeon Hyeon ),김윤경 ( Yun Gyeong Kim ),박준호 ( Jun Ho Park ),송광영 ( Kwang Young Song ),신록주 ( Rok Joo Shin ),김무상 ( Moo Sang Kim ),권기성 ( Ki Sung Kwon ),정명섭 ( Myung Sub Chung ) 한국수의공중보건학회 2011 예방수의학회지 Vol.35 No.1

This study was carried out to evaluate the microbiological characteristics of retail chicken meats stored under various conditions. Nine of whole chickens and nine of chicken breasts were used for bacterial analysis. Each chicken meat was divided into subsamples of 25 g each followed by storage at room temperature (25℃), refrigeration temperature (4 ℃), and freezing temperature (-20℃) for 180 min, 5 days, and 3 days, respectively. The standard plate counts were performed for the enumeration of the total aerobic bacteria. The number of aerobic bacteria was gradually increased by 1 log in samples held at the room temperature for 180 min. There was statistical difference in the number of bacteria between at 0 min and at 180 min of storage. For samples stored at 4℃for 5 days, the number of bacteria was increased from 5.11 to 7.26 log CFU/g in chicken breast and 3.83 to 6.04 log CFU/g in whole chicken with statistical difference. No significant changes were observed in frozen chicken. The results of this study may provide useful information to consumers for proper storage and safe handling of chicken meats.