• Title/Summary/Keyword: enrichment broth

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Comparison of Conventional Culture Method and Real-time PCR for Detection of Yersinia enterocolitica in Sausage and Vegetable Salad (소시지와 야채 샐러드에서 Yersinia enterocolitica 검출을 위한 배지법과 real-time PCR법의 비교)

  • Kim, Yun-Gyeong;Chon, Jung-Whan;Lee, Jae-Hoon;Kwak, Hyo-Sun;Hwang, In-Gyun;Seo, Kun-Ho
    • Korean Journal of Food Science and Technology
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    • v.45 no.1
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    • pp.133-136
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    • 2013
  • The purpose of this study was to compare a conventional culture method and real-time PCR for the detection of Yersinia enterocolitica (Y. enterocolitica) in sausage and in vegetable salad. Food samples inoculated with Y. enterocolitica were enriched in peptone-sorbitol bile-broth, and swabs were then streaked onto cefsulodin-irgasan-novobiocin agar. Biochemical tests for suspected colonies were performed with an API 20E strip. In parallel, real-time PCR was performed, targeting the 16S rRNA gene using 1 mL of enrichment broth. In sausage, the number of positive samples detected by culture method (49 out of 60) was similar (p>0.05) with that of real-time PCR (50 out of 60). However, the number of positive samples of real-time PCR (26 out of 60) was significantly higher (p<0.05) than that of the conventional culture method (6 out of 60) in vegetable salad. Real-time PCR could be an effective screening tool for detecting Y. enterocolitica, particularly in food samples with high levels of background flora, such as a vegetable salad.

Comparison of DNA Extraction Methods for the Detection of Foodborne Pathogenic Bacteria from Livestock Manure Composts (퇴비에서 식중독균 검출을 위한 DNA 추출 방법 비교)

  • Kim, Sung-Youn;Seo, Dong-Yeon;Moon, Ji-Young
    • Journal of Food Hygiene and Safety
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    • v.34 no.6
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    • pp.557-561
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    • 2019
  • This study investigated the efficacy of DNA extraction methods for real-time PCR detection of foodborne pathogenic bacteria in livestock manure composts. Livestock manure composts were inoculated with Escherichia coli O157:H7, Salmonella Typhimurium, Listeria monocytogenes, Bacillus cereus and incubated in enrichment broth. For DNA extraction, enriched samples were treated following boiling method, by chloroform, C18 powder, and proteinase K. As a result, 4 species of bacteria were detected by real-time PCR when subjected to boiling for 30 min and treated with proteinase K. These results suggest that detection of foodborne pathogens by real-time PCR from livestock manure composts could be applicable using effective DNA extraction methodology such as the boiling method or proteinase K.

Production of Lactic Acid from 1,2-Propanediol by Yeast (1,2-Propanediol로부터 Lactic acid의 생성)

  • Chae, Ki-Soo;Seu, Jung-Hwn
    • Microbiology and Biotechnology Letters
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    • v.9 no.2
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    • pp.51-57
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    • 1981
  • 1,2-propanediol-utilizing yeast, Y-1-4, was isolated from sludge sample by the enrichment culture technique. The product produced from 1,2-propanediol by the selected strain was identified as lactic acid by paper chromatography and infrared absorption spectrum. The strain assimilated ethanol, 1,2-propanediol, glycerine and glucose, but it produced lactic acid from 1,2-propanediol used as the sole carbon source. Under optimal conditions, the strain Y-1-4 was cultured with shaking at 3$0^{\circ}C$ for 4days in the medium containing 1,2-propanediol 20.0g, NH$_4$Cl 5.0g, KH$_2$PO$_4$ 1.0g, MgSO$_4$.7$H_2O$ 0.5g, FeSO$_4$.7$H_2O$ 0.25g, yeast extract 0.4g, CaCO$_3$ 3. 0g and tap water to one liter, and then the yield of lactic acid was about 12. 1g per liter of the culture broth.

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Xylanase Production from Bacillus safensis Isolate by Xylan or Xylan Hydrolyzed Products (Xylan과 Xylan 가수분해물에 의한 Bacillus safensis 분리균의 Xylanase 생산)

  • Jin, Hyun Kyung;Yoon, Ki-Hong
    • Microbiology and Biotechnology Letters
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    • v.44 no.3
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    • pp.324-332
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    • 2016
  • A bacterial strain capable of hydrolyzing xylan was isolated from fermented soybean paste obtained from a domestic Buddhist temple, using enrichment culture with rice straw as a carbon source. The isolate, named YB-1301, was identified as Bacillus safensis on the basis of its DNA gyrase subunit B gene (gyrB) sequence. The xylanase productivity of strain YB-1301 was drastically increased when it was grown in the presence of wheat bran or various xylans. In particular, the maximum xylanase productivity reached above 340 U/ml in the culture filtrate from LB broth supplemented with only birchwood xylan at shake-flask level. The xylanase production was significantly induced by xylans at the stationary growth phase in LB medium containing xylan, whereas only a small amount of xylanase was constitutively produced from cells grown in LB medium with no addition of xylan. Furthermore, xylanase biosynthesis was induced more rapidly by the enzymatically hydrolyzed products of xylan than by the non-hydrolyzed xylan. In addition, the xylanase in the culture filtrate of B. safensis YB-1301 was found to have optimal activity at 55℃ and pH 6.5–7.0.

Estimation of Distribution of a Commensal Thermophile in Soil by Competitive Quantitative PCR and Terminal Restriction Fragment Length Polymorphism Analysis

  • Rhee, Sung-Keun;Hong, Seung-Pyo;Bae, Jin-Woo;Jeon, Che-Ok;Lee, Seung-Goo;Song, Jae-Jun;Poo, Ha-Ryoung;Sung, Moon-Hee
    • Journal of Microbiology and Biotechnology
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    • v.11 no.6
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    • pp.940-945
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    • 2001
  • Symbiobacterium toebii has been previously reported as a novel commensal thermophile exhibiting a commensal interaction with thermophilic Geobacillus sp. SK-1. We investigated the distribution of this commensal thermophile in various soils using molecular methods, such as quantitative PCR and terminal restriction fragment polymorphism analysis. Based on a nested competitive quantitative PCR the 16S rDNA of the commensal thermophile was only detected in compost soils at about $1.0{\times}10^4$ cpoies per gram of soil, corresponding to $0.25{\times}10^4$ cells per gram of soil. However, in an enrichment experiment at $60^{\circ}C$, about $1.0{\times}10^8$ copies of 16S rDNA molecules were detected per ml of enriched culture broth for all the soils, and more than 0.1 mM indole accumulated as the product of commensal bacterial growth. When incubated at $30^{\circ}C$, neither the 16S rDNA of the commensal bacterium nor any indole accumulation was detected. Accordingly, even though the 16S rDNA of the bacterium was only detected in the compost soils by a nested PCR, the presence of the 16S rDNA molecules of commensal thermophile and accumulation of indole in all the enriched cultures appeared to indicate that the commensal thermophile is widely distributed in various soils.

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Isolation, Identification, and Characterization of Aero-Adaptive Campylobacter jejuni

  • LEE YOUNG-DUCK;MOON BO-YOUN;CHOI JUNG-PIL;CHANG HAK-GIL;NOH BONG-SOO;PARK JONG-HYUN
    • Journal of Microbiology and Biotechnology
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    • v.15 no.5
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    • pp.992-1000
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    • 2005
  • Campylobacter is one of the emerging foodborne pathogens, and its worldwide incidence rate is extremely high. This study was undertaken to isolate and identify Campylobacter strains from chicken carcasses in the local markets, and analyze their characteristics regarding oxygen tolerance. They were isolated after aerobic enrichment and identified by biochemical, physiological, and morphological characteristics, PCR, and 16S rDNA sequencing. Their oxygen tolerances were analyzed in terms of the cell surface hydrophobicity, cell fatty acid composition, and oxidoreductase. Five strains of C. jejuni were isolated and identified from 61 isolates from 50 chickens. Among them, C. jejuni IC21 grew well in Brucella broth and commercial milk under aerobic condition. However, in the aerobic exposure, the cell surface hydrophobicity of C. jejuni IC21 was almost the same as the other isolates, even though its morphology changed from the spiral-bacilli form into the coccoid form. Fatty acid analyses showed that all Campylobacter strains had a high composition of $C_{19:1}$, cyclopropane fatty acid, and that the amount of the other fatty acids were very similar between them. Interestingly, however, only oxidoreductase activities of C. jejuni IC21 increased highly under aerobic exposure even though its activities were almost the same as the other C. jejuni strains just after microaerobic culture. It had 11.8 times higher catalase activity, 4.4 times higher for SOD, and 2.0 times higher for NADH oxidase activities. Therefore, in the case of the aero-adaptive C. jejuni IC21, expression of oxidoreductase significantly increased under oxidative stressed condition, which might allow it to survive for a longer time and grow on food under aerobic exposure. Such new strain might be one of the explanations for the increase of campylobacteriosis.

Studies on The Microbial Utilization of Agricultural Wastes (Part 5) Isolation and Selection of Cellulase Producing Fungi (농산폐자원의 미생물학적 이용에 관한 연구 (제오보) 섬유소분해효소 생산 곰팡이의 분리 및 선별)

  • Bae, Moo;Kim, Byung-Hong;Lee, Gye-Jun
    • Microbiology and Biotechnology Letters
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    • v.4 no.3
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    • pp.105-110
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    • 1976
  • In the studies of microbiological utilization of cellulosic wastes, cellulolytic fungi were isolated and screened out. At the first stage, 221 cellulolytic fungi were isolated from different sources such as soils, humus, composts and rotten wood debris by enrichment culture techniques. In the second stage, 36 strains of fungi out of those previously isolated were selected for their cellulase activities estimated by means of filter paper degradation, carboxy methyl cellulose liquefaction and cup method. Activities of C$_1$-cellulase, C$\sub$x/-cellulase and filter paper activity were adopted on the final screening stage and five different strains which are tentatively identified as Aspergillus sp.(strain No. AS-9), Penicillium sp. (strain No. KNI-1-2), Trichoderma, sp. (strain No. KI-7-2, KI-7-5, KI-4-1-1B) were selected for their high potency of C$_1$ and C$\sub$x/-cellulase activities. When rice straw milled and treated with NH$_4$OH was hydrolyzed with the crude enzyme Prepared from the culture broth of Trichoderma sp. (strain No. KI-4-1-1B), saccharification rate was obtained up to 26%.

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Improvement of polymerase chain reaction methods for rapid detection of Listeria monocytogenes in raw milk (원유로부터 Listeria monocytogenes의 신속검색을 위한 종합효소 연쇄반응법의 개선)

  • Yi, Chul-hyun;Son, Won-geun;Kang, Ho-jo
    • Korean Journal of Veterinary Research
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    • v.36 no.1
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    • pp.119-129
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    • 1996
  • The present study was conducted to rapidly detect Listeria monocytogenes in raw milk. Specificity and sensitivity of polymerase chain reaction(PCR) technique, and direct PCR were examinded in raw milk, also were compared the calssical culture methods with PCR technique. This method used a pair of primers based on a unique region in the 16S rRNA sequence of L nomocytogenes. In the PCR specificity tests, each of the 10 strains of L monocytogenes tested gave a single 70-bp band. But the other six Listera spp tested gave negative results. Results of the sensitivity tests showed that as few as 2 CFU of L monocytogenes in pure cultures could be detected with 16S rRNA-based primers, L-1 and L-2. In different PCR cycles, a PCR product was detected with $10^3$ cells of L monocytogenes from 25 cycles to 50 cycles and the concentration of PCR products was cycle-dependent. Raw milk samopes added L monocytogenes cells gave negative results. However, these samplers gave a single 70-bp band by pretreatment of pronase, and PCR products were detected with $10^1$ cells of L monocytogenes. To detemine the most sensitive culture protocol to use in conjunction with the PCR assay, raw milk samples were inoculated with L monocytogenes at concentrations ranging from 1 to $5.7{\times}10^4CFU/ml$. PCR assays from Listeria enrichment broth(LEB) containing raw milk samples added L monocytogene EGD could dtect 10 cells in pronase-pretreated samples without incubation, and 1 cell of L monocytogenes in both 12 hr and 24 hr incubation, respectively. Isolation raw of PCR assays was similar to that of classical culture methods, but required time for detection of L monocytogenes could remarkably be reduced compare to culture methods.

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Detection of Campylobacter jejuni in food and poultry visors using immunomagnetic separation and microtitre hybridization

  • Simard, Ronald-E.
    • Proceedings of the Korean Society of Fisheries Technology Conference
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    • 2000.05a
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    • pp.71-73
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    • 2000
  • Campylobacter jejuni is most frequently identified cause of cause of acute diarrhoeal infections in developeed countries, exceeding rates of illness caused by both salmonella and shigilla(Skirrow, 1990 ; Lior 1994). Previous studies on campylobacter jejuni contamination of commercial broiler carcasses in u.s.(Stern, 1992). Most cases of the disease result from indirect transmission of Campylobactor from animals via milk, water and meat. In addition to Campylobactor jejuni. the closely relates species Campylobactor coli and Campylobactor lari have also been implicated as agents of gastroenteritis in humans. Campylobactor coli represented only approximately 3% of the Campylobactor isolates from patients with Campylobactor enteritis(Griffiths and Park, 1990) whereas Campylobactor coli is mainly isolated from pork(Lmmerding et al., 1988). Campylobactor jejuni has also been isolated from cases of bacteremia, appendicitis and, recently, has been associated with Guillai-Barre syndrome(Allos and Blaser, 1994; von Wulffen et al., 1994; Phillips, 1995). Studies in volunteers indicated that the infectious dose for Campylobactor jejuni is low(about 500 organisms)(Robinson, 1981). The methods traditionally used to detect Campylobactor ssp. in food require at least two days of incubation in an enrichment broth followed by plating and two days of incubation on complex culture media containing many antibiotics(Goossens and Butzler, 1992). Finnaly, several biochemical tests must be done to confirm the indentification at the species level. Therfore, sensitive and specific methods for the detection of small numbers of Campylobactor cells in food are needed. Polymerase chain reaction(PCR) assays targeting specific DNA sequences have been developed for the detection of Campylobactor(Giesendorf and Quint, 1995; Hemandex et al., 1995; Winter and Slavidk, 1995). In most cases, a short enrichment step is needed to enhance the sensitivity of the assay prior to detection by PCR as the number of bacteria in the food products is low in comparison with those found in dinical samples, and because the complex composition of food matrices can hinder the PCR and lower its sensitivity. However, these PCR systems are technically demanding to carry out and cumbersome when processing a large number of samples simutaneously. In this paper, an immunomagnetic method to concentrate Campylobactor cells present in food or clinical samples after an enrichment step is described. To detect specifically the thermophilic Campylobactor. a monoclonal antibody was adsorbed on the surface of the magnetic beads which react against a major porin of 45kDa present on the surface of the cells(Huyer et al., 1986). After this partial purification and concentration step, detection of bound cells was achieved using a simple, inexpensive microtitre plate-based hybridization system. We examined two alternative detection systems, one specific for thermophilic Campylobactor based on the detection of 23S rRNA using an immobilized DNA probe. The second system is less specific but more sensitive because of the high copy number of the rRNA present in bacterial cell($10^3-10^4$). By using specific immunomagnetic beads against thermophilic Campylobactor, it was possible to concentrate these cells from a heterogeneous media and obtain highly specific hybridization reactions with good sensitivity. There are several advantages in using microtitre plates instead of filter membranes or other matrices for hybridization techniques. Microtitre plates are much easier to handle than filter membranes during the adsorption, washing, hybridization and detection steps, and their use faciilitates the simultanuous analysis of multiple sample. Here we report on the use of a very simple detection procedure based on a monoclonal anti-RNA-DNA hybrid antibody(Fliss et al., 1999) for detection of the RNA-DNA hybrids formed in the wells.

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Comparison of Standard Culture Method and Real-time PCR Assay for Detection of Staphylococcus aureus in Processed and Unprocessed Foods (가공식품과 비가공식품에서의 황색포도상구균 검출을 위한 배지법과 Real-time PCR법의 비교)

  • Lee, Jae-Hoon;Song, Kwang-Young;Hyeon, Ji-Yeon;Hwang, In-Gyun;Kwak, Hyo-Sun;Han, Jeong-A;Chung, Yun-Hee;Seo, Kun-Ho
    • 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.