• Title/Summary/Keyword: Species detection

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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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A STUDY ON THE IDENTIFICATION OF Porphyromonas endodontalis BY PCR USING SPECIES SPECIFIC PRIMERS FOR THE 16S rDNA (16S rDNA sequence에 대한 종특이성 primer를 이용한 중합효소연쇄반응증폭에 의한 Porphyromonas endodontalis의 동정에 관한 연구)

  • Eom, Seung-Hee;Lim, Sung-Sam;Bae, Kwang-Shik
    • Restorative Dentistry and Endodontics
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    • v.24 no.1
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    • pp.13-25
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    • 1999
  • P. endodontalis which was known to be associated with the infected root canals and periapical lesions is very difficult to detect by culture methods or traditional methods. Detection of bacteria using polymerase chain reaction(PCR) for 16S ribosomal DNA(rDNA) is fast, simple, and accurate with relatively small amount of target cells. 16S rDNA consist of conserved regions those are same to all species, and variable regions which represent species specificity. The 16S rDNA sequences of P. endodontalis and P. gingivalis were aligned and two highly variable regions were selected as a pair of species specific oligonucleotide primers for P. endodontalis. And then the pair of primers for PCR amplification was synthesized to identify P. endodontalis. The sequences of the species specific primers for the 16S rDNA of P. endodontalis were as follows ; sense primer[endo1]: 5'-CTATATTCTTCTTTCTCCGCATGGAGGAGG-3' antisense primer[endo2]: 5'-GCATACCTTCGGTCTCCTCTAGCATAT-3' In this study, for the identification of P. endodontalis without culture from the mixed clinical samples, PCR was done with species specific primers for the 16S rDNA sequences of P. endodontalis. The results were as follows : 1. The species specificity of the primers for the 16S rDNA of P. endodntalis was determined by the PCR methods. About 490bp amplicon which was specific only for P. endodntalis was produced with P. endodontalis. No amplicon was produced by PCR with other strains similar to P. endodontalis. 2. The synthesized species specific primers reacted with conventionally identified P. endodontalis which we have in conservative dentistry laboratory. 3. The identification of P. endodontalis using PCR technique with samples collected from infected root canals or periapical lesions was more sensitive than that of culture methods. 4. Seven samples revealed including P. endodontalis by PCR technique. Five of them were related with pains, two of them with sinus tract, three of them with foul odor, and three of them with purulent drainage. P. endodontalis was shown to have great relation with pains.

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Overview of Preventive Measures against Invasive Alien Species in Korea and Suggestions for their Improvement (침입외래생물의 사전예방 제도 및 개선방향)

  • Kil, Jihyon;Kim, Chang-Gi
    • Korean Journal of Ecology and Environment
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    • v.47 no.4
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    • pp.239-246
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    • 2014
  • To cope with the potential risks associated with invasive alien species, the Korean Government implemented the Biodiversity Act in 2014. Among the alien species not yet introduced into that country, 24 have been designated as alert species. These include mammals, birds, fish, mollusks, insects, and plants that may invade and cause serious harm to the ecosystem. Approval from the Ministry of Environment is required to import or carry any of them. Although these measures are more advanced than those from the previous legal framework, several terms still need to be improved. First, the category of alert species should cover not only those not yet introduced but also those that are being raised or cultivated at aquariums, botanical gardens, and zoos. Second, for applicants who intend to import or carry alert species, the government must provide them with detailed standards for the ecological risk assessment of alert species as well as guidelines for their safe use in Korea to prevent their unregulated release from confinement facilities into natural environments. Third, tools and protocols should be developed for early detection and rapid responses to those escapes.

Speciation Analysis of Arsenic Species in Surface Water (수중의 비소 종 분리 분석)

  • Jeong, Gwan-Jo;Kim, Dok-Chan
    • Journal of Korean Society of Environmental Engineers
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    • v.30 no.6
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    • pp.621-627
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    • 2008
  • In this study, a technique of speciation and determination of the trace inorganic arsenic(As(III) and As(V)) in water sample using HPLC-DRC-ICP-MS has been developed. Isocratic mobile phase of 10 mM ammonium nitrate and 10 mM ammonium phosphate monobasic was used and methanol(5 v/v%) was used as flushing solvent. Selection of the best flow rate of reaction gas, O$_2$, and optimization of the parameters such as pH and flow rate of mobile phase, and injection volume of sample for the separation and detection of arsenic species were carried out. The oxygen flow rate of 0.5 mL/min, pH of 9.4 and flow rate of 1.5 mL/min of mobile phase, and injection volume of sample of 100 $\mu$L were found to be the best parameters for the speciation and determination of arsenic species. The analytical features of the method were detection limit 0.10 and 0.08 $\mu$g/L, precision(RSD) 4.3% and 3.6%, and recovery 95.2% and 96.4% for As(III) and As(V), respectively. Analysis time was 4 minutes per sample. Linear calibration graphs with r$^2$ = 0.998 were obtained for both As(III) and As(V). Speciation analysis of arsenic species in the raw water samples collected from the tributary streams to Han River and main stream of Paldnag were performed by the proposed method. The concentrations of As(III) ranged from 0.10 to 0.22 $\mu$g/L and As(V) concentrations ranged from 0.44 to 1.19 $\mu$g/L, and 93.5% of total arsenic was found to be As(V).

Detection of fish pathogens in cultured juveniles for stock enhancement in 2010 (2010년 방류용 수산종묘에 대한 병원체 검출)

  • Cho, Mi-Young;Park, Su-Young;Won, Kyoung-Mi;Han, Hyun-Ja;Lee, Soon-Jeong;Cho, Young-A;Kim, Jin-Woo
    • Journal of fish pathology
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    • v.24 no.2
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    • pp.121-129
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    • 2011
  • Aquatic animal raised in hatcheries play an important role in supplying seedling to stock enhancement and seed quality, especially, seed health is the key factor for survival in the field after release and for stocking effectiveness. We have inspected the hatchery-reared seeds of 33 marine species and 12 freshwater species for legally designated diseases in stock enhancement program in 2010. Results showed that abalone was the most abundant as 20.0% in the marine species group and then sea cucumber (15.6%), olive flounder (8.4%), rockfish (6.7%), black sea bream (6.3%) and swimming crab (6.1%) were followed. Crucian carp was the most abundant as 19.4%, and then eel (11.8%), Korean bullhead (10.9%), mandarin fish (10.8%), melanian snail (8.4%), catfish (7.7%) were followed in the freshwater species group. The total number of inspection cases for eight pathogens in this study were 2,105 and disqualification cases were 30 by detection of aquatic animals pathogens such as koi herpesvirus (KHV), red sea bream iridovirus (RSIV), white spot syndrome virus (WSSV) or viral haemorrhagic septicemia virus (VHSV).

Comparison of Conventional Culture Method, Enzyme Immune Method, and PCR for the Rapid Detection of Salmonella spp. in Pet Food (반려동물 사료의 Salmonella spp. 신속검출을 위한 증균배양법, 면역학적 검출법 및 종 특이 프라이머를 이용한 PCR 방법 비교)

  • Yun, Hyejeong;Cha, Sun Ho;Lee, Seung-Hwa;Jeong, Min-Hee;Na, Tae-Woong;Kim, Haejin;Cho, Hyunjeong;Hong, Seong-Hee
    • Journal of Food Hygiene and Safety
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    • v.37 no.1
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    • pp.15-20
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    • 2022
  • The purpose of this study was to compare the conventional culture method, enzyme immune method and the PCR method using species-specific primer in the analysis on the Salmonella spp. found in domestically distributed pet foods. For the study, Salmonella spp. were detected from 175 samples. From the conventional culture method and the PCR method, two samples (jerky and corn gluten) were determined as positive. Also, from the enzyme immune method, one sample (corn gluten) was test-positive. The study revealed that application of the PCR method with species-specific primer allows better distinguishment between the species of the strain collected from the samples than the conventional culture method and/or the enzyme immune method.

Detection of Irradiated Chicken, Pork and Beef by ESR Spectroscopy (닭고기, 돼지고기 및 쇠고기의 방사선 조사 유무 판별을 위한 ESR Spectroscopy의 활용)

  • Yang, Jae-Seung;Kim, Choong-Ki;Lee, Hae-Jung
    • Korean Journal of Food Science and Technology
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    • v.31 no.3
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    • pp.606-611
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    • 1999
  • To identify irradiated foods, studies have been carried out with electron spin resonance (ESR) spectroscopy on bone containing foods, such as chicken, pork, and beef. Bones cleaned, pieced and dried were irradiated with doses of 0, 1, 3, 5 kGys using a $Co^{60}$ irradiator. The bones were placed in a resonant quartz tube with an internal diameter of about 4.0 mm within the Bruker Win-ESR spectrometer, and the intensity of the ESR signal could be quantified by double integration of the first derivative spectrum. The irradiated bone presented an asymmetric absorption in shape, different from that of an unirradiated one. It could be possible to detect at doses lower than 1 kGy below the dose employed commercially (3 kGy) in the case of irradiated chicken bone. The signal intensity was greatest in the beef bone, intermediate in the pork bone and lowest in the chicken bone; it was normally lower for smaller animals than for larger species, and small variations were observed between samples of the same species. The intensity of the signal induced in bones increased linearly with irradiation doses in the range of 1.0 kGy to 5.0 kGy, and it was possible to distinguish between samples given low and high doses of irradiation. The signal stability for 6 weeks made them ideal for the quick and easy identification of irradiated meats.

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Development of real-time PCR for rapid detection of Mycobacterium bovis DNA in cattle lymph nodes and differentiation of M. bovis and M. tuberculosis (소 림프절에서 Mycobacterium bovis DNA의 신속 검출과 M. bovis와 M. tuberculosis 감별을 위한 real-time PCR 개발)

  • Koh, Ba-Ra-Da;Jang, Young-Boo;Ku, Bok-Kyung;Cho, Ho-Seong;Bae, Seong-Yeol;Na, Ho-Myung;Park, Seong-Do;Kim, Yong-Hwan;Mun, Yong-Un
    • Korean Journal of Veterinary Service
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    • v.34 no.4
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    • pp.321-331
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    • 2011
  • Mycobacterium bovis, a member of the M. tuberculosis complex (MTC), is the causative agent of bovine tuberculosis. Detection of M. bovis and M. tuberculosis using conventional culture- and biochemical-based assays is time-consuming. Therefore, a simple and sensitive molecular assay for rapid detection would be of great help in specific situations such as faster diagnosis of bovine tuberculosis (bTB) infection in the abattoirs. We developed a novel multiplex real-time PCR assay which was applied directly to biological samples with evidence of bTB and it was allowed to differentiate between M. bovis and M. tuberculosis. The primers and TaqMan probes were designed to target the IS1081 gene, the multi-copy insertion element in the MTC and the 12.7-kb fragment which presents in M. tuberculosis, not in the M. bovis genome. The assay was optimized and validated by testing 10 species of mycobacteria including M. bovis and M. tuberculosis, and 10 other bacterial species such as Escherichia coli, and cattle lymph nodes (n=113). The tests identified 96.4% (27/28) as M. bovis from the MTC-positive bTB samples using conventional PCR for specific insertion elements IS1081. And MTC-negative bTB samples (n=85) were tested using conventional PCR and the real-time PCR. When comparative analyses were conducted on all bovine samples, using conventional PCR as the gold standard, the relative accuracy of real-time PCR was 99.1%, the relative specificity was 100%, and the agreement quotient (kappa) was 0.976. The detection limits of the real-time PCR assays for M. bovis and M. tuberculosis genomic DNA were 10 fg and 0.1 pg per PCR reaction, respectively. Consequently, this multiplex real-time PCR assay is a useful diagnotic tool for the identification of MTC and differentiation of M. bovis and M. tuberculosis, as well as the epidemiologic surveillance of animals slaughtered in abattoir.

Development of Ultra-Rapid Multiplex PCR Detection against 6 Major Pathogens in Honeybee (꿀벌 6종 주요 병원체에 대한 초고속 다중 PCR 검출법의 개발)

  • Lim, Su-Jin;Kim, Jung-Min;Lee, Chil-Woo;Yoon, Byoung-Su
    • Journal of Apiculture
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    • v.32 no.1
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    • pp.27-39
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    • 2017
  • PCR-chip-based ultra-rapid multiplex PCRs for detection of six major infectious pathogens in honeybee were developed. The 6 kinds of major infectious pathogens in honeybee included Paenibacillus larvae causing American Foulbrood, Melissococcus plutonius causing European Foulbrood as bacteria, Ascosphaera apis (Chalkbrood), Aspergillus flavus (Stonebrood), Nosema apis and Nosema ceranae (Nosemosis) as fungi. The developed PCR-chip-based ultra-rapid multiplex PCR showed successful amplification for all six major pathogens in the presence of more than $10^3$ molecules. The time for confirming amplification (Threshold cycles; Ct-time) was about 7 minutes for two species, and about 9 minutes for four species. Total 40 cycles of PCR took 11 minutes 42 seconds and time for melting point analysis was 1 minute 15 seconds. Total time for whole PCR detection was estimated 12 minutes 57 seconds (40 cycles of PCR and melting point analysis). PCR-chip based ultra-rapid multiplex PCR using standard DNA substrates showed close to 100% accuracy and no false-amplification was found with honeybee genomic DNA. Ultra-rapid multiplex PCR is expected to be a fast and efficient pathogen detection method not only in the laboratory but also in the apiary field.