Development of a multiplex loop-mediated isothermal amplification assay to detect shiga toxin-producing Escherichia coli in cattle

동희진,Ae-Ri Cho,한태욱,조성범 대한수의학회 2014 Journal of Veterinary Science Vol.15 No.2

A multiplex loop-mediated isothermal amplification(mLAMP) assay was developed for simultaneous detection ofthe stx1 and stx2 genes and applied for detection of shigatoxin-producing Escherichia coli (STEC) in cattle farmsamples. Two target genes were distinguished based on Tmvalues of 85.03 ± 0.54oC for stx1 and 87.47 ± 0.35oC for stx2. The mLAMP assay was specific (100% inclusivity andexclusivity), sensitive (with a detection limit as low as 10fg/μL), and quantifiable (R2 = 0.9313). The efficacy andsensitivity were measured to evaluate applicability of themLAMP assay to cattle farm samples. A total of 12 (12/253;4.7%) and 17 (17/253; 6.7%) STEC O157, and 11 (11/236;4.7%) non-O157 STEC strains were isolated from cattle farmsamples by conventional selective culture, immunomagneticseparation, and PCR-based culture methods, respectively. The coinciding multiplex PCR and mLAMP results for thetypes of shiga toxin revealed the value of the mLAMP assay interms of accuracy and rapidity for characterizing shiga toxingenes. Furthermore, the high detection rate of specific genesfrom enrichment broth samples indicates the potential utilityof this assay as a screening method for detecting STEC in cattle farm samples.

오리 도체에서 등온유전자증폭기법을 이용한Salmonella spp. 신속 고감도 검출 기법 연구

조애리,동희진,조성범 한국축산식품학회 2013 한국축산식품학회지 Vol.33 No.5

In this study, a rapid and sensitive detection tool for screening Salmonella spp. by using a loop-mediated isothermal ampli-fication (LAMP) assay targeting the genomic sequence of the invA gene was developed. The inclusivity and exclusivity were both at 100% in the analysis, and the limit of detection (LOD) in a pure S. Enteritidis culture suspended in saline was 3.2×103CFU/mL at 18.17 min (R2= 0.9446). The LODs of the LAMP assay in buffered peptone water with Salmonella (BPW) and duck carcass swab sample enriched in BPW with Salmonella (BPWS) after 0 and 12 h incubation were 3.2× 103CFU/mL and 3.2×100CFU/mL, respectively. Comparing the LODs in BPW with those in BPWS, the LAMP assay was less influenced by compounds of duck carcass swab sample than the PCR assay. Sensitivity and specificity of the LAMP assay in 50 duck carcass swab samples enriched in BPW for 6 h were 96% and 84%, respectively, indicating that the LAMP assay is a rapid, simple and sensitive assay, which can be utilized as a potential screening tool of Salmonella spp. in duck carcass sample.

Meat Species Identification using Loop-mediated Isothermal Amplification Assay Targeting Species-specific Mitochondrial DNA

조애리,동희진,조성범 한국축산식품학회 2014 한국축산식품학회지 Vol.34 No.6

Meat source fraud and adulteration scandals have led to consumer demands for accurate meat identification methods. Nucleotide amplification assays have been proposed as an alternative method to protein-based assays for meat identifica-tion. In this study, we designed Loop-mediated isothermal amplification (LAMP) assays targeting species-specific mito-chondrial DNA to identify and discriminate eight meat species; cattle, pig, horse, goat, sheep, chicken, duck, and turkey. The LAMP primer sets were designed and the target genes were discriminated according to their unique annealing temper-ature generated by annealing curve analysis. Their unique annealing temperatures were found to be 85.56±0.07oC for cattle,84.96±0.08oC for pig, and 85.99±0.05oC for horse in the BSE-LAMP set (Bos taurus, Sus scrofa domesticus and Equuscaballus); 84.91±0.11oC for goat and 83.90±0.11oC for sheep in the CO-LAMP set (Capra hircus and Ovis aries); and86.31±0.23oC for chicken, 88.66±0.12oC for duck, and 84.49±0.08oC for turkey in the GAM-LAMP set (Gallus gallus,Anas platyrhynchos and Meleagris gallopavo). No cross-reactivity was observed in each set. The limits of detection (LODs)of the LAMP assays in raw and cooked meat were determined from 10 pg/µL to 100 fg/µL levels, and LODs in raw andcooked meat admixtures were determined from 0.01% to 0.0001% levels. The assays were performed within 30 min andshowed greater sensitivity than that of the PCR assays. These novel LAMP assays provide a simple, rapid, accurate, andsensitive technology for discrimination of eight meat species.

Helicobacter apodemus sp. nov., a new Helicobacter species identified from the gastrointestinal tract of striped field mice in Korea

전우진,동희진,신재훈,김일용,Hungwui Ho,오승현,윤영민,최양규,서준교,남기환,김형진,조성범,성제경 대한수의학회 2015 Journal of Veterinary Science Vol.16 No.4

A novel Helicobacter species was identified from the gastrointestinal tract of the Korean striped field mouse (Apodemus agrarius). Biochemical testing, ultrastructure characterization, and 16S rRNA gene sequence analysis suggested that this bacterium represents a distinct taxon. The bacterium was positive for urease activity, susceptible to cephalothin and nalidixic acid, and weakly positive for oxidase and catalase activity. Electron microscopy revealed that the bacterium has spirally curved rod morphology with singular bipolar nonsheathed flagella. Genotypically, the isolated bacterial strains (YMRC 000215, YMRC 000216, and YMRC 000419) were most closely related to a reference strain of Helicobacter mesocricetorum (97.25%, 97.32%, and 97.03% 16S rRNA sequence similarities, respectively). The 16S rRNA sequences of these strains were deposited into GenBank under accession numbers AF284754, AY009129, and AY009130, respectively. We propose the name Helicobacter apodemus for this novel species.

Development of a Loop-mediated Isothermal Amplification Assay for Detecting Listeria monocytogenes prfA in Milk

조애리,동희진,서건호,조성범 한국식품과학회 2014 Food Science and Biotechnology Vol.23 No.2

A rapid and sensitive loop-mediated isothermalamplification (LAMP) assay was developed for detectingListeria monocytogenes prfA in milk. The inclusivity of 23L. monocytogenes and the exclusivity of 16 non-L. monocytogenes strains were both 100% in the assay. Thelimit of detection (LoD) of the LAMP assay in Listeriaenrichment broth (LEB) was 2.22 CFU/mL after 12 h and24 h of incubation. The LoDs of the LAMP assay in LEBwith artificially contaminated milk (LEB-M) incubated for12 h (2.22×101 CFU/mL) and 24 h (2.22 CFU/mL) werelower than those of the PCR and real-time PCR assays. Comparison of the LoDs in LEB with those in LEB-Mshowed that the LAMP assay was less influenced by themilk compounds than the real-time PCR assay. Our resultsindicate that the LAMP assay can be utilized as a potentialscreening tool for L. monocytogenes in milk.

Antibiotic resistance patterns and genetic relatedness of Enterococcus faecalis and Enterococcus faecium isolated from military working dogs in Korea

방기만,안재욱,김우현,동희진,김준형,조성범 대한수의학회 2017 Journal of Veterinary Science Vol.18 No.2

Enterococcus spp. are normally present in the gastrointestinal tracts of animals and humans, but can cause opportunistic infections that can be transmitted to other animals or humans with integrated antibiotic resistance. To investigate if this is a potential risk in military working dogs (MWDs), we analyzed antibiotic resistance patterns and genetic relatedness of Enterococcus spp. isolated from fecal samples of MWDs of four different age groups. Isolation rates of Enterococcus spp., Enterococcus (E.) faecalis, and E. faecium, were 87.7% (57/65), 59.6% (34/57), and 56.1% (32/57), respectively, as determined by bacterial culture and multiplex PCR. The isolation rate of E. faecalis gradually decreased with age (puppy, 100%; adolescent, 91.7%; adult, 36.4%; and senior, 14.3%). Rates of resistance to the antibiotics ciprofloxacin, gentamicin, streptomycin, sulfamethoxazole/trimethoprim, imipenem, and kanamycin among Enterococcus spp. increased in adolescents and adults and decreased in senior dogs, with some isolates having three different antibiotic resistance patterns. There were indistinguishable pulsed-field gel electrophoresis patterns among the age groups. The results suggest that Enterococcus is horizontally transferred, regardless of age. As such, periodic surveillance studies should be undertaken to monitor changes in antibiotic resistance, which may necessitate modification of antibiotic regimens to manage antibiotic resistance transmission.