• Korean Journal of Ecology and Environment
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• v.40 no.1
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• pp.149-154
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• 2007

In the present study, we performed the PCR assay using TOX2P/TOX2M primer targeting a specific region within mcyB gene to identify potential microcystin-producing cyanobacteria. TOX2P/TOX2M primer set was effective in amplifying mcy gene in the field samples containing Microcystis spp. of 1,000 cells per mL. Moreover, the results from the PCR assay agreed with those of the ELISA analysis. Consequently, this study demonstrated that TOX2P/TOX2M primer set can be used as a genetic probe for the early detection of cyanobacterial toxigenicity in Korean water bodies.

• Journal of Microbiology and Biotechnology
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• v.23 no.4
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• pp.555-559
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• 2013

Vibrio cholerae O1 and O139 are the major serotypes associated with illness, and some V. cholera non-O1 and non-O139 isolates produce cholera toxin. The present study describes a quantitative polymerase chain reaction (qPCR) assay for the species-specific detection and quantitation of V. cholera using a primer pair based on an outer membrane lipoprotein lolB gene for the amplification of a 195 bp DNA fragment. The qPCR primer set for the accurate diagnosis of V. cholera was developed from publically available genome sequences. This quantitative PCR-based method will potentially simplify and facilitate the diagnosis of this pathogen and guide disease management.

• Korean Journal of Veterinary Service
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• v.34 no.4
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• pp.333-339
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• 2011

Loop-mediated isothermal amplification (LAMP) was developed to detect Mycobacterium tuberculosis complex (MTC) and non-tuberculous mycobacterium (NTM) genomic DNA in blood samples of Korea native cattle. A set of four primers, two outer and two inner, were designed from M. bovis and M. avium genomic DNA targeting the IS6110 and 16S rRNA gene, respectively. Based on 85 Intradermal Tuberculin Test (ITT) positive blood sample and using conventional PCR and LAMP, the agreement quotient (kappa), which measures agreement beyond chance were 0.93 (conventional PCR) and 0.97 (LAMP), respectively. The detection limit of the LAMP method was $2.0{\times}10^2$ copy/ml M. bovis and M. avium cells, compared to $2.0{\times}10^3$ copy/ml M. bovis and M. avium cells for conventional PCR. These results suggest that the LAMP is a powerful tool for rapid, sensitive, and practical detection of MTC and NTM in blood samples of Korea native cattle.

• Genomics & Informatics
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• v.20 no.4
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• pp.46.1-46.7
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• 2022

Influenza A virus (IAV) is the most widespread pathogen causing human respiratory infections. Although polymerase chain reaction (PCR)-based methods are currently the most commonly used tools for IAV detection, PCR is not ideal for point-of-care testing. In this study, we aimed to develop a more rapid and sensitive method than PCR-based tools to detect IAV using loop-mediated isothermal amplification (LAMP) technology. We designed reverse-transcriptional (RT)-LAMP primers targeting the hemagglutinin gene. RNAs from reference H1N1 and H3N2 showed specific RT-LAMP signals with the designed primers. We optimized the reaction conditions and developed universal reaction conditions for both LAMP assays. Under these conditions, the detection limit was 50 copies for both RT-LAMP assays. There was no non-specific signal to 19 non-IAV respiratory viruses, such as influenza B virus, coronaviruses, and respiratory syncytial viruses. Regarding the reaction time, a positive signal was detected within 25 min after starting the reaction. In conclusion, our RT-LAMP assay has high sensitivity and specificity for the detection of the H1 and H3 subtypes, making it suitable for point-of-care IAV testing.

• 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.

• Journal of Life Science
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• v.23 no.9
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• pp.1133-1139
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• 2013

During the collection of boar semen, bacterial contamination usually occurs. The contamination has deleterious effects both on semen quality and on sow fertility. The majority of contaminants are gram-negative bacteria, especially Serratia marcescens. In this study, we developed a PCR assay for the identification of S. marcescens targeting the luxS gene (GenBank no. EF164926). S. marcescens yielded a specific 306 bp PCR product. However, no amplification was observed in the other strains tested. The detection limit of PCR was $50pg/{\mu}l$ of template DNA of S. marcescens. The antimicrobial susceptibility patterns of S. marcescens isolated from boar semen were tested using the disk diffusion method. Gentamicin, ceftiofur, florfenicol, and neomycin showed high sensitivity in this test. The minimum inhibitory concentration (MIC) was also determined by the broth microdilution method. The $MIC_{90}$ values of ceftiofur, enrofloxacin, gentamicin, and neomycin were 8, 8, 8, and $16{\mu}g/ml$, respectively. These results indicate that PCR amplification of the luxS gene is a reliable and effective method for the identification of S. marcescens and that ceftiofur, enrofloxacin, gentamicin, and neomycin are effective semen extenders for controlling S. marcescens.

• Parasites, Hosts and Diseases
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• v.50 no.3
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• pp.229-231
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• 2012

Toxoplasma gondii is a zoonotic parasite resulting in human infections and one of the infectious pathogens leading to uveitis and retinochoroiditis. The present study was performed to assess T. gondii infection in 20 ocular patients with chronic irregular recurrent uveitis (20 aqueous humor and 20 peripheral blood samples) using PCR. All samples were analyzed by nested PCR targeting a specific B1 gene of T. gondii. The PCR-positive rate was 25% (5/20), including 5% (1) in blood samples, 25% (5) in aqueous humor samples, and 5% (1) in both sample types. A molecular screening test for T. gondii infection in ocular patients with common clinical findings of an unclear retinal margin and an inflammatory membrane over the retina, as seen by fundus examination, may be helpful for early diagnosis and treatment.

• Food Science of Animal Resources
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• v.29 no.6
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• pp.668-672
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• 2009

This paper describes the differentiation between native Korean cattle (Hanwoo) and Holsteins or imported cattle using the real-time polymerase chain reaction (PCR) by targeting the sequence of the melanocortin 1 receptor (MC1R) gene. A rapid and accurate method was developed to identify Hanwoo by genotyping the DNA extracted from 295 commercial beef samples (obtained from 5 provinces in South Korea) labeled as Hanwoo beef. The results of real-time PCR assays for the proportions of Hanwoo were 84, 85.7, 95, 91.4, and 90% in the areas of Seoul, Joongbu, Youngnam, Honam, and Chungcheong, respectively. Thus, the beef samples from 295 butcher shops, which asserted to only sell Hanwoo, showed that 259 of 295 samples were of the Hanwoo beef gene type (T-type) and 36 of 295 samples were Holsteins of imported dairy cattle gene types (C-type or C/T type). In conclusion, the proportion of Hanwoo beef was 87.8% and the proportion of Holstein or imported dairy cattle meat was 12.2% (C-type: 9.8%, C/T-type: 2.4%). Generally, most consumers can not differentiate imported meat from Hanwoo beef. Therefore, Hanwoo beef and imported dairy cattle meat that is sold in butcher shops should have mandatory identification by using MC1R genotyping based on real-time PCR.

• Genomics & Informatics
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• v.21 no.1
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• pp.13.1-13.8
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• 2023

Importance of accurate molecular diagnosis and quantification of particular disease-related pathogenic microorganisms is highlighted as an introductory step to prevent and care for diseases. In this study, we designed a primer/probe set for quantitative real-time polymerase chain reaction (qRT-PCR) targeting rgpA gene, known as the specific virulence factor of periodontitis-related pathogenic bacteria 'Porphyromonas gingivalis', and evaluated its diagnostic efficiency by detecting and quantifying relative bacterial load of P. gingivalis within saliva samples collected from clinical subjects. As a result of qRT-PCR, we confirmed that relative bacterial load of P. gingivalis was detected and quantified within all samples of positive control and periodontitis groups. On the contrary, negative results were confirmed in both negative control and healthy groups. Additionally, as a result of comparison with next-generation sequencing (NGS)-based 16S metagenome profiling data, we confirmed relative bacterial load of P. gingivalis, which was not identified on bacterial classification table created through 16S microbiome analysis, in qRT-PCR results. It showed that an approach to quantifying specific microorganisms by applying qRT-PCR method could solve microbial misclassification issues at species level of an NGS-based 16S microbiome study. In this respect, we suggest that P. gingivalis-specific primer/probe set introduced in present study has efficient applicability in various oral healthcare industries, including periodontitis-related microbial molecular diagnosis field.

• Reproductive and Developmental Biology
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• v.38 no.2
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• pp.71-77
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• 2014

The specific genetic modification in porcine somatic cells by gene targeting has been very difficult because of low efficiency of homologous recombination. To improve gene targeting, we designed three kinds of knock-out vectors with ${\alpha}1,3$-galactosyltransferase gene (${\alpha}1,3$-GT gene), DT-A/pGT5'/neo/pGT3', DT-A/NLS/pGT5'/neo/pGT3' and pGT5'/neo/ pGT3'/NLS. The knock-out vectors consisted of a 4.8-kb fragment as the 5' recombination arm (pGT5') and a 1.9-kb fragment as the 3' recombination arm (pGT3'). We used the neomycin resistance gene (neo) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. These vectors have a neo gene insertion in exon 9 for inactivation of ${\alpha}1,3$-GT locus. DT-A/pGT5'/neo/pGT3' vector contain only positive-negative selection marker with conventional targeting vector. DT-A/NLS/pGT5'/neo/pGT3' vector contain positive-negative selection marker and NLS sequences in upstream of 5' recombination arm which enhances nuclear transport of foreign DNA into bovine somatic cells. pGT5'/neo/pGT3'/NLS vector contain only positive selection marker and NLS sequence in downstream of 3' recombination arm, not contain negative selectable marker. For transfection, linearzed vectors were introduced into porcine ear fibroblasts by electroporation. After 48 hours, the transfected cells were selected with $300{\mu}g/ml$ G418 during 12 day. The G418-resistant colonies were picked, of which 5 colonies were positive for ${\alpha}1,3$-GT gene disruption in 3' PCR and southern blot screening. Three knock-out somatic cells were obtained from DT-A/NLS/ pGT5'/neo/pGT3' knock-out vector. Thus, these data indicate that gene targeting vector using nuclear localization signal and negative selection marker improve targeting efficiency in porcine somatic cells.