• Korean Journal of Veterinary Service
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• v.45 no.1
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• pp.1-11
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• 2022

A novel porcine circovirus 4 (PCV4) was recently identified in Chinese and Korean pig herds. Although several conventional polymerase chain reaction (cPCR) and real-time PCR (qPCR) assays were used for PCV4 detection, more sensitive and reliable qPCR assay is needed that can simultaneously detect PCV4 and internal positive control (IPC) to avoid false-negative results. In the present study, a duplex qPCR (dqPCR) assay was developed using primers/probe sets targeting the PCV4 Cap gene and pig (glyceraldehyde-3-phosphate dehydrogenase) GAPDH gene as an IPC. The developed dqPCR assay was specifically detected PCV4 but not other PCVs and porcine pathogens, indicating that the newly designed primers/probe set is specific to the PCV4 Cap gene. Furthermore, GAPDH was stably amplified by the dqPCR in all tested viral and clinical samples containing pig cellular materials, indicating the high reliability of the dqPCR assay. The limit of detection of the assay 5 copies of the target PCV4 genes, but the sensitivity of the assay was higher than that of the previously described assays. The assay demonstrated high repeatability and reproducibility, with coefficients of intra-assay and inter-assay variation of less than 1.0%. Clinical evaluation using 102 diseased pig samples from 18 pig farms showed that PCV4 circulated in the Korean pig population. The detection rate of PCV4 obtained using the newly developed dqPCR was 26.5% (27/102), which was higher than that obtained using the previously described cPCR and TaqMan probe-based qPCR and similar to that obtained using the previously described SYBR Green-based qPCR. The dqPCR assay with IPC is highly specific, sensitive, and reliable for detecting PCV4 from clinical samples, and it will be useful for etiological diagnosis, epidemiological study, and control of the PCV4 infections.

• Korean Journal of Veterinary Service
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• v.34 no.4
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• pp.417-428
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• 2011

Species-specific PCR assay was developed for detection of cattle, sheep, goat, horse, dog, pig, chicken, duck, goose, and turkey using mitochondrial 12S rRNA and 16S rRNA as target genes. Also, an internal positive control was used to detect possible false negatives by using 18S rRNA gene. We designed species-specific primers with amplicon length of 190, 219, 350, 467, 241, 119, 171, 229, 111 and 268 bp for cattle, sheep, goat, horse, dog, pig, chicken, duck, goose, and turkey respectively. The specificity of the primers was tested against the other 10 non-target animal species and a cross-reaction was not observed. We developed two multiplex PCR assays for the simultaneous identification of Korea's major livestock species (cattle, pig, chicken and duck) and poultry species (chicken, duck, goose and turkey) from analogous samples, retaining the same specificity. The limit of detection of the multiplex PCR assay (cattle, pig, chicken and duck) ranged between 1 pg and 0.1 pg of template DNA extracts from raw meat. Applying multiplex PCR assays to DNA extracts from experimental pork/beef and pork/chicken tested raw and heat-treated ($120^{\circ}C$ for 30 min) mixtures respectively, detection limit was 0.1% level beef in pork, pork in beef and chicken in pork and 1.0% level pork in chicken. In conclusion, this assay using gel-based capillary electrophoresis would be very useful in highly sensitive and rapid identification of animal species or ingredients in minced meat and other meat products.

• Research in Plant Disease
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• v.17 no.3
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• pp.376-380
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• 2011

The bacterial speck of tomato caused by Pseudomonas syringae pv. tomato leads to serious economic losses especially on fruits of susceptible genotype. Thus, Pseudomonas syringae pv. tomato is a plant quarantine bacterium in many countries including Korea. In this study, we developed specific PCR assays for detection of the bacterium from tomato seeds. A specific primer set is designed from the hrpZ gene for specific detection of Pseudomonas syringae pv. tomato. A 501 bp PCR product corresponding to hrpZ gene was amplified only form Pseudomonas syringae pv. tomato strains, but no PCR product was amplified from other tomato bacterial pathogens, such as Pseudomonas syringae pv. glycinea, P. syringae pv. maculicola, P. syringae pv. atropurpurea, P. syringae pv. morsprunorum, and from other P. syringae pathovar strains. The nested-PCR primer set corresponding to an internal fragment of the 501 bp sequence (hrpZ) gine was used to specific detection of Pseudomonas syringae pv. tomato in tomato seed. A 119 bp PCR product using nested PCR primer was highly specific and sensitive to detect low level of Pseudomonas syrigae pv. tomato in tomato seeds. We believe that the PCR assays developed in this study is very useful to detect Pseudomonas syringae pv. tomato from the tomato seeds.

• Journal of Microbiology and Biotechnology
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• v.32 no.10
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• pp.1344-1354
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• 2022

Laryngeal cancer is one of the highest incidence, most prevalently diagnosed head and neck cancers, making it critically necessary to probe effective targets for laryngeal cancer treatment. Here, real-time quantitative reverse transcription PCR (qRT-PCR) and western blot analysis were used to detect gene expression levels in laryngeal cancer cell lines. Fluorescence in situ hybridization (FISH) and subcellular fractionation assays were used to detect the subcellular location. Functional assays encompassing Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), transwell and wound healing assays were performed to examine the effects of target genes on cell proliferation and migration in laryngeal cancer. The in vivo effects were proved by animal experiments. RNA-binding protein immunoprecipitation (RIP), RNA pulldown and luciferase reporter assays were used to investigate the underlying regulatory mechanisms. The results showed that KIF26B antisense RNA 1 (KIF26B-AS1) propels cell proliferation and migration in laryngeal cancer and regulates the toll-like receptor 4 (TLR4) signaling pathway. KIF26B-AS1 also recruits FUS to stabilize TLR4 mRNA, consequently activating the TLR4 signaling pathway. Furthermore, KIF26B-AS1 plays an oncogenic role in laryngeal cancer via upregulating TLR4 expression as well as the FUS/TLR4 pathway axis, findings which offer novel insight for targeted therapies in the treatment of laryngeal cancer patients.

• Journal of Dairy Science and Biotechnology
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• v.31 no.2
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• pp.127-131
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• 2013

The aim of this study was to investigate adulteration of caprine milk products by bovine milk using biomolecular techniques with bovine-specific primers for the mitochondrial cytochrome b gene. Polymerase chain reaction (PCR) and real-time PCR assays were applied to caprine milk products including infant formula, city milk, and fermented milk. The results indicated that six out of the eight caprine infant formula products tested contained bovine milk components. In addition, two of the three tested caprine city milk products and two caprine fermented milk products were shown to be adulterated with bovine milk. Conventional PCR results corroborated with results obtained by real-time PCR. This study demonstrates that DNA-based species identification procedures would be useful and applicable in routine examinations of the dairy industry to ensure the quality and safety of dairy foods.

• Research in Plant Disease
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• v.16 no.2
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• pp.129-135
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• 2010

PCR primers were developed to detect Pseudomonas savastanoi pv. phaseolicola, a causal agent of halo blight that occurs in all species of common bean (Phaseolus vulgaris L.), from the bean seeds. A primer set, Psp-JHF and Psp-JH-R, specifically amplified 513 bp fragment from Pseudomonas savastanoi pv. phaseolicola only. A nested primer set, psp-JH-F-ne and psp-JH-R-ne, designed from the $1^{st}$ PCR amplicon, amplified 169 bp fragment. The primer sets did not amplify any non-specific DNA from the seed extracts of Fabaceae including 4 beans, 2 soybeans, and 2 peas. The detection sensitivity of the nested PCR method developed in this study was much higher than that of ELISA and selective medium. PCR assays developed in this study should be useful to detect Pseudomonas savastanoi pv. phasolicola from the bean seeds.

• 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.

• Food Science of Animal Resources
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• v.34 no.5
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• pp.717-723
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• 2014

A rapid and specific PCR assay for the simultaneous detection of Listeria monocytogenes, Escherichia coli O157:H7, Bacillus cereus, Salmonella spp., and Staphylococcus aureus in foods was developed to reduce the detection time and to increase sensitivity. Multiplex PCR developed in this study produced only actA, fliC, hbl, invA, ileS amplicons, but did not produce any non-specific amplicon. The primer sets successfully amplified the target genes in the multiplex PCR without any non-specific or additional bands on the other strains. The multiplex PCR assays also amplified some target genes from five pathogens, and multiplex amplification was obtained from as little as 1 pg of DNA. According to the results from the sensitivity evaluation, the multiplex PCR developed in this study detected 10 cells/mL of the pathogens inoculated in milk samples, respectively. The results suggested that multiplex PCR was an effective assay demonstrating high specificity for the simultaneous detection of five target pathogens in food system.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.599-606
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• 2004

Gram-positive antagonistic bacilli were isolated from agricultural soils for possible use in biocontrol of plant pathogenic fungi, Fusarium oxysporum, Rhizoctonia solani, and/or Pythium ultimum. Among the 65 antagonistic Gram-positive soil isolates, 22 strains were identified as Bacillus species by 16S rDNA sequence analyses. Four strains, including DF14, especially exhibited multiple antagonistic properties against the three damping-off fungi. Genotypic properties of the Bacillus isolates were characterized by rapid molecular fingerprinting methods using repetitive extragenic palindromic-PCR (REP-PCR), ribosomal intergenic spacer-length polymorphisms (RIS-LP), 16S rDNA PCR-restriction fragment length polymorphisms (PCR-RFLP), and strain-specific PCR assays. The results indicated that the REP-PCR method was more valuable than the RIS-LP and 16S rDNA PCR-RFLP analyses as a rapid and reliable approach for bacilli typing and identification. The use of strain-specific primers designed based on 16S rDNA sequence comparisons enabled it to be possible to selectively detect a strain, DF14, which is being used as a biocontrol agent against damping-off fungi.

• Pediatric Infection and Vaccine
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• v.22 no.1
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• pp.1-6
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• 2015

Purpose: This study investigated an outbreak of mumps affecting students in a high school (S high school) in Seoul, with an evaluation of the diagnostic utility of the mumps polymerase chain reaction (PCR) assay. Methods: S high school students that presented to health care providers with mumps symptoms between April 2013 and July 2013 were surveyed for the monthly distribution of symptom onset and their grade level. Mumps PCR assays were performed using buccal swabs from some of these students. Results: During the survey period, 77 students presented with suspected cases of mumps. The monthly distribution of symptom onset was as follows: one in April, 17 in May, 54 in June, and five in July. With regard to grade level, 26 students were in their first year, 28 were in their second year, and 23 were in their third year. Of the 18 students tested with PCR assays, five had positive results. Samples were collected within 3 days of symptom onset in 15 of the 18 students, and positive PCR results were obtained in five of these 15 students. The PCR results of the remaining three students from whom samples were collected more than 3 days after the onset of symptoms were negative (P=0.24). Conclusions: We evaluated the epidemiological aspects of an outbreak of mumps in a high school. Mumps PCR might be epidemiologically useful if performed within 3 days of the onset of symptoms in suspected cases.