• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.284-289
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• 2004

A PCR assay for the rapid detection of Vibrio vulnificus strains was developed using a virulence gene for elastase found in various Vibrio species. The DNA sequences in the elastase gene facilitated the identification of a species-specific probe for pathogenic V. vulnificus strains from both clinical and environmental sources. Using an elastase gene-based PCR reaction, a species-specific 507-bp PCR product was visualized by agarose gel electrophoresis. Three different DNA extraction methods were then compared to improve the simplicity and rapidity of detection. A PCR assay using the conventional DNA extraction or boiling method was able to detect as few as 25 V. vulnificus cells, making the detection limits at least 1-log-scale lower than that for the EDT A-treated DNA extraction method. In particular, the boiling method, which does not require purification of the chromosomal DNA, was very effective in terms of simple and rapid detection. Meanwhile, the detection limit in a mixed bacterial culture that included other bacteria, such as Escherichia coli or Bacillus subtilis, was two V. vulnificus cells, which was 1-log-scale lower than that for the control. Accordingly, when coupled with a new DNA extraction method, the elastase gene-based PCR can provide a rapid, specific, and sensitive method for identifying V. vulnificus in clinical and environmental samples.

• Toxicological Research
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• v.32 no.1
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• pp.81-87
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• 2016

Aflatoxin B1 (AFB1) is produced by Aspergillus flavus growing in feedstuffs. Early detection of maize contamination by aflatoxigenic fungi is advantageous since aflatoxins exert adverse health effects. In this study, we report the development of an optimized conventional PCR for AFB1 detection and a rapid, sensitive and simple screening Real-time PCR (qPCR) with SYBR Green and two pairs of primers targeting the aflR genes which involved aflatoxin biosynthesis. AFB1 contaminated maize samples were divided into three groups by the toxin concentration. Genomic DNA was extracted from those samples. The target genes for A. flavus were tested by conventional PCR and the PCR products were analyzed by electrophoresis. A conventional PCR was carried out as nested PCR to verify the gene amplicon sizes. PCR-RFLP patterns, obtained with Hinc II and Pvu II enzyme analysis showed the differences to distinguish aflatoxin-producing fungi. However, they are not quantitative and need a separation of the products on gel and their visualization under UV light. On the other hand, qPCR facilitates the monitoring of the reaction as it progresses. It does not require post-PCR handling, which reduces the risk of cross-contamination and handling errors. It results in a much faster throughout. We found that the optimal primer annealing temperature was $65^{\circ}C$. The optimized template and primer concentration were $1.5{\mu}L\;(50ng/{\mu}L)$ and $3{\mu}L\;(10{\mu}M/{\mu}L)$ respectively. SYBR Green qPCR of four genes demonstrated amplification curves and melting peaks for tub1, afIM, afIR, and afID genes are at $88.0^{\circ}C$, $87.5^{\circ}C$, $83.5^{\circ}C$, and $89.5^{\circ}C$ respectively. Consequently, it was found that the four primers had elevated annealing temperatures, nevertheless it is desirable since it enhances the DNA binding specificity of the dye. New qPCR protocol could be employed for the determination of aflatoxin content in feedstuff samples.

• Journal of Microbiology and Biotechnology
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• v.34 no.5
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• pp.1146-1153
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• 2024

The increasing economic losses associated with growth retardation caused by Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp, require effective monitoring. The internal transcribed spacer (ITS)-1 region, the non-coding region of ribosomal clusters between 18S and 5.8S rRNA genes, is widely used in phylogenetic studies due to its high variability. In this study, the ITS-1 region sequence (~600-bp) of EHP was first identified, and primers for a polymerase chain reaction (PCR) assay targeting that sequence were designed. A newly developed nested-PCR method successfully detected the EHP in various shrimp (Penaeus vannamei and P. monodon) and related samples, including water and feces collected from Indonesia, Thailand, South Korea, India, and Malaysia. The primers did not cross-react with other hosts and pathogens, and this PCR assay is more sensitive than existing PCR detection methods targeting the small subunit ribosomal RNA (SSU rRNA) and spore wall protein (SWP) genes. Phylogenetic analysis based on the ITS-1 sequences indicated that the Indonesian strain was distinct (86.2% nucleotide sequence identity) from other strains collected from Thailand and South Korea, and also showed the internal diversity among Thailand (N = 7, divided into four branches) and South Korean (N = 5, divided into two branches) samples. The results revealed the ability of the ITS-1 region to determine the genetic diversity of EHP from different geographical origins.

• 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.

• Parasites, Hosts and Diseases
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• v.51 no.6
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• pp.651-656
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• 2013

Human schistosomiasis caused by Schistosoma japonicum and Schistosoma mekongi is a chronic and debilitating helminthic disease still prevalent in several countries of Asia. Due to morphological similarities of cercariae and eggs of these 2 species, microscopic differentiation is difficult. High resolution melting (HRM) real-time PCR is developed as an alternative tool for the detection and differentiation of these 2 species. A primer pair was designed for targeting the 18S ribosomal RNA gene to generate PCR products of 156 base pairs for both species. The melting points of S. japonicum and S. mekongi PCR products were $84.5{\pm}0.07^{\circ}C$ and $85.7{\pm}0.07^{\circ}C$, respectively. The method permits amplification from a single cercaria or an egg. The HRM real-time PCR is a rapid and simple tool for differentiation of S. japonicum and S. mekongi in the intermediate and final hosts.

• Parasites, Hosts and Diseases
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• v.54 no.4
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• pp.393-397
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• 2016

Low parasitemic condition in malaria remains a diagnostic challenge; as the available diagnostic methods failed to detect. Currently, hemozoin (Hz) pigment is gaining attention in the diagnosis of malaria. The major drawback is ease of detection of Hz in routine practice. A pilot study was conducted to evaluate the role of Hz pigment and to compare the performance of quantitative buffy coat assay (QBC) and PCR in such conditions. Clinically suspected cases of malaria were examined by both Giemsa stain and immunochromatographic test (ICT). Samples positive by ICT and negative by Giemsa stain were further examined by nested PCR targeting 18S rRNA and QBC for the presence of malaria parasites and pigments. Thirty blood samples fulfilled the inclusion criteria out of which 23 were Plasmodium vivax (Pv), 4 Plasmodium falciparum (Pf), and 3 mixed (Pv and Pf) by immunochromatographic test. Twenty-one out of 30 (70%) were positive by nested PCR in comparison to 25/30 (83%) by QBC. Samples containing both malaria parasites and Hz pigment by QBC completely showed concordance with the PCR result. However, 61% of total samples containing only Hz pigment were observed positive by PCR. Hz pigment remains an important tool for malaria diagnosis. Identification of leukocytes containing pigments by QBC not only indicates recent malarial infections but also puts light on severity of the disease. QBC assay is a rapid, highly sensitive, and cost-effective method to detect malaria parasites and Hz pigment especially in low parasitemic conditions.

• Journal of Life Science
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• v.22 no.1
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• pp.110-116
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• 2012

Nuruk plays a significant role in the flavor and quality of Takju and Yakju, which are produced through saccharification and alcohol fermentation by various microorganisms. In this study, we identified microbial strains isolated from a plate count and PCR-denaturing gradient gel electrophoresis (DGGE) analysis targeting the 16S and 28S rRNA genes, in order to characterize bacterial and fungal diversity in Sansung Nuruk. The numbers of bacteria and fungi in Nuruk were $1.5{\times}10^9$ CFU/g and $2.2{\tims}10^8$ CFU/g, respectively. The 16S rRNA gene sequence indicated that the predominant bacteria in the isolates and PCR-DGGE profile of Nuruk were Kocuria spp., Pantoea spp., Lactobacillus spp., Pediococcus spp., Weissella spp., Staphylococcus spp., endophytic bacterium, uncultured Gamma-proteobacteria, uncultured Cyanobacteria, and Actinobacteria. Dominant bacteria from the PCR-DGGE profile were Pediococcous pentosaceus and uncultured Cyanobacteria. The 28S rRNA gene sequence indicated the predominant fungi in the isolates and PCR-DGGE profile to be Trichomonascus spp. Pichia spp., Torulaspora spp., Wickerhamomyces spp., Sacharomycopsis spp., Lichtheimia spp., Mucor spp., Rhizopus spp. Aspergillus spp., and Cladosporium spp. Dominant fungi from the PCR-DGGE profile were Pichia kudriavzevii and Aspergillus oryzae. The PCR-DGGE technique was used for the first time in this study to assess a microbial community in Nuruk and proved to be an effective protocol for profiling microbial diversity.

• Molecules and Cells
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• v.40 no.3
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• pp.195-201
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• 2017

In this study, qRT-PCR was employed to identify that miR-186 expression level in NSCLC tissues are highly associated with lymph node metastasis. In addition, through the application of western blotting, luciferase assay and qRT-PCR, it was found that miR-186 targeted 3'UTR of cdc42 mRNA and down-regulated cdc42 protein level in a post-transcriptional manner. Transwell assay indicated that cdc42 partially reversed the effect of miR-186 mimics. Besides, miR-186 was proved to regulate EMT by influencing biomarkers of this process and cell adhesion ability. Thus, miR-186 is a potential target for NSCLC therapy. miR-186 is proposed to be one of tumor-suppressors and may serve as a therapeutic target in NSCLC treatment.

• The Journal of Korean Society of Virology
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• v.27 no.1
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• pp.19-27
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• 1997

To develop the polymerase chain reaction (PCR) for the detection of type D simian retrovirus (SRV) infection, an oligonucleotide primer pair was designed to hybridize to the sequences within env gene of SRV subtype 1 (SRV-1). The 3' proximal env sequences annealing to the primers had been rather conserved among three different subtypes of SRV, SRV-1, SRV-2, and SRV-3 (Mason-Pfizer Monkey Virus: MPMV). The PCR using the primer pair targeting an env region successfully detected and amplified all three subtypes of SRV with excellent specificity after single round of reaction. The tests with peripheral blood mononuclear cells infected either with simian immunodeficiency virus or simian T-Iymphotropic virus type 1, major immunosuppressive viral agents together with SRV in simian, verified the specificity of the PCR by excluding any cross reactivity. Semiquantitative titration PCR, amplifying serially diluted plasmid DNA of each subtype, was performed to evaluate sensitivity limits of the reaction. Based on molecular weight of each cloned SRV genome, the PCR should be able to detect one SRV-infected cell per more than $5-7{\times}10^4$ uninfected cells after simple ethidium bromide staining of resulting products. The PCR must be very efficient screening system with its quickness, certainty, and sensitivity for SRV-infected animals used in human AIDS research model. Second round amplification of the reaction products from the first PCR, or Southern hybridization by radiolabeled probes shall render to compete its efficacy to ELISA which has been the most sensitive technique to screen SRV infection but with frequent ambiguity problem.

• Korean Journal of Veterinary Research
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• v.44 no.4
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• pp.655-662
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• 2004

The aim of this work was the validation of a rapid real-time PCR assay based on TaqMan technology for the unequivocal identification of infectious canine hepatitis (ICH) virus, to be used directly on DNA purified from blood specimens. A real-time PCR system targeting at the E3 ORFA gene sequence of canine adenovirus type 1 was optimized and validated through comparative analysis of samples using conventional PCR system. The real-time PCR assay based on TaqMan technology could disclose 23 (37.7%) out of 61 samples as PCR positive. In contrast, 18 (29.5%) samples were found PCR positive when conventional PCR was applied on these samples. The use of the ABI Prism 7700 sequence detection system allowed the efficient determination of the amplified product accumulation through a fluorogenic probe. The entire real-time TaqMan PCR assay, including DNA extraction, amplification, and detection could be completed within 3 hours. The detection method of real-time TaqMan PCR assay was 1,000 times more sensitive than conventional PCR. Real-time TaqMan probe and primer set developed and optimized in this study is a sensitive, rapid and accurate method for detection of ICH virus and can be effective screening tool for the detection of ICH in a diagnostic laboratory routines.