• Journal of Microbiology and Biotechnology
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• v.26 no.12
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• pp.2199-2205
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• 2016

Vibrio cholerae O1 serogroup Wave 3 El Tor strains are presently prevalent worldwide. The Wave 3 El Tor strains contain a TLC:RS1:CTX array on chromosome 1, and no element is integrated on chromosome 2. A multiplex PCR optimized to identify the TLC:RS1:CTX array of Wave 3 strains has been developed in this study. By using eight primers, the multiplex PCR can identify the characteristic CTX and RS1 array of Wave 3 strains from various arrays of strains belonging to other Waves. The four amplified DNA fragments of Wave 3 strains have been cloned in a vector, which could be used as a positive control for the multiplex PCR. This multiplex PCR and the positive control set could be useful tools for rapid recognition of Wave 3 El Tor strains.

• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.645-650
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• 2019

Despite differences in virulence between strains of Toxoplasma gondii, rapid and accurate genotyping methods are lacking. In this study, a method was developed to detect and genotype T. gondii in food and environmental samples using PCR and a novel peptide nucleic acid (PNA) melting array. An alignment of genome sequences for T. gondii type I, II, and III obtained from NCBI was generated, and a single nucleotide polymorphism analysis was performed to identify targets for PCR amplification and a PNA melting array. Prior to the PNA melting array, conventional PCR was used to amplify GRA6 of T. gondii. After amplification, the PNA melting array was performed using two different PNA hybridization probes with fluorescent labels (FAM and HEX) and quenchers. Melting curves for each probe were used to determine genotypes and identify mutations. A 214-bp region of the GRA6 gene of T. gondii was successfully amplified by PCR. For all T. gondii strains (type I, II, and III) used to evaluate specificity, the correct genotypes were determined by the PNA melting array. Non-T. gondii strains, including 14 foodborne pathogens and 3 protozoan parasites, such as Giardia lamblia, Cryptosporidium parvum, and Entamoeba histolytica, showed no signal, suggesting that the assay has a high specificity. Although this is only a proof-of-concept study, the assay is promising for the fast and reliable genotyping of T. gondii from food and environmental samples.

• Genomics & Informatics
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• v.19 no.3
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• pp.34.1-34.6
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• 2021

Digital PCR (dPCR) is the third-generation PCR that enables real-time absolute quantification without reference materials. Recently, global diagnosis companies have developed new dPCR equipment. In line with the development, the Lab On An Array (LOAA) dPCR analyzer (Optolane) was launched last year. The LOAA dPCR is a semiconductor chip-based separation PCR type equipment. The LOAA dPCR includes Micro Electro Mechanical System that can be injected by partitioning the target gene into 56 to 20,000 wells. The amount of target gene per wells is digitized to 0 or 1 as the number of well gradually increases to 20,000 wells because its principle follows Poisson distribution, which allows the LOAA dPCR to perform precise absolute quantification. LOAA determined region of interest first prior to dPCR operation. To exclude invalid wells for the quantification, the LOAA dPCR has applied various filtering methods using brightness, slope, baseline, and noise filters. As the coronavirus disease 2019 has now spread around the world, needs for diagnostic equipment of point of care testing (POCT) are increasing. The LOAA dPCR is expected to be suitable for POCT diagnosis due to its compact size and high accuracy. Here, we describe the quantitative principle of the LOAA dPCR and suggest that it can be applied to various fields.

• Journal of Microbiology and Biotechnology
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• v.15 no.2
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• pp.310-320
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• 2005

A membrane-based oligonucleotide array was used to detect predominant bacterial species in human fecal samples. Digoxygenin-labeled 16S rDNA probes were generated by PCR from DNA that had been extracted from fecal samples or slurries. These probes were hybridized to an array of 120 oligonucleotides with sequences specific for 40 different bacterial species commonly found in human feces, followed by color development using an alkaline phosphatase-conjugated antibody and NBT /BCIP. Twenty of the species were detected by this method, but E. coli, which was present at $\~$1 $\times 10$^5$ CFU per gram feces, was not detected. To improve the sensitivity of this assay, reverse transcriptase-PCR was used to generate probes from RNA extracted from fecal cultures. Coupled with a chemiluminescence detection method, this approach lowered the detection limit for E. coli from $\~1$ $\times 10$^6$ to ${\leq}$ 1 $\times 10$^5$ These results indicate that the membrane-array method with reverse transcriptase-PCR and chemiluminescence detection can simultaneously identify bacterial species present in fecal samples at cell concentrations as low as${\leq}$ 1 $\times 10$^5$ CFU per gram.

• Molecular & Cellular Toxicology
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• v.4 no.3
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• pp.246-252
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• 2008

Tumor tissue is usually contaminated by normal tissue components, which reduces the sensitivity of analysis for exploring genetic alterations. Although microdissection has been adopted to minimize the contamination of tumor DNA with normal cell components, there is a concern over the amount of microdissected DNA not enough to be applied to array-CGH reaction. To amplify the extracted DNA, several whole genome amplification (WGA) methods have been developed, but objective comparison of the array-CGH outputs using different types of WGA methods is still scarce. In this study, we compared the performance of non-amplified microdissected DNA and DNA amplified in 2 WGA methods such as degenerative oligonucleotide primed (DOP)-PCR, and multiple strand displacement amplification (MDA) using Phi 29 DNA polymerase. Genomic DNA was also used to make a comparison. We applied those 4 DNAs to whole genome BAC array to compare the false positive detection rate (FPDR) and sensitivity in detecting copy number alterations under the same hybridization condition. As a result microdissected DNA method showed the lowest FPDR and the highest sensitivity. Among WGA methods, DOP-PCR amplified DNA showed better sensitivity but similar FPDR to MDA-amplified method. These results demonstrate the advantage and applicability of microdissection for array-CGH analysis, and provide useful information for choosing amplification methods to study copy number alterations, especially based on precancerous and microscopically invaded lesions.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.265-270
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• 2009

An oligonucleotide array was developed to detect and genotype mollicutes based on the internal transcribed spacer (ITS) sequence. The results of the assay were compared with those of a PCR-RFLP assay. The proposed oligonucleotide array containing 5 genus- and 23 species-specific probes was able to detect Mycoplasma species, including M. penetrans and M. spermatophilum, that were not detected by the PCR-RFLP assay. Therefore, the results demonstrated that the proposed oligonucleotide array was effective for the detection and discrimination of 23 species, including an acholeplasma, 21 mycoplasmas, and a ureaplasma, and showed promise as a countermeasure to ensure that biological products are safe and of good quality.

• Journal of Chest Surgery
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• v.44 no.2
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• pp.123-130
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• 2011

Background: The aim of the present study was to identify chromosomal loci that contribute to the pathogenesis of aortic dissection (AD) in a Korean population using array comparative genomic hybridization (CGH) and to confirm the results using real-time polymerase chain reaction (PCR). Materials and Methods: Eighteen patients with ADs were enrolled in this study. Genomic DNA was extracted from individual blood samples, and array CGH analyses were performed. Four corresponding genes with obvious genomic changes were analyzed using real-time PCR in order to assess the level of genomic imbalance identified by array CGH. Results: Genomic gains were most frequently detected at 8q24.3 (56%), followed by regions 7q35, 11q12.2, and 15q25.2 (50%). Genomic losses were most frequently observed at 4q35.2 (56%). Real-time PCR confirmed the results of the array CGH studies of the COL6A2, DGCR14, PCSK6, and SDHA genes. Conclusion: This is the first study to identify candidate regions by array CGH in patients with ADs. The identification of genes that may predispose an individual to AD may lead to a better understanding of the mechanism of AD formation. Further multicenter studies comparing cohorts of patients of different ethnicities are warranted.

• Korean Journal of Microbiology
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• v.44 no.4
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• pp.346-351
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• 2008

This study develops DNA array which can detect specific sequence of human papilomavirus (HPV) by using lateral flow membrane assay which is usually used for point of care test including pregnant diagnosis. Principle of HPV DNA array is as follow; fixing DNA probe which is peculiar to HPV type 6, 11, 16, 18, 31, 45 on a surface of lateral flow membrane and inducing hybridization response between probe and HPV PCR products which is obtained by using biotin-labeled MY09/l1 primers. And then, we can see the result of DNA hybridization that streptavidin labelled colloidal gold is responded with hybrid biotin. Lateral flow membrane array developed in this study confirms major HPV type economically and conveniently compared with existing HPV DNA chip method.

• The Plant Pathology Journal
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• v.40 no.1
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• pp.40-47
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• 2024

Garlic can be infected by a variety of viruses, but mixed infections with leek yellow stripe virus, onion yellow dwarf virus, and allexiviruses are the most damaging, so an easy, inexpensive on-site method to simultaneously detect at least these three viruses with a certain degree of accuracy is needed to produce virus-free plants. The most common laboratory method for diagnosis is multiplex reverse transcription polymerase chain reaction (RT-PCR). However, allexiviruses are highly diverse even within the same species, making it difficult to design universal PCR primers for all garlic-growing regions in the world. To solve this problem, we developed an inexpensive on-site detection system for the three garlic viruses that uses a commercial mobile PCR device and a compact electrophoresis system with a blue light. In this system, virus-specific bands generated by electrophoresis can be identified by eye in real time because the PCR products are labeled with a fluorescent dye, FITC. Because the electrophoresis step might eventually be replaced with a lateral flow assay (LFA), we also demonstrated that a uniplex LFA can be used for virus detection; however, multiplexing and a significant cost reduction are needed before it can be used for on-site detection.

• Genomics & Informatics
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• v.21 no.2
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• pp.24.1-24.7
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• 2023

Assays of clinical diagnosis and species identification using molecular markers are performed according to a quantitative method in consideration of sensitivity, cost, speed, convenience, and specificity. However, typical polymerase chain reaction (PCR) assay is difficult to quantify and have various limitations. In addition, to perform quantitative analysis with the quantitative real-time PCR (qRT-PCR) equipment, a standard curve or normalization using reference genes is essential. Within the last a decade, previous studies have reported that the digital PCR (dPCR) assay, a third-generation PCR, can be applied in various fields by overcoming the shortcomings of typical PCR and qRT-PCR assays. We selected Stilla Naica System (Stilla Technologies), Droplet Digital PCR Technology (Bio-Rad), and Lab on an Array Digital Real-Time PCR analyzer system (OPTOLANE) for comparative analysis among the various droplet digital PCR platforms currently in use commercially. Our previous study discovered a molecular marker that can distinguish Hanwoo species (Korean native cattle) using Hanwoo-specific genomic structural variation. Here, we report the pros and cons of the operation of each dPCR platform from various perspectives using this species identification marker. In conclusion, we hope that this study will help researchers to select suitable dPCR platforms according to their purpose and resources.