• Journal of Microbiology and Biotechnology
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• v.31 no.3
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• pp.358-367
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• 2021

The World Health Organization (WHO) has declared the coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, which is the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (CT) of the viral RNA by RT-PCR significantly varied according to the sequences of the primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of the viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be equal to or more than that of the RT-qPCR. However, the ddPCR assay is more suitable for determining the copy number of reference materials. These findings suggest that the qPCR assay with the ddPCR defined reference materials could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.

• Journal of Ginseng Research
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• v.33 no.1
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• pp.55-58
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• 2009

Generally, the direct sequencing through PCR is faster, easier, cheaper, and more practical than clone sequencing. Frequently, standard PCR amplification is usually interpreted by mispriming internal or external regions of the target template. Normally, DNA fragments were eluted from the gel using Gel extraction kit and subjected to direct sequencing or cloning sequencing. Cloning sequencing has often troublesome and needs more time to analyze for many samples. Since touchdown (TD) PCR can generate sufficient and highly specific amplification, it reduces unwanted amplicon generation. Accordingly, TD PCR is a good method for direct sequencing due to amplifying wanted fragment. In plants the 5S-rRNA gene is separated by simple spacers. The 5S-rRNA gene sequence is very well-conserved between plant species while the spacer is species-specific. Therefore, the sequence has been used for phylogenetic studies and species identification. But frequent occurrences of spurious bands caused by complex genomes are encountered in the product spectrum of standard PCR amplification. In conclusion, the TD PCR method can be applied easily to amplify main 5S-rRNA and direct sequencing of panax ginseng cultivars.

• Tuberculosis and Respiratory Diseases
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• v.40 no.5
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• pp.509-518
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• 1993

Background: By amplifying small amount of DNA, polymerase chain reaction (PCR) can be used for the detection of very small amount of microbial agent, and may be especially useful in certain cases which are difficult to be diagnosed microbiologically or serologically. Tuberculous pleurisy is a disease that can be diagnosed in only 70% of cases by conventional diagnostic tools, and PCR would be a very rapid, easy, and sensitive diagnostic method. Method: The specificity and sensitivity of PCR to detect Mycobacterium tuberculosis DNA were evaluated using various strains of Mycobacteria. To evaluate the diagnostic usefulness of PCR in tuberculous pleurisy, we used PCR to detect Mycobacterium tuberculosis DNA in pleural fluid. The amplification target was 123 base pair DNA, a part of IS6110 fragment, 10~16 copies of which are known to exist per genome. The diagnostic yield of PCR was compared with conventional methods, including pleural fluid adenosine deaminase (ADA) activity. Also, the significance of PCR in undiagnosed pleural effusion was evaluated prospectively with antituberculosis treatment. Results: 1) Using cultured Mycobacterium tuberculosis and other strains, PCR could detect upto 1 fg DNA and specific for only Mycobacterium tuberculosis and Mycobacterium bovis. 2) Using pleural effusions of proven tuberculosis cases, the sensitivity of PCR was 80.0% (16/20), and the specificity 95.0% (19/20). 3) Among 13 undiagnosed, but suspected tuberculous effusion, the positive rate was 60% in 10 improved cases after antituberculosis medications, and 0% in 3 cases of proven malignancy later. 4) Adenosine deaminase level of proven and clinically diagnosed tuberculous pleurisy patients was significantly higher than that of excluded patients, and correlated well with PCR results. Conclusion: We can conclude that PCR detection of Mycobacterium tuberculosis in pleural effusion has acceptable sensitivity and specificity, and could be an additional diagnostic tool for the diagnosis of tuberculous pleurisy.

• Korean Journal of Clinical Laboratory Science
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• v.47 no.2
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• pp.83-89
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• 2015

Although Mycobacterium tuberculosis complex strains remain responsible for the majority of diseases caused by mycobacterial infections worldwide, the increase in HIV (human immuno deficiency virus) infections has allowed for the emergence of other non-tuberculous mycobacteria as clinically significant pathogens. M. tuberculosis was detected by two-tube nested polymerase chain reaction (PCR) and non-tuberculous mycobacteria was detected by PCR-restriction fragment length polymorphism (RFLP) with Msp I. Result of niacin test is equal to result of two-tube nested PCR after culture for M. tuberculosis. In this study, acid fast bacilli stain (AFB. stain) >2+ case, Detection of Mycobacteria is similar to result before culture and after culture. AFB. stain <1+ case, result of mycobacteria is distinguished. Conclusionly, these results suggest that identification of mycobacteria must go side by side both culture and PCR for more fast and accuracy.

• 유체기계공업학회:학술대회논문집
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• 2003.12a
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• pp.115-120
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• 2003

In this work, a continuous-flow micro-PCR system is systematically designed. From the numerical simulation based on the finite volume method, adapting oneself to a new environmental temperature without an external temperature controller is shown to be possible and a cooler as well as a heater is shown to be necessary to obtain three individual temperature zones for polymerase chain reaction. In addition, appropriate geometry of a heat sink for the cooler is determined by using a compact modeling method, the porous medium approach.

• Korean Journal of Environmental Biology
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• v.22 no.2
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• pp.329-335
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• 2004

To develop a biomarker for the monitoring of the contamination of estrogenic endocrine disrupters in the aquatic environment, reverse transcription -polymerase chain reaction (RT-PCR) analysis of vitellogenin (Vg) mRNA expression was optimized in Bombina orientalis, a Korean red bellied toad species. Based on partial cDNA sequences of both Vg and beta actin genes of B. orientalis, specific primers for RT-PCR of Vg and beta actin mRNAs were developed. Semiquantitative RT-PCR of the Vg mRNA in liver was optimized using a beta actin mRNA as an internal control in both sexes. In female RT-PCR using $1\;\mu{g}$ of the liver cDNA resulted in a linear increment in the PCR product of Vg from 18 to 34 cycles of amplification. In male, on the contrary, the RT- PCR product was first detected at 30 cycles of amplification and a linear increment was observed from 30 to 40 cycles of amplification, suggesting that male B. orientalis expresses minute amount of Vg mRNA which is a $2^{-12}$ equivalent of female. In conclusion, the optimized protocol for semiquantitative RT-PCR analysis of Vg mRNA level in B. orientalis male liver will be useful for the environmental monitoring the xenoestrogen contamination in the freshwater environment in Korea.

• BMB Reports
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• v.37 no.3
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• pp.356-361
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• 2004

To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.

• Journal of Dairy Science and Biotechnology
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• v.33 no.4
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• pp.253-262
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• 2015

The authentication and implications of misleading labeling in milk and dairy products is important to protect against cheating consumers from adulteration and to alert sensitive consumers to any undeclared potential allergens. This need to support milk and dairy products labeling has led to the development of specific analytical techniques for the analysis of milk and dairy products ingredients. Recently, several methods based on polymerase chain reaction (PCR), including restriction fragment length polymorphism (PCR-RFLP), multiplex PCR, species-specific PCR, and real-time PCR, have been proposed as useful means for identifying species of origin in milk and dairy products, as well as quantifying and detecting any adulteration. These methods have particular advantages owing to their high specificity and sensitivity, as well as rapid processing time. In this review, we provide an updated and extensive overview of the PCR-based methods used for milk and dairy products authentication with a particular focus on the application of PCR methods to detect adulteration.

• Korean Journal of Clinical Laboratory Science
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• v.36 no.1
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• pp.1-6
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• 2004

The enterobacterial repetitive intergenic consensus (ERIC)-PCR is a recently described DNA fingerprinting technique based on amplification of repetitive element distributed in bacteria. We applied of ERIC-PCR to clinical isolates of Vibrio parahaemolyticus and other bacteria associated diarrhea. Twenty isolates of V. parahaemolyticus were used for intragenic genotyping, which were isolated from 2001 to 2002 in Chungbuk National University hospital. For interspecies genotyping, V. vulnificus, V. alginolyticus, V. parahaemolyticus, Escherichia coli, Salmonella and Shigella spp. were used. The genotyping were analyzed by ERIC-PCR. The genotyping of V. parahaemolyticus were grouped two major pattern (A, B) and were subdivided into 10 subtypes (A1, A2, B1-B8) by ERIC-PCR. These method distinctly differentiated bacterial species associated diarrhea. Those results show that ERIC-PCR can be reliable and efficient method for genotyping of V. parahaemolyticus and bacteria associated diarrhea.

• Journal of Integrative Natural Science
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• v.1 no.3
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• pp.230-233
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• 2008

A simple and rapid method is described for extending the 5'- or 3'-end genomic sequence of a known partial sequence by only a single round of PCR. This method involves digesting and ligating genomic and plasmid DNAs, and amplifying the 5'-upstream or 3'-end downstream sequence of the known DNA sequence, using two primers, one gene specific and the other plasmid specific. A single round of PCR amplification is sufficient to produce gene-specific bands detectable in gels. By using this approach, 5'-end genomic sequence of the D-amoeba sams gene was extended.