• Asian Pacific Journal of Cancer Prevention
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• v.14 no.8
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• pp.4883-4889
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• 2013

Background: The most common incident cancer and cause of cancer-related deaths in women is breast cancer. The Myc gene is upregulated in many cancer types including breast cancer, and it is considered as a potential anti-cancer drug target. The present study was conducted to evaluate the Myc (gene and protein) expression pattern in an experimental mammary tumour model in rats. Materials and Methods: Thirty six Sprague Dawley rats were divided into: Experimental group (26 animals), which received the chemical carcinogen N-methyl nitrosourea (MNU) and a control group (10 animals), which received vehicle only. c-Myc oncoprotein and its mRNA expression pattern were evaluated using immunohistochemistry (IHC) and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively, in normal rat mammary tissue and mammary tumours. The rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as internal control for semi-quantitative RT-PCR. Results: Histopathological examination of mammary tissues and tumours from MNU treated animals revealed the presence of premalignant lesions, benign tumours, in situ carcinomas and invasive carcinomas. Immunohistochemical evaluation of tumour tissues showed upregulation and heterogeneous cellular localization of c-Myc oncoprotein. The expression levels of c-Myc oncoprotein were significantly elevated (75-91%) in all the tumours. Semi-quantitative RT-PCR revealed increased expression of c-Myc mRNA in mammary tumours compared to normal mammary tissues. Conclusions: Further large-scale investigation study is needed to adopt this experimental rat mammary tumour model as an in vivo model to study anti-cancer strategies directed against Myc or its downstream partners at the transcriptional or post-transcriptional level.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.39 no.4
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• pp.281-288
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• 2013

Hyaluronic acid (HA) is one of the major extracellular matrix components in skin. The HA content is reported to decline with age, which may contribute to decrease in skin moisture, wrinkle formation and the decrease in elasticity of the skin. Among the family of HA synthase genes (HAS-1, 2, 3) identified so far, HAS-2 plays crucial roles in the regulation of HA synthesis in human skin fibroblasts. In this study, we elucidated the effects of ferulic acid isolated from Cnidium officinale on HA production. Semi-quantitative RT-PCR and quantitative real-time PCR showed that ferulic acid increased mRNA level of HAS-2 gene and ELISA assay also revealed that ferulic acid increased HA production in human skin fibroblasts. Our study suggests that ferulic acid might prevent age-dependent skin deteriorations such as wrinkles, dryness and elasticity decrease, all of which could be ascribed to the reduction of the HA content in human skin.

• Journal of Environmental Health Sciences
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• v.45 no.5
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• pp.511-519
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• 2019

Objectives: The standard method for the enumeration of environmental Legionella is culturing, which has several disadvantages, including long incubation and poor sensitivity. The purpose of this study is to demonstrate the usefulness of real-time PCR and to improve the standard method. Methods: In 200 environmental water samples, a real-time PCR and culture were conducted to detect and quantify Legionella. Using with the results of the survey, we compared the real-time PCR with the culture. Results: Each real-time PCR assay had 100% specificity and excellent sensitivity (5 GU/reaction). In the culture, 36 samples were positive and 164 samples were negative. Based on the results of the culture, real-time PCR showed a high negative predictive value of 99%, 35 samples were true positive, 105 samples were true negative, 59 samples were false positive and one sample was a false negative. Quantitative analysis of the two methods indicated a weak linear correlation ($r^2=0.29$, $r^2=0.61$, respectively). Conclusions: Although it is difficult to directly apply quantitative analysis results of real-time PCR in the enumeration of environmental Legionella, it can be used as a complementary means of culturing to rapidly screen negative samples and to improve the accuracy of diagnosis.

• Microbiology and Biotechnology Letters
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• v.33 no.2
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• pp.148-153
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• 2005

Malaria is a re-emerging infectious disease that is spreading to areas where it had been eradicated, such as Eastern Europe and Central Asia. To avoid the mortality from malaria, early detection of the parasite is a very important issue. The peripheral blood smear has been the gold standard method for the diagnosis of malaria infection. Recently, several other methods have been introduced for quantitative detection of malaria parasites. Real time PCR that employs fluorescent labels to enable the continuous monitoring of PCR product formation throughout the reaction has recently been used to detect several human malaria parasites. 18S rRNA sequences from malaria parasites have been amplified using Taqman real time PCR assay. Here, a SYBR Green-based real time quantitative PCR assay for the detection of malaria parasite-especially, Plasmodium vivax - was applied for the evaluation of 26 blood samples from Korean malaria patients. Even though SYBR Green-based real time PCR is easier and cheaper than Taqman-based assay, SYBR Green-based assay cannot be used because 18S rRNA cannot be specifically amplified using 1 primer set. Therefore, we used DBP gene sequences from Plasmodium vivax, which is specific for the SYBR Green based assays. We amplified the DBP gene from the 26 blood samples of malaria patients using SYBR Green based assay and obtained the copy numbers of DBP genes for each sample. Also, we selected optimal reference gene between ACTB and B2M using real time assay to get the stable genes regardless of Malaria titer. Using selected ACTB reference genes, we successfully converted the copy numbers from samples into titer, ${\sharp}$ of parasites per microliter. Using the resultant titer from DBP based SYBER Green assay with ACTB reference gene, we compared the results from our study with the titer from Taqman-based assay. We found that our results showed identical tendency with the results of 18S rRNA Taqman assay, especially in lower titer range. Thus, our DBP gene-utilized real time assay can detect Plasmodium vivax in Korean patient group semi-quantitatively and easily.

• Journal of Korean society of Dental Hygiene
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• v.17 no.2
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• pp.319-330
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• 2017

Objectives: The purpose of this study was to compare SYBR Green qPCR, TaqMan, and bacterial selective medium cultures for accurate quantitative analysis of oral microorganisms. Methods: The SYBR Green method is widely used to analyze the total amount of oral microorganisms in oral saliva. However, in this study, MTR-PCR method based on TaqMan method was performed using newly developed primers and probes. In addition, it was designed to confirm the detection agreement of bacteria among bacteria detection method. Results: As a result of MRT-PCR and SYBR Green qPCR analysis, more than 40 times (0.9-362.9 times) bacterium was detected by MRT-PCR. In addition, more bacteria were detected in saliva in the order of MRT-PCR, SYBR Green qPCR, and bacterium culture, and the results of MRB-PCR and SYBR Green qPCR showed the highest agreement. The agreement between the three methods for detecting P. intermedia was similar between 71.4 and 88.6%, but the agreement between MRT-PCR and SYBR Green qPCR was 80% for S. mutans. Among them, the number of total bacteria, P. intermedia and S. mutans bacteria in saliva was higher than that of SYBR Green qPCR method, and bacterium culture method by MRT-PCR method. P. intermedia and S. mutans in saliva were detected by MRT-PCR and MRT-PCR in 88.6% of cases, followed by the SYBR Green qPCR method (80.0%). Conclusions: The SYBR Green qPCR method is the same molecular biology method, but it can not analyze the germs at the same time. Bacterial culturing takes a lot of time if there is no selective culture medium. Therefore, the MRT-PCR method using newly developed primers and probes is considered to be the best method.

• Research in Plant Disease
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• v.27 no.3
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• pp.120-127
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• 2021

Plant viruses cause significant yield losses, continuously compromising crop production and thus representing a serious threat to global food security. Tomato spotted wilt virus (TSWV) is the most harmful plant virus that mainly infects horticultural crops and has a wide host range. Reverse-transcription quantitative real-time PCR (RT-qPCR) has been widely used for detecting TSWV with high sensitivity, but its application is limited owing to the lack of standardization. Therefore, in this study, a sensitive and accurate reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) method was established for TSWV detection. Additionally, we compared the sensitivities of RT-qPCR and RT-ddPCR for TSWV detection. Specificity analysis of RT-ddPCR for TSWV showed no amplification for main pepper viruses and negative control. TSWV transcripts levels measured by RT-ddPCR and RT-qPCR showed a high degree of linearity; however, the former yielded results that were at least 10-fold more sensitive and detected lower TSWV copy numbers than the latter. Collectively, our findings show that RT-ddPCR provides improved analytical sensitivity and specificity for TSWV detection, making it suitable for identifying low TSWV concentrations in field samples.

• Microbiology and Biotechnology Letters
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• v.36 no.3
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• pp.173-181
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• 2008

Bovine blood, cell, tissue, and organ are used as raw materials for manufacturing biologics such as biopharmaceuticals, tissue-engineered products, and cell therapy. Manufacturing processes for the biologics have the risk of viral contamination. Therefore viral validation is essential in ensuring the safety of the products. Bovine parvovirus (BPV) is one of the common bovine pathogens and has widely been known as a possible contaminant of biologics. In order to establish the validation system for the BPV safety of biologics, a real-time PCR method was developed for quantitative detection of BPV contamination in raw materials, manufacturing processes, and final products. Specific primers for amplification of BPV DNA were selected, and BPV DNA was quantified by use of SYBR Green 1. The sensitivity of the assay was calculated to be $1.3{\times}10^{-1}\;TCID_{50}/mL$. The real-time PCR method was validated to be reproducible and very specific to BPV. The established real-time PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with BPV. BPV DNA could be quantified in CHO cell as well as culture supernatant. Also the real-time PCR assay could detect $1.3{\times}10^0\;TCID_{50}/mL$ of BPV artificially contaminated in bovine collagen. The overall results indicated that this rapid, specific, sensitive, and robust assay can be reliably used for quantitative detection of BPV contamination during manufacture of biologics.

• BMB Reports
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• v.37 no.1
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• pp.1-10
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• 2004

DNA and RNA quantifications are widely used in biological and biomedical research. In the last ten years, many technologies have been developed to enable automated and high-throughput analyses. In this review, we first give a brief overview of how DNA and RNA quantifications are carried out. Then, five technologies (microarrays, SAGE, differential display, real time PCR and real competitive PCR) are introduced, with an emphasis on how these technologies can be applied and what their limitations are. The technologies are also evaluated in terms of a few key aspects of nucleic acids quantification such as accuracy, sensitivity, specificity, cost and throughput.

• Journal of Microbiology and Biotechnology
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• v.22 no.8
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• pp.1113-1117
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• 2012

Shigella sonnei is a causal agent of fever, nausea, stomach cramps, vomiting, and diarrheal disease. The present study describes a quantitative polymerase chain reaction (qPCR) assay for the specific detection of S. sonnei using a primer pair based on the methylase gene for the amplification of a 325 bp DNA fragment. The qPCR primer set for the accurate diagnosis of Shigella sonnei 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.

• Journal of Microbiology and Biotechnology
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• v.22 no.11
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• pp.1575-1579
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• 2012

Paenibacillus polymyxa is known to be a plant-growth-promoting rhizobacterium. The present study describes a quantitative polymerase chain reaction (qPCR) assay for the specific detection and quantitation of P. polymyxa using a primer pair based on the sequence of a membrane-fusion protein for the amplification of a 268 bp DNA fragment. This study reports that the qPCR-based method is applicable for the rapid and sensitive detection of P. polymyxa and can be used as an alternative method for agricultural soil monitoring.