• International Journal of Oral Biology
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• v.46 no.3
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• pp.140-145
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• 2021

This study aimed to develop Lautropia mirabilis-specific quantitative real-time polymerase chain reaction (qPCR) primers based on the sequence of DNA-directed RNA polymerase subunit beta gene. The PrimerSelect program was used in designing of the qPCR primers, RTLam-F4 and RTLam-R3. The specificity of the qPCR primers were performed by conventional PCR with 37 strains of 37 oral bacterial species, including L. mirabilis. The sensitivity of the primers was determined by qPCR with the serial dilution of purified genomic DNA of L. mirabilis KCOM 3484, ranged from 4 ng to 4 fg. The data showed that the qPCR primers could detect only L. mirabilis strains and as little as 40 fg of genome DNA of L. mirabilis KCOM 3484. These results indicate that this qPCR primer pair (RTLam-F4/RTLam-R3) may be useful for species-specific detection of L. mirabilis in epidemiological studies of oral bacterial infectious diseases such as periodontal disease.

• International Journal of Oral Biology
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• v.44 no.3
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• pp.96-100
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• 2019

The purpose of this study was to develop Peptoniphilus mikwangii-specific quantitative real-time polymerase chain reaction (qPCR) primers based on the 16S ribosomal RNA (16S rDNA) gene. The specificity of the primers was determined by conventional PCR using 29 strains of 27 oral bacterial species including P. mikwangii. The sensitivity of the primers was determined by qPCR using the purified genomic DNA of P. mikwangii KCOM $1628^T$ (40 ng to 4 fg). The data showed that the qPCR primers (RTB134-F4/RTB134-R4) could detect P. mikwangii strains exclusively and as little as 40 fg of the genomic DNA of P. mikwangii KCOM $1628^T$. These results suggest that the developed qPCR primer pair can be useful for detecting P. mikwangii in epidemiological studies of oral bacterial infectious diseases.

• International Journal of Oral Biology
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• v.41 no.3
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• pp.149-154
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• 2016

The purpose of this study was to develop Streptococcus sobrinus-specific qPCR primers based on the nucleotide sequence of the RNA polymerase ${\beta}-subunit$ gene (rpoB). The specificity of the primers was determined by conventional polymerase chain reaction (PCR) with 12 strains of S. sobrinus and 50 strains (50 species) of non-S. sobrinus bacteria. The sensitivity of the primers was determined by quantitative real-time PCR (qPCR) with serial dilutions of the purified genomic DNAs (40 ng to 4 fg) of S. sobrinus ATCC $33478^T$. The specificity data showed that the S. sobrinus-specific qPCR primers (RTSsob-F4/RTSsob-R4) detected only the genomic DNAs of S. sobrinus strains with a detection limit of up to 4 fg of S. sobrinus genomic DNA. Our results suggest that the RTSsob-F4/RTSsob-R4 primers are useful in detecting S. sobrinus with high sensitivity and specificity for epidemiological studies of dental caries..

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.8
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• pp.3669-3673
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• 2012

A role for the xenotropic murine leukemia virus (XMRV) in prostate cancer development has been postulated. To answer questions regarding the prevalence of XMRV in Iranian patients with prostate cancer and its association with the RNASEL R462Q polymorphism, we here investigated a series of cases in Kerman, in the Southeast of Iran, and sought to verify the association with the R462Q using Real Time PCR Method. Prostate tissue specimens of 200 patients with prostate cancer were genotyped for R462Q by real time polymerase chain reaction allelic discrimination and were screened for XMRV proviral DNA by real time polymerase chain reaction specific for the envelope gene. Of 200 patients in this study 8 (4%) cases were positive for XMRV, the QQ allele being the most frequenct regarding the R426Q polymorphism while in negative patients it was the RQ allele. There was significant correlation between high pathological scores and XMRV positive samples. No significant relationship was found between age groups and XMRV results. XMRV was only found in patients with QQ and RQ alleles, not RR. XMRV is detectable in tumor prostate tissue from some patients with prostate cancer, independent of R462Q.

• Journal of the korean academy of Pediatric Dentistry
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• v.42 no.4
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• pp.312-318
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• 2015

The aim of this study was to investigate the possibility of the supernumerary teeth for the stem cell source in dentistry. The Real Time Quantitative Reverse Transcription Polymerase Chain Reaction (Real Time qRT-PCR) method was used to evaluate the differentiation toward the odontoblast of the supernumerary dental pulp stem cells (sDPSCs). Supernumerary dental pulp stem cells were obtained from 3 children (2 males and 1 female, age 7 to 9) diagnosed that the eruption of permanent teeth was disturbed by supernumerary teeth. The common genes for odontoblasts are alkaline phosphatase (ALP), osteocalcin (OC), osteonectin (ON), dentin matrix acidic phosphoprotein 1 (DMP-1), dentin sialophosphoprotein (DSPP). The sDPSCs were treated for 0 days, 8 days and 14 days with additives and then Real Time qRT-PCR was performed in intervals of 0 days, 8 days and 14 days. The alizarin-red solution staining was performed to visualize the stained color for the degree of calcification at 7 days, 14 days, 21 days and 28 days after treating additives to the sDPSCs. From the result of the Real Time qRT-PCR, the manifestation exhibit maximum value at 8 days after additive treatment and shifted to a decrease trend at 14 days. Alizarin-red solution staining exhibit light results at 7 days after staining and generalized dark result at 14 days. Consequently, in studies with sDPSCs, appropriate treatment time of additives for Real Time qRT-PCR is 8 days. Also, a suitable period of Alizarin-red solution staining is 14 days.

• Journal of Embryo Transfer
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• v.25 no.2
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• pp.111-116
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• 2010

Sperm chromatin integrity is essential for successful fertilization and development of an embryo. Reported here is a quantification of DNA fragments which is intimately associated with reproductive potential to provide one of criteria for sperm chromatin integrity. Three sperm populations were considered: CONTROL (no treatment), UV irradiation (48mW/$cm^2$, 1h) and $H_2O_2$ (oxidative stress induced by hydrogen peroxide, 10 mM, 50 mM and 100 mM). DNA fragments in boar sperm were evaluated by using ligation-mediated quantitative real-time polymerase chain reaction (LM-qPCR) assay, which relies on real-time qPCR to provide a measure of blunt 5' phosphorylated double strand breaks in genomic DNA. The results in agarose gel electrophoresis showed no significant DNA fragmentation and no dose-dependent response to $H_2O_2$. However, the remarkable difference in shape and position was observed in melting curve of LM-qPCR. This result supported that the melting curve analysis of LM-qPCR presented here, could be more sensitive and accurate than previous DNA fragmentation assay method.

• Environmental Engineering Research
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• v.14 no.1
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• pp.63-67
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• 2009

Escherichia coli K-12 (E. coli K-12) is a representative indicator globally used for distinguishing and monitoring dynamic fates of pathogenic microorganisms in the environment. This study investigated how to most critically quantify lacZ ($\beta$-galactosidase) gene in E. coli K-12 by two different real-time polymerase chain reaction (real-time PCR) in association with three different DNA extraction practices. Three DNA extractions, i.e., sodium dodecyl sulfate (SDS)/proteinase K, magnetic beads and guanidium thiocyanate (GTC)/silica matrix were each compared for extracting total genomic DNA from E. coli K-12. Among them, GTC/silica matrix and magnetic beads beating similarly worked out to have the highest (22-23 ng/${\mu}L$) concentration of DNA extracted, but employing SDS/proteinase K had the lowest (10 ng/${\mu}L$) concentration of DNA retrieved. There were no significant differences in the quantification of the copy numbers of lacZ gene between SYBR Green I qPCR and QProbe-qPCR. However, SYBR Green I qPCR obtained somewhat higher copy number as $1{\times}10^8$ copies. It was decided that GTC/silica matrix extraction or magnetic beads beating in combination with SYBR Green I qPCR can be preferably applied for more effectively quantifying specific gene from a pure culture of microorganism.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.3
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• pp.1655-1659
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• 2013

Background: HER-2/neu is a proto-oncogene that encodes a transmembrane tyrosine kinase growth factor which is crucial for stimulating growth and cellular motility. Overexpression of HER-2/neu is observed in 10-35% of human breast cancers and is associated with pathogenesis, prognosis as well as response to therapy. Given the imperative role of HER-2/neu overexpression in breast cancer, it is important to determine the magnitude of amplification which may facilitate a better prognosis as well as personalized therapy in affected patients. In this study, we determined HER-2/neu protein expression by immunohistochemistry (IHC) concurrently with HER-2/neu DNA amplification by quantitative real time-polymerase chain reaction (Q-PCR). Materials and Methods: A total of 53 paired tissue samples from breast cancer patients were frozen-sectioned to characterize the tumour and normal tissues. Only tissues with 80% tumour cells were used in this study. For confirmation, Q-PCR was used to determine the HER-2/neu DNA amplification. Results: We found 20/53 (37.7%) of the tumour tissues to be positive for HER-2/neu protein overexpression using IHC. Out of these twenty, only 9/53 (17%) cases were in agreement with the Q-PCR results. The concordance rate between IHC and Q-PCR was 79.3%. Approximately 20.7% of positive IHC cases showed no HER-2/neu gene amplification using Q-PCR. Conclusion: In conclusion, IHC can be used as an initial screening method for detection of the HER-2/neu protein overexpression. Techniques such as Q-PCR should be employed to verify the IHC results for uncertain cases as well as determination of HER-2/neu gene amplification.

• Korean Journal of Veterinary Service
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• v.46 no.2
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• pp.123-135
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• 2023

Feline calicivirus (FCV) is considered the main viral pathogen of feline upper respiratory tract disease (URTD). The frequent mutations of field FCV strains result in the poor diagnostic sensitivity of previously developed molecular diagnostic assays. In this study, a more sensitive real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for broad detection of currently circulating FCVs and comparatively evaluated the diagnostic performance with previously developed qRT-PCR assay using clinical samples collected from Korean cat populations. The developed qRT-PCR assay specifically amplified the FCV p30 gene with a detection limit of below 10 copies/reaction. The assay showed high repeatability and reproducibility, with coefficients of intra-assay and inter-assay variation of less than 2%. Based on the clinical evaluation using 94 clinical samples obtained from URTD-suspected cats, the detection rate of FCV by the developed qRT-PCR assay was 47.9%, which was higher than that of the previous qRT-PCR assay (43.6%). The prevalence of FCV determined by the new qRT-PCR assay in this study was much higher than those of previous Korean studies determined by conventional RT-PCR assays. Due to the high sensitivity, specificity, and accuracy, the new qRT-PCR assay developed in this study will serve as a promising tool for etiological and epidemiological studies of FCV circulating in Korea. Furthermore, the prevalence data obtained in this study will contribute to expanding knowledge about the epidemiology of FCV in Korea.

• Proceedings of the Korean Vacuum Society Conference
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• 2016.02a
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• pp.375.1-375.1
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• 2016

Polymerase chain reaction (PCR) has revolutionized genetics and become one of the most popular techniques in modern biological and medical sciences. It can be used not only as an in vitro DNA amplification method but also used in many bioassay applications. The PCR can be used to exponentially produce a large number of DNA copies from a small quantity of DNA molecules in a few hours. However, as unwanted DNA fragments are also often manufactured, the amplification efficiency of PCR is decreased. To overcome this limitation, several nanomaterials have been employed to increase the specificity of the PCR reaction. Recently, graphene has attracted a great interest for its excellent electron transfer, thermal and biocompatibility. Especially, gold nanoparticle-coated graphene oxide (GO/AuNPs) led to enhance electron and thermal transfer rate and low-charge transfer resistance. Therefore, we report the development of a demonstration for the PCR efficiency using a large-scale production of the GO and combination of gold nanoparticles. Because a thermal conductivity is an important factor for improving the PCR efficiency in different DNA polymerases and different size samples. When PCR use GO/AuNPs, the result of transmission electron microscopy and real-time quantitative PCR (qPCR) showed an enhanced PCR efficiency. We have demonstrated that GO/AuNPs would be simply outperformed for enhancing the specificity and efficiency of DNA amplification procedure.