• Journal of Korean society of Dental Hygiene
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• v.15 no.6
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• pp.1063-1071
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• 2015

Objectives: The purpose of the study is to investigate the quantitative detection of periodontal pathogens before and after scaling by real-time polymerase chain reaction. Methods: Participants were voluntarily recruited at D university, and saliva samples were extracted before and after scaling. Multiple real-time polymerase chain reactions were used to analyze characteristics and the amount of nine kinds of periodontal pathogens; Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Prevotella intermedia, Fusobacterium nucleatum, Parvimonas micra, Campylobacter rectus, and Eikenella corrodens. Results: After scaling, most periodontal pathogens except Eikenella corrodens were significantly decreased in all subjects(p<0.05). In addition, the percentage of microorganisms associated with disease, the microorganism risk index of periodontitis and the prevalence of red complex, orange complex, and Aggregatibacter actinomycetemcomitans was also significantly reduced after scaling(p<0.05). Conclusions: Scaling decreased in the amount of major periodontal pathogens and periodontitis prevalence rate.

• 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.

• Animal Bioscience
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• v.34 no.12
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• pp.1995-2002
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• 2021

Objective: Detection of adulteration in processed meats is an important issue for some countries due to substitution of beef with a cheaper source of protein like poultry. In this study, the presence of chicken meat was investigated using real-time polymerase chain reaction (real-time PCR) and enzyme-linked immunosorbent assay (ELISA) techniques to verify adulteration of fermented sausage samples. Methods: A total of 60 commercial samples were collected from 20 establishments in three replicates including 10 fermented sausage manufacturers and 10 butchers to investigate the presence of chicken meat with the sequential use of real-time PCR and ELISA techniques. In addition, pH, moisture content, water activity and color values of the samples were determined. Results: Both real-time PCR and ELISA showed agreement on the presence or absence of chicken meat in 55 out of 60 fermented sausage samples and chicken meat was identified with both methods in 16 samples. Five samples produced inconsistent results for the presence of chicken meat in the first run. Nevertheless, the presence of chicken meat was verified with both methods when these samples were analyzed for the second time. In addition, the average physico-chemical values of the fermented sausage samples tested positive for chicken meat were not significantly different from some of those fermented sausage samples tested negative for the chicken meat. Conclusion: The sequential use of real-time PCR and ELISA techniques in fermented sausages could be beneficial for the government testing programs to eliminate false negatives for detection of adulteration with chicken meat. Furthermore, consumers should not rely on some of the quality cues including color to predict the adulteration of fermented sausages with chicken meat since there were no statistical differences among some of the samples tested positive and negative for chicken meat.

• Fisheries and Aquatic Sciences
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• v.24 no.11
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• pp.351-359
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• 2021

The red sea bream iridovirus (RSIV) belonging to genus Megalocytivirus is responsible for red sea bream iridoviral disease (RSIVD) in marine and freshwater fishes. Although several diagnostic assays for RSIV have been developed, diagnostic sensitivity (DSe) and specificity (DSp) of real-time polymerase chain reaction (PCR) assays are not yet evaluated. In this study, we developed a TaqMan probe-based real-time PCR method and evaluated its DSe and DSp. To detect RSIV, the probe and primers were designed based on consensus sequences of the major capsid protein (MCP) genes from megalocytiviruses including RSIV, infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV). The probe and primers were shown to be specific for RSIV, ISKNV, and TRBIV-types megalocytiviruses. A 95% limit of detection (LOD_95%) was determined to be 5.3 viral genome copies/µL of plasmid DNA containing the MCP gene from RSIV. The DSe and DSp of the developed real-time PCR assay for field samples (n = 112) were compared with those of conventional PCR assays and found to be 100% and 95.2%, respectively. The quantitative results for SYBR Green and TaqMan probe-based real-time PCR were not significantly different. The TaqMan probe-based real-time PCR assay for RSIV may be used as an appropriate diagnostic tool for qualitative and quantitative analysis.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.10
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• pp.1411-1416
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• 2014

Gender selection is important in livestock industries; for example, female calves are required in the dairy industry. Sex-sorted semen is commonly used for the production of calves of the desired gender. However, assessment of the sex ratio of the sorted semen is tedious and expensive. In this study, a rapid, cost effective and reliable method for determining the sex ratio was developed using a multiplex real-time polymerase chain reaction (PCR) assay. In this assay, the X and Y chromosome-specific markers, i.e., bovine proteolipid protein (PLP) gene and sex-determining region Y (SRY) were simultaneously quantified in a single tube. The multiplex real-time PCR assay was shown to have high amplification efficiencies (97% to 99%) comparable to the separated-tube simplex real-time PCR assay. The results obtained from both assays were not significantly different (p>0.05). The multiplex assay was validated using reference DNA of known X ratio (10%, 50%, and 90%) as templates. The measured %X in semen samples were the same within 95% confidence intervals as the expected values, i.e., >90% in X-sorted semen, <10% in Y-sorted semen and close to 50% in the unsorted semen. The multiplex real-time PCR assay as shown in this study can thus be used to assess purity of sex-sorted semen.

• Tuberculosis and Respiratory Diseases
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• v.78 no.4
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• pp.473-473
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• 2015

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.10
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• pp.1683-1690
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• 2020

Objective: The rapid and reliable detection of the African swine fever virus (ASFV) plays an important role in emergency control and preventive measures of ASF. Some methods have been recommended by FAO/OIE to detect ASFV in clinical samples, including realtime polymerase chain reaction (PCR). However, mismatches in primer and probe binding regions may cause a false-negative result. Here, a slight modification in probe sequence has been conducted to improve the qualification of real-time PCR based on World Organization for Animal Health (OIE) protocol for accurate detection of ASFV in field samples in Vietnam. Methods: Seven positive confirmed samples (four samples have no mismatch, and three samples contained one mutation in probe binding sites) were used to establish novel real-time PCR with slightly modified probe (Y = C or T) in comparison with original probe recommended by OIE. Results: Both real-time PCRs using the OIE-recommended probe and novel modified probe can detect ASFV in clinical samples without mismatch in probe binding site. A high correlation of cycle quantification (Cq) values was observed in which Cq values obtained from both probes arranged from 22 to 25, suggesting that modified probe sequence does not impede the qualification of real-time PCR to detect ASFV in clinical samples. However, the samples with one mutation in probe binding sites were ASFV negative with OIE recommended probe but positive with our modified probe (Cq value ranked between 33.12-35.78). Conclusion: We demonstrated for the first time that a mismatch in probe binding regions caused a false negative result by OIE recommended real-time PCR, and a slightly modified probe is required to enhance the sensitivity and obtain an ASF accurate diagnosis in field samples in Vietnam.

• Journal of Sensor Science and Technology
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• v.14 no.5
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• pp.337-342
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• 2005

A silicon-based micro-reactor to amplify small amount of deoxyribonucleic acid (DNA) has been fabricated using micro-electro-mechanical systems (MEMS) technology. Polymerase chain reaction (PCR) of DNA requires a precise and rapid temperature control. A Pt sensor is integrated directly in the chamber for real-time temperature measurement and an infrared lamp is used as external heating source for non-contact and rapid heating. In addition to the real-time temperature sensing, PCR needs a rapid thermocycling for effective PCR. For a fast thermal response, the thermal mass of the reactor chamber is minimized by removal of bulk silicon volume around the reactor using double-side KOH etching. The transparent optical property of silicon in the infrared wavelength range provides an efficient absorption of thermal energy into the reacting sample without being absorbed by silicon reactor chamber. It is confirmed that the fabricated micro-reactor could be heated up in less than 30 sec to the denaturation temperature by the external infrared lamp and cooled down in 30 sec to the annealing temperature by passive cooling.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.4
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• pp.467-472
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• 2018

Objective: Molecular cloning and bioinformatics analysis of annexin A2 (ANXA2) gene in sika deer antler tip were conducted. The role of ANXA2 gene in the growth and development of the antler were analyzed initially. Methods: The reverse transcriptase polymerase chain reaction (RT-PCR) was used to clone the cDNA sequence of the ANXA2 gene from antler tip of sika deer (Cervus Nippon hortulorum) and the bioinformatics methods were applied to analyze the amino acid sequence of Anxa2 protein. The mRNA expression levels of the ANXA2 gene in different growth stages were examined by real time reverse transcriptase polymerase chain reaction (real time RT-PCR). Results: The nucleotide sequence analysis revealed an open reading frame of 1,020 bp encoding 339 amino acids long protein of calculated molecular weight 38.6 kDa and isoelectric point 6.09. Homologous sequence alignment and phylogenetic analysis indicated that the Anxa2 mature protein of sika deer had the closest genetic distance with Cervus elaphus and Bos mutus. Real time RT-PCR results showed that the gene had differential expression levels in different growth stages, and the expression level of the ANXA2 gene was the highest at metaphase (rapid growing period). Conclusion: ANXA2 gene may promote the cell proliferation, and the finding suggested Anxa2 as an important candidate for regulating the growth and development of deer antler.

• Food Science and Biotechnology
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• v.18 no.3
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• pp.788-792
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• 2009

Gamma $({\gamma})-irradiation$ can be used to control pathogens such as Vibrio vulnificus in seafood. The effects of irradiation on microbial cell populations (%) have been studied in order to develop detection methods for irradiated foods. The method used in this study was ethidium bromide monoazide (EMA) real-time polymerase chain reaction (PCR), using V. vulnificus specific primer, EMA, and $SYBR^{(R)}$ Green to discriminate between ${\gamma}-irradiated$ and non-irradiated cells. Confocal microscope examination showed that ${\gamma}-irradiation$ damaged portions of the cell membrane, allowing EMA to penetrate cells of irradidated V. vulnificus. ${\gamma}-Irradiation$ at 1.08 KGy resulted in log reduction ($-1.15{\pm}0.13$ log reduction) in genomic targets derived from EMA real-time PCR. The combination cold/heat shock resulted in the highest ($-1.74{\pm}0.1$ log reduction) discrimination of dead irradiated V. vulnificus by EMA real-time PCR.