• Food Science of Animal Resources
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• v.31 no.6
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• pp.851-857
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• 2011

This study was conducted to develop a real time reverse transcriptase-PCR (RT-PCR) method for the detection of viable Enterobacteriaceae in milk using primers based on the genes of ribosomal proteins S11 and S13 and to determine effects of heating and subsequent treatments on the threshold cycle (Ct) of the real time RT-PCR. Total RNA was isolated from 17 strains of bacteria including 11 strains of Enterobacteriaceae suspended in milk using a modified Tri reagent method. SYBR Green Master Mix was added to the RNA and the mixture was subjected to the real time RT-PCR. The Cts of eleven type strains of the Enterobacteriaceae in milk ($10^7$ cells) in the real time RT-PCR ranged from 21.5 to 24.6. However, the Cts of Pseudomonas fluorescens, Acinetobacter calcoaceticus, and three gram-positive bacteria were more than 40. The real time RT-PCR detected as low as $10^3$ cells in agarose gel electrophoresis. The Cts increased from 22.0 to 34.2 when milk samples contaminated with Escherichia coli ($10^7$ cells/mL) were heated at $65^{\circ}C$ for 30 min. In addition, subsequent incubation at $37^{\circ}C$ for 6 and 24 h increased the Cts further up to 36.2 and 37.2, respectively. Addition of RNase A to the bacterial suspension obtained from the heated milk and subsequent incubation at $37^{\circ}C$ for 1 h increased the Cts to more than 40. The results of this study suggests that pretreatment of bacterial cells heated in milk with RNase A before RNA extraction might enhance the ability to differentiate between viable and dead bacteria using real time RT-PCR.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.3
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• pp.873-877
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• 2012

MicroRNAs (miRNAs) are short non-coding RNA molecules characterized by their regulatory roles in cancer and gene expression. We analyzed the expression of miR-21, miR-205, and miR-342 in 59 patients with breast cancer. Samples were divided into three different groups according to their immunohistochemistry (IHC) classification: ER- positive and/or PR-positive group ($ER^+$ and/or $PR^+$; group I); HER2-positive group ($HER^{2+}$; group II); and ER/ PR/ HER2- negative ($ER^-$/ $PR^-$/ $HER^{2-}$; group III) as the triple negative group. The expression levels of the 3 miRNAs were analyzed in the tumor samples and the compared with the normal neighboring dissected tumor (NNDT) samples in all three groups. The expression of miR-21 was similar in all three groups. In patients positive for P53 by IHC, positive for axillary lymph node metastasis and higher tumor stages, it appeared to have significantly elevated. However, significant increase was not found among the 18 fibroadenoma samples. Both miR-205 and miR-342 expressions were significantly down regulated in group III. We conclude that miR-21 does not discriminate between different breast cancer groups. In contrast, miR-205 and miR-342 may be used as potential biomarkers for diagnosis of triple negative breast cancer.

• Journal of Microbiology and Biotechnology
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• v.18 no.11
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• pp.1841-1847
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• 2008

Vibrio vulnificus is a causative agent of serious diseases in humans, resulting from the contact of wound with seawater or consumption of raw seafood. Several studies aimed at detecting V. vulnificus have targeted vvh as a representative virulence toxin gene belonging to the bacterium. In this study, we targeted the rpoS gene, a general stress regulator, to detect V. vulnificus. PCR specificity was identified by amplification of 8 V. vulnificus templates and by the loss of a PCR product with 36 non-V. vulnificus strains. The PCR assay had the 273-bp fragment and the sensitivity of 10 pg DNA from V. vulnificus. SYBR Green I-based real-time PCR assay targeting the rpoS gene showed a melting temperature of approximately $84^{\circ}C$ for the V. vulnificus strains. The minimum level of detection by real-time PCR was 2 pg of purified genomic DNA, or $10^3$ V. vulnificus cells from pure cultured broth and $10^3$ cells in 1 g of oyster tissue homogenates. These data indicate that real-time PCR is a sensitive, species-specific, and rapid method for detecting this bacterium, using the rpoS gene in pure cultures and in infected oyster tissues.

• International Journal of Oral Biology
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• v.43 no.1
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• pp.5-11
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• 2018

Recent findings indicate that Type 2 taste receptors (T2Rs) are expressed outside the gustatory system, including in the gastrointestinal tracts and the exocrine glands, such as the submandibular (SM), parotid (P), lacrimal (L) glands and pancreas (PC). Specifically, T2Rs are found in some of the gastrointestinal endocrine cells, and these cells secreted peptide hormones in response to stimulation by bitter-tasting compounds. The results show that T2Rs may have significant physiological roles besides bitter taste reception. The functions of the T2Rs in the exocrine glands remain poorly understood. An expression levels analysis of T2Rs will help to determine those functions in the exocrine glands. The expression levels of the T2Rs in the exocrine glands were discovered via the qPCR. C57BL/6J mice of 42~60-day-old were used. Messenger RNAs were extracted from S, P, L and PC. Cloned DNAs were synthesized by reverse transcription. Quantitative PCRs were performed using the SYBR Green method. The expression levels of the T2Rs were calculated as relative expression levels to that of the GAPDH. The statistical significance among the observed exocrine glands was tested using the variance analysis (ANOVA test). Tas2r108, out of murine 35 T2Rs, was the most highly expressed in every observed exocrine gland. This finding was similar to previous results from tongue papillae, but the expression levels were lower than those of the tongue papillae. Tas2r137 of SM, P, L and PC were expressed a little lower than that of tongue papillae. The T2Rs in the exocrine glands may play slightly different roles from those in the tongue. We suggest that physiological studies such as a patch clamp and functional $Ca^{2+}$ imaging of acinar cells are necessary for understanding the Tas2r108 functions.

• Journal of Korean Academy of Oral Health
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• v.42 no.4
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• pp.224-228
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• 2018

Objectives: As a first step to study the anticaries effect of ethanol alone, we investigated the effects of ethanol on the expression levels of the atpB gene and proton permeability of Streptococcus mutans in suspension cultures. Methods: S. mutans UA159 was grown in brain heart infusion medium at either pH 4.8 or 6.8. The total extracted RNA was reverse-transcribed into cDNA using a $Superscript^{TM}$ First-Strand Synthesis System. The resulting cDNA and negative controls were amplified by ABI PRISM 7700 real-time PCR system with SYBR Green PCR Master Mix. For proton flux assay, bacterial suspensions were titrated to pH 4.6 with 0.5 M HCl, and then additional 0.5 M HCl was added to decrease the pH values by approximately 0.4 units. The subsequent increase in pH was monitored using a glass electrode. Ten percent (v/v) butanol was added to the suspensions at 80 min to disrupt the cell membrane. Results: In a concentration-dependent manner, ethanol alone not only decreased the growth rate of S. mutans and the expression of the atpB gene but also increased the proton permeability at both pH 4.8 and 6.8. Conclusions: These findings suggest that ethanol has the potential for an anticaries ingredient. We believe that ethanol may be used together with fluoride and/or other cariostatic agents in order to develop better anticaries toothpastes and/or mouthrinses.

• The Plant Pathology Journal
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• v.34 no.6
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• pp.499-505
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• 2018

Huanglongbing (HLB, Citrus greening disease) is one of the most devastating diseases that threaten citrus production worldwide. Although HLB presents systemically, low titer and uneven distribution of these bacteria within infected plants can make reliable detection difficult. It was known loop-mediated isothermal amplification (LAMP) method has the advantages of being highly specific, rapid, efficient, and laborsaving for detection of plant pathogens. We developed a new LAMP method targeting gene contained tandem repeat for more rapid and sensitive detection of Candidatus Liberibacter asiaticus (CLas), putative causal agent of the citrus huanglongbing. This new LAMP method was 10 folds more sensitive than conventional PCR in detecting the HLB pathogen and similar to that of real-time PCR in visual detection assay by adding SYBR Green I to mixture and 1% agarose gel electrophoresis. Positive reactions were achieved in reaction temperature 57, 60 and $62^{\circ}C$ but not $65^{\circ}C$. Although this LAMP method was not more sensitive than real-time PCR, it does not require a thermocycler for amplification or agarose gel electrophoresis for resolution. Thus, we expect that this LAMP method shows strong promise as a reliable, rapid, and cost-effective method of detecting the CLas in citrus and can be applied for rapid diagnosis is needed.

• ALGAE
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• v.34 no.2
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• pp.71-90
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• 2019

A new encrusting red alga was found growing abundantly on glass debris items that drifted ashore along the coasts of Oregon and Washington. These included discarded fluorescent tubes, incandescent light bulbs, capped liquor bottles, and ball-shaped fishing-net floats. Field collections and unialgal cultures of the alga revealed that it consisted of two morphological phases: a young loosely aggregated turf and a mature consolidated mucilaginous crust. The turf phase consisted of a basal layer of globose cells that produced erect, rarely branched, uniseriate to multiseriate filaments up to $500{\mu}m$ long with closely spaced cells lacking pit-plugs. These filaments expanded in size from their bases to their tips and released single cells as spores. At maturity, a second phase of growth occurred that produced a consolidated crust, up to $370{\mu}m$ thick. It consisted of a basal layer of small, tightly appressed ellipsoidal-to-elongate cells that generated a mucilaginous perithallial matrix containing a second type of filament with irregularly spaced cells often undergoing binary division. At the matrix surface, the original filaments continued to grow and release spores but often also eroded. Individual cells, examined using confocal microscopy and SYBR Green staining, were found to contain a central nucleus, a single highly lobed peripheral chloroplast without a pyrenoid, and numerous chloroplast nucleoids. Morphological data from field and culture isolates and molecular data (rbcL, psbA, and SSU) show that this alga is a new genus and species which we name Viator vitreocola, "a traveller on glass."

• Journal of Animal Science and Technology
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• v.63 no.2
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• pp.426-439
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• 2021

It is already well known that castration improves marbling quality but exact timing of castration is still highly debated in beef cattle production industry. After castration, blood hormonal changes occur in steer and objective of this study was to investigate the effects of growth hormone (GH) levels on adipocyte differentiation in stromal vascular cells (SVCs) and transdifferentiation into adipocytes in C2C12 myoblasts. Total GH concentrations were measured via enzyme-linked immunosorbent assay (ELISA) in 24 male calves and 4 female calves. Cell proliferation, cellular triglyceride (TG) accumulation, and the cell's lipolytic capability were measured in C2C12 myoblasts and SVCs. Myogenic, adipogenic, and brown adipocyte-specific gene expression was measured via real-time polymerase chain reaction (PCR) using SYBR green. Serum GH levels were the highest in late-castrated calves. Treatment with 5 ng/mL GH resulted in greater TG accumulation as well as increased CCAAT-enhancer-binding protein (C/EBP)α and peroxisome proliferator-activated receptor (PPAR)γ expression compared to that after treatment with 15 ng/mL GH. Treatment with 5 ng/mL GH also resulted in lower myogenin (myo)G and myoD expression compared to that after treatment with 15 ng/mL GH. The expression of bone morphogenetic protein (BMP) 7 after treatment with 5 ng/mL GH was higher than that after treatment with 15 ng/mL GH. But carcass characteristics data showed no significant difference between early and late castrated steers. Therefore, our results indicate that castration timing does not seem to be inevitable determinate of carcass qualities, particularly carcass weight and marbling score in Hanwoo beef cattle.

• 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.

• KSBB Journal
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• v.23 no.4
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• pp.303-310
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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 parainfluenza virus type 3 (BPIV3) is one of the common bovine pathogens and has widely been known as a contaminant of biologics. In order to establish the validation system for the BPIV3 safety of biologics, a real-time RT-PCR method was developed for quantitative detection of BPIV3 contamination in raw materials, manufacturing processes, and final products. Specific primers for amplification of BPIV3 RNA was selected, and BPIV3 RNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be 2.8 $TCID_{50}/mL$. The real-time RT-PCR method was validated to be reproducible and very specific to BPIV3. The established real-time RT-PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with BPIV3. BPIV3 RNA could be quantified in CHO cell as well as culture supernatant. Also the real-time RT-PCR assay could detect 7.8 $TCID_{50}/mL$ of BPIV3 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 BPIV3 contamination during the manufacture of biologics.