• 한국축산식품학회지
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• 제31권6호
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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-Australasian Journal of Animal Sciences
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• 제31권4호
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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.

• Korean Journal of Radiology
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• 제21권4호
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• pp.505-508
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• 2020

The epidemic of 2019 novel coronavirus, later named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still gradually spreading worldwide. The nucleic acid test or genetic sequencing serves as the gold standard method for confirmation of infection, yet several recent studies have reported false-negative results of real-time reverse-transcriptase polymerase chain reaction (rRT-PCR). Here, we report two representative false-negative cases and discuss the supplementary role of clinical data with rRT-PCR, including laboratory examination results and computed tomography features. Coinfection with SARS-COV-2 and other viruses has been discussed as well.

• Journal of Microbiology and Biotechnology
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• 제17권3호
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• pp.461-467
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• 2007

Staphylococcus aureus strains are important foodborne pathogens that produce various toxins. To evaluate the risk of the enterotoxins, four S. aureus strains from kimbap and two clinical samples were isolated and identified, and their expression of the enterotoxin genes were analyzed using a reverse transcription real-time PCR. Various enterotoxin genes were detected, including sea, seg, seh, sei, sen, seo, and sem, where each isolate contained one or two. When the mRNA detection of the enterotoxin genes was analyzed using a reverse transcriptase PCR, various levels of expression were found depending on the species and enterotoxin gene. Therefore, it is reasonable to suggest that the poisoning risk of S. aureus can be effectively evaluated based on the gene expression at the mRNA level.

• Asian Pacific Journal of Cancer Prevention
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• 제16권7호
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• pp.2923-2928
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• 2015

Background: Gallbladder carcinoma (GBC) has been stated as an Indian disease, with the highest number of cases being reported from certain districts of northeast India, which has an ethnically distinct population. Unfortunately there are no scientific reports on the underlying molecular mechanisms associated with the pathogenesis of the disease from this region. Aim: The present study evaluated the role of differential expression of human telomerase reverse transcriptase (hTERT) in the development of gall bladder anomalies. Materials and Methods: Blood and tissue samples were collected from patients undergoing routine surgical resection for clinically proven cases of gallbladder disease {cholelithiasis (CL, n=50), cholecystitis (CS, n=40) and GBC (n=30) along with adjacent histopathologically proved non-neoplastic controls (n=15)} with informed consent. Whole blood was also collected from age and sex matched healthy controls (n=25) for comparative analysis. Differential hTERT mRNA expression was evaluated by semi-quantitative rt-PCR and real-time PCR based analysis using ${\beta}$-actin as an internal control. Evaluation of differential hTERT protein expression was studied by Western blot analysis and immunoflourescence. Statistical analysis for differential expression and co-relation was performed by SPSSv13.0 software. Results: Gallbladder anomalies were mostly prevalent in females. The hTERT mRNA and protein expression increased gradiently from normal

• 한국임상수의학회지
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• 제27권5호
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• pp.508-513
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• 2010

싸이토카인은 염증 및 면역 반응의 평가에 있어서 매우 중요한 요소이다. 따라서 이들의 mRNA 수준을 정량하고 평가하는 것은 염증 및 면역 반응을 평가하는데 있어서 그 민감도가 매우 높은 방법으로 알려져 있다. 본 연구의 목적은 SYBR green dye를 이용하여 개의 싸이토카인 mRNA를 정량적 실시간 역전사 중합효소연쇄반응(real-time reverse transcriptase PCR; qRT-PCR)으로 분석을 할 수 있도록 함에 있다고 할 수 있다. 제작된 시발체(primer)의 최적화된 붙임 온도(annealing temperature; $T_a$)는 인터루킨(interleukin; IL)-$1{\beta}$, IL-6, IL-10이 각각 $62^{\circ}C$, glyceraldehyde 3-phosphate dehydrogenase (GAPDH)와 tumor necrosis factor (TNF)-${\alpha}$가 $60^{\circ}C$ 그리고 high mobility group box 1 (HMGB1)이 $58^{\circ}C$였다. 표준 정량 곡선을 이용하여 측정한 시발체의 효율성은 97.1%에서 102.%로 매우 높았고, 2차 구조물(secondary structure)과 시발체-이합체 형성(primer-dimer formation)은 융해곡선(melt-curve)분석과 전기영동을 통해 확인하였다. 이렇게 정립된 qRT-PCR 분석법은 민감도와 특이도가 매우 높은 개 싸이토카인 유전자 정량법으로 활용될 수 있을 것이다.

• Molecules and Cells
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• 제23권3호
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• pp.287-303
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• 2007

Expressed sequence tags (ESTs) and differentially expressed cDNAs from the self-fertilizing fish, Kryptolebias marmoratus were mined to develop alternative biomarkers for endocrine-disrupting chemicals (EDCs). 1,577 K. marmoratus cDNA clones were randomly sequenced from the 5'-end. These clones corresponded to 1,518 and 1,519 genes in medaka dbEST and zebrafish dbEST, respectively. Of the matched genes, 197 and 115 genes obtained Unigene IDs in medaka dbEST and zebrafish dbEST, respectively. Many of the annotated genes are potential biomarkers for environmental stresses. In a differential display reverse transcriptase-polymerase chain reaction (DD RT-PCR) study, 56 differential expressed genes were obtained from fish liver exposed to bisphenol A. Of these, 16 genes were identified after BLAST search to GenBank, and the annotated genes were mainly involved in catalytic activity and binding. The expression patterns of these 16 genes were validated by real-time RT-PCR of liver tissue from fish exposed to bisphenol A. Our findings suggest that expression of these 16 genes is modulated by endocrine disrupting chemicals, and therefore that they are potential biomarkers for environmental stress including EDCs exposure.

• 한국동물위생학회지
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• 제38권2호
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• pp.107-116
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• 2015

In this study, we developed a rapid, sensitive and specific reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for detection of swine influenza viruse (SIV) including major subtypes of swine influenza viruses H1N1, H1N2 and H3N2, and a novel subtype of influenza A virus that accidentally infected in pig population. The RT-LAMP was completed in 40 min at $58^{\circ}C$ and the sensitivity of the RT-LAMP ($1copy/{\mu}L$) was 10-fold higher than conventional reverse transcription-polymerase chain reaction (RT-PCR) ($10copy/{\mu}L$) and the same to real time RT-PCR ($1copy/{\mu}L$). Also, the result of the RT-LAMP can be confirmed without any detection system. Therefore, the RT-LAMP could be a alternative diagnostic method for SIV detection in national SIV monitoring system and clinical diagnostic laboratory in the future.

• Journal of Microbiology and Biotechnology
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• 제30권3호
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• pp.459-468
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• 2020

This study established a new polymerase spiral reaction (PSR) that combines with reverse transcription reactions for HCV detection targeting 5'UTR gene. To avoid cross-contamination of aerosols, an isothermal amplification tube (IAT), as a separate containment control, was used to judge the result. After optimizing the RT-PSR reaction system, its effectiveness and specificity were tested against 15 different virus strains which included 8 that were HCV positive and 7 as non-HCV controls. The results showed that the RT-PSR assay effectively detected all 8 HCV strains, and no false positives were found among the 7 non-HCV strains. The detection limit of our RT-PSR assay is comparable to the real-time RT-PCR, but is more sensitive than the RT-LAMP. The established RT-PSR assay was further evaluated for detection of HCV in clinical blood samples, and the resulting 80.25% detection rate demonstrated better or similar effectiveness compared to the RT-LAMP (79.63%) and real-time RT-PCR (80.25%). Overall, the results showed that the RT-PSR assay offers high specificity and sensitivity for HCV detection with great potential for screening HCV in clinical blood samples.

• 한국동물위생학회지
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• 제41권2호
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• pp.111-118
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• 2018

This study was carried out to investigate the prevalence of honeybee (Apis mellifera) diseases in Gwangju area. From November 2016 to August 2017, 89 samples were collected from 33 apiculture farms and reverse transcriptase-polymerase chain reaction (RT-PCR), polymerase chain reaction (PCR), and real time PCR were conducted. 14 infectious pathogens, including seven viruses, two bacteria, three fungi, and two parasites, were investigated from random apiculture farms in Gwangju. The percentage of infectious pathogens were as follows: Stonebrood (76.4%), Deformed wing virus (51.7%), Nosema (27.0%) in PCR and RT-PCR. This result indicated that Stonebrood was most prevalent disease in Gwangju area. And we could get similar results from real time PCR. 84.8% of farms have more than two of infectious pathogens. Stonebrood and Deformed wing virus were major diseases in almost all seasons and Black queen cell virus disease was especially prevalent in May.