• Title, Summary, Keyword: SYBR Green Quantitative RT-PCR

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Development of Real-time Quantitative PCR Assay based on SYBR Green I and TaqMan Probe for Detection of Apple Viruses (사과 바이러스 검정을 위한 SYBR Green I 및 TaqMan probe 기반의 real-time PCR 검사법 개발)

  • Heo, Seong;Chung, Yong Suk
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.65 no.4
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    • pp.496-507
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    • 2020
  • Virus infections of apples result in lowered commercial qualities such as low sugar content, weakened tree vigor, and malformed fruits. An effective way to control viruses is to produce virus-free plants based on the development of an accurate and sensitive diagnostic method. In this study, real-time PCR assays based on SYBR Green I and TaqMan probes were developed for detecting ASGV, ASPV, and ApMV viruses. These methods can detect and quantify 103 to 1011 RNA copies/μL of each virus separately. Compared with methods with two different dyes, the SYBR Green I-based method was efficient for virus detection as well as for assay using the TaqMan probe. Field tests demonstrated that real-time PCR methods developed in this study were applicable to high-throughput diagnoses for virus research and plant quarantine.

Development and Evaluation of a SYBR Green Real-time PCR Assay for Canine Cytokine Gene Expression (SYBR Green 실시간 역전사 중합효소연쇄반응을 이용한 개 싸이토카인 유전자 발현의 정량)

  • Yu, Do-Hyeon;Ihn, Dong-Chul;Park, Chul;Park, Jin-Ho
    • Journal of Veterinary Clinics
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    • v.27 no.5
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    • pp.508-513
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    • 2010
  • Cytokines are important mediators of the immune response, and quantitating cytokine mRNA is a highly sensitive and attractive method for measuring cytokine production. The objective of the current study was to develop and validate a SYBR green quantitative real-time reverse transcriptase PCR (qRT-PCR) assay for measuring canine cytokine mRNA. The optimal annealing temperatures ($T_a$) of the designed primers were $62^{\circ}C$ for interleukin (IL)-$1{\beta}$, IL-6 and IL-10; $60^{\circ}C$ for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tumor necrosis factor (TNF)-${\alpha}$; and $58^{\circ}C$ for high mobility group box 1 (HMGB1). Primer efficiencies of all primers calculated for standard curve samples were between 97.1% and 102.6%. No evidence of secondary structure or primer-dimer formation was seen via melt-curve analysis or gel electrophoresis. The developed qRT-PCR assays are highly specific and sensitive and can be used to quantify gene expression levels of canine cytokines.

Quantitative Real-time PCR using Lactobacilli as Livestock Probiotics (Real-time PCR을 이용한 가축생균제용 유산균 정량분석)

  • Choi, Yeon-Jae;Kim, Sun-Ho;Gu, Min-Jeong;Choe, Han-Na;Kim, Dong-Un;Cho, Sang-Bum;Kim, Su-Ki;Jeon, Che-Ok;Bae, Gui-Seok;Lee, Sang-Seok
    • Journal of Life Science
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    • v.20 no.12
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    • pp.1896-1901
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    • 2010
  • This study was conducted using quantitative real-time PCR using Lactobacilli as probiotics. Quantitative real-time PCR (RT PCR) was conducted via a method involving SYBR Green 1 and a probe. Plasmid DNA was cloned using the 16S-23S rRNA intergenic species region. Gene clones were diluted from $10^2$ to $10^{10}$. Standard curves were constructed via Ct values obtained from the results of Real-time PCR via the aforementioned SYBR Green 1 and probe method. Plasmid DNA was also cloned using the 16S-23S rRNA intergenic species region and the gene clones were diluted from $10^2$ to $10^{10}$ copy numbers via the probe method. Using RT PCR, a standard curve of plasmid DNA copy numbers was also determined. The slope value for the Y-axis intercept and $R^2$ value were measured as -3.346, 33.18, and 0.993, respectively, via the first method. For the second method, the slope value for the Y-axis intercept and $R^2$ were -3.321, 31.10 and 0.995, respectively. The PCR inhibitor could not express the detection curve at a copy number over $10^{10}$ via either method, owing to high DNA density. The DNA extract from probiotics was diluted without pre-culturing, and 16 products were amplified via both methods. The Ct value was 11.06~18.12 in the first method and 16.74~22.11 in the second method. Measured probiotics and log copy values were largely similar among the methods used. It was concluded that both methods are effective for analysis, but further research will be required to verify the optimal method.

Real-Time RT-PCR for Quantitative Detection of Bovine Viral Diarrhoea Virus during Manufacture of Biologics (생물의약품 제조공정에서 Bovine Viral Diarrhoea Virus 정량 검출을 위한 Real-Time RT-PCR)

  • Cho, Hang-Mee;Lee, Dong-Hyuck;Kim, Hyun-Mi;Kim, In-Seop
    • Microbiology and Biotechnology Letters
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    • v.36 no.1
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    • pp.34-42
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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 using bovine materials have the risk of viral contamination. Therefore viral validation is essential in ensuring the safety of the products. Bovine viral diarrhoea virus (BVDV) is the most common bovine pathogen and has widely been known as a contaminant of biologics. In order to establish the validation system for the BVDV safety of biologics, a real-time RT-PCR method was developed for quantitative detection of BVDV contamination in raw materials, manufacturing processes, and final products. Specific primers for amplification of BVDV RNA was selected, and BVDV RNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be 1 $TCID_{50}/mL$. The rent-time RT-PCR method was validated to be reproducible and very specific to BVDV. The established real-time RT-PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with BVDV. BVDV RNA could be quantified in CHO cell as well as culture supernatant. Also the real-time RT-PCR assay could detect $10TCID_{50}/mL$ of BVDV artificially contaminated in bovine collagen.

Gene Microarray Analysis for Porcine Adipose Tissue: Comparison of Gene Expression between Chinese Xiang Pig and Large White

  • Guo, W.;Wang, S.H.;Cao, H.J.;Xu, K.;Zhang, J.;Du, Z.L.;Lu, W.;Feng, J.D.;Li, N.;Wu, C.H.;Zhang, L.
    • Asian-Australasian Journal of Animal Sciences
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    • v.21 no.1
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    • pp.11-18
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    • 2008
  • We created a cDNA microarray representing approximately 3,500 pig genes for functional genomic studies. The array elements were selected from 6,494 cDNA clones identified in a large-scale expressed sequence tag (EST) project. These cDNA clones came from normalized and subtracted porcine adipose tissue cDNA libraries. Sequence similarity searches of the 3,426 ESTs represented on the array using BLASTN identified 2,790 (81.4%) as putative human orthologs, with the remainder consisting of "novel" genes or highly divergent orthologs. We used the gene microarray to profile transcripts expressed by adipose tissue of fatty Chinese Xiang pig (XP) and muscley Large White (LW). Microarray analysis of RNA extracted from adipose tissue of fatty XP and muscley LW identified 81 genes that were differently expressed two fold or more. Transcriptional differences of four of these genes, adipocyte fatty acid binding protein (aP2), stearyl-CoA desaturase (SCD), sterol regulatory element binding transcription factor 1 (SREBF1) and lipoprotein lipase (LPL) were confirmed using SYBR Green quantitative RT-PCR technology. Our results showed that high expression of SCD and SREBF1 may be one of the reasons that larger fat deposits are observed in the XP. In addition, our findings also illustrate the potential power of microarrays for understanding the molecular mechanisms of porcine development, disease resistance, nutrition, fertility and production traits.

Real-Time AT-PCR for Quantitative Detection of Bovine Parainfluenza Virus Type 3 during the Manufacture of Biologics (생물의약품 제조공정에서 Bovine Parainfluenza Virus Type 3 정량 검출을 위한 Real-Time RT-PCR)

  • Lee, Dong-Hyuck;Kim, Chan-Kyong;Kim, Tae-Eun;Kim, In-Seop
    • 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.

Gene Expression of Heart and Adipocyte Fatty Acid-binding Protein in Chickens by FQ-RT-PCR

  • Tu, Yunjie;Su, Yijun;Wang, Kehua;Zhang, Xueyu;Tong, Haibing;Gao, Yushi
    • Asian-Australasian Journal of Animal Sciences
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    • v.23 no.8
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    • pp.987-992
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    • 2010
  • This study was to detect the expression of heart fatty acid-binding protein (H-FABP) and adipocyte fatty acid-binding protein (A-FABP) gene mRNA in different tissues of Rugao and Luyuan chickens at 56 d and 120 d by real-time fluorescence quantitative reverse transcription polymerase-chain reaction (FQ-RT-PCR). The primers were designed according to the sequences of HFABP, A-FABP and GAPDH genes in Gallus gallus, which were used as target genes and internal reference gene, respectively. The levels of H-FABP and A-FABP gene expression were detected by SYBR Green I FQ-RT-PCR. The relative H-FABP and A-FABP gene mRNA expression level was calculated with 2-$^{{\Delta}Ct}$. Melting curve analysis showed a single peak of three genes. Intramuscular fat (IMF) content in breast muscle and leg muscle of the two chicken breeds at 120 d was higher than at 56 d. IMF content in breast muscle and leg muscle at 56 d and 120 d in Luyuan was significantly higher than in Rugao, however, abdominal fat of Luyuan was significantly lower than that of Rugao. The relative H-FABP gene mRNA expression level in cardiac muscle was the highest in both chicken breeds. The relative H-FABP and A-FABP gene expression of different tissues in Luyuan was higher than in Rugao. H-FABP gene mRNA expression had a negative effect on IMF of leg and breast muscles, and was significantly negatively correlated with IMF content. The relative A-FABP gene mRNA level in abdominal fat was higher than in liver. The A-FABP gene mRNA was not expressed in leg, breast and cardiac muscles. A-FABP gene mRNA expression level was significantly positively correlated with abdominal fat and had a significant effect on abdominal fat but not IMF content.

Real-Time RT-PCR for Validation of Reovirus Type 3 Safety During the Manufacture of Mammalian Cell Culture-Derived Biopharmaceuticals (세포배양 유래 생물의약품 생산 공정에서 Reovirus Type 3 안전성 검증을 위한 Real-Time RT-PCR)

  • Lee, Dong-Hyuck;Jeong, Hyo-Sun;Kim, Tae-Eun;Oh, Seon-Hwan;Lee, Jung-Suk;Kim, In-Seop
    • Korean Journal of Microbiology
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    • v.44 no.3
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    • pp.228-236
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    • 2008
  • Validation of viral safety is essential in ensuring the safety of mammalian cell culture-derived biopharmaceuticals, because numerous adventitious viruses have been contaminated during the manufacture of the products. Mammalian cells are highly susceptible to Reovirus type 3 (Reo-3), and there are several reports of Reo-3 contamination during the manufacture of biopharmaceuticals. In order to establish the validation system for the Reo-3 safety, a real-time RT-PCR method was developed for quantitative detection of Reo-3 in cell lines, raw materials, manufacturing processes, and final products as well as Reo-3 clearance validation. Specific primers for amplification of Reo-3 RNA was selected, and Reo-3 RNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be $3.2{\times}10^0\;TCID_{50}/ml$. The real-time RT-PCR method was proven to be reproducible and very specific to Reo-3. The established real-time RT-PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with Reo-3. Reo-3 RNA could be quantified in CHO cell as well as culture supernatant. When the real-time RT-PCR assay was applied to the validation of virus removal during a virus filtration process, the result was similar to that of virus infectivity assay. Therefore, it was concluded that this rapid, specific, sensitive, and robust assay could replace infectivity assay for detection and clearance validation of Reo-3.