SYBR Green I-based RT-PCR Assay for Detecting <i>IFN-β</i> mRNA in Duck

FANG Tiehui; DONG Hui; XIAO Shifeng; WANG Shao; CHENG Xiaoxia; LIN Fengqiang; ZHU Xiaoli; CHEN Xiuqin; ZHENG Min; CHEN Shilong; CHEN Shaoying

doi:10.19303/j.issn.1008-0384.2021.10.004

Volume 36 Issue 10

Oct. 2021

Turn off MathJax

Article Contents

Article Navigation > Fujian Journal of Agricultural Sciences > 2021 > 36(10): 1139-1144

FANG T F, DONG H, XIAO S F, et al. SYBR Green I-based RT-PCR Assay for Detecting IFN-β mRNA in Duck [J]. Fujian Journal of Agricultural Sciences，2021，36（10）：1139−1144 doi: 10.19303/j.issn.1008-0384.2021.10.004

Citation:

FANG T F, DONG H, XIAO S F, et al. SYBR Green I-based RT-PCR Assay for Detecting IFN-β mRNA in Duck [J]. Fujian Journal of Agricultural Sciences，2021，36（10）：1139−1144 doi: 10.19303/j.issn.1008-0384.2021.10.004

Citation:

PDF( 895 KB)

SYBR Green I-based RT-PCR Assay for Detecting IFN-β mRNA in Duck

doi: 10.19303/j.issn.1008-0384.2021.10.004

FANG Tiehui^1
,,
DONG Hui^{1, 2
,},
XIAO Shifeng^{1, 2},
WANG Shao^{1, 2},
CHENG Xiaoxia^{1, 2},
LIN Fengqiang^{1, 2},
ZHU Xiaoli^{1, 2},
CHEN Xiuqin^{1, 2},
ZHENG Min^{1, 2},
CHEN Shilong^{1, 2
,
,},
CHEN Shaoying^{1, 2
,
,}

1.
Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350013, China
2.
Fujian Animal Diseases Control Technology Development Center, Fuzhou, Fujian　350013, China

Received Date: 2021-08-09
Rev Recd Date: 2021-09-18

Available Online: 2021-10-23

Publish Date: 2021-10-28

Abstract

Abstract

Objective A method for detecting IFN-β in duck using SYBR Green I-based RT-PCR was developed. Methods A pair of specific primers was designed according to the GenBank nucleotide sequence on IFN-β of duck (KT428159). The gene was cloned into a pET-30a vector, and the recombinant plasmid pET-30a-IFN-β severed to establish a standard curve. The specificity, sensitivity, and repeatability of the new methodology were determined. Result The melting curves of measurement showed a sharp single peak at Tm=87.94±0.16 ℃ without non-specific amplification and primer dimers, indicating high specificity of the methodology. The Ct value ranged from 8.9 to 34.0 with a standard curve showing a linearity with R²＞99.5%. The detection limit on IFN-β was 2.84 copies/μL. The repeatability on the Ct data for the intra- and inter-groups had coefficients of variation below 0.13% and 1%, respectively. Conclusion The newly developed assay was specific, sensitive, repeatable, and suitable for the quantitative detection of IFN-β mRNA in ducks.
- Duck,
- IFN-β,
- qRT-PCR,
- SYBR Green I,
- mRNA

FullText(HTML)

References(17)

References

[1]	WALTER M R. The role of structure in the biology of interferon signaling [J]. Frontiers in Immunology, 2020, 11: 606489. doi: 10.3389/fimmu.2020.606489
[2]	RAI K R, SHRESTHA P, YANG B C, et al. Acute infection of viral pathogens and their innate immune escape [J]. Frontiers in Microbiology, 2021, 12: 672026. doi: 10.3389/fmicb.2021.672026
[3]	万春和, 朱海侠, 陈红梅, 等. 番鸭IFN-α mRNA实时荧光定量RT-PCR检测方法的建立 [J]. 中国动物传染病学报, 2012, 20(4):63−68. doi: 10.3969/j.issn.1674-6422.2012.04.012 WAN C H, ZHU H X, CHEN H M, et al. Development of a quantitative real-time PCR for detection of IFN-α mRNA of Muscovy ducks [J]. Chinese Journal of Animal Infectious Diseases, 2012, 20(4): 63−68.(in Chinese) doi: 10.3969/j.issn.1674-6422.2012.04.012
[4]	刘澜澜, 庄艳娜, 于晓红, 等. 绿头鸭IFN-α的可溶性表达及其活性分析 [J]. 生物技术通报, 2014(11):142−146. LIU L L, ZHUANG Y N, YU X H, et al. Soluble expression and activity analysis of mallard IFN-Α [J]. Biotechnology Bulletin, 2014(11): 142−146.(in Chinese)
[5]	杨发龙, 谢秀兰, 汤承, 等. 鸭瘟病毒疫苗株与强毒株诱导雏鸭IFN-α mRNA在肝脏中表达的动态定量研究 [J]. 中国预防兽医学报, 2008, 30(8):647−650. YANG F L, XIE X L, TANG C, et al. Dynamics of IFN-α mRNA expression in liver of ducks infected with duck plague virus of different virulence [J]. Chinese Journal of Preventive Veterinary Medicine, 2008, 30(8): 647−650.(in Chinese)
[6]	高全新, 刘云霞, 程玉强, 等. 鸭IFN-β启动子双荧光素酶报告基因系统的构建及活性检测 [J]. 上海农业学报, 2018, 34(3):66−71. GAO Q X, LIU Y X, CHENG Y Q, et al. Construction and activity detection of dual luciferase reporter gene system for duck IFN-β promoter [J]. Acta Agriculturae Shanghai, 2018, 34(3): 66−71.(in Chinese)
[7]	CHEN Z L, LUO G F, WANG Q X, et al. Muscovy duck reovirus infection rapidly activates host innate immune signaling and induces an effective antiviral immune response involving critical interferons [J]. Veterinary Microbiology, 2015, 175(2/3/4): 232−243.
[8]	LI N, WANG Y, LI R, et al. Immune responses of ducks infected with duck Tembusu virus [J]. Frontiers in Microbiology, 2015, 6: 425.
[9]	张盼涛, 曾显营, 杨婧, 等. 鸭IFN-γ, IL-2 mRNA荧光定量RT-PCR方法的建立及应用 [J]. 中国预防兽医学报, 2013, 35(6):472−476. doi: 10.3969/j.issn.1008-0589.2013.06.11 ZHANG P T, ZENG X Y, YANG J, et al. Establishment and application of a real-time assay for detecting of IFN-γ and IL-2 mRNA in ducks [J]. Chinese Journal of Preventive Veterinary Medicine, 2013, 35(6): 472−476.(in Chinese) doi: 10.3969/j.issn.1008-0589.2013.06.11
[10]	PIZZATO M, ERLWEIN O, BONSALL D, et al. A one-step SYBR Green I-based product-enhanced reverse transcriptase assay for the quantitation of retroviruses in cell culture supernatants [J]. Journal of Virological Methods, 2009, 156(1/2): 1−7.
[11]	TAKEUCHI O, AKIRA S. Innate immunity to virus infection [J]. Immunological Reviews, 2009, 227(1): 75−86. doi: 10.1111/j.1600-065X.2008.00737.x
[12]	陈超, 池晓娟, 白庆玲, 等. 甲型流感病毒感染过程中干扰素介导的天然免疫应答机制 [J]. 生物工程学报, 2015, 31(12):1671−1681. CHEN C, CHI X J, BAI Q L, et al. Mechanisms underlying interferon-mediated host innate immunity during influenza A virus infection [J]. Chinese Journal of Biotechnology, 2015, 31(12): 1671−1681.(in Chinese)
[13]	LI N, HONG T Q, LI R, et al. Cherry valley ducks mitochondrial antiviral-signaling protein-mediated signaling pathway and antiviral activity research [J]. Frontiers in Immunology, 2016, 7: 377.
[14]	ZHANG H H, SONG X D, LI T X, et al. DDX1 from Cherry valley duck mediates signaling pathways and anti-NDRV activity [J]. Veterinary Research, 2021, 52(1): 9. doi: 10.1186/s13567-020-00889-4
[15]	LI N, JIANG S N, ZHAO J, et al. Molecular identification of duck DDX3X and its potential role in response to Tembusu virus [J]. Developmental and Comparative Immunology, 2020, 106: 103599. doi: 10.1016/j.dci.2019.103599
[16]	HE T Q, WANG M S, CHENG A C, et al. Duck enteritis virus pUL47, as a late structural protein localized in the nucleus, mainly depends on residues 40 to 50 and 768 to 777 and inhibits IFN-β signalling by interacting with STAT1 [J]. Veterinary Research, 2020, 51(1): 1−12. doi: 10.1186/s13567-019-0731-2
[17]	ZHANG W, JIANG B W, ZENG M, et al. Binding of duck tembusu virus nonstructural protein 2A to duck STING disrupts induction of its signal transduction cascade to inhibit beta interferon induction[J]. Journal of Virology, 2020, 94(9). DOI: 10.1128/jvi.01850-19.