鸽白细胞介素8基因实时荧光定量PCR检测方法的建立

doi:10.16431/j.cnki.1671-7236.2016.10.006

Abstract

Abstract:

To develop a quantitative Real-time PCR method for detection of pigeon interleukin-8 (IL-8),a pair of specific primers was designed based on the conserved region of IL-8 gene sequence published on GenBank.A fragment of IL-8 gene was amplified from the pigeon lymphocytes template and cloned into pMD18-T vector.Plasmid DNA was extracted from the bacteria and was serially diluted to serve as a standard.A standard curve for the SYBR Green Ⅰ of quantitative Real-time PCR was established and the specificity,sensitivity and reproducibility of this assay were investigated.The results showed that the quantitative Real-time PCR melting curve only had one single melting peak.The assay was linear with R² values was 1.0;The reaction efficiency for the pigeon IL-8 gene was 103%.The detection limit of this assay was 16 copies per reaction.Other interleukin including IL-1β,IL-6 and IL-18 and double distilled water control were tested by this assay and the results were all negative.The CV values of intra- and inter-assay were less than 1.52%.The established quantitative Real-time PCR assay of this study was suitable for the detection of pigeon IL-8.It would provide basis for analyzing cytokine expression quantitatively after the host cell was infected by the virus.

Key words: pigeon; interleukin-8; quantitative Real-time PCR

CLC Number:

S854.4

PAN Chao, WEI Tian-chao, NONG Hai-lian, LI Xiu-feng, JIANG Gui-lin, YUAN Ya-dong, CHENG Er-cai, MO Mei-lan, WEI Ping. Development of a Quantitative Real-time PCR Method for Detection of Pigeon IL-8 Gene[J]. , 2016, 43(10): 2547-2552.

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Development of a Quantitative Real-time PCR Method for Detection of Pigeon IL-8 Gene

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