猪星状病毒4型SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立

doi:10.11843/j.issn.0366-6964.2020.02.023

Abstract

Abstract: In order to establish a SYBR Green Ⅰ fluorescence quantitative RT-PCR method for the rapid detection of Porcine astrovirus 4 (PAstV4), a pair of specific primer was designed based on the conservative ORF1a gene. The amplified fragment was cloned into pMD19-T vector. The acquared recombinant plasmid, as a standard, was detected by SYBR Green Ⅰ fluorescence quantitative RT-PCR assay. And the sensitivity, specificity and repeatability were verified. The results showed that the minimum detectable amount of the method was 50.3 copies·μL^-1, which was 100 times more sensitive than the traditional PCR, and there was no cross reaction with CSFV, PRRSV, PRV, PEDV and PDCoV. The established standard curve showed a good linear relationship with R²=1.00. The established method was used to detect 43 clinical samples collected from 2018 to 2019, with a positive rate of 18.6%. These results indicated that a convenient, specific and sensitive qPCR assay was successfully established for the detection of PAstV4.

Key words: Porcine astrovirus, RT-PCR, SYBR Green Ⅰ

CLC Number:

S852.659.6

CHEN Shaojie, LIU Lihui, WU Zhiyong, XIAO Na, YUAN Guangfu, WANG Jing, XING Yaru, LIU Ruyue, FAN Jinghui, ZUO Yuzhu. Establishment and Application of a SYBR Green Ⅰ Real-time PCR Technique for the Detection of Porcine Astrovirus 4[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(2): 404-408.

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Establishment and Application of a SYBR Green Ⅰ Real-time PCR Technique for the Detection of Porcine Astrovirus 4

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