猪伪狂犬病病毒SYBR GreenⅠ实时荧光定量PCR方法的建立

doi:10.16431/j.cnki.1671-7236.2016.07.009

Abstract

Abstract:

In this study,a SYBR GreenⅠ dye based on Real-time quantitative PCR was established using the specific primers-pair according to gE gene characterization of porcine pseudorabies virus(PRV).The optimized results demonstrated that the detection assay with good linear determination range from 7.53×10¹ to 7.53×10⁶ copies per reaction.There was no cross-reaction occurred with nucleic acids extracted from the common porcine infectious diseases,such as porcine circovirus 2 (PCV2),porcine parvovirus (PPV),Haemophilus parasuis and Streptococcus susi,no amplification signals were detected.The results showed that the melting curve analysis with only one specific peaked with a melting temperature (Tm),which was (92.9±0.1)℃ at detecting PRV positive samples,also no specific peak could be detected for common porcine infectious diseases described above.Series experiments were carried out in order to assess the sensitivity,specificity and reproducibility for the method,following by the intra-assay and inter-assay CVs for Ct values obtained with the standard plasmids.The intra-assay and inter-assay statistics were 0.31% to 1.14% and 0.42% to 1.74%,respectively.All the results showed that the established method was sensitive,specific and reproducible,which meaned it could be used for the research of pathogenic mechanism of porcine pseudorabies virus.

Key words: porcine pseudorabies virus; gE gene; SYBR Green Ⅰ; Real-time quantitative PCR

CLC Number:

S852.65⁺5

CHEN Ru-jing, WU Xue-min, CHEN Qiu-yong, YAN Shan, CHE Yong-liang, WANG Chen-yan, WANG Long-bai, ZHOU Lun-jiang. Establishment of SYBR GreenⅠReal-time Quantitative PCR for Detection Porcine Pseudorabies Virus[J]. , 2016, 43(7): 1717-1722.

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Establishment of SYBR GreenⅠReal-time Quantitative PCR for Detection Porcine Pseudorabies Virus

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