禽多瘤病毒SYBR Green荧光定量PCR检测方法的初步建立  

Preliminary establishment of a SYBR Green Quantitative PCR detection method for avian polyomavirus

作　　者：张杰 吕涵婧 刘云涛 李可 张芷若 张超凡 麻荣耀 ZHANG Jie;LYU Hanjing;LIU Yuntao;LI Ke;ZHANG Zhiruo;ZHANG Chaofan;MA Rongyao(School of Biology and Brewing Engineering,Taishan University,Taian Shandong,271000)

机构地区：[1]泰山学院生物与酿酒工程学院,山东泰安271000

出　　处：《现代牧业》2024年第4期1-7,共7页Modern Animal Husbandry

基　　金：泰安市科技发展计划项目(2021NS120)。

摘　　要：禽多瘤病毒主要感染鹦鹉幼雏,导致羽毛脱落和生长受阻,目前尚无有效治疗措施,早期准确的诊断对防控该病毒至关重要。本研究建立了一种针对禽多瘤病毒的SYBR Green荧光定量PCR检测方法。具体方法如下:选取禽多瘤病毒大T抗原编码基因作为靶标设计特异性引物,并构建含有大T抗原部分编码基因的重组质粒作为阳性标准品,通过梯度稀释建立标准曲线,公式为y=-3.621x+46.443,相关系数为0.9964,熔解曲线呈单一峰。在特异性试验中,分别应用该方法检测含有鹦鹉疱疹病毒、鹦鹉腺病毒和鹦鹉喙羽病病毒基因片段的3种假病毒以及含有禽多瘤病毒大T抗原基因的阳性标准品,结果显示除阳性标准品外,其他假病毒均未出现特异性扩增曲线,表明该方法特异性良好。在灵敏度试验中,分别应用该方法检测含有禽多瘤病毒大T抗原基因的5个阳性质粒标准品,浓度分别为5.9×10^(4)copies/μL、5.9×10^(3)copies/μL、5.9×10^(2)copies/μL、5.9×10^(1)copies/μL和5.9×10^(0)copies/μL,结果显示该方法的理论检测下限为5.9×10^(1)copies/μL。综上所述,本研究所建立的SYBR Green荧光定量PCR检测方法具有较高的特异性和灵敏度,可作为防控禽多瘤病毒感染的重要技术储备。Avian polyomavirus primarily infects young parrots,leading to feather loss and growth retardation.Currently,there are no effective treatments for this virus,making early and accurate diagnosis crucial for its control and prevention.This study established a SYBR Green quantitative PCR(qPCR)method for detecting avian polyomavirus.Specific primers targeting the large T antigen-encoding gene of the virus were designed,and a recombinant plasmid containing part of this gene was constructed as a positive standard.A standard curve was established using gradient dilutions,yielding the equation y=-3.621x+46.443 with a correlation coefficient of 0.9964,and the melting curve showed a single peak.In specificity tests,the method was applied to detect three pseudoviruses containing gene fragments of Psittacid herpesvirus,Psittacid adenovirus,and Psittacine beak and feather disease virus,along with a positive standard containing the large T-antigen gene of avian polyomavirus.Results showed that only the positive standard produced a specific amplification curve,demonstrating good specificity.Sensitivity tests involved detecting five positive plasmid standards with avian polyomavirus large T-antigen gene concentrations of 5.9×10^(4)copies/μL,5.9×10^(3)copies/μL,5.9×10^(2)copies/μL,5.9×10^(1)copies/μL,and 5.9×10^(0)copies/μL.The theoretical detection limit was determined to be 5.9×101 copies/μL.In conclusion,the SYBR Green qPCR method developed in this study is highly specific and sensitive,providing an important tool for the control and prevention of avian polyomavirus infections.

关 键 词：禽多瘤病毒 荧光定量PCR 特异性 灵敏度 

分 类 号：S852.65[农业科学—基础兽医学]