猪繁殖与呼吸综合征(PRRS)诊断DNA微阵列的构建  

Development of PRRS diagnostic DNA microarray

作　　者：肖驰[1] 曹三杰[1] 文心田[1] 

机构地区：[1]四川农业大学基因芯片实验室,四川雅安625014

出　　处：《中国兽医学报》2007年第1期6-12,共7页Chinese Journal of Veterinary Science

基　　金：教育部长江学者和创新团队发展计划资助项目(IRT0555-9)

摘　　要：为构建猪繁殖与呼吸综合征（PRRS）诊断DNA微阵列，应用RT—PCR从猪繁殖与呼吸综合征病毒（PRRSV）C14—2分离株和欧洲型弱毒疫苗株VP046扩增并克隆获得戋洲型重组质粒C14P9（基因号：AY775132）、PRRS—PS9（基因号：AY775134）、Ulb（基因号：AY775135）、Y11（基因号：AY775133）、Y12（基因号：AY775136）和欧洲型基因重组质粒E6和E8。PCR法制备的靶基因和探针纯化后，质量浓度分别为300～600mg／L和200-400mg／L。芯片杂交动力学研究表明，杂堑信号强度随靶基因和探针浓度的增加而增强，最佳靶基因质量浓度为200mg／L，探针适宜范围为3～3000μg／L，系统灵敏性为3μg／L。靶基因杂交特异性研究表明，除个别靶基因在某一温度下存在交叉反应和杂交信号弱外，大多数靶基因在40～56℃下杂交信号强。采用AY775133、AY775134、E6和E8靶基因构建PRRS诊断DNA微阵列在40℃下预杂交1h、48℃湿盒避光杂交6h后经洗涤和扫读分析，杂交信号强、斑点明显、特异性高，按SNR≥1．5为标准能够区分PRRSV蔓洲型和欧洲型。应用PRRS诊断DNA微阵列检测了AMERVAC-PRRS、PRRSV弱毒疫苗、C14—2株、SCU株和临床样本，能正确区分PRRSV基因型。研究结果表明构建的PRRS诊断DNA微阵列可用于PRRS临床诊断和基因分型。The present study was conducted to construct diagnostic PRRS DNA microarray for detecting PRRSV and distinguishing its genotype. Preparing the targets and probes by PCR,screening the best concentration of printing target,hybridizing each target at different temperature and detecting the DNA microarray, the PRRS diognostic DNA microarray was developed. The results were as follows： 1）AY775133, AY775132, AY775135, AY775133,AY775136,E6,E8 targets were amplified by One-step RT-PCR from C14-2 isolate and Spain VP046 vaccine strain and cloned using pMDT-18 vector. Targets were prepared by PCR with AY775133, AY775132, AY775135,AY775133,AY775136,E6,E8 recombinant plasmids and purified by isopropanol with the concentration of 300-600 mg/L. Probes were labeled by two-step RT-PCR with fluorescence Cy3 and its concentration was 200- 400 mg/L. 2）Curve of hybridization signal intensity step fast when target concentration was 50-200 mg/L or 3- 3 000 μg/L （probe）, and tailed off when concentration was〉 200 mg/L （target） or 3 000 μg/L （probe）. System sensitivity was 3 μg/L of probe concentration. 3）Targets was specific at 40-56℃ ,very few target had cross-reactivity due to its high identity of overlap fragment and non-stringency hybridization conditions. Diagnostic PRRS DNA microarray was fabricated by spotting 200 mg/L of AY775133,AY775134,E6 and E8 on aminated glass slides with SpotArray^TM 24 Microarray Printing System. After pre-hybridizing at 40℃ for 1 h and hybridizing at 48℃ for 6 h, the microarray was washed,scanned and analyzed using QuantAray 3.0. Its positive standard is ≥1. 5 of Signal-tonoise（SNR）. 4）The probe labeled by two-step RT-PCR with American and European type mixture primers and detected the samples with PRRS DNA microarray. The results indicated that it can distinguish genotype of PRRSV, and that PRRS diagnostic DNA microarray should be an useful tool for detecting PRRS and it＇s molecular epidemiological survey.

关 键 词：猪繁殖与呼吸综合征病毒 DNA微阵列 克隆 

分 类 号：S852.65[农业科学—基础兽医学]