摘要
为建立检测猪源牛病毒性腹泻病毒(BVDV)抗体的ELISA方法,经ELISA反应条件优化、特异性试验、重复性试验及与中和法、IDEXX ELISA对比试验建立了特异性ELISA。用此方法对福州市周边20个猪场的222份猪血样进行了BVDV抗体检测。结果显示,本ELISA反应条件为:BVDV Oregon C24V与NADL株联合包被(质量浓度均为31.25μg/L,每孔加50μL)的条件为37℃1h加4℃8h,家兔抗猪瘟病毒(CSFV)超免疫血清(每孔血清效价可中和400RID CSFV)阻断时间为2.0h,封闭剂和血清稀释液中NaCl的浓度和Tween-20的体积分数分别为0.7mol/L和3.00mL/L,100mL/L马血清封闭剂、1∶50稀释的待检血清、0.625 mg/L HRP-家兔抗猪IgG酶标二抗(100μL/孔)及底物的反应时间分别为2.0、0.5、0.5h及15min;检测CSFV、伪犬病病毒、猪圆环病毒2型、猪繁殖与呼吸综合征病毒阳性血清均为阴性;4份血样进行5次板内、板间重复试验结果均一致;参照中和试验,本ELISA方法的符合率(97.9%)高于IDEXX BVDV ELISA抗体检测试剂盒(93.8%);福州市周边猪群BVDV抗体阳性率为34.7%,猪场阳性率为80.0%,其中母猪群阳性率(44.6%)高于商品猪群(26.4%)。结果表明,本ELISA方法特异性强、符合率高、重复性好,适合用于猪源BVDV的血清学监测。
The study was aimed at establishing an ELISA to detect bovine viral diarrhea virus(BVDV)specific antibody in pig sera.With the established ELISA,222 blood samples from 20 swine farms in Fuzhou Prefecture of Fujian Province were tested for the infections of BVDV.Reactive conditions,specificity and conformity of ELISA were tested,together with contrast test with Neutralization method and IDEXX ELISA.The results showed that 31.25μg/L BVDV Oregon C24V(50μL/well)and 31.25μg/L BVDV NADL(50μL/well)were optimally concentration for co-coating as antigen at 37℃for 1hand at 4℃for 8h.The rabbit anti-classical swine fever virus(CSFV)antibody neutralizing 400 RID CSFV was blocking for 2.0h.The concentration of NaCl and Tween-20 in blocking solution and serum dilution was 0.7mol/L and 3.00mL/L.Reaction time of 100mL/L equine serum confining liquid,50×diluted serum sample,0.625 mg/L rabbit anti-porcine IgG secondary antibody labeled with HRP(100μL/well)and substrate was 2.0,0.5,0.5hand 15 min respectively.The ELISA had no reaction with the positive sera against CSFV,pseudorabies virus,porcine circovirus type 2,porcine reproductive and respiratory syndrome virus.The same results were observed from 5times repeated iner and intra plate test with four blood samples.With the comparation of neutralization test,coincidence rate of the established ELISA(97.9%)was higher than IDEXX BVDV ELISA(93.8%).In the field of Fuzhou Prefecture,the positive rate for BVDV antibody of swine,swine farm,sow and commercial swine was 34.7%,80.0%,44.6%and 26.4%respectively.These findings indicated that the developed ELISA with high coincidence rate,specificity and reproducibility which could provide an efficient tool for detection of swine BVDV antibody.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2014年第9期944-950,共7页
Chinese Veterinary Science
基金
福建省科技计划重大项目(2012N3004)
福州市区域科技重大项目(2011-Q-25)
福建农业职业技术学院科研专项(14-ZM-02)
