摘要
目的建立中蜂囊状幼虫病病毒(chinese sacbrood,CSBV)依赖解旋酶扩增(helicase-dependent amplific ation,HDA)检测方法,并对该方法进行优化及验证。方法提取感染CSBV的中蜂囊状幼虫RNA,以其为模板进行HDA,对建立的HDA的引物浓度(原始浓度为50μmol/L,进行10、20、40、80、160、240、320倍稀释)、反应温度(60、65及70℃)及时间(60、90及120 min)进行优化,并对优化的方法进行特异性、敏感性及稳定性验证。经电镜观察、RT-PCR及优化的HDA法对37份临床样品进行检测。结果 HDA最佳引物浓度、反应温度及时间分别为5μmol/L、65℃及90 min;CSBV与健康幼虫、蜜蜂急性麻痹病毒(acute bee paralysis virus,ABPV)、蜜蜂慢性麻痹病毒(chronic bee paralysis virus,CBPV)、黑蜂王台病毒(black queen cell virus,BQCV)和残翅病毒(deformed wing virus,DWV)cDNA均无交叉反应,HDA的最低检出限为10-2ng,检测试剂保存12个月后,仍可扩增出目的条带;37份临床样品中,有6份样品的RT-PCR和HDA结果为阳性,但电镜观察仅有4份为阳性。结论建立了CSBV HDA检测方法,该方法具有较高的敏感性、特异性及稳定性,为检测其他蜂病毒及快速鉴定其他动物病毒提供了参考。
Objective To develop,optimize and verify a helicase-dependent amplification（HDA）method for detection of Chinese sacbrood virus（CSBV）. Methods RNA was extracted from larvae infected with CSBV and used as a template for HDA. The primer concentration（original concentration was 50 μmol / L, diluted by 10, 20, 40, 80, 160, 240 and 320 folds）, reaction temperature（60, 65 and 70 ℃）and reaction time（60, 90 and 120 min） for HDA were optimized, and the optimized method was verified for specificity, sensitivity and stability. Thirty-seven clinical samples were observed by electron microscopy, and tested by RT-PCR and the developed HDA method. Results The optimal reaction tempera- ture, reaction time and primer concentration were 65 ℃, 90 min and 5 μmol / L respectively. No cross reactions with the cDNAs of healthy larvae, ABPV, CBPV, BQCV and DWV were observed by the developed HDA method, with a mini- mum detection limit of 10-2ng. Target gene fragment was amplified by the HDA kit after storage for 12 months. Of the 37 clinical samples, 6 were positive for CSBV by RT-PCR and HDA, while only 4 by electron microscopy. Conclusion The HDA method for detection of CSBV was developed, which showed high sensitivity, specificity and stability. It pro- vided a good reference for detection of other bee viruses as well as rapid identification of other animal viruses.
出处
《中国生物制品学杂志》
CAS
CSCD
2014年第2期267-271,共5页
Chinese Journal of Biologicals
基金
国家自然科学基金(31372435)
辽宁省人力资源和社会保障厅资助(2011921021)
关键词
中蜂囊状幼虫病病毒
依赖解旋酶扩增
Chinese sacbrood virus（CSBV）
Helicase-dependent amplification（HDA
