应用PCR技术快速鉴定熊蜂微孢子虫Nosema bombi  

Rapid detection of Nosema bombi by using PCR technique

作　　者：张体银[1] 刘蓓[2] 田国宁[3] 白泉阳[1] 郑腾[1] 王武军[1] 张志灯[1] 于师宇[1] 

机构地区：[1]福建出入境检验检疫局福建省检验检疫技术研究重点实验室,福建福州350001 [2]福建农林大学蜂学学院,福建福州350002 [3]山东省潍坊出入境检验检疫局,山东潍坊261041

出　　处：《中国兽医学报》2017年第9期1715-1719,1730,共6页Chinese Journal of Veterinary Science

基　　金：福建省科技计划重点资助项目(2014N0001);国家质检总局科技计划资助项目(2014IK244);福建检疫局科技计划资助项目(FK2015-JS001)

摘　　要：基于熊蜂微孢子虫16SrRNA基因序列设计了1对引物,并对引物退火温度和引物浓度等反应条件进行了优化。对所建立方法的敏感性、特异性和稳定性进行了验证后,使用该方法对40份样品进行检测。结果显示,本研究建立了一种能够特异、敏感鉴定熊蜂微孢子虫的PCR方法,对熊蜂微孢子虫总DNA的敏感性达到2.86×10-5 mg/L,可以将熊蜂微孢子虫从西方蜜蜂微孢子虫和东方蜜蜂微孢子等其他可以感染熊蜂的寄生虫中区分出来,具有良好的特异性和稳定性。通过对40份熊蜂样品进行检测结果表明,整个检测过程可以在4h内完成并具有良好的适用性。本研究建立的熊蜂微孢子虫检测方法可用于进境熊蜂的检验检疫。This study was conducted to develop a PCR method for detecting Nosema bornbi and ap- ply to the inspection and quarantine of the imported bumble bees. A pair of primers, Nos-F and Nos-R,was designed based on the 16S ribosomal RNA gene of Noserna bombi, and the reaction conditions including the annealing temperature and concentration of primers were further opti- mized. The sensitivity, specificity and stability of developed detection method were evaluated, and 40 samples were detected by the method. The results showed that a polymerase chain reaction method was developed for the specific and sensitive diagnosis of the Nosema bombi in bumblebees. The sensitivity of the method to the total DNA of Nosema bombi was approximately 2. 86 ×10^-5 mg/L,and it can distinguish Nosema bombi from these other bee parasites including the Noserna apis and Nosema cerana. Moreover,40 samples from a apiary were detected by the meth- od and the test can be finished within 4 hours and has a good applicability. The method established in this study can be applied in the inspection and quarantine of Nosema bornbi from the imported bumble bees.

关 键 词：熊蜂微孢子虫 PCR 16S核糖体RNA 进境熊蜂 

分 类 号：S852.66[农业科学—基础兽医学] S895.34[农业科学—兽医学]