柑橘溃疡病菌LAMP实时浊度检测方法的建立及应用  

Rapid Detection of Xanthomonas axonopodis pv. citri by LAMP Real-time Turbidity Method

作　　者：封立平[1] 孔德英[2] 孙涛[2] 孙冬[1] 纪瑛[1] 王简[1] 

机构地区：[1]山东出入境检验检疫局检验检疫技术中心,山东青岛266002 [2]重庆出入境检验检疫局检验检疫技术中心,重庆400020

出　　处：《热带作物学报》2015年第6期1153-1160,共8页Chinese Journal of Tropical Crops

基　　金：质检总局科研项目(No.2009IK254;2011IK186;2011IK286);"十二五"国家科技支撑计划课题(No.2012BAK11B02)

摘　　要：以柑橘溃疡病菌特异性高的假定蛋白基因(登录号:AE008923.1)为靶标,设计并筛选4条引物来特异性识别靶标序列中的6个独立区域,建立柑橘溃疡病菌LAMP实时浊度检测方法。该方法利用实时浊度仪检测反应过程中产生的焦磷酸镁白色沉淀,实现对LAMP整个反应过程的实时监控。从镁离子浓度、甜菜碱浓度、d NTPs浓度、引物浓度和反应温度5个变量参数对LAMP检测体系进行优化,并对方法的特异性、灵敏度、稳定性及实际样品检测效果进行评价。结果显示,该体系经优化后稳定性良好,可在66℃恒温条件下,60 min内完成检测;DNA检测下限可达0.02 ng/μL,灵敏度与荧光定量PCR方法相同,比常规PCR方法高1个数量级;能快速、准确地对田间疑似样品进行检测,与荧光定量PCR方法的检出情况一致,没有漏检。结果说明柑橘溃疡病菌实时浊度LAMP检测方法操作简便、所需时间短,特异性与灵敏度高,为柑橘溃疡病菌的快速筛查提供较好的途径。The conserved hypothesis protein sequence published in GenBank （accession no.AE008923,1） of Xanthomonas axonopodis pv. cirri was used to design a set of four special primers that recognized six distinct sequences of the conserved region of X. axonopodis pv. citri genome, and a real-time turbidity method for rapid detection of X. axonopodis pv, citri by loop-mediated isothermal amplification （LAMP） was established. Real-time monitoring of magnesium pyrophosphate produced from the LAMP reaction was achieved by the turhidimeter.The LAMP detection system was optimized by five variable parameters： magnesium ion concentration, dNTPs concentration, betaine concentration, primer concentration and reaction temperature, and the specificity, sensitivity, stability, and effect on real samples of this method were evaluated. Results showed that after optimizing the detection system the stability was good and detection was completed at 66℃ in 60 minutes.The lowest DNA detection limit was 0.02 ng/μL, with the same sensitivity as the real time Q-PCR method, but 10 times higher than the PCR method. This method could do field detection of suspected samples quickly and accurately, consistent with the fluorescence PCR detection methods without any missing cases. The LAMP real-time turbidity detection method is easy and quick to operate, with high specificity and sensitivity, and provides a good method for the rapid screening of X. axonopodis pv. cirri. LAMP Real time turbidity method established in this study is a new method of LAMP detection of X. axonopodis pv. citri, has not been reported at home and abroad.

关 键 词：LAMP实时浊度检测方法 柑橘溃疡病菌 检测 

分 类 号：S436.661.1[农业科学—农业昆虫与害虫防治]