柑橘溃疡病菌的普通LAMP及快速LAMP检测方法的建立  

Regular LAMP and fast LAMP for the detection of Xanthomonas axonopodis pv.citri

作　　者：张仑[1] 殷幼平[1] 吴瑜佳[1] 王中康[1] 

机构地区：[1]重庆大学生物工程学院,基因工程研究中心,重庆400030

出　　处：《植物保护》2013年第3期95-101,共7页Plant Protection

基　　金：国家自然科学基金(30971875);公益性行业(农业)科研专项(201003067)

摘　　要：建立柑橘溃疡病菌的普通LAMP和快速LAMP检测方法,使其能应用于基层检验检疫部门对病害的快速检测。利用柑橘溃疡病菌基因组特有的保守区域设计LAMP引物,通过优化反应条件,建立柑橘溃疡病菌的普通LAMP检测体系;在普通LAMP引物的基础上设计一对环引物,建立柑橘溃疡病菌的快速LAMP检测体系,并以多种参比菌DNA以及健康柑橘叶片基因组DNA为模板对普通LAMP和快速LAMP检测体系的特异性进行了验证,利用柑橘溃疡病菌菌液和DNA溶液梯度稀释液对普通LAMP和快速LAMP检测体系的灵敏度进行了验证。普通LAMP检测体系菌体和DNA检测灵敏度分别达到了2.25×104cfu和2.03×10-1 ng,快速LAMP检测体系菌体和DNA检测灵敏度分别达到了2.25cfu和2.03×10-5 ng。在特异性测试中,普通LAMP检测体系与快速LAMP检测体系均仅对柑橘溃疡病菌进行扩增,对非靶标菌和柑橘叶片基因组DNA不产生扩增,普通LAMP与快速LAMP检测体系特异性测试结果一致。快速LAMP检测体系在0.5h内就可以达到普通LAMP检测体系的扩增量,是普通LAMP检测体系反应时间的一半,大大提高了检测的效率;快速LAMP检测体系菌悬液和DNA检测灵敏度均比普通LAMP检测体系提高了10 000倍。成功地建立了柑橘溃疡病菌的普通LAMP及快速LAMP检测方法,为柑橘溃疡病菌的检测提供了一种新的简便、快速的检测手段。This study is aimed to establish the regular LAMP and fast LAMP for the detection of Xanthomonas ax- onopodis pv. cirri and make it useful for the grassroots quarantine departments. A specific region of the genome of X. axonopodis pv. citri was used for the design of regular LAMP and fast LAMP primers; the regular LAMP and fast LAMP detection system were established by optimization of reaction conditions; the specificity of the sys- tem was verified using genomic DNA of several reference strains and healthy citrus leaves by using a series of gra- dient dilution of DNA solution of X. axonopodis pv. citri and bacterial suspension. Bacterial suspension and DNA sensitivity of regular LAMP system reached 2.25 X 10~ cfu and 2.03）〈 10 1 ng, respectively, while those of fast LAMP system reached 2.25 cfu and 2.03）〈 10-s ng, respectively. In the specificity test, both regular LAMP and fast LAMP system showed high and identical specificity. The total time for the fast LAMP reaction was within 30 minutes,only half of that for the regular LAMP, greatly improving the efficiency of the analysis. The fast LAMP system was ten thousand times as sensitive as the regular LAMP system. Regular LAMP and fast LAMP for the de- tection of X. axonopodis pv. citri was successfully established. Regular LAMP and fast LAMP system provide anew, fast and easy way for the detection of X. axonopoclis pv. citri.

关 键 词：柑橘溃疡病菌 环介导等温扩增 LAMP 检疫 

分 类 号：S436.661.1[农业科学—农业昆虫与害虫防治]