柑橘溃疡病菌LAMP实时浊度检测方法的建立及应用
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。
关键词 LAMP实时浊度检测方法；柑橘溃疡病菌；检测
中图分类号 S 436.661.1 文献标识码 A
Abstract The conserved hypothesis protein sequence published in GenBank（accession no.AE008923.1）of Xanthomonas axonopodis pv. citri 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 turbidimeter.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. citri. 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.
Key words LAMP real-time turbidity method； Xanthomonas axonopodis pv. citri； Detection
由地毯草黄单胞柑橘致病变种[Xanthomonas axonopodis pv. citri（Hasse） Vauterin et al.，简称柑橘溃疡病菌]引起的柑橘溃疡病（citrus bacterial canker disease，CBCD）是中国重要的检疫性有害生物，对柑橘生产危害极大，世界大部分国家对该病菌都有严格的检疫要求。该病目前广泛分布于亚洲、太平洋、印度洋岛国、非洲、南美洲和北美洲的30多个国家和地区。在中国已有14个省市（包括台湾）的柑橘产区有该病发生，尤以甜橙、柚和柑发生较严重[1-2]。
柑橘溃疡病菌，即旧的细菌分类系统中Xanthomonas campestris pv. citri中的A菌系，目前在Vauterin的新分类系统中被划分在地毯草黄单胞杆菌（Xanthomonas axonopodis）[3]。柑橘溃疡病菌侵染未老化的叶片、新梢和幼果形成病斑，严重时引起落叶、落果和枝梢枯死。同时，果实受害后还会影响外观、降低商品价值。柑橘溃疡病菌还有潜伏侵染现象，给此病的检疫、采穗和防治工作带来困难。柑橘溃疡病发生后，没有有效的防治办法，只能通过砍树烧毁等措施杜绝病害蔓延，对果农的利益造成极大的冲击[4]。因此迫切需要建立一种简单可靠的快速筛选方法，以便更好地对该菌进行检疫和监测，为柑橘溃疡病的防控提供可靠的资料和数据。