烟草疫霉菌LAMP检测方法的建立及验证

为了建立烟草疫霉菌（Phytophthora parasitica）可视化快速检测方法，试验以羟基萘酚蓝（HNB）为指示剂，以烟草疫霉菌特异的核糖体转录间隔区（Internal transcribed spacer，ITS）为目的DNA片段，应用LAMP设计软件设计4条引物，通过优化LAMP反应体系和反应条件，建立一种准确快速的LAMP检测方法，并对该检测方法的特异性、灵敏度及实际应用效果进行验证。结果表明，本试验建立的烟草疫霉菌LAMP反应体系具有较好特异性且灵敏度较高。特异性检测只有烟草疫霉菌株LAMP产物为阳性（蓝色），且电泳结果能够产生梯形条带；灵敏度验证在DNA水平上可达到100 fg，是普通PCR检测方法的1000倍。对田间疑似黑胫病病株及其根际土样提取的DNA进行LAMP检测，各有3份样本为阳性，且病株检测结果与组织分离法对烟草疫霉的检测结果相一致；接种2 d后，病害症状还不明显的烟株中即可检测到烟草疫霉菌。本研究建立的烟草疫霉LAMP检测体系，可快速、简捷地检测到病株及其携带土壤中的烟草疫霉菌。

Establishment of the Loop-mediated Isothermal Amplification Assay for Detection of Phytophthora parasitica

In order to establish a rapid detection method for Phytophthora parasitica,hydroxynaphthol blue(HNB)was used as an indicator for the Phytophthora nicotiana specific transcribed spacer(ITS)DNA sequence.The primers were designed with the LAMP design software.The LAMP reaction system and reaction conditions were optimized and the specificity,sensitivity and practical application of the detection method were verified.The results showed that the LAMP reaction system of Phytophthora nicotianae established in this experiment has good specificity and high sensitivity.Specific detection showed that only the LAMP product of the Phytophthora nicotiana strain is positive(blue),and the electrophoresis can produce trapezoidal bands.The sensitivity verification can reach 100 fg at the DNA level,which is 1000 times of the detection sensitivity of the common PCR method.The LAMP test was carried out on the DNA extracted from the suspected black smut disease strain and its rhizosphere soil samples,and each of the three samples were positive,and the test results of the diseased plants and the tissue separation method were consistent with the detection results of Phytophthora nicotianae.After 2 days of inoculation,Phytophthora nicotianae can be detected in tobacco plants with no obvious symptoms of tripping.The Phytophthora nicotiana LAMP detection system established in this study can quickly and easily detect the Phytophthora nicotianae in the diseased plants and their carrying soil.