摘要
为了建立一种能够快速、准确、高效地检测山核桃干腐病菌的技术,本研究根据茶藨子葡萄座腔菌的β-tubulin基因序列设计外引物、内引物和环引物,并对扩增体系和条件进行了优化、并分析其特异性和灵敏度。结果使用羟基萘酚蓝(HNB)作为反应指示剂,反应体系(Bst DNA聚合酶0.16U/μL,外引物F3和B3均为0.2μmol/L,内引物FIP和BIP均为1.6μmol/L,环引物LB为0.8μmol/L,Mg^(2+)为4 mmol/L,dNTP为1mmol/L,甜菜碱Betaine为0.6mmol/L)在等温(64℃)条件下反应1h能特异性检测山核桃干腐病菌:以山核桃干腐病菌DNA为模板的反应液呈阳性(变为蓝色),而其余的病菌均为阴性(仍为紫色),该技术的最低检测限为1pg/μL。
The aim of this study is to set up a fast,accurate and efficient way of detecting Botryosphaeria dothidea.Outer primers,internal primers and the loop primer are designed according to the gene ofβ-tubulin for Loop-mediated isothermal amplification.Besides,the system of amplification and the condition are also optimized.The system of reaction includes0.16 U/μL Bst DNA polymerase,0.2μmol/L Outer primers：F3 and B3,1.6μmol/L internal primers：FIP and BIP,0.8μmol/L loop primer：LB,4 mmol/L Mg^（2＋）,1 mmol/L dNTP,0.6 mmol/L Betaine.The specificity and sensitivity were then analyzed.The LAMP assay efficiently amplified the target DNA in 1 hour under isothermal conditions at 64℃.A positive color（blue）was only observed in the presence of Botryosphaeria dothidea by addition of hydroxy naphthol blue as an indicator reaction prior to amplification,however none of otherpathogens changed color（still purple）.The limitation of detection of the LAMP assay for Botryosphaeria dothidea was 1 pg/μL of genomic DNA per reaction.
作者
童琪
张佳星
张宇
张传清
吴莹莹
王叶青
TONG Qi;ZHANG Jia-xing;ZHANG Yu;ZHANG Chuan-qing;WU Ying-ying;WANG Ye-qing(Department of Plant Protection,Zhejiang Agricultural and Forestry University,Lin＇an 311300,China;College of Forestry and Bio technology,Zhejiang Agricultural and Forestry University,Lin＇an 311300,China;College of Agriculture and Food Sciences,Zhejiang Agricultural and Forestry University,Lin＇an 311300,China)
出处
《河北农业大学学报》
CAS
CSCD
北大核心
2018年第4期87-91,100,共6页
Journal of Hebei Agricultural University
基金
杭州市重大科技项目(20172015A01)
国家级大学生创新训练项目(201610341026)
