苹果属3种检疫性疫霉的四重PCR分子检测  

Quadruple-PCR Molecular Detection of Three Quarantine Fungal Diseases of Malus

作　　者：朱林慧 廖芳 曹保红 李碧娟[1] 张豆豆[1] 罗加凤 李关荣[1] 

机构地区：[1]西南大学农学与生物科技学院,重庆400176 [2]天津市出入境检验检疫局,天津300461

出　　处：《西南大学学报（自然科学版）》2017年第5期7-14,共8页Journal of Southwest University(Natural Science Edition)

基　　金：国家质检总局项目(2012IK286;2013IK290);国家"十二五"科技支撑计划(2012BAK11B02)

摘　　要：为建立苹果属冬生疫霉Phytophthora hibernali、丁香疫霉P.syringae和栗黑水疫霉P.cambivora 3种检疫性疫霉的同步分子检测方法,根据疫霉属18S rRNA,ITS,HSP90和Ypt1基因分别设计通用引物,冬生疫霉、丁香疫霉及栗黑水疫霉特异引物,建立了四重PCR检测方法,并进行了灵敏度和模拟带菌测试.建立了可同时检测苹果属上冬生疫霉、丁香疫霉和栗黑水疫霉的特异四重PCR检测体系:在20μL反应体系中,最佳引物浓度组合为10μmol/L的18SUF/18SUR,PHSF/PHSR,PCSF/PCSR和PSSF/PSSR分别为0.2,0.6,0.8,1.0μL;最佳退火温度和退火时间分别为63℃和20s.该体系扩增冬生疫霉出现884bp的18S rRNA条带和232bp的ITS基因特异带,丁香疫霉出现884bp的18S rRNA条带和683bp的HSP90基因特异带,扩增栗黑水疫霉出现884bp的18S rRNA条带和314bp的Ypt1基因特异带,对照菌只出现18S rRNA条带;四重PCR反应体系检测灵敏度低于单重PCR;模拟带菌试验可同时扩增出4个片段.表明该四重PCR检测方法能实现冬生疫霉、丁香疫霉和栗黑水疫霉的同步特异检测,实现苹果属类水果及种苗检疫性疫霉的快速检测.To establish a synchronous molecular detection method for the three China entry-forbidden quar antine fungal Phytophthora pathogens of Malus （P. hibernalis, P. syringae and P. cambivora）, uni versal primers 18SUF/18SUR and specific primers PHSF/PHSR for P. Hibernalis, PSSF/PSSR for P syringae and PCSF/PCSR for P. cambivora, were designed based on the 18S rRNA, the ITS, HSP90 and Ypt 1 genes respectively, to establish a quadruple PCR detection system and to carry out its sensitivity and simulation carrier tests. The established specific quadruple PCR reaction system simultaneously detec- ting P. Hibernalis, P. syringae and P. cambivora of Malus was as follows： in a 20μL reaction system, the optimal primer combination of 18SUF/18SUR, PHSF/PHSR, PCSF/PCSR and PSSF/PSSR was 0.2, 0.6, 0.8, 1.0μL, respectively, of its 10μmol/L stock solution, and the optimal annealing temperature and time was 63℃ and 20 s, respectively. Application of this system for P. hibernalis detected an 884 bp band of 18S rRNA and a 232 bp specific band to the ITS gene, for P. syringae detected an 884 bp band of 18S rRNA and a 683 bp specific band to the HSP90 gene, and for P. cambivora detected also an 884 bp band of the 18S rRNA and a 314 bp specific band of the Ypt1 gene, while the control only detected the band of the 18S rRNA. The sensitivity of the quadruple PCR was lower than that of conventional PCR. The simulated carrier test detected the four bands simultaneously. It showed that the established quadruple PCR could achieve the synchronous and specific detection of P. hibernalis, P. syringae and P. cambivora, and thus can be used effectively to promote the quick detection of the quarantine Phytophthora patho gens of Malus fruits and seedlings.

关 键 词：苹果属 检疫性真菌 冬生疫霉 丁香疫霉 栗黑水疫霉 四重PCR分子检测 

分 类 号：S436.611.1[农业科学—农业昆虫与害虫防治]