柑橘黄龙病常规PCR与qPCR检测体系的比较  

Comparison of Conventional PCR and Real-Time Fluorescent Quantitative PCR Detection Systems for Citrus Huanglongbing (HLB)

作　　者：邹剑锋 郝晨星[2] 汤丹[2] ZOU Jian-feng;HAO Chen-xing;TANG Dan(College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,PRC;College of Horticulture,Hunan Agricultural University,Changsha 410128,PRC)

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128 [2]湖南农业大学园艺学院,湖南长沙410128

出　　处：《湖南农业科学》2022年第3期1-5,12,共6页Hunan Agricultural Sciences

基　　金：湖南农业大学培育基地项目(湘农培育基地[2018]1号,17KFXM05)。

摘　　要：为促进湖南省柑橘产业的可持续发展,加强对柑橘黄龙病(Huanglongbing,HLB)的监测预警,根据黄龙病菌耐热性亚洲种“Candidatus Liberibacter asiaticus”(CLas)DNA不同保守区域设计了8组引物,分别采用常规PCR和qPCR进行检测,比较2种方法的灵敏度。结果表明:常规PCR供试的4组引物中,In1/In2引物未扩增出目的条带,其他3组引物的灵敏度由高到低排列依次为OI1/OI2>LB1/LB2=OL1/OL2,OI1/OI2这组引物对HLB菌的检测下限为98 pg/μL;qPCR供试的4组引物的灵敏度由高到低排列依次为LJ900f/LJ900r>Las-I-f/Las-I-r>HLBas/HLBr>Las-O-f/Las-O-r(根据可检测到最低浓度样本的Ct值排序),其中LJ900f/LJ900r这组引物对HLB菌的检测下限为9.8 pg/μL。另外,以湖南省郴州市汝城县农业局提供的11个柑橘HLB疑似样品为材料进行检测,引物LJ900r/LJ900f进行的qPCR检测,检出率为72.7%;引物OI1/OI2进行的常规PCR,检出率为45.5%;表明引物LJ900r/LJ900f结合qPCR方法对于HLB菌的检测效率更高。The detective efficiency of two PCR systems (conventional PCR and qPCR) to verify citrus Huanglongbing (HLB) with eight pairs of primers designed according to the conservative sequences of Candidatus Liberibacter asiaticus (CLas) DNA had been compared in this paper.The results showed as follows:In conventional PCR,primer pair In1/In2 failed to amplify the target band.The sensitivity order of the other three pairs of primers was OI1/OI2>LB1/LB2=OL1/OL2.The premier primer pair was OI1/OI2 with the detection limit of Clas DNA:98 pg/μL.In qPCR,the sensitivity order of the four pairs of primers was LJ900f/LJ900r>Las-I-f/Las-I-r>HLBas/HLBr>Las-O-f/Las-O-r (counting on Ct values of the detectable lowest concentration samples).The premier primer pair was LJ900r/LJ900f with the detection limit of Clas DNA:9.8 pg/μL.Moreover,in the detection of 11 suspected citrus HLB samples provided by Rucheng County Agriculture Bureau,Chenzhou City,Hunan Province,the detection rate of qPCR with primer LJ900r/LJ900f was 72.7%,but that of conventional PCR with primer OI1/OI2 was only 45.5%.The above results indicated that qPCR with primer LJ900r/LJ900f had a higher detection rate for HLB than conventional PCR.

关 键 词：柑橘黄龙病 分子生物学 实时荧光定量PCR 常规PCR 引物设计 

分 类 号：Q789[生物学—分子生物学]