PDF《苹果茎痘病毒TaqMan 探针实时荧光定量》

园艺学报,2015,42 (7):1400C1408.

Acta Horticulturae Sinica

1400 doi:10.16420/j.issn.0513-353x.2015-0161;

http://www. ahs. ac. cn 收稿日期:2015C03C04;

修回日期:2015C05C20 基金项目:国家现代农业产业技术体系建设专项资金项目(CARS-28) * 通信作者 Author for correspondence(E-mail:[email protected];

[email protected]) 苹果茎痘病毒 TaqMan 探针实时荧光定量 RT-PCR 检测方法的建立 秦子禹 1,2 ,孙建设 1,* ,王娜2,* ,邵建柱

1 (1 河北农业大学园艺学院,河北保定 071001;

2 河北科技师范学院园艺科技学院,河北昌黎 066600) 摘要:为建立一种检测苹果茎痘病毒(Apple stem pitting virus,ASPV)的TaqMan 探针实时荧光定 量RT-PCR 方法,根据 ASPV 外壳蛋白基因(coat protein,cp)保守序列设计了特异性引物和 TaqMan 探针,以体外转录的 RNA 为标准品构建标准曲线,并对该方法的特异性、灵敏性、重复性进行检验.建立 的标准曲线决定系数达 0.996,扩增效率为 99%;

该方法特异性好,与苹果茎沟病毒(ASGV) 、苹果褪绿 叶斑病毒(ACLSV) 、苹果锈果类病毒(ASSVd)均无交叉反应;

其最低检测限为 1.31 *

102 copies ・ μL-1 , 灵敏度比常规 RT-PCR 高100 倍;

批内和批间变异系数均小于 1.85%.该方法具有特异性强、灵敏度高、 重复性好等优点,适用于田间样品中 ASPV 的快速定量检测. 关键词:苹果茎痘病毒;

TaqMan 探针;

实时荧光定量 RT-PCR 中图分类号:S 661.1 文献标志码:A 文章编号:0513-353X(2015)07-1400-09 Development of a TaqMan Real-time RT-PCR Method for Detecting Apple stem pitting virus(ASPV) QIN Zi-yu1,2 ,SUN Jian-she1,* ,WANG Na2,* ,and SHAO Jian-zhu1 (1 College of Horticulture,Agricultural University of Hebei,Baoding,Hebei 071000,China;

2 Horticultural College of Science &

Technology,Hebei Normal University of Science &

Technology,Changli,Hebei 066600,China) Abstract: This study established a TaqMan real-time RT-PCR method for detecting Apple stem pitting virus(ASPV). A pair of primers and a TaqMan probe were designed based on the conserved nucleotide sequence of ASPV coat protein gene(cp). The RNA standard of ASPV was prepared with transcription in vitro and the standard curve was plotted. The specificity,sensitivity and reproducibility of this method were evaluated. The results showed that the coefficient of determination(R2 )was 0.996 and the amplification efficiency(E)was 99%. There was no crossing reaction with Apple stem grooving virus (ASGV) ,Apple chlorotic leaf spot virus(ACLSV)and Apple scar skin viroid(ASSVd) ,indicating a strong specificity. The detection limit of this method was 1.31 *

102 copies ・ μL-1 and the sensitivity was

100 times higher than the conventional RT-PCR. The coefficients of variation between the intra- and inter-assay were both within 1.85%. This method could be used to detect ASPV rapidly and quantitatively in field samples with strong specificity,high sensitivity and reliable reproducibility. 秦子禹,孙建设,王娜,邵建柱. 苹果茎痘病毒 TaqMan 探针实时荧光定量 RT-PCR 检测方法的建立. 园艺学报,2015,42 (7):1400C1408.

1401 Key words:Apple stem pitting virus(ASPV) ;

TaqMan probe;

real-time RT-PCR 苹果茎痘病毒(Apple stem pitting virus,ASPV)为单链正义 RNA 病毒(李丽丽 等,2010a) , 是凹陷病毒属(Foveavirus)的代表种(Martelli &

Jelkmann,1998) ,世界各地均有分布,是苹果和 梨树上普遍发生的潜隐性病毒之一,近年也有侵染樱桃(Dhir et al.,2010)和XK(Mathioudakis et al.,2006)的报道.该病毒能引起苹果茎痘病、梨脉黄病、梨石痘病等多种病害,可致苹果和梨产 量下降,品质变劣(Jelkmann,1994) ,严重影响生产.ASPV 主要通过嫁接传播,目前尚无有效的 治疗药剂和治疗措施, 繁育无病毒苗木是其防治的最有效途径 (廖明安和冷怀琼, 1993;