Crops ›› 2020, Vol. 36 ›› Issue (3): 79-84.doi: 10.16035/j.issn.1001-7283.2020.03.013

Previous Articles     Next Articles

Establishment of RT-qPCR Detection System for Potato Spindle Tuber Viroid

Qiu Cailing1, Fan Guoquan1, Shen Yu1, Gao Yanling1, Zhang Wei1, Han Shuxin2, Zhang Shu1, Dong Xuezhi1, Ma Ji1, Bai Yanju1()   

  1. 1Potato Research Institute, Heilongjiang Academy of Agricultural Sciences/Supervision and Testing Center for Virus-Free Seed Potatoes Quality of Ministry of Agriculture and Rural Affairs (Harbin), Harbin 150086, Heilongjiang, China
    2Plant Protection Research Institure of Heilongjiang Academy of Land Reclamation Sciences, Harbin 150000, Heilongjiang, China
  • Received:2019-11-21 Revised:2020-03-01 Online:2020-06-15 Published:2020-06-10
  • Contact: Yanju Bai E-mail:yanjubai@163.com

RichHTML

5

PDF (PC)

348

Abstract:

Potato spindle tuber viroid (PSTVd) is an important disease in potato production. The disease currently relies mainly on the use of virus-free seed potatoes and isolation measures to control. For PSTVd control, it is very important to explore a detection technology with high efficiency, sensitivity and specificity. In this study, three pairs of primers and one probe were designed and the optimal combination was selected from them. By use of these primer pairs and the probe PSTV 251T, marked with 5'FAM and 3'TAMRA, a PSTVd RT-qPCR detection system was established and 22 potato samples were tested successfully using this system. Compared with the RT-PCR technology with relatively high sensitivity, the sensitivity of this RT-qPCR system was increased 100-1 000 times.

Key words: Potato spindle tuber viroid (PSTVd), RT-qPCR, Detection technology

Table 1

The sources of potato samples"

样品编号
No. of sample		 样品来源
Source of sample		 样品类型
Sample type		 PSTVd
（NASH）
1 黑龙江 试管苗 +
2 黑龙江 试管苗 +
3 黑龙江 试管苗 +
4 吉林 试管苗 +
5 内蒙古 试管苗 -
6 黑龙江 试管苗 +
7 吉林 试管苗 +
8 黑龙江 休眠块茎 +
9 内蒙古 试管苗 -
10 山东 试管苗 +
11 内蒙古 试管苗 -
12 内蒙古 试管苗 -
13 山东 休眠块茎 +
14 内蒙古 试管苗 -
15 内蒙古 试管苗 +
16 内蒙古 试管苗 -
17 内蒙古 试管苗 -
18 内蒙古 试管苗 -
19 黑龙江 休眠块茎 +
20 陕西 试管苗 +
21 吉林 试管苗 +
22 内蒙古 试管苗 +

Table 2

Design of primers and probes"

引物/探针
Primer/Probe		 方向
Direction		 序列 (5′-3′)
Sequence (5′-3′)		 备注
Note		 反应体系及程序
Reaction system and procedure
PSTV 231F 正向引物 GCCCCCTTTGCGCTGT 参考Boonham等[9] 反应体系（10μL）: 引物1（10μmol/L）1μL+引物2（10μmol/L）1μL+探针（10μmol/L）0.4μL+Master Mix（2×）10μL+PCR Grade H2O 5.6mL+质粒/cDNA 2μL
反应程序: 95℃ 10 min,1个循环;95℃ 10s,55℃ 50s,72℃ 1s,45个循环
PSTV 296R 反向引物 AAGCGGTTCTCGGGAGCTT
PSTV 234F 正向引物 CCCTTTGCGCTGTCGCTT
PSTV 350R 反向引物 TGCGGTTCCAAGGGCTAA
PSTV 356R 反向引物 ACCAACTGCGGTTCCAAG 正向引物也使用PSTV 234F
PSTV 251T 探针 CAGTTGTTTCCACCGGGTAGT 5′FAM,3′TAMRA,3对引物均用此探针

Fig.1

The PCR results of the positive colonies M: 100bp DNA ladder, the amplification product fragment is 359bp; 1-18: transformed colonies"

Fig.2

The Real-time PCR standard curve for primer pair of PSTV 231F and PSTV 296R The plasmid concentration in the standard are 3.11×1010, 3.11×109, 3.11×108, 3.11×107, 3.11×106, and 3.11×105 copies/μL, respectively. The same below"

Fig.3

The Real-time PCR standard curve for primer pair of PSTV 234F and PSTV 350R"

Fig.4

The Real-time PCR standard curve for primer pair of PSTV 234F and PSTV 356R"

Fig.5

Sensitivity detection of three primer sets The concentration of each standard is 3.11×107-3.11×10-1 copies/μL"

Table 3

Stability analysis for PSTVd detection by RT-qPCR"

质粒浓度（拷贝/μL）
Plasmid concentration (copies/μL)		 Cp1 Cp2 Cp3 Cp平均值
Cp mean		 标准差
Standard deviation		 变异系数
Variation coefficient(%)
3.11×107 17.96 17.88 17.42 17.75 0.29 1.64
3.11×106 19.89 19.94 20.12 19.98 0.12 0.61
3.11×105 21.28 21.81 22.04 21.71 0.39 1.80
3.11×104 24.79 24.60 24.81 24.73 0.12 0.47
3.11×103 28.22 28.07 27.72 28.00 0.26 0.92
3.11×102 32.05 31.36 31.58 31.66 0.35 1.11
3.11×101 33.99 34.60 34.98 34.52 0.50 1.45
3.11×100 37.71 36.98 37.40 37.36 0.37 0.98

Fig.6

The amplification curve established by RT-qPCR system for detection of PSTVd"

[1] Diener T O . Potato spindle tuber 'virus'. Ⅵ. A replicating,low molecular weight RNA. Virology, 1971(45):411-428.
[2] Singh R P, Finnie R E, Bagnall R H . Losses due to the potato spindle tuber virus. American Potato Journal, 1971,48(7):262-267.
doi: 10.1007/BF02861592
[3] Hooker W L . Compendium of potato diseases. Minnesota:American Phytopathological Society, 1981: 89-90.
[4] Badilla R, Hammond R, Rivera C . First report of potato spindle tuber viroid in Costa Rica. Plant Disease, 1999,83(11):1072.
[5] 邱彩玲 . 马铃薯纺锤块茎类病毒的qRT-PCR检测及其遗传进化分析. 哈尔滨:东北农业大学, 2015: 14-16.
[6] Balka G, Hornyak A, Balint A , et al. Development of a one-step real-time quantitative PCR assay based on primer-probe energy transfer for the detection of porcine reproductive and respiratory syndrome virus. Journal of Virological Methods, 2009,158(1):41-45.
doi: 10.1016/j.jviromet.2009.01.014
[7] 谢丽基, 谢芝勋, 庞耀珊 , 等. WSSV和IHHNV二重实时荧光PCR检测方法的建立. 水生生物学报, 2009,33(1):22-27.
[8] 谢丽基, 谢芝勋, 刘加波 , 等. 禽流感和新城疫病毒二重荧光定量RT-PCR检测方法的建立. 生物技术通讯, 2008,19(3):410-413.
[9] Boonham N, Pérez L G, Mendez M S , et al. Development of a real-time RT-PCR assay for the detection of potato spindle tuber viroid. Journal of Virological Methods, 2004,116(2):139-146.
doi: 10.1016/j.jviromet.2003.11.005
[10] Singh R P, Boucher A . Comparative detection of mild strains of potato spindle tuber viroid from the dormant potato tubers by return-polyacrylamiee gel electrophoresis and nucleic acid hybridization. Potato Research, 1988,31(1):159-166.
doi: 10.1007/BF02360033
[11] 吕典秋, 李学湛, 于德才 , 等. 马铃薯类病毒(PSTVd)非放射性标记cDNA探针的制备及其在检测上的应用. 植物病理学报, 2005,35(2):104-108.
[12] 何小源, 周广和, 成卓敏 , 等. 用聚合酶链式反应(PCR)检测马铃薯纺锤块茎类病毒. 中国病毒学, 1992,7(3):362-366.
[13] 宋静静, 蒙姣荣, 邹承武 , 等. 用小RNA 深度测序鉴定广西冬种马铃薯病毒. 中国农业科学, 2013,46(19):4075-4081.
doi: 10.3864/j.issn.0578-1752.2013.19.013
[14] 路秉翰, 汤亚飞, 于琳 , 等. 基于小RNA深度测序和RT-PCR检测侵染广东省冬种马铃薯的病毒. 植物保护学报, 2019,46(3):604-610.
[15] 张丽, 于沛侠, 齐永红 , 等. 小RNA深度测序技术分析西瓜花叶病毒蜀葵分离物. 中国生物化学与分子生物学报, 2019,35(3):324-332.
[16] 赵晓丽, 周琦, 孙宁 , 等. 啤酒花矮化类病毒实时荧光定量RT-PCR检测方法的建立与应用. 植物保护学报, 2013,40(4):309-314.
[17] 郭立新, 段维军, 张祥林 , 等. 啤酒花潜隐类病毒的实时荧光RT-PCR检测. 植物病理学报, 2012,42(5):466-473.
No related articles found!
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!