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

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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

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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"

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