Duplex realtime RT-PCR assay for the detection of Potato spindle ...

Duplex realtime RT-PCR assay for the detection of Potato spindle tuber viroid (PSTVd) along with ef 1-α gene of potato A. Jeevalatha, Ravinder Kumar, Baswaraj Raigond, S. Sundaresha, Sanjeev Sharma & B. P. Singh Phytoparasitica ISSN 0334-2123 Phytoparasitica DOI 10.1007/s12600-014-0452-z

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Author's personal copy Phytoparasitica DOI 10.1007/s12600-014-0452-z

Duplex realtime RT-PCR assay for the detection of Potato spindle tuber viroid (PSTVd) along with ef 1-α gene of potato A. Jeevalatha & Ravinder Kumar & Baswaraj Raigond & S. Sundaresha & Sanjeev Sharma & B. P. Singh

Received: 20 August 2014 / Accepted: 29 December 2014 # Springer Science+Business Media Dordrecht 2015

Abstract SYBR green based realtime RT-PCR assay coupled with melt curve analysis was developed for the detection of Potato spindle tuber viroid (PSTVd) along with and without internal control from potato. The amplification of the specific targets was identified by their melting points viz., 85.93±0.22, 85.62±0.34 and 82.07 ±0.23 for primer pairs PSTVd-1F/PSTVd-1R, PSTVdNFP/PSTVd-NRP and PSTVd-QFP1/PSTVd-QRP1, respectively. The realtime RT-PCR assay was 1×104 times more sensitive than RT-PCR assay and the assay could detect up to 20 copies of the target using serially diluted plasmid and up to 0.025 fg of total RNA from infected tissues diluted in healthy plant RNA. Duplex realtime RT-PCR assay was also standardized to detect PSTVd along with elongation factor 1- α (ef-1α) gene, a stable housekeeping gene of potato. Duplex realtime RT-PCR assay showed two melt peaks indicating the successful amplification both the PSTVd RNA and internal control RNA. The developed assays could consistently detect PSTVd and proved to be highly sensitive and rapid for the detection of PSTVd in post entry quarantine testing.

Keywords qRT-PCR . internal control . Melt curve analysis . standard curve A. Jeevalatha (*) : R. Kumar : B. Raigond : S. Sundaresha : S. Sharma : B. P. Singh Central Potato Research Institute, Shimla 171 001 Himachal Pradesh, India e-mail: [email protected]

Introduction The type species of Pospiviroidae, Potato spindle tuber viroid (PSTVd) consists of single stranded 356 to 361 nts RNA molecule. PSTVd RNA is extremely stable due to its complex secondary structure (Gast et al. 1996) and it is highly infectious, spread by contact through pollen and true seed. It is reported to be prevalent in temperate countries like United States, Canada, China and Argentina etc. (Diener 1979). In India, PSTVd has been reported in some of the germplasms of wild Solanum spp. imported/ introduced from other countries (Owens et al. 1992). In potato, symptoms of PSTVd are strain/cultivar/environment-dependent and may vary from severe symptoms (reduction in plant size, changes in plant growth habit) to mild and symptomless infection. Tubers may be reduced in size and may be misshapen to spindle and dumbbell shape and with conspicuous prominent eyes which are evenly distributed. Mild strains of PSTVd are reported to reduce 17-24 % yield while severe strains can reduce up to 64 % (Pfannenstiel and Slack 1980). Accurate and rapid detection of PSTVd in germplasms imported for breeding purpose is crucial to avoid introduction of the pathogen. PSTVd RNA does not contain open reading frames (ORFs) and is not known to code for any protein products. Hence, serology based diagnostic techniques are not applicable. The samples are being tested for PSTVd infection by NASH technique at post entry quarantine testing but this technique has disadvantage of using hazardous radioactive materials and also time consuming. Therefore, SYBR green based realtime RT-PCR assay was

Author's personal copy Phytoparasitica

developed and sensitivity of the assay was compared with conventional RT-PCR and validated for its routine use in post entry quarantine testing.

QRP2 were designed using Primer Express 3.0 software. The ef-1α gene specific primers (EF1-α-F/ EF1-α-R) developed by Nicot et al. (2005) was used to develop duplex realtime RT-PCR assay.

Materials and methods

RNA extraction and cDNA synthesis

Source of viroid and plant samples

Total RNA was extracted using Spectrum™ Plant Total RNA kit (Sigma-Aldrich, Missouri, USA), digested with DNase I and the integrity of the RNA was checked by running the total RNA in gel. Then first strand cDNA was synthesized using Revert aid™ first strand cDNA synthesis kit (Fermentas, USA). Three μl of template RNA, 1 μl of random hexamer primer (100μM), 4 μl of 5X reaction buffer (250 mM Tris-HCl (pH8.3), 250 mM KCl, 20 mM MgCl2, 50 mM DTT), 1 μl of RNase inhibitor (20u/ μl ), 2 μl of 10mM dNTP Mix and 1 μl of M-MuLV Reverse transcriptase (200u/ μl) and 8 μl of DEPC treated water was added, mixed gently and incubated at 25°C for 5 min followed by 60 min at 42°C and the reaction was terminated by heating at 70°C for 5 min.

PSTVd was intercepted in some of the germplasm accessions of Solanum spp. imported from other countries during post entry quarantine testing using nucleic acid spot hybridization. Some of the infected tubers of these germplasms were planted and maintained in the insect proof glass house under an average of 14h day length during summer and day/night temperatures of 25-30°C/ 15-20°C. Leaf and tuber samples from these plants were used for developing the assays. Post entry quarantine samples, tissue culture raised micro plants/mericlones of infected germplasms and positive samples maintained in glass house were used for validation. Primer designing

Realtime PCR assay Three pairs of primers amplifying the partial sequence of PSTVd RNA were designed using the complete RNA sequence of some of the PSTVd isolates from GenBank (GQ853464, AY372400, EF044304, EF044303, M88681, HQ639697, HQ639698, HQ639699, HQ639700 and HQ639701) and software Primer3 Input version 0.4.0 (Table 1). The primer pairs, PSTVd-QFP1/PSTVd-QRP1 and PSTVd-QFP2/PSTVd-

The assays were carried out in a Step one plus realtime PCR machine (Applied Biosystems). Each 20μl reaction mixture contained 10μl of Power SYBR Green PCR Master mix, 0.5μl each of 10μM forward and reverse primers, 2 μl of cDNA templates and 7μl of nuclease free water. Cycling conditions were as follows: 95 °C for 10 min followed by 40 cycles consisting of 95 °C for 15

Table 1 Primers used for RT-PCR and realtime RT-PCR detection of PSTVd Primer

Polarity

Sequence

Product size Product Tm qRT-PCR efficiency R2 (mean±S.D.)

PSTVd-1F

Sense

5’-TTCCTGTGGTTCACACCTGA-3’

246 bp

85.93±0.22

101.363

0.987

PSTVd-1R PSTVd-2F

Antisense 5’-GGGTAATATCCGAAGCGACA-3’ Sense 5’-ACTCGTGGTTCCTGTGGTTC-3’

179 bp

84.67±0.15

73.006

0.988

PSTVd-2R

Antisense 5’-GGAAGG GTGAAAACCCTGTT-3’

PSTVd-NFP PSTVd-NRP

Sense 5’- TCGGAGGAGCGCTTCAGGGATCC-3’ 267 bp Antisense 5’- CTGCGGTTCCAAGGGCTAAACACCC-3’

85.62±0.34

85.483

0.993

PSTVd-QFP1 Sense 5’- CTAAACTCGTGGTTCCTGTGGTT-3’ PSTVd-QRP1 Antisense 5’- GCCAGTTCGCTCCAGGTTT-3’

111 bp

82.07±0.23

104.582

0.995

PSTVd-QFP2 Sense 5’- TACTACCCGGTGGAAACAACTGA-3’ PSTVd-QRP2 Antisense 5’- TGCGGTTCCAAGGGCTAA-3’

95 bp

79.61±0.30

99.785

0.995

EF1-α-F EF1-α-R

101 bp

78.87±0.15

-

-

Sense 5’-ATTGGAAACGGATATGCTCCA-3’ Antisense 5’-TCCTTACCTGAACGCCTGTCA-3’


s, 60 °C (PSTVd-QFP1/PSTVd-QRP1, PSTVd-QFP2/ PSTVd-QRP2, PSTVd-1F/PSTVd-1R and PSTVd-2F/ PSTVd-2R) / 64 °C (PSTVd-NFP/ PSTVd-NRP) for 30 s and 72 °C for 30 s. After 40 amplification cycles, melt curve analysis was carried out to verify the amplicon by its melting temperature. The temperature range for the melting curve analysis was from 60 °C to 95 °C, raising by 0.3 °C each step. Sensitivity and specificity analysis The complete RNA sequence of PSTVd was amplified using PSTVd-FP (5'-CGGAACTAAACTCGTGGTTC CTG-3') and PSTVd-RP (5'-AGGAACCAACTGCG GTTCCAAGG-3') and cloned into the T/A cloning vector, pTZ57R/T (Fermentas, USA) by following the instructions of the manufacturer. Number of copies per ng of plasmid DNA harbouring the PSTVd genome was calculated using a dsDNA copy number calculator ( h t t p : / / w w w. u r i . e d u / r e s e a r c h / g s c / r e s o u r c e s / cndna.html). Concentration of the plasmid was measured using a Nanodrop 2000 spectrophotometer (Thermoscientific, Leon-Rot, Germany in triplicates and average value was taken to prepare normalized final stock solution of 1×1010 copies/ μl. Then the plasmid was serially diluted in sterile nuclease free water to get 109 to 101 copies of plasmid/μl. Two μl of diluted plasmid (2×109 to 2×100 copies) mixed with 2 μl of cDNA obtained from healthy potato leaves and used as standard for realtime PCR. Standard curve was prepared by plotting the Ct values against the amount of plasmid copy number and the viroid load was quantified in infected plants. The sensitivity of the assay was determined by using total RNA from PSTVd infected tissues along with healthy plant total RNA in realtime RTPCR. For this the infected plant RNA was serially diluted (100 ng to 0.1 fg) in sterile distilled water. Then the diluted infected RNA was mixed with 100 ng of healthy plant total RNA and converted to 20 μl cDNA. Five μl of cDNA per reaction was used further for realtime PCR assay in which the final concentration of infected plant total RNA ranged from 25 ng to 0.025 fg per reaction. Specificity of the assay was established by carrying out melting curve analysis and agarose gel electrophoresis of the realtime PCR product. Reproducibility of the assay was confirmed by repeating the assay thrice. The assay was also validated by testing the post entry quarantine

samples, mericlones and infected germplasm collections. Optimization of duplex realtime RT-PCR Duplex realtime RT-PCR assay was standardized to detect PSTVd along with elongation factor 1-α (ef-1 α) gene of potato. Two primer pairs, PSTVd-1F/ PSTVd-1R and PSTVd-QFP1/PSTVd-QRP1 were selected based on their melting peak and efficiency and used along with ef-1 α gene primer, EF-1α-F/EF-1α-R. The assay was standardized with different primer concentrations (0.2μl to 1.0μl) and modifying the cycle conditions with a range of annealing/extension temperatures (60- 64 °C) and times (30s to 60s) to get successful amplification of both the targets. Each optimized 30μl reaction mixture contained 15μl of Power SYBR Green PCR Master mix, 0.5μl each of 10μM PSTVd specific forward and reverse primers, 0.3 μl each of 10μM internal control forward and reverse primers, 2 μl of cDNA/ templates and 11.4 μl of nuclease free water. Cycling conditions were as follows: 95 °C for 10 min followed by 40 cycles consisting of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. After 40 amplification cycles, melt curve analysis was carried out to verify the amplicon by its melting temperature. The temperature range for the melting curve analysis was from 60 °C to 95 °C, raising by 0.3 °C each step.

Results Realtime RT-PCR assay and sensitivity analysis The amplicons of realtime RT-PCR assays were identified by their melting peaks at Tm (melting temperatures) 85.93±0.22, 85.62±0.34 and 82.07±0.23 for primer pairs PSTVd-1F/PSTVd-1R, PSTVd-NFP/PSTVdNRP and PSTVd-QFP1/PSTVd-QRP1, respectively (Fig. 1). Expected 246 bp, 267 bp and 111 bp amplicons were observed in agarose gel electrophoresis of the realtime RT-PCR products that further confirmed the assay specificity and reliability. A linear relationship between threshold cycle and plasmid copy number was observed (Fig. 1a, 1c and 1e) and the efficiency of the realtime RT-PCR assays using different primer pairs were 101.363 (PSTVd-1F/ PSTVd-1R), 85.483 (PSTVd-NFP/PSTVd-NRP), 104.582 (PSTVd-QFP1/PSTVd-QRP1) and 99.785


2x10 9

y = - 3.29x+40.477

2x10 7

2x10 5

CT value

2x10 4

2x10 3 2x10 2

2x10 1

2x10 0

Quantity, copy number

a

b 2x10 9 2x10 7

y = - 3.727x+41.206 2x10 5

CT value

2x10 4

2x10 3

Primer dimers

2x10 2

2x10 1

2x10 0


c

d 2x10 9

y = - 3.217x+37.476

2x10 7

2x10 5

CT value

2x10 4

2x10 3

2x10 2

2x10 1

2x10 0


e

f


Fig. 1

realtime RT-PCR assay using primer pair, PSTVdQFP1/PSTVd-QRP1. The developed realtime RTPCR assay using PSTVd-QFP1/PSTVd-QRP1 primers were 1×104 times more sensitive and the detection limit was 0.025 fg of total RNA (Table 2; Fig. 2) while the detection limit of conventional RT-PCR assay using PSTVd-NFP/ PSTVd-NRP was 0.25 pg.

Standard curves for realtime RT-PCR amplification of PSTVd with primer pair, PSTVd-1F/PSTVd-1R(a), PSTVdNFP/PSTVd-NRP(c), PSTVd-QFP1/PSTVd-QRP1(e) using 10fold serial dilutions containing 2×109 to 2×100 copies of plasmid per reaction and melt curve analysis showing the specific melting peaks of amplicons for PSTVd-1F/PSTVd-1R (b), PSTVd-NFP/ PSTVd-NRP (d), PSTVd-QFP1/PSTVd-QRP1 (f)

(PSTVd-QFP2/PSTVd-QRP2). While the primer pair, PSTVd-2F/PSTVd-2R showed lower efficiency of 73.006. The correlation coefficients (R 2), slope and intercept values of the standard curves are 0.987, -3.29, 40.477 for primers, PSTVd-1F/ PSTVd-1R; 0.993, -3.727, 41.206 for primers, PSTVd-NFP/PSTVd-NRP and 0.995, -3.217, 37.476 for primers, PSTVd-QFP1/PSTVd-QRP1, respectively. In melt curve analysis, an additional melting peak with a lower Tm of ≈78 was observed in the products of primers, PSTVd-NFP/ PSTVd-NRP (Fig. 1b) at lower concentration of the targets. Perhaps, this peak is due to primer dimers (unused primers form dimers) since no non specific products were observed in agarose gel electrophoresis of the products. While no primer dimers were observed in the amplification products of primers, PSTVd-QFP1/PSTVd-QRP1 and PSTVd-1F/PSTVd-1R. The viroid load in the infected plant tissues ranged from 7.0 × 102 to 9.2 × 106 viroid copies/ng of total RNA in

Duplex realtime RT-PCR assay In duplex realtime RT-PCR assay detection of PSTVd along with ef-1 α gene of potato, melt curve analysis showed two peaks viz., 78.87±0.15 for ef-1 α gene (EF1-α-F/EF1-α-R) and 85.93±0.22 (PSTVd-1F/ PSTVd-1R) or 82.07±0.23 (PSTVd-QFP1/PSTVdQRP1) for PSTVd RNA indicating the successful amplification of both PSTVd RNA and internal control RNA (Fig. 3a and 3b). To determine the sensitivity, duplex realtime RT-PCR assay was carried out using mixture of serial dilutions of plasmids in healthy plant cDNA and total RNA extracts of infected tissues diluted in healthy plant RNA. The results indicated that at higher dilutions of plasmid/ concentrations of total RNA from infected plants, the amplification of internal control was inhibited and while at lower dilutions/ concentrations both the peaks were observed (Fig. 2d and 3d). The sensitivity of duplex realtime RT-PCR was as

Table 2 Comparison of sensitivity of realtime RT-PCR with conventional RT-PCR Dilutions

Concentration of total RNA

realtime RT-PCR

RT-PCR

PSTVd1F/PSTVd1R

PSTVd-QFP1/PSTVd-QRP1

Uniplex

Uniplex

Duplex

Duplex

D1

25ng

+

+

+

+

+

D2

2.5 ng

+

+

+

+

+

D3

0.25 ng

+

+

+

+

+

D4

0.025 ng

+

+

+

+

+

D5

0.0025 ng

+

+

+

+

+

D6

0.25 pg

+

-

+

+

+

D7

0.025pg

-

-

+

+

-

D8

0.0025pg

-

-

+

+

-

D9

0.25fg

-

-

+

+

-

D10

0.025fg

-

-

+

+

-

(+) – Positive (-)- Negative


high as uniplex realtime RT-PCR assay and higher than RT-PCR assay. Validation of RT-PCR assay The realtime RT-PCR assay was validated using 40 random samples including 14 known positive

Internal control

samples, 10 mericlones derived from infected germplasms and 16 post entry quarantine samples and out of which all the positive samples, 10 mericlones and 8 post entry quarantine samples were found positive. Realtime RT-PCR assay could detect the viroid in few samples in which the RTPCR failed to detect the viroid (Table 3).

PSTVd

Fig. 2 Sensitivity analysis of the uniplex and duplex realtime assay using RNA of PSTVd infected tissues serially diluted in RNA extract from healthy plants- Melt curve analysis of realtime RT-PCR amplification using PSTVd-1F/PSTVd-1R (a & c) and

PSTVd Internal control

PSTVd-QFP1/PSTVd-QRP1 (b & d) primers. Dilutions D1-D10 (D1-25 ng, D2-2.5 ng, D3- 0.25 ng, D4- 0.025ng, D5- 0.0025 ng, D6-0.25 pg, D7- 0.025 pg, D8- 0.0025 pg, D9- 0.25 fg, D100.025 fg of total RNA from infected tissues; HC-Healthy control)


Internal control

Internal control

PSTVd

PSTVd

Primer dimers NC NC

PSTVd-1F/PSTVd-1R and EFα-1F/EFα-1R

PSTVd-QFP1/PSTVd-QRP1 and EFα-1F/EFα-1R

Internal control

PSTVd

PSTVd

PSTVd-1F/PSTVd-1R and EFα-1F/EFα-1R

Fig. 3 Duplex realtime RT-PCR detection of PSTVd along with internal control, ef-1 α gene. Melt curve analysis of products in infected samples (a & b) and in 10-fold serial dilutions of plasmid

Internal control

PSTVd-QFP1/PSTVd-QRP1 and EFα-1F/EFα-1R

containing 2×109 to 2×100 copies along with cDNA from healthy plants (c & d) showing two melt peaks viz., 78.87±0.15, 85.93 ±0.22 (a & c) and 78.87±0.15, 82.07±0.23 (b & d)

Table 3 Validation of realtime RT-PCR using random samples Samples

Tissues

Plant material

Positive samples/total samples realtime RT-PCR

RT-PCR

Known positive samples

Leaves/tubers

Potato/Tomato

14/14

14/14

Mericlones of infected germplasms

Leaves

Potato/Solanum spp.

10/10

7/10

Quarantine samples

Leaves

Potato/Solanum spp.

8/16

6/16

Tissue culture micro plants

Leaves

Potato

0/15

0/15


Discussions SYBR green based realtime RT-PCR assay coupled with melt curve analysis was developed for the detection of Potato spindle tuber viroid (PSTVd). Earlier, NASH techniques based on radioactive and non radioactive probes (Fluorescein labelled probes) were used to detect PSTVd which had a detection limit of 10 pg and 10 ng, respectively (Verma et al. 2006). In case of purified PSTVd RNA, sensitivity of digoxigenin labelled probe was up to 2.5 pg (Welnicki and Hiruki 1992). But in this study, using realtime RT-PCR assay we could detect up to 0.025 fg total RNA, which indicates that the developed realtime RT-PCR highly sensitive than NASH assay. These assays are also comparatively less time consuming and does not involve any hazardous chemicals. Taqman probe based realtime PCR assays for the detection pospiviroids including PSTVd have been developed and validated (Boonham et al. 2004; Roenhorst et al. 2005; Monger et al. 2010; Botermans et al. 2013). Boonham et al. (2004) found that the real time RT-PCR was 1000 times more sensitive than the chemiluminescent hybridization detection of PSTVd and it could detect up to 1 ×10-6 dilutions of infected RNA. Later, the same method was used by Lenarcic et al. (2012) who reported that the sensitivity was found up to 10 PSTVd RNA copies. Similarly, the detection limit of the realtime RT-PCR assay developed by Botermans et al. (2013) was 2.5× 10-5 dilutions (serial dilutions of infected tomato leaf sap in healthy tomato leaf sap). In this study, the detection limit of newly developed SYBR green based realtime RT-PCR assay was up to 20 copies of plasmid which is as good as the Taqman based realtime RT-PCR assays reported by Boonham et al. (2004) and Botermans et al. (2013). Moreover, Taqman probe based assays require high cost probe and it would be unaffordable in case of large scale testing. SYBR green based realtime PCR does not require any fluorescent probes and hence cost effective. In our study, we observed that SYBR green based realtime RT-PCR assay was 1×104 times more sensitive than RTPCR assay. RT-PCR protocols were also used for the specific detection of PSTVd or along with other viroids (Matsushita et al. 2010; Olivier et al. 2014). Olivier et al. (2014) observed the generic RT-PCR assay could detect at least up to a 1000-fold dilution of RNA extracts PSTVd infected tomato and analytical sensitivity was up to 10,000 times lower than the realtime RT-PCR assay proposed by Botermans et al. (2013).

Duplex realtime RT-PCR assay was also standardized to detect PSTVd along with ef-1 α gene from potato as internal control to avoid false negative results. RTPCR protocols to detect PSTVd along with plant internal controls like ND2, nad5, and ndhB gene have already been reported (MA Hui et al. 2007; Seigner et al. 2008; Hataya 2009). However, Nicot et al. (2005) reported that ef-1 α gene was the most stable housekeeping gene out of seven housekeeping genes tested during both biotic and abiotic stress conditions in potato. Hence, in this study duplex realtime RT-PCR assay was developed using ef-1 α gene as plant internal control. In support to these findings, amplification of internal control gene was observed in all healthy and infected leaf and tuber samples during validation. It shows the reliability of using this gene as internal control in duplex realtime RT-PCR detection of PSTVd. However, in duplex realtime RT-PCR, the amplification of internal control was interfered when the viroid concentration was higher. The developed realtime RT-PCR assays were more sensitive as it could detect the PSTVd up to 0.025 fg of total RNA from infected tissues. SYBR green based realtime RT-PCR assays are also cost effective and time saving. These assays will be an alternative to NASH technique and could be used in rapid screening of imported germplasms in post entry quarantine testing.

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