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Feb 5, 2011 - Abstract A specific and sensitive reverse transcriptase- nested polymerase chain reaction assay (RT-nPCR) was developed for the detection of ...

Curr Microbiol (2011) 62:1455–1459 DOI 10.1007/s00284-011-9883-7

A Sensitive and Reliable RT-Nested PCR Assay for Detection of Citrus tristeza Virus from Naturally Infected Citrus Plants Charith Raj Adkar-Purushothama • P. K. Maheshwar • Teruo Sano • G. R. Janardhana

Received: 5 June 2010 / Accepted: 17 January 2011 / Published online: 5 February 2011 Ó Springer Science+Business Media, LLC 2011

Abstract A specific and sensitive reverse transcriptasenested polymerase chain reaction assay (RT-nPCR) was developed for the detection of Citrus tristeza virus (CTV) from naturally infected citrus samples. Two sets of primer pairs were designed by alignment of nucleotide sequences available in GenBank database for different genotypes of CTV. RT-nPCR reaction components and thermal cycling parameters were optimized and reaction conditions were standardized. Sequencing of the PCR products from direct and nested-PCR reactions confirmed the specificity of both primer pairs. Presence of CTV specific amplicons in asymptomatic samples which were collected from diseased orchards indicated the sensitivity of the test. As RT-nPCR technique, developed in the present study, is specific and efficient in detecting CTV, this could be envisioned for diagnostic applications and surveillance.

Introduction Citrus is one of the most important fruit crops worldwide. Based on the previous reports [5], along with recent Citrus tristeza virus (CTV) outbreaks in Florida (USA) and in C. R. Adkar-Purushothama  G. R. Janardhana (&) Molecular Phytodiagnostic Laboratory, Department of Studies in Botany, University of Mysore, Manasagangothri, Mysore 570 006, Karnataka, India e-mail: [email protected] P. K. Maheshwar Department of Microbiology, Yuvaraja’s College, University of Mysore, Mysore 570 005, Karnataka, India T. Sano Laboratory of Plant Pathology, Faculty of Agriculture and Life Sciences, Hirosaki University, Hirosaki, Japan

some Mediterranean countries, tristeza has infected and killed ca. 38 million trees in America, [55 million in the Mediterranean Basin, especially in Spain, and ca. 5 million elsewhere [15]. Citrus tristeza virus is a phloem-limited Closterovirus of the family Closteroviridae, occurs in most citrus producing regions of the world, and is the most economically important virus of citrus [3]. Numerous biological strains or isolates have been found which cause a variety of symptoms such as decline and death of sweet orange as well as grapefruit and on sour orange rootstock stem pitting, seedling yellows, vein clearing, and vein corking [12], resulting in reduced vigor and small fruits on infected trees regardless of the rootstock. Most of the citrus cultivars are grown as grafted plants and CTV is commonly disseminated by grafting with virus-infected plant material and by certain aphid species in a semi-persistent manner [8]. Various detection methods have been used for CTV, such as ELISA [7], immunofluorescence [4], cDNA probe [18], RT-PCR [10], and mPCR [21]. However, in certain cases citrus viruses may be difficult to detect because of their low titer or uneven distribution. Since tristeza disease transmits through grafting and vector, the propagation of clean nursery stock, growing healthy trees and the control of the vector, are some of the important measures in managing the disease. Furthermore, trees in commercial citrus groves and private yards need to be tested regularly, so that diseased trees can be removed in order to prevent the spread of CTV. Hence, highly specific, sensitive and reliable diagnostic technique is required for monitoring the disease. Recently, reverse transcription quantitative realtime PCR assays have been developed [2, 22, 24]. These techniques are extremely rapid, specific, and sensitive. Unfortunately, machinery and chemicals for real-time PCR are still very expensive and several laboratories, especially

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homogenate of 0.5 ml was transferred to 1.5 ml tube and incubated at 60°C for 15 min with intermediate vortexing. Nucleic acids were extracted with an equal volume of phenol:chloroform (1:1), followed by precipitation through centrifugation at 10,000 rpm for 15 min at 4°C with 2.5 vol of ice-cold absolute alcohol. Obtained nucleic acid pellet was rinsed with 70% ethanol and air dried. Nucleic acid was re-suspended in 100 ll of nucleic acid-free sterile DEPC treated water.

those in underdeveloped countries, need sensitive diagnostic tools more affordable. In the present work, two sets of primer pairs were designed by aligning coat protein gene sequences of all the genotypes of Citrus tristeza virus available in GenBank. This allowed the development of a reverse transcriptasenested PCR (RT-nPCR) assay for the detection of CTV in symptomatic and asymptomatic citrus plants infected with the pathogen. Specificity of both primer pairs was checked by sequence analysis of PCR products of both the steps. The feasibility of this RT-nPCR approach technique would be very useful to select the plants to be grafted, to manage further spread of the disease, as well as for the citrus certification programs in the underdeveloped countries.

Primers, PCR amplification, and Cloning For the RT-nPCR, two pairs of primers specific for CTV coat protein gene were designed by aligning respective sequences of CTV genotypes available in GenBank using CLUSTAL W program [23]. Four primers dCTVf/dCTVr and nCTVf/nCTVr (dCTVf : 50 -ATGTTGTTGCAGCWG AGTCT-30 ); (dCTVr: 50 -AGCTCCGGTCAAGAAATCT G-30 ); (nCTVf: 50 -GATGAACGATGTGCGTCAGT-30 ); (nCTVr: 50 -CTTCAACACCCTCCCGAGT-30 ) were selected for use in a RT-nPCR assay. These primers compatibility was initially confirmed by performing BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi). First strand cDNA was synthesized through Reverse Transcription (RT) reaction using 5 ll of total nucleic acids, 1.0 ll of 10 lM of the dCTVr primer, incubated for 10 min at 95°C, and immediately quenched on ice for 5 min. A cocktail mixture of 59 1st strand buffer, 10 mM dNTP, 20 U RNasinÒ Ribonuclease Inhibitor, 200 U of M-MuLV-RT (Promega, Madison, WI, USA) was prepared according to manufacturer’s instructions. The mixture was then equally distributed to each tube containing the nucleic acid templates, and was mixed gently. Reaction mixture without any template was used as negative control. The tube was incubated at 37°C for 1 h followed by 72°C for 15 min in an Advanced Thermus 25 Thermocycler (Peqlab, Erlangen, Germany).

Materials and Methods Collection of Plant Material Ninety-one leaf samples were collected from 44 Tristeza disease symptomatic citrus plants and from 45 asymptomatic citrus plants from orchards in the citrus growing areas in Kodagu region, India. Healthy leaf samples were collected from two citrus plants maintained in greenhouse (Table 1). Isolation and Purification of Total Nucleic Acids Total nucleic acid extraction for cDNA synthesis was carried out from leaf samples as previously described by Adkar-Purushothama et al. [1] with minor modifications. Briefly, veins weighing approximately 0.3 g were chopped to a fine mince from midribs of leaf samples with a razor blade and homogenized with 2 ml of pre-heated extraction buffer (2% CTAB, 1.4 M NaCl, 20 mM EDTA pH 8.0, 100 mM Tris–Cl pH 8.0) using mortar and pestle. Clear

Table 1 Comparison between RT-PCR and RT-nPCR assay developed in this study for the presence of CTV in both disease symptomatic and asymptomatic plants Sl. No

Place of sample collection

Asymptomatic sample No of samples

RT-PCR

Symptomatic sample a,b

a,c

RT-nPCR

No of samples

RT-PCRa,b

RT-nPCRa,c

01

Virajpet

10

02

06

12

12

12

02

Madikeri

08

00

02

10

10

10

03

Somavarpet

15

01

06

08

08

08

04

Polibetta

07

02

04

08

08

08

05

Shanivarsanthe

05

00

01

06

06

06

a

Number of samples showing positive results

b

dCTVf/dCTVr primers used in RT-PCR

c

dCTVf/dCTVr primers used in first round RT-PCR followed by nested PCR using second set of primer nCTVf/nCTVr

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1457

First round of PCR detection was performed by using 2 ll of cDNA in a 25 ll reaction volume containing 2 units of Taq DNA polymerase, 109 PCR buffer, 1.5 mM MgCl2, 0.2 mM each of dATP, dCTP, dGTP, and dTTP and 10 mM each of dCTVf and dCTVr primers. All the PCR reagents were procured from Genei (Bangalore, India). PCR was performed using the following parameters; one cycle at 94°C for 2 min, 35 cycles at 94°C for 45 s, 57°C for 45 s, and 72°C for 60 s, followed by one cycle at 72°C for 10 min. The amplified PCR products were separated on 1.5% agarose containing ethidium bromide, visualized and documented in a Biorad UV Trans illuminator. Initial RT-PCR product was diluted to 1:25 before using as template for nested PCR. In nested PCR, reaction mixture (25 ll) contained 1 ll of the template DNA, 2 units of Taq DNA polymerase, 109 PCR buffer, 1.5 mM MgCl2, 0.2 mM each of dATP, dCTP, dGTP, and dTTP and 10 mM each of nCTVf and nCTVr primers. PCR was performed in a thermal cycler having one cycle at 94°C for 3 min, 35 cycles at 94°C for 45 s, 58°C for 45 s, and 72°C for 60 s, followed by one cycle at 72°C for 10 min. The amplified PCR products were separated on 2.0% agarose containing ethidium bromide and documented as described above. Amplicons of the three representative samples obtained in first PCR and nested PCR were eluted from agarose gel using the Qiagen gel purification kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Purified DNA was ligated to pGEM-T (Promega, Madison, WI, USA), cloned into Escherichia coli JM109 cells according to the manufacturer’s protocol and sequenced. Sequence comparisons against international GenBank databases were performed using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). To evaluate the sensitivity of RT-nPCR, total nucleic acid extracted for RT-nPCR (44 symptomatic samples, 45 asymptomatic samples and 2 healthy samples) was subjected for RT real-time PCR as described by Saponari et al. [22].

Result and Discussion

Fig. 1 Agarose gel (1.5%) electrophoresis of RT-PCR amplicons obtained with primer pairs dCTVf/dCTVr (expected amplicon size, 491 bp). Lane 1–15 Citrus samples, Lane 16 Healthy citrus samples

collected from green house, Lane 17 Negative control (PCR reagents with 1 ll of RT reagents without any nucleic acids), Lane M 100 bp NEB (New England Biolabs, MA) molecular marker

Total nucleic acids extracted from 91 (44 symptomatic, 45 asymptomatic, and two healthy) citrus leaf samples were subjected for reverse transcription reaction for the synthesis of cDNA followed by nested PCR detection. During diagnosis of CTV in die-back affected citrus plants by RTPCR, experiments using previously described techniques resulted in amplification of host DNA along with CTV. BLAST analyses highlighted that primers used in previous studies shared significant sequence identity with host nucleic acid. Hence, designing of highly specific new set of primers to diagnose CTV from plants was crucial. Primer pairs and RT-nPCR conditions optimized in the present work are found to be very promising in detecting CTV in both symptomatic as well as in asymptomatic plants. For RT-nPCR, new primer pairs were designed on conserved segments of CTV coat protein gene sequences and their specificity was checked by BLAST analysis. First round of PCR was done by using primer pair dCTVf/dCTVr, expected product size of 491 bp (Fig. 1), and reaction was analyzed for amplicons in agarose gel electrophoresis. All the disease symptomatic samples along with five asymptomatic samples showed amplification (Table 1). In nested PCR reactions with nCTVf/nCTVr primer pair, expected amplicons of 212 bp (Fig. 2), all symptomatic and 19 out of 45 asymptomatic samples showed amplification for the coat protein gene of CTV (Table 1). Amplification was not visualized in other 26 asymptomatic samples, two healthy samples collected from green house as well as in PCR negative control (PCR reagent without any template). Specificity of direct RT-PCR was confirmed by analyzing sequences obtained from one symptomatic and two asymptomatic samples. Moreover, as nested PCR can give false positive results, amplicons from one diseased and two asymptomatic samples, which were negative in direct RTPCR, were sequenced. All the three nucleotide sequences were identical in both the PCRs and one sequence representative of each PCR reaction was deposited in GenBank

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Fig. 2 Agarose gel (2.0%) electrophoresis of RT-PCR amplicons obtained with primer pairs nCTVf/nCTVr (expected amplicon size, 212 bp). Lane 1–15 Citrus samples, Lane 16 Healthy citrus samples

collected from green house, Lane 17 Negative control (PCR reagents with 1 ll of first PCR negative control), Lane M 100 bp NEB (New England Biolabs, MA) molecular marker

(GenBank accession no. GQ221020 for dCTVf/dCTVr and GenBank accession no. GQ221021 for nCTVf/nCTVr). In BLAST searches, these sequences showed 92–99% homology with coat protein gene of Citrus tristeza virus, highlighting the specificity of RT-nested PCR products. As some isolates of CTV are mild and do not elicit symptoms in infected plants [8], sometimes due to low titer and uneven distribution of citrus viruses it may be difficult to detect these viral particles [21]. It has been demonstrated that sweet orange and grapefruit plants that get infected with both decline-inducing as well as non decline-inducing isolates of CTV, may carry the decline-inducing isolates at a concentration that easily eluded detection by ELISA with MCA 13 [20]. Other studies have also reported that PCR methods are more sensitive than ELISA for the detection of CTV and Cucumber mosaic virus (CMV) [9, 11, 16]. Moreover, IC-RT-PCR, RT-PCR, RT-nested PCR, and RT real-time PCR techniques have been also described to detect CTV in a vector and non-vector aphid species [2, 6, 15, 17, 19, 22, 24]. Above all, real-time PCR has gained popularity over conventional PCR because it uses reduced number of cycle and there is no need for electrophoresis, staining, and gel documentation. It also has increased sensitivity, reproducibility, and the reduced risk of carryover contamination since the amplicon production is monitored in a closed tube. Amplicon production is monitored during amplification by labeling the primers, probes, or amplicons with fluorogenic molecules [14]. It is being used as a powerful tool for the rapid and sensitive detection and strain differentiation of woody plant pathogens [13]. Though the RT real-time PCR is a very highly sensitive, real-time PCR machinery and chemicals are too expensive for several laboratories especially in developing and underdeveloped countries. Hence, an equally sensitive and reliable technique is required for such laboratories for routine diagnosis. In RT-directPCR (RT-dPCR), only few asymptomatic samples (five samples) showed amplification, whereas in RT-nPCR more asymptomatic (19 samples) showed positive bands for CTV, thus proves sensitivity of the technique (Table 1). Further, sensitivity of RT-nPCR was evaluated by comparing with RT

Table 2 Comparison between RT-nPCR and RT-real-time PCR

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SI. No.a No of samplesb RT-nPCR

Realtime RT-PCR

Positive Negative Positive Negative 1

22

18

4

18

4

2

18

12

6

12

6

3

23

14

9

14

9

4

15

12

3

12

3

5

11

7

4

7

4

a

Samples numbers are given as in table 1

b

No of samples = asymptomatic sample ? symptomatic sample

real-time PCR. Results from RT real-time PCR were completely consistent with those obtained by RT-nPCR (Table 2). Moreover, when RT-nPCR experiment was repeated with the same samples under the same conditions, results were in agreement with previous data thus underscoring the reproducibility of the technique. In conclusion, RT-nPCR assay developed in this study is a specific, sensitive, and reliable diagnostic tool for CTV in both diseased symptomatic and asymptomatic plants. Hence, it can be employed alternatively to RT real-time PCR for CTV detection in plants and in insect vectors, allowing to manage the disease epidemics in citrus orchards. In addition, RT-nPCR, combined with nucleic acid extraction described here (a simple and efficient method usable on a large number of samples in a short period of time), can be employed for routine assays, for the production of pathogen-free citrus plants and for certification programs.

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