Citrus tristeza virus (CTV) diagnosis and strain typing by PCR- based methods Nolasco G. in

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Citrus tristeza virus (CTV) diagnosis and strain typing by PCR- based methods

Nolasco G.

in

D' Onghia A.M. (ed.), Menini U. (ed.), Martelli G.P. (ed.).

Improvement of the citrus sector by the setting up of the common conservation strategies for the free exchange of healthy citrus genetic resources

Bari : CIHEAM

Options Méditerranéennes : Série B. Etudes et Recherches; n. 33 2001

pages 95-107

Article available on lin e / Article dispon ible en lign e à l’adresse :

--- http://om.ciheam.org/article.php?ID PD F=2001699

--- To cite th is article / Pou r citer cet article

--- Nolasco G. Citru s tristeza viru s ( CTV) diagn osis an d strain typin g by PCR -based meth ods. In : D 'Onghia A.M. (ed.), Menini U. (ed.), Martelli G.P. (ed.). Improvement of the citrus sector by the setting up of the common conservation strategies for the free exchange of healthy citrus genetic resources. Bari : CIHEAM, 2001. p. 95-107 (Options Méditerranéennes : Série B. Etudes et Recherches; n. 33) ---

http://www.ciheam.org/

http://om.ciheam.org/

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methods

G. Nolasco Universidade do Algarve

Faro, Portugal

Decline of citrus plants grafted on sour orange rootstock is the commonest aspect usually associated to citrus tristeza virus. Manifestation of decline may range from being almost un-noticeable, extending over a period of years, to quick decline in which the tree dies in a matter of months. Slow decline is very frequent on the Mediterranean Basin. In some citrus producing areas outside the Mediterranean region, where sour orange is not extensively used as a rootstock, it is common to find plants exhibiting stem-pitting symptoms on the branches which are associated to a size reduction of the plants and fruits.

The existence of virus strains is one cause for this wide range of symptoms.

which differ heavily on their economic importance.

The current tendency to use tolerant rootstocks has allowed the virus to spread covering vast regions where a multiplicity of strains may develop.

Diagnosis of CTV which since the onset of ELISA has been regarded as a matter of detection. aiming to know if the virus is present or not, is now insufficient. From a practical point of view the adoption of efficient control measures requires the knowledge about the strains that are present on a certain region or that are being introduced.

In this communication it is presented a two-step method for diagnosis of CTV.

The first step aims at the detection and may also be accomplished by ELISA.

The viral particles, if present in the sample to be analysed, are captured by antibodies and the coat protein (CP) gene is reverse transcribed and amplified by the polymerase chain reaction (immunocapture / RT-PCR). During the PCR reaction the DNA molecules that are produced are labelled with a rare molecule digoxigenin. These PCR products are mainly single stranded due to an imbalance of the primers concentration (asymmetric PCR). The single stranded DNA molecules are captured by hybridisation to a short capture probe immobilized on a microtitre plate and detected by digoxigenin specific antibodies. This results in a highly sensitive ability to detect the virus.

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The second step aims at typing the CTV strains that may be present on the sample. The positive samples are re-amplified in a similar way and hybridised to an array of strain specific capture probes in a microtitre plate.

Design of the strain specific probes was based on the alignment of a large number (160) of CP gene sequences. It was verified that these sequences clustered in 7 groups according to the biological properties (symptoms induced) of the virus. These probes have approximately 20 nucleotides and explore single nucleotide differences that exist in certain regions of the of the CP gene, resulting in cluster specific efficiencies of hybridisation. The pattern of reaction of each sample is analysed in an excel spreadsheet enabling the identification of the strains present and with their approximate proportion.

The two examples shown refer to very different epidemiological conditions.

On Madeira island CTV was detected on 1995, possibly introduced from South America. Besides CTV is also present its most efficient vector Toxoptera citricida. Most of the samples analysed on the island contain strains from groups 3A, 4 and 5. These strains which are not widespread on the Mediterranean region produce quick decline and stem-pitting symptoms and constitute a serious threat if introduced in the Mediterranean basin. Recent results have shown that some of these strains have been in a certain moment present in Morocco, without spreading.

The second example refers to Portugal mainland where CTV has been introduced in the recent years with budwood coming from Spain. The strains present on these samples are usually of the mild type which do not produce stem-pitting or quick-decline. However it can be seen also that in some cases were detected severe strains, in varietal collections or in budwood from unknown origin present on some nurseries, for which eradication was a first priority.

Options Méditerranéennes, Série B n. 33 96

Citrus tristeza virus (CTV) diagnosis and strain typing by PCR-based methods

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CTV Diagnosis and strain typing by PCR based methods

• Praxis 2159-95, FCT, Portugal

• New approach for diagnosis and prevention of tristeza outbreaks (FIGCF 03)

Infection by CTV may originate different aspects besides tristeza in strict sense – decline of plants on sour orange:

Mediterranean basin:

no symptoms on mexican lime or other indicators mexican lime symptoms, slow decline mexican lime symptoms, quick decline Exotic strains

Stem-pitting of sweet orange and/or grapefruit and/or decline

http://www.ecoport.org> /resources/slide shows

Madeira island + toxoptera citricida

in some point in the future, the actual concept of CTV diagnosis based on broad spectrum antibodies will be useless due to the change to tolerant rootstocks and increase of natural transmission of CTV

Objectives

Problems

High volume of samples Low technical skill

Methods that:

are user-friendly are cheap require low degree of

manipulation Detection High sensitivity, broad spectrum Typing Strain discrimination

CTV Diagnosis 1

3

5

7

2

4

6

8

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Biological

Indexing Electron Microscopy

Imunological ELISA Immunoprinting

Genomic Target Amplification

Routine Diagnosis

Unifying methodology

Diagnostic Methods for Virus and Virus-like agents

Purifying the target Sample preparation by:

Nucleic acid extraction Probe capture Immunocapture Target amplification Not a bottleneck: PCR, LCR, NASBA, ...

Getting the results Assays for amplicon detection:

Electrophoresis Heterogeneous colorimetric Homogeneous Fluorescent

5’-exonuclease Sunrise Molecular beacons

Bottlenecks in Amplification Based Methods

Solid phase coated with IgG

Immunocapture

Incubate for cDNA synthesis

PCR amplification Add plant sap

Wash, add RT-PCR reagents

IC/RT-PCR IC/RT-PCR – in practice

Total duration: 6h30 Incubate in thermocycler Immunocapture

IgG coated tubes

(8 x 12 format) Wash, add RT-PCR

reagents Add aqueous

plant extract

Purifying the target Sample preparation by:

Nucleic acid extraction Probe capture Immunocapture Target amplification Not a bottleneck: PCR, LCR, NASBA, ...

Getting the results Assays for amplicon detection:

Electrophoresis Heterogeneous colorimetric

Asymmetric PCR-ELISA Homogeneous Fluorescent

5’-exonuclease (taqman) Sunrise

Molecular beacons

Bottlenecks in Amplification Based Methods

Nolasco, G., Sequeira, Z., Bonacalza, B., Mendes, C., Torres, V., Sanchez, F.

Urgoiti, B., Ponz, F., Febres, V., Cevik, B., Lee, R.F., Niblett, C.L.,(1997) Sensitive CTV diagnosis using immunocapture reverse transcriptional Polymerase chain reaction and exonuclease fluorescent probe assay.

Fruits, 52 391-396

Nolasco, G., Sequeira, Z., Sabino, J., F., Febres, V., Cevik, B., Lee, R.F., Niblett, C.L.,(1999) PCR-based detection and strain typing of citrus tristeza virus. Petria 9: 131-135

Nolasco, G., Sequeira, Z., Soares, C., Mansinho, A., Bailey, A.M., Niblett Asymmetric PCR ELISA: increased sensitivity and reduced costs for the detection of plant viral nucleic acids.

European Journal of Plant Pathology (accepted w/ minor revision)

Asymmetric PCR - ELISA

Asymmetric PCR

DIG - dUTP

9

11

13

15

10

12

14

16

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0 500 1000 1500 2000 2500

1 1+ 9 1+ 99 1+ 999

A405 x 1000

Taqman Asymmetric PCR Detection in pooled samples

ELISA

17

19

21

23

18

20

22

24

Coat ELISA plate with Streptavidin

Add the biotinylated capture probe (broad spectrum probe – 150 CP sequences)

Add the asymmetric RT-PCR Dig labelled product, hibridise.

Add the anti-DIG-f(ab’)2 alkaline phosphatase conjugate

Asymmetric PCR ELISA

wash

Add substrate. Absorbance readings at 405 nm.wash

0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0

0 1 2 3 4

-log [amount of target DNA(pg)]

Absorbanceor1/1000xFluorescence

Comparison of the fluorescent exonuclease assay (Taqman system, closed circles) and asymmetric PCR ELISA (after 15 min of substrate incubation – closed squares and 1 h - closed triangles; note that at the highest concentrations the absorbance readings at 1h were already higher than 4) for the detection of decreasing amounts of a cloned coat protein gene of CTV isolate B53. Each point represents the mean of three replicates. The values of the mean of three negative controls (non-template) have been subtracted. The positive / negative threshold is represented at its approximated position as the horizontal dashed line for Taqman (threshold = 0.143) and the dotted line for asymmetric PCR ELISA (threshold = 0.014). The values obtained with the lowest amount of target were 0.023 and 0.075 for Taqman and asymmetric PCR ELISA respectively.

0 0 , 5 1 1 , 5 2 2 , 5 3 3 , 5 4 4 , 5

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88

0 0 ,5 1 1 ,5 2 2 ,5 3 3 ,5 4 4 ,5

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88

ELISA

Asymmetric PCR ELISA

Objectives

Problems

High volume of samples Low technical skill

Methods that:

are user-friendly are cheap require low degree of

manipulation Detection High sensitivity, large spectrum

Typing Strain discrimination

CTV Diagnosis

0.01

B185ARF3

B128 10C

10D 13-117a 8a 15a

8.38.18.2 13 117b13C

398A B165

B185crf1 s27S23 t388

ssperf5 sspfrf1 B31arf3

130A 28-121novo13A 28C 34a M97BRF5

ACHT225A Hedt Mikt VTa

ST ST4 MortVT B7 ssphrf5 OSPARF1 B53 CU17b 19-121

TR23 TR12TR13TR5 TR22 K

B31-12 B31BRF4TNEGRF5TNEFRF5B1BRF6 TNJRF5PA2ARF8OSPBRF1 SSPBRF3

SSPDRF5B30ARF1SSPCRF3 PB88FRF1 163BRF1

RB2FRF5 P192DRF1RB2IRF5SSPARF1 201a2 ML94-3-5b ML-B.2202b1 ML94-3-8a P53DRF1

201a3 ML94-3-5c ML94-3-8bML-J.1219j1202b2T10aT10b217h1FS84

Ht T36 2513bcp1T36-15B23-7 4838cp2 T66D11-5FL14215f1

ML94-3-5aML-A.1 D9-5 D12-12D15-16D20-8 D32-5

B23-2 D16-3D10-10 15b 216g1

Ctv2 9815-118novo 11a

134B

B15-30

B249 Mt

MLE-1 214E1 214e1FL7

1174cp2 1174cp11174 2773cp 2773cp1 2773cp2B57-31B14-62012cp2

2012cp3 Treunion11a

B1ARF6 SSPJRF5 Pb51erf

T3134A B16arf4 B16brf4

B220 B227

B57-7

B67 B213B188

B215aB215 203c1 203C-1 HG-C1 T30 T26

B52

B23-15 B52-27 204D-1 204d1HG-D1 204D-2 204d2D16-43D16-44

T4

T41B155ARF3 B155BRF3 25-120 2-93B274 B272

120B S10S9 20.2

T306 40g 40d B35 B5B32

Gr 1

Gr 2 Gr 3b

Gr 3a Gr M Gr 4

Gr 5

0.01

B185ARF3

B128 10C

10D 13-117a 8a 15a

8.38.18.2 13 117b13C

398A B165

B185crf1 s27S23 t388

ssperf5 sspfrf1 B31arf3

130A 28-121novo13A 28C 34a M97BRF5

ACHT225A Hedt Mikt VTa

ST ST4 MortVT B7 ssphrf5 OSPARF1 B53 CU17b 19-121

TR23 TR5 TR12TR13 TR22 K

B31-12 B31BRF4TNEGRF5TNEFRF5B1BRF6 TNJRF5PA2ARF8OSPBRF1 SSPBRF3

SSPDRF5B30ARF1SSPCRF3 PB88FRF1 163BRF1

RB2FRF5 P192DRF1RB2IRF5SSPARF1 201a2 ML94-3-5b ML-B.2202b1 ML94-3-8a P53DRF1

201a3 ML94-3-5c ML94-3-8bML-J.1219j1202b2T10aT10b217h1FS84

Ht T36 2513bcp1T36-15B23-7 4838cp2 T66D11-5FL14215f1

ML94-3-5aML-A.1 D9-5 D12-12D15-16D20-8 D32-5

B23-2 D16-3D10-10 15b 216g1

Ctv2 9815-118novo 11a

134B

B15-30

B249 Mt

MLE-1 214E1 214e1FL7

1174cp2 1174cp11174 2773cp 2773cp1 2773cp2B57-31B14-62012cp2

2012cp3 Treunion11a

B1ARF6 SSPJRF5 Pb51erf

T3134A B16arf4 B16brf4

B220 B227

B57-7

B67 B213B188

B215aB215 203c1 203C-1 HG-C1 T30 T26

B52

B23-15 B52-27 204D-1 204d1HG-D1 204D-2 204d2D16-43D16-44

T4

T41B155ARF3 B155BRF3 25-120 2-93B274 B272

120B S9 S10 20.2

T306 40g 40d B35 B5B32

Coat protein gene – 672 nt 160 sequences

Stem-pitting, reduction of fruit size even on tolerant rootstocks

Decline on sour orange Gr 1

Gr 2 Gr 3a Gr 3b Gr 4 Gr 5 Gr M

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25

27

13

31

26

28

30

32

5’ 3’

Strain specific probes

Samples to be tested

1 2 3b 3a 4 5 8 -

Coat protein gene

Probe / sample pl1 pl2 pl3 pl4 pl5 pl6 pl7 pl8 pl9 pl10 pl11 C-

B1 0,007 0,004 0,003 0,006 0,003 0,004 0,004 0,005 0,004 0,004 0,005 0,004

B2 0,14 0,187 0,003 0,164 0,009 0,006 0,004 0,259 0,005 0,006 0,005 0,004

B3a 0,249 0,006 0,005 0,004 0,004 0,004 0,092 0,007 0,005 0,008 0,006 0,004

B3b 0,004 0 0,002 0 0,215 0,089 0,003 0,001 0,062 0,005 0,002 0

III 0,147 0,004 0,077 0,004 0,176 0,004 0,004 0,009 0,004 0,006 0,005 0,006

B4 0,006 0,005 0,006 0,006 0,004 0,008 0,317 0,005 0,006 0,005 0,006 0,005

B5 0,005 0,003 0,003 0,004 0,16 0,004 0,003 0,003 0,004 0,006 0,006 0,006

B8 0,016 0,021 0,004 0,025 0,009 0,353 0,005 0,027 0,241 0,088 0,031 0,013

probe / strainsGR 1 GR2 GR3a GR3b Gr4 Gr5 Gr M

B1 0,466 0,006 0,006 0,006 0,007 0,007 0,007

B2 0,004 0,155 0,005 0,005 0,005 0,007 0,005

B3a 0,305 0,013 0,24 0,012 0,108 0,006 0,208

B3b 0,041 0,004 0,004 0,276 0,018 0,063 0,169

III 0,004 0,006 0,016 0,462 0,007 0,005 0,006

B4 0,004 0,004 0,011 0,006 0,472 0,008 0,028

B5 0,005 0,004 0,004 0,004 0,005 0,31 0,006

B8 0,235 0,008 0,005 0,016 0,005 0,016 0,543

Resultados pl1 pl2 pl3 pl4 pl5 pl6 pl7 pl8 pl9 pl10 pl11

Gr 1 0 0 2 0 0 0 0 0 0 0 2

Gr 2 41 99 6 98 2 0 0 99 0 12 24

Gr 3a 46 0 1 0 0 0 11 0 0 0 0

Gr 3b 13 0 82 0 42 0 0 0 0 5 8

Gr 4 0 0 5 0 0 0 89 0 0 0 5

Gr 5 0 0 4 0 56 0 0 0 1 8 16

Gr M 0 1 0 2 0 100 0 1 98 74 44

Strain composition (%) Maximum reaction speed

Pattern of reaction of type strains with probes

T. citricida, 1994? ... CTV 1995 Gr 1

Gr 2 Gr 3a Gr 3b Gr 4 Gr 5 Gr M

gr2

gr2 gr5

gr5

Gr 1 Gr 2 Gr 3a Gr 3b Gr 4 Gr 5 Gr M

gr2 gr2

Identification to the Species Level of the Plant Pathogens Phytophthora and Pythium by Using Unique Sequences of the ITS1 Region of Ribosomal DNA as Capture Probes for PCR ELISA

A. M. Bailey¹, D. J. Mitchell², K. L. Manjunath³, G. Nolasco⁴ and C.

L. Niblett³

FEMS Microbiology letters 2002 (in press)

Fungal species P. aphanidermatum

P. arrhenomanes P. myriotylum P. palmivora P. capsici P. infestans Pythium spp Phytophthora spp A B C D E

G H F

Probe number N 18 21 24 13 17 27 0

1 2 3 4 5 6 7 8

PCR ELISA reactions of the DIG- labeled amplified DNA of the ITS1 regions of three species of Pythium (P.

aphanidermatum, P. arrhenomanes and P.

myriotylum) and three species of Phytophthora (P. palmivora, P. capsici and P. infestans) and mixtures of the DNAs of two Pythium species [P.

aphanidermatum and P. myriotylum]

and two Phytophthora species [P.

palmivora and P. infestans] with their homologous and heterologous capture probes. The DNA samples were placed horizontally in rows A through H. The capture probe numbers are shown above the plate. They were placed vertically in columns 2 through 7. Column N received neither capture probe nor DIG-labeled DNA. Column 0 received the DIG-labeled DNAs, but no capture probes.

3’5’

R

5’

3’ 3’

5’

3’ 3’

5’

Q Q

Q

RR Q RR

RR

RR Q

Q

5’ Exonuclease Fluorescent Probe Assay

Polymerase Chain Reaction 29

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Protocols

CTV detection by Immunocature Reverse

Transcriptional-Polymerase Chain Reaction (IC/RT-PCR)

1. Coat 0.2 ml PCR tubes with CTV specific antibodies as for ELISA. (e.g. use 50 µl of a 1:1000 dilution of polyclonal antibodies in carbonate buffer to each tube/well). Incubate plates overnigth at 4 ºC or alternatively 3 - 4 hours at 37ºC.

2. Wash tubes by flooding with PBS-Tween (3 x 3 minutes). Finally empty the tubes/plate by manual shaking. Use immediately or keep frozen (up to several weeks).

3. Prepare citrus extract as for ELISA. (grind at 1/10 -1/20 (w/v) using carborundum or a mechanical grinder). Clarify the extracts by centrifuging at 5000 g for 5 min. Keep on ice until use. Extracts may be kept frozen for several months.

4. Add 50 µl of plant extract to each coated tube/well. Incubate for immunocapture for 3-4 hours at room temperature or at 4ºC overnight.

5. Wash tubes twice with PBS-Tween and one last time with sterile distilled water (3 minutes each). Empty the tubes by manual shaking.

6. Add 50 µl of reverse transcriptional - polymerase chain reaction mix (asymmetric, with Dig-dUTP) to each tube.

7. Close the tubes, put on the thermocycler and run the following program:

1. 38ºC for 45 min.

2. 94ºC for 2 min.

3. 92ºC for 30 s 4. 52 ºC for 30 s

5. 72 ºC for 45 s.Repeat steps 3 to 5 for 50 cycles.*

6. 72 ºC for 5 min.

(*)- note the higher number of cycles that compensates for the lower yield of asymmetric PCR.

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Composition of RT-PCR mixtures

RT-PCR mix (50ml) 1 sample 1.1 x n samples

H20 (MiliQ) 34.9

Stock solution 1-10 14.6

RT-PCR Enzyme mix 0.5

Stock solution 1-10

1 sample

PCR Buffer (without Mg) 10´ 5

MgCl2 (25 mM) 8

Dig labelling mix 0.5

Primer 1 (10mM) (forward) 0.1

Primer 10 (10mM) (reverse) 1

RT-PCR enzyme mix 1 sample

RNA guard (Pharmacia 27-0815 – 01, 27.2 U/ml) 0.14 Transcriptase reverse (Perkin Elmer N808-0018 50 U/ml) 0.16 Taq polymerase (MBI Fermentas, 5 U/ml) 0.20

Dig Labelling Mix

H2O 82.1 ml

dATP, dCTP, dGTP (100 mM) 2.0 ml/each

dTTP (100 mM) 1.9 ml

Dig-11-dUTP (Roche 1093088, 1 mM) 10.0 ml

Primer Sequences Position in CP gene

CTV 1 (5’ to 3’) ATG GAC GAC GAA ACA AAG AA 1 CTV 10 (5’ to 3’) ATC AAC GTG TGT TGA ATT TCC 653

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Other Solutions Coating buffer, pH 9.6

NaHCO3 2.93 g/l

Na2CO3 1.59 g/l

NaN3 0.2 g/l

PBS 1x pH 7.4

NaCl 8 g/l

KH2PO4 0.2 g/l

Na2HPO4 1.15 g/l

KCl 0.2 g/l

NaN3 0.2 g/l

Stock solution can be prepared 10x concentrated and kept at 4ºC.

PBS-Tween: PBS containing 0.05 % Tween 20. Keep at 4ºC CTV Extraction Buffer

PBS 1x PVP 2%

0.05% Tween 20

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Colorimetric detection of IC/RT-PCR amplified products.

1. Streptavidin coating of ELISA plate: add 100 ml per well of Streptavidin (Roche 1721666, 1 mg /ml in PBS) diluted 1:100 in coating buffer. Incubate overnight 4ºC or 1.30 h at 37ºC

2. Wash 3 3 minutes with PBS-Tween

3. Add biotinylated capture probe 1:1000 in hybridization buffer (100 ml/well).

Incubate for 30 minutes at 37ºC 4. Wash 3 3 minutes with PBS-Tween.

5. Add to each well 65 l of hybridization buffer and 35 ml of IC/RT-PCR Dig- labeled asymmetric product from each sample. Incubate for 90 minutes at 37ºC 6. Wash 3 3 minutes with PBS-Tween

7. Add anti-DIG-F(ab') , Roche 1 093 274 conjugated alkaline phosphatase (1:1000 2

from a 150U/200 ml stock) in ELISA conjugate buffer, 100 ml/well. Incubate for 30 min at 37 ºC

9. Add substrate (p-nitrophenyl phosphate) 1 mg/ml in ELISA substrate buffer, 100 ml/well.

Read the plate in an ELISA reader at OD 405 nm

Determine negative /positive threshold as for ELISA and analyze the results.

Biotinylated probe sequence (stock 200 mM)

CTV ABC (5' to 3') Biotin-AAA AAA CTG ATA GCG ATG AAC GAT GTG CGT CA Solutions

ELISA coating buffer, pH 9.6

NaHCO3 2.93 g/l

Na2CO3 1.59 g/l

NaN3 0.2 g/l

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PBS pH 7.4

NaCl 8 g/l

KH2PO4 0.2 g/l

Na2HPO4 1.15 g/l

KCl 0.2 g/l

NaN3 0.2 g/l

PBS– Tween

PBS plus 0.5 ml Tween 20 per litre

Hybridization Buffer

n-laurylsarcosine 1 g

20x SSPE (see below) 250 ml

5 M NaCl 100 ml

Complete the volume to 1l with double-distilled water

20x SSPE pH7.4

NaCl 175.3 g/l

NaH2PO4, H2O 27.6 g/l

EDTA 7.4 g/l (or 40 ml of 500 mM stock)

Adjust the pH to 7.4 with NaOH

ELISA Substrate Buffer pH 9.8 1 litre

Diethanolamine 97 ml

NaN3 0.2 g

Adjust pH to 9.8 with concentrated HCl

ELISA conjugate Buffer

PBS 10x 100 ml

Tween20 500 ml

PVP-40 20 g

BSA 2 g

Add water to 1l.

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Typing of CTV strains in an array of strain specific probes.

1. Set up a PCR reaction for the reamplification of the previously amplified products that will be typed. The reamplification will be done with internal primers 42-43.

2. Distribute the PCR mix on the tubes and add 1 ml of each IC/RT-PCR to be typed.

3. Close the tubes, put on the thermocycler and run the following program:

1. 94ºC for 2 min.

2. 92ºC for 30 s 3. 52 ºC for 30 s

4. 72 ºC for 30 s. Repeat steps 2 to 4 for 50 cycles.

5. 72 ºC for 5 min.

Stock solution 42-43

PCR mix (50ml) 1 sample 1.1 x n samples

H20 (MiliQ) 30.7

Stock solution 42-43 19.1

Taq DNA polymerase (MBI Fermentas, 5 U/ml) 0.2

1 sample

PCR Buffer (without Mg) 10´ 5

MgCl2 (25 mM) 8

Dig labelling mix 5

Primer 42 (10mM) (reverse) 0.1

Primer 43 (10mM) (forward)

Dig Labelling Mix

H2O 82.1 ml

dATP, dCTP, dGTP (100 mM) 2 ml/each

dTTP (100 mM) 1.9 ml

Dig-11-dUTP (Roche 1 093 088, 1 mM) 10 ml

Primer Sequences Position in CP

CTV42 (5’ to 3’) CTC AAA TTG CGR TTC TGT CT 454 CTV43 (5’ to 3’) ATG TTG TTG CNG CNG AGT C 59 Citrus tristeza virus (CTV) diagnosis and strain typing by PCR-based methods

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Colorimetric detection of the CTV strains.

1. Streptavidin coating of ELISA plate: add 100 ml per well of Streptavidin (Roche 1721666, 1 mg /ml in PBS) diluted 1:100 in coating buffer. Incubate overnight at 4ºC or 1h30 at 37ºC

3. Add each of the biotinylated strain specific probes to each row of the plate (use each row for one probe); 1:1000 in hybridization buffer (100 µl/well). Incubate for 30 minutes at 37ºC

4. Wash 3 3 minutes with PBS-Tween.

5. Mix 45 ml of PCR product with 855 µl of hybridization buffer. Distribute 100 µl per well of each column (use one column for each sample). Incubate for 90 minutes at 37ºC

7. Add anti-DIG-F(ab') , Roche 1093274 conjugated alkaline phosphatase (1:1000 2

from a 150U/200 ml stock) in ELISA coating buffer, 100 µl/well. Incubate for 30 min at 37 ºC

9. Add substrate (p-nitrophenyl phosphate) 1 mg/ml in ELISA substrate buffer, 100 µl/well.

Read the plate in an ELISA reader at OD 405 nm at 15 min intervals up to 1h30.

Compare the values with a set of values obtained with specific clones.

Sample

1 Sample

2 Sample

3 Sample

4 Sample

5 Sample

6 Sample

7 Sample

8 Sample

9 Sample

10 Sample

11 Sample

12 B1B2

B3aB3b IIIB4 B5 B8