Effective detection of MLN pathogens in leaf tissues and seed lots:

Immuno-capture technique as a new option

Monica Mezzalama Head of Seed Health Laboratory

PROGRESS ON MLN DIAGNOSTIC SUMMARY of year 1 We defined, described and agreed on: • Detection • Diagnosis • Surveillance • Characteristics of the detection method • Sampling procedure:

• Plants in the field • Seed lots

• Tolerance level • Risk management • Detection methods: leaves and dry seed

“. . .surveillance is the process of searching, detection is the process of finding, and diagnosis is the process of determining

and/or verifying what is found.” (Stack & Fletcher, 2007)

TECHNIQUE ASSESMENT REPRODUCIBILITY TIME LABOR COST/SAMPLE

ELISA VIRAL

ANTIGEN GOOD HOURS MODERATE MODERATE-HIGH

IMMUNOSTRIPS

VIRAL

ANTIGEN

GOOD LESS THAN 30

MINUTES MODERATE MODERATE-HIGH

Loop mediated

isothermal amplification

(LAMP)

VIRAL GENE EXCELLENT HOURS LOW, EXPERIENCED MODERATE-HIGH

PCR VIRAL GENE EXCELLENT HOURS HIGH,

EXPERIENCED HIGH

SUMMARY: ASSESSMENT OF SELECTED DETECTION METHODS

FEW REMARKS ON DETECTION IN DRY SEED WITH SEROLOGICAL METHODS • Immunostrips: not advisable for detection in dry seed

• ELISA: soaking dry seed for different lengths of time and grinding the soaked seed at the end of

the soaking step

RESULTS OD VALUES AT 405 nm

DESCRIPTION soaked

30' ground

soaked 1h

ground soaked

2h ground

soaked 4h

ground soaked

6h ground

soaked 8h

ground soaked

15h ground

Kenya 288(1) 0.50 1.41 0.51 1.17 0.54 2.11 0.50 2.38 0.71 0.50 1.01 1.33 1.36 2.14

Peru (1055) 0.27 0.83 0.21 0.91 0.36 1.42 0.41 2.01 0.50 1.41 0.62 1.31 0.57 1.20

MEX 408(1) 0.10 0.34 0.12 0.28 0.13 0.49 0.09 0.10 0.15 0.28 0.09 0.11 0.25 0.75

MEX 408(7) 0.12 0.34 0.11 0.22 0.09 0.41 0.21 1.19 0.08 0.09 0.22 0.37 0.10 0.09

Neg seed 0.09 0.10 0.08 0.09 0.08 0.08 0.08 0.12 0.09 0.10 0.08 0.09 0.09 0.08

Neg BIOREBA 0.08 0.08 0.09 0.08 0.08 0.09 0.08 0.09 0.08 0.09 0.09 0.08 0.10 0.09

Pos BIOREBA 2.83 2.81 3.06 2.99 2.56 2.83 2.60 2.74 3.77 2.18 3.50 2.25 2.61 2.80

REMARK on SEED TRANSMISSION TO PROGENY • 9 maize entries with 1000 seeds each • Testing dry seed according to SHL procedures: 10% sampling intensity; all entries tested positive to MCMV (with

ELISA-AGDIA; PCR with Adams and Zhang primers)

• Planting of 900 seeds per each entry • Total of 8040 seedlings • Bulks of 10 plants = 804 bulks tested with ELISA • 1 positive: 0.01% comparable to 0.04% (17/4200 seedlings) Jensen et al. (1991)

ImmunoCapture-Reverse Transcription Polymerase Chain Reaction

• IC-RT-PCR: has been used for more than a decade for plant virus detection

• It combines: ELISA and PCR techniques

• It exploits the specific binding capabilities of an antibody to recognize a molecule or a group of similar molecules, in this case a viral particle, trapped by an antibody.

• These captured viral particles are used directly for PCR detection.

• In the case of RNA viruses the cDNA synthesis is done before the PCR on the captured viral particles

ImmunoCapture Reverse Transcription-PCR (Le Provost, G. et al., 2006; Mallik, I. et al., 2012)

1. ImmunoCapture (= coating as in ELISA ) in PCR tubes (0.2 µL) with specific IgG

2. Grind sample (plant tissue or seed) in ELISA extraction buffer

3. Clarification of the sample extracts by centrifugation (7000 rpm for 5’)

4. Incubate the samples in the coated PCR tubes

5. Elute RNA from the sample with RNAse free water and heat the elute 70°C for 15 min

6. Make the cDNA synthesis during the PCR protocol, this can be made in the same tube with the captured virus or use another tube

7. PCR test for detection can now be carried out

DETECTION OF MCMV: ONE-STEP IMMUNOCAPTURE REAL TIME RT-PCR (Jin-Guang Y. et al. 2012)

• The following samples were tested: 1. Maize leaves positive to ELISA test for MCMV (Kenya isolate) 2. Dry seed positive to ELISA test for MCMV (Mexican strain) 3. Maize leaves positive to ELISA test for SCMV (Mexican isolate) 4. Healthy dry Seed 5. RNA from maize leaves positive to MCMV (Kenya isolate) used as positive control 6. Extraction buffer as negative control

1. ImmunoCapture (= coating as in ELISA ) in PCR tubes (0.2 µL) with specific IgG IC-RT-qPCR was performed with the specific capture antibody Bioreba (Cat. #140772)

2. Incubate 3 hours at 37oC

3. Wash the PCR tubes 3 times with 100 µl PBS-T (pH 7.4)

4. Grind sample (plant tissue and ground dry seed) in ELISA extraction buffer and store at 4oC until time to be used

5. Clarification of the sample extracts by centrifugation (7000 rpm for 5’)

6. Add 25 µl of clarified samples extracts to the coated PCR tubes

7. Incubate 1.5 hours at 37oC

8. Wash the tubes 3 times with PBS-T (pH 7.4)

9. Elute the sample with 20 µl RNAse free water and heat the eluate 70° C for 15 min

10. Prepare 20 µl of mastermix which includes: 10.3 µl of iTaq Universal Probes One Step Mix (BIORAD), 0.9 µl of Adams et al. (2013) primers for MCMV detection, 8.8 µl of eluted sample

11. Perform the One-Step IC Real Time RT-PCR

Results Sample ID Cq

1 Purified RNA (from maize leaves infected with MCMV Kenya isolate) 4.55

2 Maize leaves with MCMV Kenya isolate 17.67

3 Dry maize seed positive to ELISA for MCMV 25.05

4 Healthy dry seed 35.03

5 Maize leaves infected with SCMV (Mexican isolate) 36.84

6 Extraction buffer 38.39

1 2 3 4 5 6

Advantages • No need of RNA extraction

• Higher specificity than the two separate techniques

• No presence of PCR inhibitors

• Less time to make the detection than the two separate techniques because IC-RT-PCR can be carried out it in a Real Time PCR platform

• No cost of an RNA extraction kit, but cost of the capture antibody

• Fast

• Safe for the operator

Disadvantages

• Medium-high laboratory infrastructure

• Medium-high technical skills

• A specific capture antibody must be available

• Reverse transcriptase is expensive, can have limited availability and short shelf life

TECHNIQUE ASSESMENT REPRODUCIBILITY TIME LABOR COST/SAMPLE

ELISA VIRAL

ANTIGEN GOOD HOURS MODERATE MODERATE-HIGH

IMMUNOSTRIPS

VIRAL

ANTIGEN

GOOD LESS THAN 30

MINUTES MODERATE MODERATE-HIGH

Loop mediated

isothermal amplification

(LAMP)

VIRAL GENE EXCELLENT HOURS LOW, EXPERIENCED MODERATE-HIGH

PCR VIRAL GENE EXCELLENT HOURS HIGH,

EXPERIENCED HIGH

IC-RT-PCR VIRAL GENE Not assessed yet HOURS LOW, EXPERIENCED Not assessed yet

SUMMARY: ASSESSMENT OF SELECTED DETECTION METHODS

References

• Adams, I.P. et al. 2013. Use of next-generation sequencing for the identification and characterization of Maize Chlorotic Mottle Virus and Sugarcane Mosaic Virus causing Maize Lethal Necrosis in Kenya. Plant Pathology, 62(4),741–749.

• Le Provost, G. et al. 2006. Improved detection of episomal Banana Streak Viruses by multiplex immunocapture-PCR. Journal of Virological Methods, 137(1),7–13.

• Mallik, I.et al. 2012. Detection and Differentiation of Potato Virus Y Strains from potato using Immunocapture Multiplex RT-PCR. American Journal of Potato Research, 89,184–191.

• Mulholland, V. 2008. Immunocapture-PCR for Plant Virus Detection. In: Plant Pathology Techniques and Protocols, 508,183-192.

• Rowhani, A. et al.1998. Development of a sensitive colorimetric-PCR assay for detection of viruses in woody plants. Plant Disease, 82(8), p.880–884.

• Jin-Guang Y. et al. 2012. Development of a One-Step Immunocapture Real-Time RT-PCR Assay for Detection of Tobacco Mosaic Virus in Soil. Sensors, 12, 16685-16694; doi:10.3390/s121216685.

Noemi Valencia

Benjamin Martinez

Gabriela Juarez