Details on the this workshop on maize lethal necrosis (MLN) disease at: http://bit.ly/1GZ6KoX

1. MLN DIAGNOSTIC METHODS
Anne Wangui
Training Workshop on MLN Diagnosis and Management
MLN Screening Facility Naivasha -17th- 19th March 2015

2. Is pathogen detection necessary?
• Correct diagnosis and identification of the causal
agent in any disease management is critical.
• Globally, agricultural trade and germplasm
exchange pose a potential risk of spreading seed-
borne and seed-transmitted pathogens across
borders.
• Proper and timely diagnosis of these pathogens will
ensure safe trade and germplasm exchange across
borders.

3. Detection methods
• Various methods available for detection of plant
viruses:
Ø Use of symptoms
Ø Indicator plants
Ø In vitro properties of the virus (thermal
inactivation point, dilution end point and
longevity in vitro)
Ø Electron microscopy
Ø Serological methods and
Ø Molecular (nucleic acid)based methods.

4. How do you choose your detection
method?
• The type of detection method adopted depend on:
Ø Availability of resources
Ø Facilities available
Ø Availability of reagents
Ø Required level of specificity and sensitivity
Ø Expertise and skills available
Ø Type and sample sizes
Ø Information available on the virus
Ø Time required for completing the test.

5. Detection methods for MLN
causing viruses (SCMV & MCMV)
• Two methods mostly used:
Ø Serological methods e.g. ELISA
Ø Molecular based methods e.g. PCR.

6. Serological methods
• Serological methods are based on:
Ø surface properties of virus protein and
Ø antibody- antigen binding ability.
• Are conducted on a solid surface (microtitre
plate or nitrocellulose membrane) and the
antigen- antibody reaction is visualized using
enzyme-labeled antibody.

7. Enzyme-Linked Immunosorbent
Assay (ELISA)
• ELISA is the most popular technique in detection of
plant viruses.
• Introduced by Clark and Adams (1977).
• Principle:
Product
Substrate
2nd antibody (enzyme labeled)
Antigen
Capture (1st ) antibody
Solid surface (Micro titre plate)

8. Enzyme-Linked Immunosorbent
Assay (ELISA) con’t
• Types:
• “Indirect” ELISA- polyclonal antibody conjugate not
virus-specific but specific to virus antibody.
Ø A single antibody conjugate (rabbit anti mouse)-
detects a wide range of viruses.
Ø Useful in disease surveys eg.PTA, TAS and
PAS- ELISA.
• “Direct” ELISA- virus specific monoclonal antibodies
Ø Highly specific -detecting antibody conjugated to
an enzyme
Ø Example: DAS-ELISA.

9. SCMV & MCMV detection: Why
DAS-ELISA?
Advantages:
Ø Highly specific since two antibodies (capture &
detection) are used.
Ø Antigen does not require purification before use.
Ø Eliminates cross-reactivity between other
antibodies.
Ø Easy to use and adapt.
Ø Faster and cost effective in testing large number of
samples.
Ø Readily available reagents and ELISA kits.

10. Double Antibody Sandwich (DAS)-
ELISA
• Requirements:
Ø Micro titre plates(polyestrene /polyvinyl chloride)
Ø Buffers (wash,coating, conjugate, substrate).
Ø Capture antibody
Ø Antibody-enzyme conjugate
Ø PNP substrate tablets
Ø ELISA reader
Ø Micro pipettes
Ø Weighing balances
Ø Freezer (-20oC )
Ø Refrigerator

11. DAS-ELISA procedure
1. Coat the plate with capture antibody
2. add the sample extract (Antigen)
3.Add enzyme conjugated antibody
4. Add PNP substrate (for colour dev’t)
5. Read results at 405nm: Colour change (yellow)= positive,
OD values above 2x average value of negative control=
positive; equal or less than= negative.
NB: step a & b = wash plate 3 times with PBST and incubate at
37oC and RT for 1hour respectively.
1
2 3 4
5
a a a b

12. What influences ELISA results?
• Reagents and buffers preparation: Use distilled
water, correct pH and molarity.
• Glassware and micropipettes must be clean to avoid
contamination.
• Sample extraction: sample extracts must be kept at
low temp to avoid denaturation. Addition of PVP 24-
40,000 in extraction buffer helps bind polyphenols.
• Use of Tween 20 in wash buffer facilitate antibody –
antigen interaction while blocking agents (BSA,
NDM PVP prevent formation of non-specific
reactions.

13. What influences ELISA results?
Con’t
• Virus distribution in the sample: Viruses are
unevenly distributed thus need to use composite
samples.
• Incubation condition (stationary/shaking)affect the
interaction of reagents in an assay. Incubation all
plate at the same time and condition but not stack
together.
• Antibody variations: polyclonal antibodies-
variation in antigenic reaction between animals.
Monoclonal antibodies-hybridoma cells are immortal
and can be stored are low temp for long periods
giving specific and consistent results.

14. What is Polymerase Chain
Reaction (PCR)?
• In vitro method for amplifying target DNA sequence in
a complex mixture of DNA.
• Invented by Kary Mullis in 1980s.
• Viruses are composed of proteins and nucleic acid.
• Nucleic acid carry genetic information for virus
multiplication.
• NA (DNA/RNA) comprise of nucleosides(sugar +
base) and a phosphate group joined together by
hydrogen bonds.
• Nucleotides: Purines= Guanine and Adenine,
Pyrimidines= Cytosine and Thiamine (Uracil).
NB:A purine always pairs with pyrimidine: G=C, A=T in
a double stranded NA.

15. Why PCR ?
• Advantages:
Ø Increased sensitivity, versatility, speed and
specificity.
• Principle:
• PCR uses Taq Polymerase and primers (short
single stranded DNA sequences complementary to
a target DNA made in the lab) to amplify the target
DNA.
• The primers (forward and reverse) anneal to
opposite strand sequences of the target DNA,
Taq polymerase extends the primers along the
target DNA (5’ to 3’) doubling the amount of the
target DNA sequence.

16. PCR principle: thermal cycling
1. Denaturation at high
temperatures (94-95oC)
2. Primer annealing
(temp dep. on primer
nucleotide composition
and length usually 35-
65oC)
3. Primer extension
along the target region
using Taq polymerase
at 72oC
• 3 major steps repeated 35-40 cycles in a thermal
cycler

17. SCMV & MCMV detection: RT-PCR
technique
• Reverse Transcription(RT)-PCR, amplify viruses
with RNA as their genome.
• SCMV and MCMV have +ssRNA: convert to
complementary DNA (cDNA) through reverse
transcription to serve as a template for synthesis of
a new DNA strand.
• Commonly used reverse transcriptase enzymes are:
AMV (Avian Myoblastosis virus) or MMLV (Molony
Murine Leukemia virus) from retroviruses.

18. Reverse transcription (RT)PCR
• RT-PCR can either be carried-out as one-step RT-
PCR or two-step RT-PCR.
• One-step RT-PCR: all PCR reagents are put in one
tube. RT-step is performed before PCR cycling.
• Preferred for diagnostics- simpler and less chances
of contamination because the tube is opened post-
PCR.
• Two- step RT-PCR: RT step (cDNA) is done
separately in a reverse transcription reaction and
added to PCR reaction.

19. Reverse transcription (RT)PCR
con’t
Ø 96 well PCR plates
Ø Plate seals
Ø PCR set up cabinet
Ø Micro pipettes
Ø Pipette tips
Ø PCR thermal cycler
Ø Reagents(dNTPs,PCR
buffer, MMLV-RT, Taq
polymerase, MgCl2)
Ø DNA template,
Ø Primers (forward and
reverse)
Ø Nuclease free water
Ø DNA ladder
Ø Agarose
Ø DNA loading dye
Ø Tris acetate EDTA
buffer
Ø SYBR green/ ethidium
bromide
Ø UV-transilluminator

20. Setting up a PCR reaction
• Separate designated areas for pre and post-PCR,
have dedicated equipment for each area/work and
use aerosol resistant tips to prevent contamination
which can lead to false-positive results.
• Prepare RT-PCR mastermix: how many reactions
are needed?; what type of PCR (Conventional or
real-time PCR)
NB: Each sample is tested in duplicate and require
24ul of the mastermix and 1ul of sample per well
(final vol. 25ul). Always add extra reactions (2) to
cater for volume loss during pipetting.

21. One-step RT-PCR mastermix for
conventional PCR:
Reagent Volume Final Conc. in
25μl
Sterile, nuclease-free water 18.5 μl final volume of
50ul
10 x reaction buffer 5 μl (1 x)
25 mM MgCl2 3 μl (1.5 mM)
10 mM dNTP mix (each
10mM)
1 μl (0.2 mM each )
5pmol/ul primers 2 2.0 μl (0.2pmol/ul)
200 Units/μl MMLV RT 1 μl (0.4 U/μl)
5 units/μl DNA Taq
Polymerase
0.25 μl (1.25 U)
RNA 1.0 μl

22. PCR product visualization
• Gel electrophoresis:
Ø The amplified PCR products are observed
through agarose gel electrophoresis.
Ø DNA molecules move towards anode (+)
because its negatively charged.
Ø Agarose gel is stained by use of ethidium
bromide or SyBr green which chelates in the
DNA and fluorescence under UV illumination.
• Agarose gel
DNA bands
DNA ladder

23. Real- time PCR using Taqman®
chemistry
• In real time PCR, amplification of the target DNA is
monitored at each PCR cycle (real time) through
fluorescence emission.
• Uses fluorescent probe labeled with a reporter dye (FAM
or TET/JOE/VIC) and a quencher dye (TAMRA or black
hole (BHQ).
• Probe: single-stranded DNA with a specific base
sequence.
• Used to detect the complementary base sequence of
target DNA/RNA by hybridization
• Flourescence Resonance Energy Transfer (FRET)
process -quencher absorbs fluorescence from reporter
when in close proximity to each other.

24. TaqMan® Probe-Based Assay
Chemistry.
Reporter Fluorescence
Fluorescence absorbed
Probe
primer
Probe cleavageTAQ
Q
R
TAQ
R Q
Polymerization and 5’ nuclease activity of TAQ enzyme

25. TaqMan® Probe-Based Assay
Chemistry con’t
• Polymerization: A fluorescent reporter ® dye and
a quencher (Q) are attached to the 5’ and 3’ of a
Taqman® probe respectively.
• Strand displacement: when the probe is intact, the
reporter dye emission is quenched.
• Cleavage: During each extension cycle, the DNA
taq polymerase cleaves the reporter dye from the
probe.
• Polymerization completed: Once separated from
the quencher, the reporter dye emits flourescence.

26. One step RT-PCR mastermix for
real-time PCR
Reagent Volume Final Conc. in 25μl
Sterile, nuclease-free water 11.325 μl
10 x reaction buffer A 2.5 μl (1 x)
25 mM MgCl2 5.5 μl (5.5 mM)
6.25 mM dNTPs 2.0 μl (0.5 mM)
7.5 μM Forward primer 1.0 μl (300 nM)
7.5 μM Reverse primer 1.0 μl (300 nM)
5μM TaqMan probe 0.5 μl (100 mM)
200 Units/μl MMLV RT-ase 0.05 μl (0.4 U/μl)
5 units/μl AmpliTaq Gold DNA
Polymerase
0.125 μl (0.625 U)
RNA 1.0 μl

27. Real-time PCR con’t
ΔRn
Rn+
Rn-
Threshold
Ctvalue
Cycle
Baseline
• Red line= positive
sample (Ct value = 22)
• Purple line =
negative sample(Ct
value recorded = 45).
• Real time PCR
results are in form of Ct
values. Negative and
positive controls must
be included in an assay
as proof reactions have
worked. Taqman amplification plot

28. Definition of terms in a real-time
PCR
• Ct: is the threshold cycle. PCR cycle at which an
increase in reporter fluorescence can first be detected.
• ΔRn: is the normalised reporter signal minus the
baseline signal established in the first few cycles of
PCR. This value also increases with target amplification,
until the reaction reaches a plateau.
• Rn: normalised reporter which is calculated by dividing
the reporter signal by the passive reference signal
(ROX). During PCR, Rn increases as target nucleic acid
is amplified until the reaction approaches a plateau.

29. Factors that affect a PCR reaction
• Mg2+:Mg2+ ions are affected by DNA template conc,
presence of chelating agents (EDTA) and proteins
in the sample, dNTP conc.
• Taq polymerase is inactive in absence of enough
Mg2+ ions.
• Excess mg++ increases non-specific amplification
and can inhibit PCR amplification.
• Taq polymerase is usually supplied with free 25mM
MgCl2 .
• Set up a series of PCR with 1.0-5.0 mM Mg2+ in 0.5-
1mM increments to determine the optimal level for
each reaction.

30. Factors that affect a PCR reaction
con’t
• Annealing temperature (Ta): Optimizing Ta
prevent formation of primer dimers and non-specific
amplification.
• Use Ta slightly below the primer melting
temperature (Tm). Try several Ta in 2-3oC steps
starting 5-10oC below Tm.
• Tm is the temperature at which 50% of the
complementary DNA molecules will be annealed.
• Cycling conditions can also be optimized which
include Ta, Number of cycles and primer extension
time.

31. Factors that affect a PCR reaction
con’t
• Contamination: All equipment and reagents used in
PCR must be sterile. DNA must be free from
contamination, including detergents such as SDS,
ethanol, phenol and salts.
• DNA quality and quantity: the optimal amount will
depend on the size of the DNA molecule. Too little
DNA will affect amplification while too much
template can cause non- specific amplification

32. Primers and probes for MCMV &
SCMV detection: real-time PCR
Zhang, Y. et al. (2011) J. Virol. Methods 171, 292–4
MCMV. Adams, I. P. et al. (2013). Plant Pathol. 62, 741–749
Primers
Name Sequence 5’-3’ Target gen Specific for
MCMVf CGTATCACTTGGGAAACA
Coat protein MCMV
MCMVr CAGAGAGGAATGCCATGGA
Taqman Probe
MCMVpe FAM- TCACAGCAGACACCACTAGCGGATACA
Primers
Name Sequence 5’-3’ target gen Specific for
MCMVf CCGGTCTACCCGAGGTAGAAA
- MCMV
MCMVr TGGCTCGAATAGCTCTGGATTT
Taqman Probe
MCMVpe FAM-CAG CGC GGA CGT AGC GTG GA-BHQ1

33. Primers and probes for MCMV &
SCMV detection:real-time RT-PCR
Primers
Name Sequence 5’-3’ target gen Specific for
SCMVf CCA GGC CAA CTT GTA ACA AAG C
- SCMV
SCMVr CAT CAT GTG TGG ATA AAT ACA GTT GAA
Taqman Probe
SCMVpe FAM-TGT CGT TAA AGG CCC ATG TCC GCA-BHQ1
Adams, I. P. et al. (2013). Plant Pathol. 62, 741–749
Primers
Name Sequence 5’-3’
target
gene
Specific for
COX f CGT CGC ATT CCA GAT TAT CCA
-
Cox (internal
control)
COXr CAA CTA CGG ATA TAT AAG RRC CRR AAC TG
Taqman Probe
COX Pe VIC] - AGG GCA TTC CAT CCA GCG TAA GCA - [TAMRA]

34. Primers for MCMV & SCMV
detection: conventional RT-PCR
Primers
Name Sequence 5’-3’ Target gen Specific for
MCMVf (268IF) ATGAGAGCAGTTGGGGAATGCG
Coat protein MCMV
MCMVr (3226R) CGAATCTACACACACACTCCAGC
Wangai et al., 2012
Primers
Name Sequence 5’-3’ Target gen Specific for
SCMVf (8679F) GCAATGTCGAAGAAAATGCG
SCMV
SCMVr (9595R) GTCTCTCACCAAGAGACTCGCAGC

35. Sources of antisera for SCMV &
MCMV
Commercially available ELISA kits can be supplied by
among others:
• Agdia (www.agdia.com)
• Neogen (www.neogen.com)
• DSMZ: www.dsmz.de
• Bioreba: www.bioreba.ch
• Ac diagnostics: www.acdiainc.com

36. Sources of primers and probes
• Bio-rad laboratories:www.bio-rad.com
• Africa bio-systems:
www.appliedbiosystems.com
• Sigma –Aldrich : www.sigmaaldrich.com
• Invitrogen:http://www.lifetechnologies.com

37. THANK YOU