Difficulties with conventional
phytoplasma diagnostic using
PCR/RFLP analyses
Jana Fránová
Dept of Plant Virology
Dept.
BC ASCR v.v.i. IPMB
Branišovská 31
České Budějovice
Czech Republic
PCR difficulties:
Nonspecific reactions: – contamination (use powder free gloves
gloves, pipets
with filter, sterile tips with filter, close the windows)
– from phytoplasma positive samples
M Ep Ep W W W W
– from phytoplasma positive controls
– and/or with other micro-organisms
– amplification of plant DNA
– some primers
i
can iinduce
d
di
dimers, b
band
d off nonspecific
ifi
sizes (Heinrich et al., 2001)
F1/R0→F1(III)/R2(III)
f l positives
false
iti
inclusion of negative controls is necessary (DNA isolated from
corresponding healthy plants, sterile water, amplification without template)
M AP CP AY W W W W W W AP CP AY W W W W
P1/P7  F1/B6
M AP CP AY W W W W W AP CP AY W W W W W
P1/P7F1/B6  F2/R2
false negatives – check DNA quality and quantity (20ng/reaction,
use higher dilution of DNA)
– use more sensitive PCR method (nested PCR
PCR,
second nested PCR, group specific primers)
– include positive control (different from those,
which are expected to be present in examined samples)
M
1
2 3
4
5
6
7 8
9 10 11 12 13 14 15 16
M 17 18 PK W W W W
P1/P7
P1/P7
M
1
2
3 4
5
6 7
8
9 10 11 12 13 14 15 16
P1/P7F1/B6  F2/R2
M 17 18 PK
W W W
P1/P7F1/B6  F2/R2
false negatives or week positives, smears
– use primers from different company
– change Taq polymerase or PCR Mix
M
M
1
2
3
1
2
1
2
3
4
5
6
7
W
3
PCR Mix of firm III
P1/P7→P1A/P7A→F2n/R2
fi I
firm
firm
fi II
M
1
2
3
4
P1/P7
PCR Mix of firm IV
5
6
7
W
PCR difficulties – different PCR Mix
– different primer combination
M 1
2
3
4 5 6
7
8
9 10 11 12 13 14 15 16
M
PCR Mix of firm III
M
1
2
3
P1/P7F1/B6  F2/R2
4
5
6
7
8
W
P1A/P7A→F1/B6→F2/R2
PCR Mix of firm IV
17 18 19 20 CP AY AP W W
P1/P7F1/B6  F2/R2
PCR Mix of firm III
PCR difficulties – change Taq polymerase or PCR Mix
– different primer combination
M
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16
P1/P7 F2/R2
PCR Mix of firm III
M 1 1
2 2 3 3 3 W 1 1 1
2 2 2 W
– repeat and/or optimize
PCR ((hot start,, gradient
g
PCR)
– try direct PCR
P 399/P 1694
Pc399/Pc1694
PCR Mix of firm IV
rpAP15f/rpAP15r
false positives
confirmation of phytoplasma presence by RFLP (at least with
2 or more endonucleases) is necessary and/or using other
primer
i
combination
bi ti
RFLP difficulties
- unusual or illegible profiles → repeat and/or optimize PCR
- secondary bands → optimization of digestion (time
elongation)
M I-B I-C II-A III-A X-A
d
i
g
e
s
t
t.
20
hours
AluI
X-B XII-A
P1/P7→F2/R2
M I-B I-C II-A III-A X-A
X-B XII-A
AluI P1/P7→P1A/P7A→F2/R2
d
i
g
e
s
t.
60
hours
DNA digestion with more enzymes for phytoplasma
identification
M
I-B I-C II-A III-A X-A
X-B
XII-A
HhaI
M
M
I-B I-C II-A III-A X-A
RsaI
X-B
II-B
B II-C
C II
II-A
A III
III-A
A
XII-A
MseI
X
X-A
A
X
X-B
B
XII
XII-A
A
Evaluation of different primers
DNA iisolation
l ti
( h
(phenol/chloroform)
l/ hl f
) ffrom
• phytoplasma reference strains (PnWB, CX, GSFY/1,
GSFY/2,, MOL,, AY,, CPh,, CYE,, AP,, PD))
• 18 healthy Catharanthus roseus plants
PCR
PCR:
P1/P7→ P1A/P
P1/P
P1A/P71
1
→ F1/B6
→ F2n/R2
→ fU5/rU3
→ fU5/P7
→ fU2/P7
→ 16R 758F/16R1232R
→ R16F1/R0
→ Pc399/Pc1694
→ R16 F1(I)/R1(I)
P1A/P7A →F1/B6
→ F2n/R2
•PCR: P1/P7→ P1A/P71 → F2n/R2
P1/P7 → F1/B6 → F2n/R2
P1/P7 → F1/B6 → 16R758F/16R1232R
P1/P7 → F1/B6 → fU5/rU3
P1/P7→ F2n/R2 → R16 F1(I)/R1(I)
•RFLP:
RFLP: AluI,
AluI HhaI,
HhaI MseI,
MseI RsaI (R16F2n/R2
amplicons)
•DNA sequencing (1 healthy looking C.
plant),
), comparison
p
with data
roseus p
available in the GenBank (BLAST)
Results:
M I-B I-C II-A III-A X-A
Specific amplification: P1/P7→ P1A/P7A
→ F1/B6
→ F1/R0
M AY CP PnwB CX
AP GSFY S
→ R16F2n/R2
U5/
→ fU5/P7
P1/P7→ F1/B6 → R16F2n/R2
P1/P7→ P1A/P7A → R16F2n/R2
P1A/P7A → R16F2n/R2
P1/P7→F2n/R2
M
1
2
3
4
5
6
7
8 PC
P1/P7→F2n/R2
X-B
XII-A
MseI
-specific amplification of DNA from
positive controls including RFLP
confirmation of phytoplasmas
identity
- usually no product with DNA
from 18 C. roseus plants and
water controls
Results:
Lower specificity, contamination or nonspecific
amplification:
P1/P7→ F1(I)/R1(I)
→ fU5/rU3
→ 16R758F/16R1232R (M1/M2)
- DNA iisolated
l t d ffrom 5 h
healthy
lth C.
C roseus plants
l t gave positive
iti
results in PCR with some primer combinations (in our hands)
M 1 2
M 1
2
3 4 5 6
7 8
3 4 NC PC
9 10 11 12 13 NC PC
P1/P7→R16F2n/R2
M 1 2 3 4 5 NC NC NC PC
P1/P7→fU5/rU3
P1/P7→M1/M2
Results:
RFLP of F2/R2 amplicons after digestion with
MseI and AluI obtained from healthy C. roseus
M
9
14 18 NC PC M
I-B I-C II-A III-A
X-A
X-B
XII-A
MseI
MseI
Positive controls
DNA sequencing (sample no. 9) exclude phytoplasma presence
M
3
6
9
14
AluI
Conclusion:
• In the case of phytoplasma positive samples,
primers p
preferentially
y amplified
p
p
phytoplasma
y p
the p
sequence of expected sizes, exceptionally,
additional bands could be observed
• In the case of DNA isolated from healthyy p
plants,
some primers can react probably with sequences of
plant genome or dimers and false positives could be
observed
b
d
Including DNA from corresponding healthy plant
and water controls is necessary
Conclusion:
•PCR
PCR alone is not sufficient enough for
phytoplasma detection. Confirmation of
phytoplasma presence and its identification
must be accomplished at least by RFLP, using
two or more endonucleases
•Critical samples: different primer pair
combination, RFLP with more enzymes and
sequencing should be used for elucidation of
phytoplasma
p
y p
p
presence
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