Real time PCR by Sujata Dandale

1. A new technique to detect plant pathogens
Real-time PCR:
SUJATA DANDALE

2. ““Perfect as is the wing of a bird, itPerfect as is the wing of a bird, it
could never raise the bird upcould never raise the bird up
without resting on air. Facts arewithout resting on air. Facts are
the air of a scientist. Withoutthe air of a scientist. Without
them you never canthem you never can fly.”fly.”
- Ivan Pavlov- Ivan Pavlov

3. PCR
• 2nd
most celebrated discovery in molecular biology.
• Allows trace amounts of a DNA sequence to be
amplified giving enough DNA for cloning,
sequencing or other analyses.
• Needs primers to start DNA synthesis.
• Involving multiple cycles of DNA strand separation,
binding of primers, and synthesis of new DNA.

4. Limitations of PCR
• Low resolution
• Poor precision
• Low sensitivity
• Non automated
• Size based discrimination only
• Results are not expressed as Numbers
• EtBr for staining is not very quantitative
• Post PCR processing
• EtBr is carcinogenic

5. Real-Time PCR
A technique used to monitor the progress of a
PCR reaction in real time (i.e., as it
happens).
PCR product (DNA, cDNA or RNA) can be
quantified.
Also known as kinetic PCR, qPCR, qRT-PCR
and RT- qPCR.

6. The evolution of PCR to Real-time PCR
Fig: PCR to Real-time PCR evolution

7. Real-Time PCR:Real-Time PCR:
PrinciplePrinciple1.1. The real time PCR is based on the detection and theThe real time PCR is based on the detection and the
quantification of a fluorescent transmitter during the process ofquantification of a fluorescent transmitter during the process of
amplification.amplification.
2. The increase in the fluorescent signal is directly proportional to2. The increase in the fluorescent signal is directly proportional to
the quantity of amplicons produced during the reaction.the quantity of amplicons produced during the reaction.
3. Two general principles for the quantitative detection of amplicons:3. Two general principles for the quantitative detection of amplicons:
- agents binding to the double-stranded-DNA (SYBR Green)- agents binding to the double-stranded-DNA (SYBR Green)
- fluorescent probes (FAM, TAMRA, JOE, ROX,…)- fluorescent probes (FAM, TAMRA, JOE, ROX,…)
4. For the fluorescent probes, there are 44. For the fluorescent probes, there are 4 mainmain technologies:technologies:
- probe hydrolysis- probe hydrolysis
- hybridisation of 2 probes- hybridisation of 2 probes
- molecular beacons- molecular beacons
- scorpion primer- scorpion primer

8. SYBR Green
• The free SYBR Green exhibits
little fluorescence at the time of
the denaturation.
• SYBR Green dye intercalates
into ds-DNA & produces a
fluorescent signal.
• The intensity of signal is
proportional to the amount of
ds-DNA in the reaction.
• During polymerisation step,
more and more of molecules bind
to the nascent strand and the
increase in fluorescence can be
followed in real time.
• Binds non-specifically.
Clark, 2005

9. TaqMan Probe
-During the denaturing step,
the probe is free on
solution.
-During the annealing step,
probe hybridises to their
target sequence. The
proximity of the
fluorochrome allows the
inhibition of fluorescence.
-The polymerase moves and
hydrolyses the probe. The
transmitting fluorochrome
is released from the
environment of the
suppressor thus allowing
the emission of
fluorescence.
Clark, 2005

10. TaqMan Probe In Action

11. Hybridization Probes
- 4 oligo method
2 PCR primers & 2 seq.
soecific probes.
- 3 oligo method
2 PCR primers & 1 seq.
specific probe.
D - Donor fluorophore
A - Accepter fluorophore
Wong & Medrano, 2005

12. Molecular beacon
• Has two engineered
regions at the ends of
probe sequence.
• Stem and loop
conformation – no
fluorescence.
• Central region is
complementary to the
target sequence of 20 to
30 bases.
• After binding it gets
linearized and fluoresce.
• High temp. may cause
unpairing and give a
false positive response.
Clark, 2005

13. Scorpion Primers
• Molecular Beacon joined to a
ss DNA primer by an inert
hexethylene glycol (blocks
duplication )
• Extension step, primers
portion anneals & makes new
DNA.
• Denaturation step, probe+
new DNA become ss.
• Loop anneals to ss target DNA,
separating fluorophore &
quencher.
• Duplex scorpion.
• Specific for allelic
discrimination. Clark, 2005
Wong & Medrano, 2005

15. Sunrise Primers
-Similar to Scorpions except
inert linker mol.
-Dual labeled hairpin loop on
5’ end and 3’ end acting as
primer.
-Integration into PCR
product, separation of two
groups & emission.
-Non-specific flu. due to
duplication of primer during
formation of primer-dimer.
Wong & Medrano, 2005
Mackay et al., 2002
Q - Quencher fluorophore
R - Reporter fluorophore

16. LUX Primers
-Light upon extension
primers.
-Single fluorophore labeled.
-secondary str. of 3’end
reduces initial fluorescence.
-does not require a quencher
dye – self quenched.
Wong & Medrano, 2005

17. Characteristics of detection chemistries
Wong and Medrano, 2005

18. Primer & probe design
• Tm-58-60 oC for primer & 10 oC higher for probe.
• 15-30 bases in length
• G+C content – 30-80% ; if higher – high annealing &
melting temperatures, cosolvents glycerol, DMSO, or 7-
deaza dGTP.
• No run of an identical ntd.
• Total no. of Gs & Cs in the last 5 ntds. at 3’ end – 2.
• Primers spanning exon-exon jn. in the cDNA sequence.
• TaqMan – Allelic discrimination, mismatching ntds.
should be in the middle.
• Probe - No runs of identical ntds.; G+C – 30-80%.
• More Cs than Gs & no G at 5’ end.
• Amlicon size – 400bp (50-150bp).
Primer Express Software
Dorak MT, 2006

19. Fig: Real-time PCR
machine/thermocycler

22. Phases of Amplification curvePhases of Amplification curve

23. Threshold cycle (Ct)
Fig:-Threshold cycle (Ct)
The concept of the threshold cycle is at the heart of accurate and
reproducible quantification using fluorescence-based PCR.
It corresponds to the cycle from which one observes a statistically
significant increase in standardized fluorescence.

24. Melting curve

25. Detection of non-specific amplification

26. Real-Time PCR :
ApplicationsViral quantitation
Quantitation and gene expression
Array verification
Drug therapy efficacy
DNA damage measurement
Quality control and assay validation
Genotyping
Pathogen detection

27. Detection of PLRV in dormant potato
tubers
Schoen et al., 1996.

28. Primer and probe design
a PLRV coat protein ORF.
b Down= downstream; Up= upstream.
c from Hadidi et al.
d PLRV coat protein open reading frame
e At 5’ end FAM dye & at 3’ TAMRA dye.
Contd.

29. Detection of PLRV by IMC/RT-PCR * & verification by ELISA.
Cultivar
No. of
tubers
tested
Positive by
ELISA
on leaves #
Positive by
IMC-RT-
PCR
Positive by
ELISA
(shoot test) $
Bintje
Desiree
Kennebec
Total
12
11
9
32
7
1
5
13
11
8
9
28
11
9
9
29
# leaves from the PLRV-inoculated plants that produced the tubers tested.
$ Shoots grown from the tested tubers.
* Found that IMC/RT-PCR is 10 times more efficient than IC/RT-PCR.
Contd.

30. Detection of PLRV in pr. Infected tubers after
8 wks. storage(4 0C) by IMC/RT-PCR &
verification by ELISA
Cultivars
No. of
tubers
tested
+ve by
IMC/RT-PCR
+ve by
ELISA #
(shoot test)
Bintje
Desiree
Kennebec
Total
4
4
4
12
2
0
4
6
4
4
4
12
# Shoots grown from the tested tubers was tested for ELISA.
Contd.

31. Cultivar No. of
tubers
tested
Positive
by 5’
nuclease
assay
Range of
del-RQ
values
Positive
by EtBr
Positive
by
ELISA
Bintje
Desiree
Kennebec
Spounta
Total
11
14
8
4
37
11
14
8
4
37
1.59-2.57
1.06-3.11
2.06-3.15
2.45-2.94
11
14
8
4
37
11
14
8
4
37
Detection of PLRV in dormant tubers after 12 wks.
Storage by IMC followed by real-time PCR &
verification by ELISA
ELISA was done after shoot test
Contd.

32. Fig : Comparison of real-time PCR & GE after
IMC-RT-PCR
Del-RQ =
RQ+ - RQ-
Contd.

33. Finding
Testing time of seed potato reduced from
a minimum of 5 weeks for the current
indexing method to 1 day by Real-time
IMC/RT- PCR – based assay.
Schoen et al., 1996.

34. Quantification of P. capsici in
different pepper genotypes
Silver et al., 2005.

35. Fig: Disease
progress curves of
pepper cultivars
inoculated with
A, UDC196Pc or
B, UDC248Pc.
Plotted points - mean
value of 20 plants
from two
independent
experiments.
Padron , Yolo
Wonder ,
P1201234 , and
SCM331 .
Contd.

36. Fig: Standard curve for real-time PCR analysis of a 10-fold serial
dilution of Phytophthora capsici DNA
Contd.

37. Fig: P. capsici
DNA
quantification in
leaves
inoculated with
A, UDC196Pc
or B,
UDC248Pc
Contd.

38. Fig: P. capsici DNA
quantification in
stems inoculated
with A,
UDC196Pc or B,
UDC248Pc.
Contd.

39. Fig: P. capsici DNA
quantification in
roots inoculated
with A, UDC196Pc
or B, UDC248Pc
Silver et al., 2005

40. Findings
• Detected pathogen DNA before first
symptoms of the disease were
observed in plants (8hrs. ; 4 days).
• Selection of most resistance cultivar.
Silver et al., 2005

41. Detection of Bacteria in seven minutes
Belgrader et al., 1999.

42. Portable
ANAA
• Can be used in the
field and offers real-
time monitoring.
• Consists of an array of
ten reaction modules
and a laptop
computer.
• Each reaction module
comprising a silicon
reaction chamber with
efficient integral thin-
film heaters & a low-
power optical system.
• Software
automatically informs
the user via an audible
alert and green-to-red
indicator.
Contd.

43. Table - Thermal cycling settings and detection times for
analyzing bacteria cells with the ANAA.
Denature time and anneal and extend time represent the set
point values at 96 and 56 degree Celsius, respectively.
Contd.

44. Fig: Detection profiles
obtained by rapid,
real-time PCR
analysis of Erwinia
cells
(A) Effect of
decreasing the
cycle time for the
analysis of 500
cells.
(B) Quantitative PCR
performed with the
17-s cycle time.
Contd.

45. Fig: temperature profile of a reaction
performed with a 17-s cycle time
Contd.

46. Finding
• Bacteria can be detected as
early as in seven minutes.
Belgrader et al., 1999

47. Simultaneous detection of four
viruses from dormant potato tubers
Agindotan et al., 2007

48. Primer & Probe design
Contd.

49. Testing for primer-probe specificity and comparison of uniplex
and quadruplex two-step real-time PCR for detection of potato
viruses
a Mean of three repititions.
b Reverse primer in each reverse transcription reaction, and a primer pair and probe of each
virus in a PCR reaction.
c All the four reverse primers of the four viruses in each reverse transcription reactions, and all
the four virus primer pairs and four probes in each PCR reaction.
d Templates were total RNAs from infected potato tubers.
e Templates were viral RNAs purified from virions
Contd.

50. Comparison of real-time uniplex RT-PCR detection of potato
viruses from purified total RNAs and saps obtained from
the same dormant potato tubers
n: number of potato tested; S.D.: standard deviation.
a Total RNAs of four tubers each infected with PLRV, PVA, PVX or PVY were
used as positive controls.
b The negative control was total RNA extracted from virus-free tissue culture
minituber.
Contd.

51. Multiplex detection of potato viruses
n: number of repititions.
a Composite saps obtained from dormant
tubers infected with three or four
viruses.
b Composite samples of three viruses.
c Composite total RNAs obtained from
dormant tubers.
d These were viral RNAs purified from
infected potato leaf.
Contd.

52. Findings
• Detected four viruses
simultaneously.
• Saving of time & money.
Agindotan et al., 2007

53. Advantages
1. Collects data in the exponential growth phase.
2. Wide dynamic range of quantification(7-8 log
decades).
3. High precision(< 2% CV of Ct values).
4. Extremely sensitive (ng to pg).
5. No post-PCR steps.
6. High throughput.
7. Field use.
8. Multiplex approach.
Contd.

54. Contd.
9. Most accurate & reproducible technique for
NA quant.
10. Absolute quantification.
11. Selection of resistance cultivar.
12. Can analyze low expression level genes from
limited samples.
13. Study of changes in gene expression in
response to phytopathogenic & antagonistic
fungi.
14. Detection is capable down to 2-fold change.
15. Rapid detection (7 to 30 minutes).

55. Limitations
• PCR products increase exponentially.
• Variation increases with cycle no.
• Increased variation after transformation to
linear value.
• Need of optimization.
• Overlap of emission spectra.
• Maximal four simultaneous reaction.
• Increased risk of false negative results.

56. Conclusion
- Improved ability to detect & monitor plant
pathogens.
- Comparison of results from different labs easier &
more reliable.
- With a well-designed experiment performed with
the proper controls, can be one of most sensitive,
efficient, fast & reproducible method.
- More effort is required to resolve problems in
regard to cost, reproducibility & feasibility for
large scale testing.

57. In Future…
- Potential to discriminate an increasing no. of targets
by further improvement to the hairpin primers and
hairpin & nuclease oligoprobes.
- Further exploration towards the av. of fluorescence dye
combination can monitor more than 4 or 5 different
targets simultaneously.
- Improvement in instrumentation can lead to reduction
in cost.
- Can be applied for assessing the samples directly from
the farmers’ field.
Contd.

58. • Plant clinics should be established in each
& every block of the country.
• Real-Time PCR machine should be kept
there.
• Samples from farmers’ field should be
monitored.
• Accordingly the management practices
should be recommended.
Contd.

59. THANK YOU