NIB activities in Workpackage 7
Rok Lenarčič, Dany Morisset, Maja Ravnikar
February, 2013
NIB activities in Workpackage 7
PSTVd RT-LAMP
• Single tube reaction, 25’ to final answer
• Sensitivity 10x better than RT-PCR, 100x lower than RTqPCR
• Detects all tested PSTVd isolates,
• Paper accepted published in Plant Pathology journal
Ralstonia solanacearum LAMP
• 3 LAMP sets evaluated (fliC, 16S rRNA, egl - 3 primer sets)
• LAMP on endoglucanase gene was selected as the best
• Detects all isolates from Phylotype I, II and III and most of Phylotype IV (negative proposed to form a new
phylotype/species)
• Total running time 30’
• Boiling without DNA extraction is sufficient before LAMP
• Paper in preparation
Multiplexing
• Simultaneous detection of RNA (PSTVd) and DNA (Rs) targets works in one tube. Work in progress
Ligation based Universal LAMP (done in collaboration with FERA, UK)
• Ligation optimised
• Problematic ‘Loop’ primers swaped for ‘Stem’ primers (Gandelman et al., 2011)
• Exonuclease treatment tested (probably not worth regarding gain vs. time+cost)
• Multiplexing on DNA targets works
• Biotinylation works for detection on arrays
PSTVd detection by RT-LAMP
Paper published in Plant Pathology
Reverse transcription combined with LAMP→ single tube reaction
Run time 25 mins
Faster and more efficient amplification than with previously reported RT-LAMP
Specificity of the PSTVd RT-LAMP
Detects all tested PSTVd and TPMVd and some TCDVd isolates (similar
sequence, same origin)
PSTVd has different Tm than TPMVd.
16 PSTVd isolates and 42 Pospiviroid isolates (TPMVd, TCDVd, TASVd, CSVd, CEVd, CLVd). One isolate from the genus
Cocadviroid (Pospiviroidae), and five isolates from the family Avsunviroidae tested.
Sensitivity of the PSTVd RT-LAMP
• 10x more sensitive than RT-PCR
• 100x less sensitive than real time RT-PCR
Total plant
Estimated PSTVd
Real-time RT-
RNA dilution
RNA copy
PCR (Cq)
RT-LAMP (Tp)
(this study)
Tsutsumi RT-LAMP
RT-PCR
assay (Tp)
number
1x10-2
10000-
+ (23.6 ± 0.2)
+ (13.0 ± 0.1)
+ (26.2 ± 1.1)
+
100000
1x10-3
1000-10000
+ (27.6 ± 0.5)
+ (14.1 ± 0.4)
+ (28.5 ± 1.8)
+
1x10-4
100-1000
+ (30.9 ± 0.1)
+ (15.5 ± 0.2)
-
-
1x10-5
10-100
+ (33.9 ± 0.2)
-
-
-
1x10-6
1-10
+ (37.6 ± 0.3)
-
-
-
1x10-7
0
-
-
-
-
1x10-8
0
-
-
-
-
1x10-9
0
-
-
-
-
Comparison of different LAMP assays for
Ralstonia solanacearum detection
R. solanacearum detection by LAMP
• LAMP on 3 targets:
– fliC (Kubota et al., 2008) additional loop primer, optimised primer mix
– 16S rRNA. Based on Weller’s qPCR assay
– egl (3 primer sets designed, one chosen for further validations)
Assay characteristic
egl
LAMP
fliC
LAMP
16S rRNA LAMP
16S rRNA qPCR
Target gene
endoglucanase
flagellar subunit
16S rRNA
16S rRNA
Running temperature
60°C
65°C
65°C
cycling
Running time
30 min
40 min
60 min
90 min
Analytical sensitivity*
104-106 CFU/ml
105-107 CFU/ml
ND
102-104 CFU/ml
Diagnostic sensitivity**
105 CFU/ml
105 CFU/ml
103-104 CFU/ml
102-104 CFU/ml
* depends on phylotype
** phylotype IIB
R. solanacearum isolates
Phylotype
(nr. of isolates)
egl LAMP
fliC LAMP
16SrRNA
LAMP
qPCR
Phylotype I (20)
20
20
20
20
Phylotype IIA (17)
17
17
17
17
Phylotype IIB (24)
24
22
24
24
Phylotype III (15)
15
15
15
15
Phylotype IV* (8)
4**
8
8
7**
Unknown (4)
4
3
4
4
R. pickettii (2)
-
1
-
-
R. mannitolilytica (1)
-
-
-
1
Other strains (10)
-
-
-
-
6***
4***
29
4
Potato extracts (56)
*Phylotype IV including Blood Disease Bacterium and R. syzygii which were proposed to form a separated species (Remenant
et al. 2011)
** 2 R. solanacearum detected + 2 R. syzygii, except from a genetically distant strain RUN14 that is proposed to form a new
phylotype V
*** Observed signal is not reproducible, and can be distinguished from real positive based on Tm
Sensitivity of egl LAMP for R. solanacearum detection
CFU
Phyl I
Phyl IIA
Phyl IIB
Phyl III
Phyl IV
108
12.7
15.8
12.9
12.2
19.4
107
14.2
16.5
14.7
13.8
18.8
106
17.0
18.1
16.3
15.7
19.3
105
18.1
23.3a
17.7b
17.4
21.3a
104
20.8
26.3a
21.3a
20.5
-
103
-
-
-
-
-
102
-
-
-
-
-
10
-
-
-
-
-
0
-
-
-
-
-
qPCR sensitivity marked with bold line
a detected
once out of three replicates
bdetected twice out of three replicates
egl LAMP Tm
Phylotypes I and III:
94.6°C
Phylotypes IIA, IIB, IV:
93.8°C
Multiplex LAMP (DNA & RNA)
Multiplexing R. solanacearum (DNA) and PSTVd (RNA).
Dilutions made in dH2O (extracts might cause problems).
Usually one product is detectable, rarely both (example below)
Triplex testing in progress (R.solanacearum, C. michiganensis sepedonicus,
PSTVd)
Legend
Sample
tp
Tm
RESULT
108 RS + 10-5 PSTVd
29:11
92.66
RS
107 RS + 10-4 PSTVd
27:56
92.48
RS
106 RS + 10-3 PSTVd
27:41
90.68
PSTVd
105 RS + 10-2 PSTVd
24:41
90.79
PSTVd
104 RS + 10-1 PSTVd
22:41
90.82
PSTVd
NTC
/
/
/
Future work
• Complete multiplexing story
– sensitivity
– specificity
• Ligation universal LAMP:
– To be discussed between developers?
NAIMA
• Initially proposed in Qdetect
• NAsba Integrated Multiplex Amplification
•
•
•
•
•
PSTVd
Starting material: RNA
Same region as for Boonham’s one step RT-PCR (2004) (used in routine lab)
Amplification up to 7.107 in 45min
About 60x less sensitive than qPCR
Specificity OK
– Test done on several isolates (PSTVd and other viroids)
Family
Genus
Viroid name
NAIMA
Real-time RT-PCR
Pospiviroidae
Pospiviroid
PSTVd
+
+
TCDVd
+
+
TPMVd
+
+
TASVd
-
+/-*
CSVd
-
-
CEVd
+
+/-*
CLVd
-
+/-*
14
Ralstonia solanacearum
•
•
•
•
•
Starting material: DNA
Targeting 16S RNA gene.
Very good amplification rates for all (108 to 109 in 45min).
Sensitivity comparable to that of qPCR (Weller).
Specificity OK
– Ralstonia solanacearum strains from three phylotypes + BDB and R. syzygii
(phyIV) tested and detected.
– Cross reaction strains requested by EU regulation collected (other Ralstonia
sp., Bacillus polymixa, Burkholderiaceae ...). All negative except R.
mannitolilytica (also detected by qPCR)
– No amplification in potato extract used as negative samples
15
Duplex PSTVd-Ralstonia
• Starting material: DNA, RNA or mixture of
both
• Duplex primers mixture
– Sensitivity for a single target is same as in
singleplex reactions
– When both targets are present in different
amounts, amplification is good but the target at
low concentration is sometimes not amplified
NAIMA products detection
• Adding of biotinylated-UTP (up to 1/3) in the
reaction does not affect the amplification
• Clondiag Array Tubes for confirmation
– 110 min from product hybridization to data analysis (to
be improved)
– Multiplexing is conserved
– Array tube (14x14) just received
– Test in progress
17
Acknowledgements
NIB:
• Dany Morisset
• Rok Lenarčič
• Maja Ravnikar
•
•
•
•
Nataša Mehle
Tanja Dreo
Manca Pirc
David Dobnik (NAIMA)
FERA:
• Jenny Tomlinson
• Neil Boonham
PRI:
• Cor Schoen