quantitative real-time pcr for diagnosis and identifi
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Quantitative Real-time PCR for diagnosis and identifi-cation of Xanthomonas citri pv. citri pathotypes,
causal agents of Asiatic Citrus CankerAsiatic Citrus Canker is induced by • Xanthomonas citri pv. citri (Xcc) (EPPO List A1)
Two pathotypes with different economical impacts: •
X. citri - pv. citri-A : wide host range of citrus and other related genera, worldwide distribution X. citri - pv. citri-A*: narrow host range : limes (C. aurantifolia) and alemow (C. macrophylla), li-mited areasX. citri - pv. citri -Aw : a junior synonym of X. citri pv. citri -A* (1)
Preliminary study: evaluation of different Xcc •primers previously published for PCR diagnosis:
Collection of - Xcc (n=37) and other Xantho-monas species and pvs. (n=24)Lack of specificity of PCR-based diagnostic -tools (included primers proposed in the EPPO standard protocol)
Detection of X. citri pv. citri and identification of pathotypes by qPCR
Hydrolysis probes: Taqman-MGB •
Duplex qPCR •
Markers •
Xcc-Chem: - Xcc specific sequenceXcc-hK: - Xcc-A specific sequence from a housekeeping gene
Detection of Xcc by PCR in pure cultures
With three PCR primers designed on genes •identified based on from the genome of strain IAPAR 306 (Xcc-A):
involved in pathogenicity (Flm) - related to chemotaxism (Chem) -
Specific of • X. citri pv. citri
No pathotype specific mutation valuable •for qPCR
Perspectives
Development of internal standard
Validation of the assay •
Calibration of the extraction •
Citrus-18S: a sequence of the 18S gene from Citrus plant •
Sensitivity
Detection of 10 • 7 to103 bacteria/ml
Detection of 2.5x10 • 6 to 2.5x103 bacteria/mg of lime leaves
Specificity
Assays on pure cultures •
No other • Xanthomonas (pathogenic or not on Citrus) amplified with Xcc-Chem system, spe-cific to X. citri pv. citri
Extraction method
Conclusion
Rapid detection of the bacterium in tissues with internal stan- •dard
Distinguish pathotypes with different economic incidence •
Useful for indexing propagation material in nurseries and for •surveillance of international movement of X. citri pv. citri
Further assays are necessary to validate the internal standard •
Delcourt S., Robène-Soustrade I., Vernière C., Boyer C., Pruvost O.CIRAD- UMR Peuplements Végétaux et Bioagresseurs en Milieu Tropical, Saint-Pierre, La Réunion, France
Develop a reliable diagnostic tool to :
Detect • X. citri pv. citri = pathovar specific
Identify both pathotypes = pathotype specific •
Exclude other Xanthomonads including those pa- •thogenic on Citrus: X. citri pv. aurantifolii, X. citri pv. bilvae, X. alfalfae pv. citrumelonis
100 µl of extract
Wizard® Genomic DNA purification ( Promega)
(1) Bui Thi Ngoc L, et al. (2010) IJSEM vol 60:515-525
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Detection of Xanthomonas strains by primers used for Xcc diagnosis
Primers
Freq
uenc
y of
det
ecti
on
2/3 4/7J−p
thJ−R
xKing
VM
XAC01/02
XACF/XACR
XCF/XCR
Target
Non target
40 mg of healthy lime leaves at -80°C crushed with a mortar
+108 bacteria
Xcc-A (n=21)
Non-target Xanthomonas
(n=24)
Xcc-A* (n=16)
Xcc-Chem
Xcc-Chem
Xcc-Chem
Xcc-hK (variable)
Xcc-hK
Xcc-hKPure cultures of Xcc IAPAR 306
Xcc-hK efficacity 81% R2=0.996
Xcc-Chemefficacity 94%R2=0.994
Plant extract contaminated with Xcc IAPAR 306
Xcc-hKefficacity 74%R2=0.997
Xcc-Chemefficacity 87%R2=0.997
This work was funded by the program POSEIDOM interDOM
Citrus-18S
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