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Rapid identification of Bovine Mastitis pathogens by High Resolution Melt Analysis of 16S rDNA sequences. CAHLN 2010. Praseeda Ajitkumar Jeroen De Buck Herman Barkema Department of Production Animal Health. Background.
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Rapid identification of Bovine Mastitis pathogens by High Resolution Melt Analysis of 16S rDNA sequences CAHLN 2010 PraseedaAjitkumar Jeroen De Buck Herman Barkema Department of Production Animal Health
Background • Mastitis: persistent problem and the most expensive disease of dairy cows • Coagulase-negative staphylococci (CNS) are a frequent cause of bovine mastitis in many countries. • CNS are not identified further by species but are treated as a uniform group
Identification of mastitis pathogens • Bacteriological culture- gold standard • PCR based assays- to complement or replace conventional identification methods • DNA sequencing
High Resolution Melt (HRM) • Rapid molecular technique introduced in 2002 • Generation of melting curves after PCR amplification • Based on differences in the thermal stability of DNA • Genotyping of several organisms (Chlamydia psittaci, Mycoplasmapneumoniae, Mycobacterium tuberculosis , M. aviumsubsp.paratuberculosis(Castellanos et al.,2010a and 2010b), Influenza A virus)
HRM versus Melt curve HRM is an extended analysis of melt curve • Requires additional analysis software - Normalize melt curves - Apply an optional temperature shift - Plot curves in a difference graph for easy visualization - Clusters curves into groups representing different genotypes/sequences
HRM versus Melt curve • “Saturation” dyes are less inhibitory to PCR than SYBR (Evagreen, LC green dyes) • Observed melting behaviour is characteristic of a particular DNA sample • Target - 16S rRNA gene • Gold standard for broad-range microbial identification • Feasibility of using high-precision melting for bacterial speciation (Cheng et al., 2006) • Highly specific species identification of clinically relevant biothreat bacterial agents (Yang et al., 2009)
Hypothesis • High resolution analysis of melting curves generated after PCR amplification can lead to rapid speciation of mastitis pathogens
Objective • Development of novel and rapid assays to speciate major and minor mastitis pathogens based on real-time PCR and HRM
Pathogens in Clinical Mastitis n-3,024 OldeRiekerink et al., 2008
Materials & Methods Extraction of bacterial genomic DNA • 7 bacterial strains from ATCC and 6 isolates from mastitis milk samples subjected to DNA extraction • 14 coagulase-negative staphylococci isolates from CBMRN • Genomic DNA extracted with the DNeasy Blood and Tissue Kit (Qiagen)
Materials& methods (contd…) • Amplification of 16S rRNA gene using real-time PCR • Real-time PCR amplification of 16S rRNA gene using BioRad CFX thermal cycler • Cycling conditions 1: 98.0°C for 2:00 min 2: 98.0°C for 0:05 3: 55.0°C for 0:10 Plate Read 4: GOTO 2, 39 more times 5: 95.0°C for 1:00 6: 70.0°C for 1:00 7: Melt Curve 70°C to 95°C : Increment 0.2°C for 0:10 Clustering
Result HRM-common mastitis pathogens • 1. A. pyogenes • 2. C. bovis • 3. S. agalactiae • 4. S. dysgalactiae • 5. E. coli • 6. K. pneumoniae • 7. S. uberis • 8. P. melaninogenica • 9. F. necrophorum • 10. S. aureus • 11. B. fragilis • 12. M. bovis 1 3 2 4 5 6,7 8 9 12 10,11 13
Result HRM- coagulase-negative staphylococci • 1. S. auricularis • 2. S. chromogenes • 3. S. intermedius • 4. S. hyicus • 5. S. aureus • 6. S. capitis • 7. S. epidermidis • 8. S. sciuri • 9. S. simulans • 10. S. warneri • 11. S. saprophyticus • 12. S. cohnii • 13. S. xylosus • 14. S. haemolyticus • 15. S. hominis
Advantages of HRM • Inexpensive • High sensitivity & specificity • Rapid-completed in about 1 h 30 min
Conclusions • High resolution melt analysis is a rapid molecular tool for the identification of mastitis pathogens • Validation of the technique is necessary • Applicability of the technique in speciation of pathogens in mastitis milk samples needs to be evaluated
Future Directions • Test and validate HRM assays on DNA extracts of subclinical and clinical mastitis cases (CNS and other mastitis pathogens) • Culture-negative mastitis samples
Acknowledgements • Supervisors Herman Barkema & JeroenDe Buck • John Middleton, Faculty of Vet. Med, Missouri • Lab mates Elena Castellanos Amanda Reith Nick Mackenzie VineetSaini RienskeMortier Joel David • Faculty of Veterinary Medicine, University of Calgary for the UCVM scholarship • National Mastitis Research Foundation