Evaluation of the Capacity of PCR and High-Resolution Melt Curve Analysis for Identification of Mixed Infection with Mycoplasma gallisepticum Strains

Pathogenicity and presentation of Mycoplasma gallisepticum (MG) infection may differ from one strain to another and this may have implications on control measures. Infection of individual birds with more than one MG strain has been reported. A PCR followed by high resolution melt (HRM) curve analysi...

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Bibliographic Details
Published in:PloS one 2015-05, Vol.10 (5), p.e0126824-e0126824
Main Authors: Ghorashi, Seyed A, Kanci, Anna, Noormohammadi, Amir H
Format: Article
Language:English
Subjects:
Adhesins, Bacterial - genetics
Animals
Birds
Coinfection - diagnosis
Coinfection - microbiology
Coinfection - veterinary
Deoxyribonucleic acid
DNA
Genes
Genes, Bacterial
Genetic aspects
Health aspects
High resolution
Identification and classification
Infections
Lectins - genetics
Mixed infection
Molecular Diagnostic Techniques
Mutation
Mycoplasma
Mycoplasma gallisepticum - genetics
Mycoplasma Infections - diagnosis
Mycoplasma Infections - microbiology
Mycoplasma Infections - veterinary
Pathogenicity
Pathogens
Polymerase Chain Reaction
Poultry
Poultry Diseases - diagnosis
Poultry Diseases - microbiology
Strains (organisms)
Transition Temperature
Turkeys - microbiology
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Summary:Pathogenicity and presentation of Mycoplasma gallisepticum (MG) infection may differ from one strain to another and this may have implications on control measures. Infection of individual birds with more than one MG strain has been reported. A PCR followed by high resolution melt (HRM) curve analysis has been developed in our laboratory and routinely used for detection and differentiation of MG strains. However the potential of this test for identification of MG strains in a mixed specimen has not been evaluated. In the present study, the capability of PCR-HRM curve analysis technique, targeting vlhA and pvpA genes was assessed for identification of individual MG strains in a mixed population. Different DNA ratios of two MG strains from 1 to 10(-4) ng were tested with some generated conventional and normalized curves distinct from those of individual strains alone. Using genotype confidence percentages (GCP) generated from HRM curve analysis, it was found that vlhA PCR-HRM was more consistent than pvpA PCR-HRM for the detection of MG ts-11 vaccine strain mixed with any of the MG strains 6/85, F, S6 or a field isolate. The potential of vlhA PCR-HRM to detect mixed MG strains in a specimen was found to be primarily dependent on quantity and proportion of the target DNAs in the mixture. This is the first study examining the capacity of PCR-HRM technique for identification of individual MG strains in a mixed strain population.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0126824