Authors
Alagarasu, K., Katendra, S., Prabhakar, M., Viswanathan, R.
Abstract
Background: Real time PCR provides rapid and accurate laboratory confirmation of clinically suspected pertussis and pertussis like illness (PLI). However, it does not inform the viability as it detects bacterial DNA from both viable and degraded bacterial cells. We report development of platinum based viability real time PCR assays for Bordetella pertussis, B.parapertussis and B.holmesii which cause pertussis and PLI. Material and Methods: ATCC strains of the three organisms were grown on selective media, and cell counts determined. Untreated (control) and Sodium dodecyl sulphate (SDS) lysed cultures were treated with varying concentrations of platinum chloride (PtCl4) and genomic DNA extracted. Real time PCR was performed for the targets of B.pertussis (IS481), B.parapertussis (pIS1001) and B.holmesii (hIS1001) respectively, and quantitative results obtained. threshold cycle values after PtCl4 exposure and before PtCl4 exposure ({Delta} Cq) were calculated for both untreated and SDS treated cultures. Results: For all three species, increase in {Delta}Cq values was noted with corresponding reduction in DNA copy numbers, as PtCl4 concentration was increased, in untreated and SDS treated cultures. Untreated cultures of all three species exposed to PtCl4showed increase in Cq values, suggesting that liquid cultures contain a mixture of live and dead bacteria. Conclusion: Performance of viability q PCR assay incorporating PtCl4 at a concentration range of 6 to 10 mM would inform the viability of the infecting organism in the clinical sample. This assay would improve the diagnostic reliability, help select samples for culture as well as adoption of mitigation strategies among contacts of pertussis cases.
Preprint server:
bioRxiv
The authors list and abstract were imported from bioRxiv on 06 Nov 2025.
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