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Confirmed identification and toxin profiling of Campylobacter jejuni using a thermostabilized multiplex PCR formulation
Author(s) -
Ramachandran Nitya,
Ramlal Shylaja,
Batra Harsh Vardhan
Publication year - 2017
Publication title -
apmis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.909
H-Index - 88
eISSN - 1600-0463
pISSN - 0903-4641
DOI - 10.1111/apm.12700
Subject(s) - campylobacter jejuni , microbiology and biotechnology , cytolethal distending toxin , toxin , multiplex , campylobacter , multiplex polymerase chain reaction , biology , polymerase chain reaction , bacteria , microbial toxins , gene , bioinformatics , genetics
Cytolethal distending toxin ( CDT ) producing Campylobacter jejuni species are one of the leading causes of human gastroenteritis worldwide. The main intent of the study was to develop a multiplex PCR assay for the confirmed identification and toxin profiling of C. jejuni . The genes targeted were rpo B as genus specific, hip O for species; cdt A, cdt B, cdt C encoding respective subunit proteins of CDT with Internal Amplification Control ( IAC ). To enhance its application as a pre‐mixed ready‐to‐use format, the master mix of developed mPCR was dried by lyophilization and stability was assessed. Thermostabilized reagents showed stability of 1.5 months at room‐temperature and upto six months at 4 °C without any loss of functionality. The assay was evaluated on a number of presumptive Campylobacter isolates along with biochemical tests. Results obtained indicated the accurate identification of C. jejuni by developed mPCR format in contrast to misconception associated with biochemical assays. The assay was also tested on spiked samples for its real‐time utility. Altogether, the room‐temperature storable and ready‐to‐ use mPCR format developed in this study could be preferred for rapid detection and confirmed identification of toxigenic strains of C. jejuni in place of conventional biochemical assays.