A Thermostabilized, One-Step PCR Assay forudSimultaneous Detection of Klebsiella pneumoniae andudHaemophilus influenzae

摘要

Klebsiella pneumoniae and Haemophilus influenzae are two common pathogens associated with respiratory tract infections.udThe identification of these pathogens using conventional molecular diagnostic tests requires trained personnel, cold-chainudtransportation, and storage-dependance, which does not render them user-friendly. The aim of this study was to develop audthermostabilized, cold-chain-free, one-step multiplex PCR for simultaneous detection of K. pneumoniae and H. influenzae. Theudmultiplex PCR assay was designed to amplify the php gene of K. pneumoniae (202 bp) and p6 gene of H. influenzae (582 bp).udIn addition, the specific primer to amplify glm gene of Helicobacter pylori (105 bp) was included as an internal amplificationudcontrol. Subsequently, the designed primers and all PCR reagents were thermostabilized by lyophilization. The stability of theudthermostabilized PCR was evaluated using the Q10 method. The sensitivity and specificity of performances for thermostabilizedudPCR were evaluated using 127 clinical isolates and were found to be 100% sensitive and specific.The thermostabilized PCR mix wasudfound to be stable for 30 days and the Q10 accelerated stability was found to be 3.02 months. A cold-chain-free, PCR assay for easy,udrapid, and simultaneous detection of K. pneumoniae and H. influenzae was successfully developed in this study.