title: A Thermostabilized, One-Step PCR Assay for Simultaneous Detection ofKlebsiella pneumoniaeandHaemophilus influenzae.
abstract: Klebsiella pneumoniaeandHaemophilus influenzaeare two common pathogens associated with respiratory tract infections. The identification of these pathogens using conventional molecular diagnostic tests requires trained personnel, cold-chain transportation, and storage-dependance, which does not render them user-friendly. The aim of this study was to develop a thermostabilized, cold-chain-free, one-step multiplex PCR for simultaneous detection ofK. pneumoniaeandH. influenzae.The multiplex PCR assay was designed to amplify thephpgene ofK. pneumoniae(202 bp) andp6gene ofH. influenzae(582 bp). In addition, the specific primer to amplifyglmgene ofHelicobacter pylori(105 bp) was included as an internal amplification control. Subsequently, the designed primers and all PCR reagents were thermostabilized by lyophilization. The stability of the thermostabilized PCR was evaluated using theQ10method. The sensitivity and specificity of performances for thermostabilized PCR were evaluated using 127 clinical isolates and were found to be 100% sensitive and specific. The thermostabilized PCR mix was found to be stable for 30 days and theQ10 accelerated stability was found to be 3.02 months. A cold-chain-free, PCR assay for easy, rapid, and simultaneous detection ofK. pneumoniaeandH. influenzaewas successfully developed in this study.
authors.affiliation: Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Kota Bharu, Malaysia.
authors.forename: Nur Amalina
authors.initials: NA
authors.lastname: Khazani
authors.affiliation: Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Kota Bharu, Malaysia.
authors.forename: Nik Zuraina Nik Mohd
authors.initials: NZ
authors.lastname: Noor
authors.affiliation: Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Kota Bharu, Malaysia.
authors.forename: Chan
authors.initials: C
authors.lastname: Yean Yean
authors.affiliation: Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Kota Bharu, Malaysia.
authors.forename: Habsah
authors.identifier: 0000-0002-4948-2157
authors.initials: H
authors.lastname: Hasan
authors.affiliation: Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Kota Bharu, Malaysia.
authors.forename: Siti
authors.identifier: 0000-0003-3115-8479
authors.initials: S
authors.lastname: Suraiya
authors.affiliation: School of Dental Sciences, Universiti Sains Malaysia, Kota Bharu, Kelantan, Malaysia.
authors.forename: Suharni
authors.identifier: 0000-0001-8539-718X
authors.initials: S
authors.lastname: Mohamad