Development of real-time fluorescence quantitative PCR for the detection of Moraxella catarrhalis

Abstract

Objective To developed A laboratory diagnosis of Moraxella catarrhalis by an laboratories diagnostic method real-time fluorescence quantitative PCR assay. Methods The specific primers and probes were designed based on the sequence of outer membrane protein CopB(copB)gene in Moraxella catarrhalis, and the Taqman probe RT-PCR method was developed to detect the Moraxella catarrhalis.The standard plasmids extracted from the Moraxella catarrhalis standard strains were used to constitute the standard samples, and compared with these standard samples, the sensitivity of the fluorescence quantitative PCR assay was tested by the established standard curves.The specificity of the fluorescence quantitative PCR assay was tested by the DNA samples of other bacterias in the laboratory.Meanwhile, 321 throat swab samples from inpatient and outpatient child patients, with asthma infection were collected as clinical samples to validate the fluorescence quantitative PCR assay. Results The standard curve was drawn in the real-time PCR by the Taqman fluorescence reporter.During the sensitivity tests, the newly-developed real-time fluorescence PCR could detect at least 10 copies of Moraxella catarrhalis, and could successfully distinguish several DNAs of the pathogens.On the basis of the validation result of the 321 throat swab samples, there are 25 Moraxella catarrhalis with 7.79 % positive rate. Conclusion The fluorescence quantitative PCR assay is of great sensitivity and specificity, and it can be widely used for the detection of Moraxella catarrhalis. Key words: Moraxella catarrhalis; Respiratory tract infections in children; Real-time fluorescence quantitative PCR