Development of a diagnostic assay by three-tube multiplex real-time PCR for simultaneous detection of nine microorganisms causing acute respiratory infections

Acute respiratory infections are widespread in vulnerable populations of all ages and are characterized by a variety of symptoms. The underlying infection can be caused by a multitude of microorganisms, including viruses and bacteria. Early detection of respiratory infections through rapid pathogen screening is vital in averting infectious respiratory disease epidemics. This study utilized a multiplex real-time PCR system to develop a three-tube reverse transcription-PCR (RT-PCR) assay, enabling simultaneously detect nine respiratory pathogens, including: influenza A and B, adenovirus, respiratory syncytial virus (RSV), Streptococcus pneumoniae, Legionella pneumophila, Haemophilus influenzae, Chlamydia pneumoniae, and Mycoplasma pneumoniae. This technique utilizes a one-step assay, with specifically designed TaqMan primer–probe sets combined in the same tube. This assay provided rapid and simplified detection of the nine prevalent pathogens, as well as increased sensitivity and reduced cross-contamination. This assay was evaluated using 25 related viral/bacterial strains as positive references, the other 25 irrelevant strains as negative controls, and clinical specimens from 179 patients. All positive strains were detected with no amplification of the non-target microorganism mixtures and the assay’s detection limits ranged between 250–500 copies/ml (1.25–2.5 copies/reaction). A total of 167 (93.3%) samples tested positive for at least one of the pathogens identified; 109 of these samples were from patients confirmed to have RSV infections. The diagnostic accuracy of our assay was further confirmed by matching results from classical direct immunofluorescence assay and nucleotide sequencing. These data demonstrate the innovative multiplex real-time PCR assay as a promising alternative to the current approaches used for early screening of acute respiratory infections.