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

Abstract

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.

A multiplex recombinase polymerase amplification assay combined with CRISPR/Cas12a for the detection of respiratory syncytial virus and respiratory adenovirus

“…48 Compared with the multiple reverse transcription-PCR approach conducted by Jiang et al., our method detected comparatively few types of pathogens, but involved relatively simple procedures and has better detection performance. 49…”

“…48 Compared with the multiple reverse transcription-PCR approach conducted by Jiang et al, our method detected comparatively few types of pathogens, but involved relatively simple procedures and has better detection performance. 49 Considering the traditional pathogen detection methods, our assay has several advantages. First, the RPA-CRISPR/ Cas12a reaction is performed at a lower temperature that is constant without the need for complex temperature control devices.…”

Are ELISA and PCR Discrepancies in the Identification of Chlamydia pneumoniae Caused by the Presence of “Chlamydia-Related Bacteria”?

“…Apparently, this assay is not suitable for routine diagnosis in practice. Therefore, we made an attempt to adjust the panNPCR to one tube and for real-time PCR [ 40 , 41 , 42 , 43 , 44 ]. Unfortunately, this modification provided good sensitivity in dilutions of C. pneumoniae TW-183 culture, but in sputum samples, DNA could be amplified only if they were “spiked” with 10× more cells above the detection limit in panNPCR.…”

Advances in the application of molecular diagnostic techniques for the detection of infectious disease pathogens (Review)

“…Bennett and Gunson ( 23 ) developed an MqPCR that could simultaneously detect adenovirus, astrovirus, rotavirus and sapovirus in stool samples; this had a reduced turnaround time and overall cost compared with qPCR. Recently, Jiang et al ( 24 ) developed an MqPCR assay capable of concurrently detecting nine respiratory pathogens with no cross-reactivity and a limit of detection (LoD) of 250–500 copies/ml (1.25–2.5 copies/reaction), which is a promising alternative for the early screening of acute respiratory tract infections; however, qPCR is the preferred method for the quantitative detection of common pathogens in general laboratories. Despite the low cost and mature nature of the technology ( Table I ), qPCR is prone to nucleic acid contamination, primer dimer formation, improper baseline setting and a number of other issues which can lead to false-positives.…”