Abstract
We utilized one-step multiplex reverse transcription-PCR (RT-PCR) and Luminex xMAP technology to develop a respiratory multiplex liquid-chip assay (rMLA) for simultaneous detection of 6 common respiratory viruses, including influenza virus type A (FluA) and type B (FluB), para-influenza virus type 3 (PIV-3), respiratory syncytial virus (RSV), human metapneumovirus (MPV) and a threatening virus to China, Middle East Respiratory Syndrome coronavirus (MERS-CoV). Performance of rMLA was evaluated by comparing with real-time RT-PCR. Detection data from clinical specimens showed that the rMLA had diagnostic sensitivities of 97.10% for FluA, 94.59% for FluB, 98.68% for PIV-3, 94.87% for RSV and 95.92% for MPV (No Data for MERS-CoV due to the lack of positive specimens). Data of analytical sensitivities showed that the detection limits of the rMLA assay were 5–25 viral RNA copies per μl for FluA, FluB, PIV-3 and MERS-CoV, approximate to the real-time RT-PCR assay; while the values were 8 and 22copies/μl for MPV and RSV, lower than the real-time RT-PCR(78 and 114 copies/μl respectively). The results indicated that the rMLA is a sensitive, specific detection tool and comparable to real-time RT-PCR, especially suitable for high-throughput detection of respiratory specimens.
Highlights
Human respiratory tract infection, mostly caused by respiratory viruses, gives rise to a considerable socioeconomic burden in medical care and social productivity due to the significant morbidity [1,2,3,4,5]
The analytical sensitivities of the Luminex-based respiratory multiplex liquid-chip assay (rMLA) and multiplex real-time RT-PCR assay developed in this study were assessed by testing in duplicate 10-fold serial dilutions of positive standards ranging from 106 to 101 copies/μl of viral RNA transcripts for each target
The standard curves were drawn by the log10 values of standard concentrations versus the MFI values for rMLA (Figure 1A) and the CT values for real-time RT-PCR (Figure 1B)
Summary
Mostly caused by respiratory viruses, gives rise to a considerable socioeconomic burden in medical care and social productivity due to the significant morbidity [1,2,3,4,5]. A rapid, sensitive, specific and multi-target assay for detecting major and common respiratory viruses is desirable. Respiratory viral infections have been diagnosed by cell culture, rapid antigen/antibody test, or direct fluorescent assay [10, 11]. They are time-consuming, unable to give an early diagnosis. Real-time PCR/RT-PCR shows significant superiorities due to its higher sensitivity and shorter turnaround time [16,17,18,19]. The limited multiplexing capacity of real-time PCR disables it to detect more targets and more clinical specimens in one assay simultaneously [12, 20]. The flexible multi-analyte profiling (xMAP) technology developed by Luminex www.impactjournals.com/oncotarget
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