# Simultaneous Detection of Nine Key Bacterial Respiratory Pathogens Using Luminex xTAG(r) Technology
## 4 Discussion
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline \multirow{2}{*}{**Pathogens**} & \multirow{2}{*}{**Real-Time**} & \multicolumn{2}{c}{**MPL Assay**} & \multicolumn{2}{c}{**Performance of the MPLT Assay**} & \multicolumn{1}{c}{**Measures of**} \\ \cline{3-8}  & & & & & **Consistency** & **Sensitivity** & **Specificity** & **Agreement** \\  & **PCR Assay** & **Positive** & **Negative** & **(\%)** & **(\%)** & **(\%)** & **(\%)** & **Kappa Values** \\ \hline \multirow{2}{*}{_S. pneumoniae_} & Positive & 7 & 0 & 98.8 & 87.5 & 100 & 0.927 (\(p\) \(<\) 0.001) \\  & Negative & 1 & 78 & & & & \\ \hline \multirow{2}{*}{_M. catarrhalis_} & Positive & 0 & 0 & - & - & - & * \\  & Negative & 0 & 86 & & & & \\ \hline \multirow{2}{*}{_S. aureus_} & Positive & 11 & 0 & 100 & 100 & 100 & 1.000 (\(p\) \(<\) 0.001) \\  & Negative & 0 & 75 & & & & \\ \hline \multirow{2}{*}{_S. pyogenes_} & Positive & 0 & 0 & - & - & - & * \\  & Negative & 0 & 86 & & & & \\ \hline \multirow{2}{*}{_H. influenzae_} & Positive & 8 & 0 & 100 & 100 & 100 & 1.000 (\(p\) \(<\) 0.001) \\  & Negative & 0 & 78 & & & & \\ \hline \multirow{2}{*}{_M. pneumoniae_} & Positive & 1 & 0 & 100 & 100 & 100 & 1.000 (\(p\) \(<\) 0.001) \\  & Negative & 0 & 85 & & & & \\ \hline \multirow{2}{*}{_Legionella_ spp.} & Positive & 0 & 0 & - & - & - & * \\  & Negative & 0 & 86 & & & & \\ \hline \multirow{2}{*}{_P. aeruginosa_} & Positive & 10 & 0 & 100 & 100 & 100 & 1.000 (\(p\) \(<\) 0.001) \\  & Negative & 0 & 76 & & & & \\ \hline \multirow{2}{*}{_K. pneumoniae_} & Positive & 5 & 0 & 100 & 100 & 100 & 1.000 (\(p\) \(<\) 0.001) \\  & Negative & 0 & 81 & & & \\ \hline \multicolumn{8}{l}{* No statistics were computed because the MPLT assay and real-time PCR assay for _M. catarrhalis, S. pyogenes,_ and _Legionella_ spp. are constants.} \\ \end{tabular}
\end{table} 
Table 4: Performance of the MPLT assay compared to the real-time PCR assay. successfully generated this MPLT assay to investigate nine bacterial pathogens responsible for LRTI using this Luminex xTAG(r) technology. Other analysis platforms, such as the flow-based microarray analysis platform MCR 3 [46] and stopped polymerase chain reaction combined with chemiluminescence flow-through DNA microarray analysis platform [47], have been built. Compared with our Luminex platform, all these platforms are characterized by low limits of detection, high reproducibility and reduced assay time [46; 47]. Although multiplex real-time PCR is a direct format and is cheaper than Luminex technology, the throughput of it is low. Furthermore, due to the limitation of its fluorescence, it can detect only a limited number of bacterial pathogens in one reaction [10; 12; 13; 14]. The results of our study showed that this MPLT assay had high specificity, sensitivity and excellent consistency compared to the real-time PCR reaction. Furthermore, we found the detection limits for these nine pathogens were as low as \(10^{2}\)-\(10^{3}\) CFU/mL (\(10^{-3}\)-\(10^{-4}\) ng/\(\,\mu\)L). These results strongly suggest that the development of the MPLT assay for identification of these nine pathogens is both feasible and useful. The sensitivity and specificity of Luminex technology is closely related to primers [48; 49; 50; 51], and good, specific primers can avoid non-specific amplification while ensuring stability of the assay. Consequently, the primers used in our assay were carefully selected from different primers, according to the GC content of the primers and the annealing temperature. In addition, the results of the MPLT assay also showed that all primers were chosen appropriately and the nine target pathogens could be detected effectively. A previous study [52] showed that a ten-plex PCR-coupled liquid bead array system had lower detection limits than a single PCR. Therefore, it was important in this study to optimise the conditions of the multiplex PCR reactions.