Detection of Common Respiratory Viruses and Mycoplasma pneumoniae in Patient-Occupied Rooms in Pediatric Wards

Wan, Gwo-Hwa; Huang, Chung-Guei; Chung, Fen-Fang; Lin, Tzou-Yien; Tsao, Kuo-Chien; Huang, Yhu-Chering

Real-Time Quantitative PCR Assay for Enterovirus, RSV, Influenza A Virus, Adenovirus, and M pneumoniae Table 1 shows the sequences of the primers and probes for enterovirus, RSV, influenza A virus, adenovirus, and M pneumoniae. For enterovirus detection, the PCR reaction mixtures contained 5 mL RNA, 20 mL 2Â TaqMan universal master mix (Applied Biosystems, Waltham, MA), 10 mM of each primer, and 5 mM probe. The PCR thermal profile was 30 min at 488C, 10 min at 958C, and 45 cycles of 15 s at 958C and 1 min at 588C. The PCR products were amplified and detected by using an ABI Prism 7900 sequence detection system (Applied Biosystems). For influenza A virus and RSV detection, the PCR reaction mixtures contained 5 mL RNA, 20 mL reverse transcription-PCR buffer (OneStep RT-PCR kit; Qiagen), 10 mM dNTPs, 25 mM MgCl 2 , 2000 U RNAsin (Invitrogen, Paisley, Scotland), 50 mM of each primer, and 10 mM probe. The PCR thermal profile was 30 min at 508C, 10 min at 958C, and 50 cycles of 30 s at 958C, 30 s at 558C, and 30 s at 728C. The PCR products were amplified and detected by using the CFX96 real-time detection system (Bio-Rad, Veenendaal, The Netherlands). The concentrations of adenovirus and M pneumoniae in the filter samples were quantitatively measured as described previously. 41 In this study, the detection limits in the real-time PCR assay were 1 and 100 copies/mL for RSV and enterovirus, respectively, and 10 copies/mL for the other viruses and M pneumoniae.