Detection of respiratory viral and bacterial pathogens causing pediatric community-acquired pneumonia in Beijing using real-time PCR

Zhang, Tie-Gang; Li, Ai-Hua; Lyu, Min; Chen, Meng; Huang, Fang; Wu, Jiang

The duplex real-time PCR kit for detecting S. pneumoniae and H. influenzae, and the multiplex combined real-time PCR detection kit for respiratory viruses were purchased from Jiangsu Uninovo Biological Technology Co., LTD. The duplex real-time PCR was performed using the Applied Biosystems 7500 Fast Real-Time PCR System (ABI, California, USA). The reaction conditions were: initial denaturation at 95 C for 2 min, followed by 40 cycles of denaturation of 95 C for 10 s and annealing and extension at 60 C for 50 s. Fam and Hex fluorescence was acquired at the end of the annealing and extension step. The multiplex combined Real-time PCR was also performed using the Applied Biosystems 7500 Fast Real-Time PCR System (ABI, California, USA). The reaction conditions were: (1) reverse transcription at 42 C for 10 min, (2) preliminary denaturation at 95 C for 10 s, and (3) 45 cycles of denaturation of 95 C for 5 s, annealing at 56 C for 50 s, extension at 72 C for 12 s. Fam, Hex, Rox and Cy5 fluorescence was acquired at the end of the annealing step. The results were analyzed according to the kit user's manuals.