Simultaneous Detection of Key Bacterial Pathogens Related to Pneumonia and Meningitis Using Multiplex PCR Coupled With Mass Spectrometry

Zhang, Chi; Xiu, Leshan; Xiao, Yan; Xie, Zhengde; Ren, Lili; Peng, Junping

To design the assay, we used Assay Design 4.0 software (Agena Bioscience, Inc., San Diego, CA, USA) according to the user's manual. For each of the 11 pathogens, a target gene was chosen to design the detection assays. Human β-globin (HBB) was selected as an internal control, and BLASTn was used to check the specificity of each target gene. For each target gene, sequences in GenBank database were downloaded and aligned. Conserved regions were chosen to design the assays. In each assay, forward and reverse PCR primers were used to amplify the template and an extension primer was used for single base extension (SBE) reaction. To avoid interference in the mass spectra, a 10-base mass tag (ACGTTGGATG) was added to the 5 ′ end of each PCR primer. PCR and extension primers were synthesized by Tsingke Biological Technology (Beijing, China).