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

All of the nasopharyngeal swabs were collected according to a standard protocol in general bacteria collection tubes with maintenance medium (Yocon, Beijing, China) and then stored at −80 • C. For DNA extraction, samples were thawed at room temperature, 200 µl of maintenance medium was added, and the mixture was centrifuged at 5,000 × g for 10 min. The bacterial pellets were then resuspended in 150 µl of enzyme cocktail containing 30 U of lysostaphin, 6 mg of lysozyme, 37.5 U of mutanolysin, and 30 U of lyticase (Millipore Sigma, Darmstadt, Germany) in lysis buffer with working concentrations of 20 mM Tris-HCI (pH 8), 2 mM EDTA, and 1.2% Triton. To lyse the rigid cell walls of gram-positive bacteria, the resuspended bacteria were incubated at 37 • C for 30 min. After incubation, 20 µl of proteinase K and 200 µl of buffer AL (QIAgen, Hilden, Germany) were added, and the mixture was incubated for 20 min at 56 • C and then 15 min at 95 • C. Finally, the DNA was purified using a QIAamp DNA Mini Kit (QIAgen, Hilden, Germany) according to the user's guide.