Lu Xi Jiang1, Hong Yu Ren2, Hai Jian Zhou2, Si Hong Zhao3, Bo Yan Hou3, Jian Ping Yan4, Tian Qin2, Yu Chen3. 1. Department of Respiratory Medicine, Shengjing Hospital of China Medical University, Shenyang 110004, Liaoning, China; National Institute for Communicable Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, Chinese Center for Disease Control and Prevention, Beijing 102206, China; Department of Respiratory Medicine, Zhejiang Provincial People's Hospital, Hangzhou 310014, Zhejiang, China. 2. National Institute for Communicable Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, Chinese Center for Disease Control and Prevention, Beijing 102206, China. 3. Department of Respiratory Medicine, Shengjing Hospital of China Medical University, Shenyang 110004, Liaoning, China. 4. Department of Respiratory Medicine, Zhejiang Provincial People's Hospital, Hangzhou 310014, Zhejiang, China.
Abstract
OBJECTIVE: Lower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction (PCR) and capillary electrophoresis (MPCE) assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebacterium diphtheriae, and Streptococcus pyogenes. METHODS: Three multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. RESULTS: The detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10-7 to 10-2 ng/μL. Furthermore, analysis of the 152 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. CONCLUSION: This study revealed that the MPCE assay is a rapid, reliable, and high-throughput method with high specificity and sensitivity. This assay has great potential in the molecular epidemiological survey of respiratory pathogens.
OBJECTIVE: Lower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction (PCR) and capillary electrophoresis (MPCE) assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebacterium diphtheriae, and Streptococcus pyogenes. METHODS: Three multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. RESULTS: The detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10-7 to 10-2 ng/μL. Furthermore, analysis of the 152 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. CONCLUSION: This study revealed that the MPCE assay is a rapid, reliable, and high-throughput method with high specificity and sensitivity. This assay has great potential in the molecular epidemiological survey of respiratory pathogens.
Authors: Claire Healy; Natalia Munoz-Wolf; Janné Strydom; Lynne Faherty; Niamh C Williams; Sarah Kenny; Seamas C Donnelly; Suzanne M Cloonan Journal: Respir Res Date: 2021-04-29
Authors: Olga V Kovaleva; Daniil Romashin; Irina B Zborovskaya; Mikhail M Davydov; Murat S Shogenov; Alexei Gratchev Journal: J Immunol Res Date: 2019-07-29 Impact factor: 4.818