Application of Multiplex Fluorescence Polymerase Chain Reaction for Detecting Pathogenic Bacteria in Sputum Samples from Patients with Lower Respiratory Tract Infection

Abstract

Deyong Tan,1– 4 Jianfeng Han,5 Qingzhi Sun,5 Xing Cheng,5 Juan Liu,5 Jia Liu,5 Qing Li,1– 4 Lizhong Dai5 1Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha, 410008 People’s Republic of China; 2Institute of Clinical Pharmacology, Central South University, Hunan Key Laboratory of Pharmacogenetics, Changsha, 410078 People’s Republic of China; 3Engineering Research Center of Applied Technology of Pharmacogenomics, Ministry of Education, Changsha, 410078 People’s Republic of China; 4National Clinical Research Center for Geriatric Disorders, Changsha, Hunan, 410008 People’s Republic of China; 5Sansure Biotech Inc, Changsha, Hunan Province, People’s Republic of ChinaCorrespondence: Qing Li, Department of Clinical Pharmacology, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan, People’s Republic of China, Tel [phone], Fax [phone], Email [email protected] Lizhong Dai, Sansure Biotech Inc, No. 680 Lusong Road, Changsha, Hunan, People’s Republic of China, Tel [phone], Email [email protected]: In this study, we conducted a multi-center research on six common lower respiratory tract pathogens using novel multiplex fluorescence quantitative polymerase chain reaction (PCR), and investigated the additional diagnostic value of this method, to provide a molecular diagnostic basis for clinical practice.Methods: From March 2019 to October 2021, a total of 2047 respiratory sputum samples were collected from Hunan Provincial People’s Hospital (the First Affiliated Hospital of Hunan Normal University), Hunan Provincial Children’s Hospital, Jiangxi Provincial Children’s Hospital, and Wuhan Infectious Disease Hospital. The samples were analyzed using a novel multiplex fluorescence quantitative PCR method for Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Legionella pneumophila, and Staphylococcus aureus. The results were compared to the results of bacterial culture and sequencing, as well as the results of third-party kits.Results: Compared to the bacterial culture method, 2047 samples were detected with a sensitivity of 100%, a specificity of 72.22%, and an overall compliance rate of 81.91%. Compared to the sequencing method, the positive agreement percentage was 99.88%, the negative agreement percentage was 97.72%, and the overall agreement rate was 98.84%. Compared to similar control reagents, the positive agreement percentage was 100%, negative agreement percentage was 79.79%, and overall compliance rate was 96.19%.Conclusion: The multiplex fluorescence PCR method has the advantages of simultaneously detecting multiple pathogenic bacteria and reducing the duration of pathogen culture identification. Combined detection can increase the detection rate, which has favorable performance and application prospects.Keywords: bacterial infection, lower respiratory tract, melting curve, multiplex PCR, rapid diagnosis

Keywords

• bacterial infection
• lower respiratory tract
• melting curve
• multiplex pcr
• rapid diagnosis