Multiplex Droplet Digital PCR for the Detection and Quantitation of Streptococcus pneumoniae, Mycoplasma pneumoniae and Haemophilus influenzae

Yaling Cao¹Junwen Wang²Yitong Jiang³Ling Xu¹Yuan Tian¹Zihao Fan¹Zhenzhen Pan¹Yinkang Mo¹Xianru Zhu¹Xiangying Zhang¹Jing Huang⁴Feng Ren^1*

• ¹Institute of Liver Diseases, Beijing Youan Hospital, Capital Medical University, Beijing, China
• ²Department of Clinical Laboratory, Chui Yang Liu Hospital, Beijing, China
• ³Yanjing Medical College, Capital Medical University, Beijing, China
• ⁴Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Department of Infection Control, Beijing, China

Background: Lower respiratory tract infection is one of the major causes of disease and death worldwide. Streptococcus pneumoniae, Mycoplasma pneumoniae, and Haemophilus influenzae are important pathogens responsible for lower respiratory tract infection. Here, we established a multiplex droplet digital polymerase chain reaction (ddPCR) method for the simultaneous detection of S. pneumoniae, M. pneumoniae and H. influenzae DNA.Methods: Specific primers and probes were designed for ddPCR. The sensitivity and specificity of the ddPCR assay were evaluated using standard strains, positive samples and 26 common pathogenic bacteria. One hundred and sixty-seven clinical samples were collected and tested via ddPCR, qPCR, bacterial culture and microfluidic chip technology.The limits of detection (LoDs) of ddPCR were 2.5, 2.8 and 2.0 copies/μL for S. pneumoniae, M. pneumoniae and H. influenzae, respectively, which were approximately tenfold lower than the LoDs of qPCR. For 167 clinical samples, the positivity rates of ddPCR and microfluidic chip for S. pneumoniae and M. pneumoniae were 27.5% and 22.8%, respectively, which were higher than those of qPCR 25.7% and 21.6%. The positive rate of H. influenzae detection via ddPCR and microfluidic chip method was 29.9%, which was higher than that of qPCR (28.7%). The clinical sensitivity for S. pneumoniae, M. pneumoniae and H. influenzae improved from 97.4%, 94.7% and 95.1% for qPCR to 100% for ddPCR. Moreover, ddPCR showed less inhibition by the inhibitor in respiratory specimens than qPCR.The multiplex ddPCR assay established in this study can accurately detect S. pneumoniae, M. pneumoniae and H. influenzae DNA and can be used as an auxiliary tool for the clinical identification of pathogens and guidance of antibiotic therapy.