Precise and simultaneous enumeration of multiplex pathogens using multiplex polymer chain reaction coupled with a novel quantitative capillary electrophoresis

High throughput, reliable and low cost methodologies for parallel pathogen counting are demanded in the diagnosis of infections, but still technically challenging. This work proposed a precise, simultaneous process of enumerating multiplex periodontal pathogens by using multiplex polymer chain reaction (mPCR) and quantitative capillary electrophoresis (qCE). A self-developed qCE protocol, which was time modified internal standard (TMIS) method, was employed to quantitatively study mPCR reactions. The optimal mPCR conditions were obtained for the amplification of three important periodontal pathogens, Porphyromonas gingivalis (P.g.), Treponema denticola (T.d.) and Tannerella forsythia (T.f.). The simultaneous quantitative determination of genome copies of P.g., T.d. and T.f. was demonstrated with a dynamic range from 0.2 to 1.5 × 105 unit/μL. Results showed that this mPCR-qCE assay had run-to-run and day-to-day variations of 6.1-10.9% and 2.3-17.4%, respectively. As little as 4 pathogen cells would be sufficient to have a positive result. Finally, a clinical sample was determined and its P.g., T.d. and T.f. concentrations were 39720 unit/μL, 5214 unit/μL, and 1140 unit/μL, respectively. This mPCR-qCE assay was proved to have comparable precision and sensitivity with quantitative PCR technology. Besides, it was more cost-effective, time saving and more capable of simultaneous detection.