Multiplex real-time PCR using temperature sensitive primer-supplying hydrogel particles and its application for malaria species identification

PLoS One. 2018 Jan 2;13(1):e0190451. doi: 10.1371/journal.pone.0190451. eCollection 2018.

Abstract

Real-time PCR, also called quantitative PCR (qPCR), has been powerful analytical tool for detection of nucleic acids since it developed. Not only for biological research but also for diagnostic needs, qPCR technique requires capacity to detect multiple genes in recent years. Solid phase PCR (SP-PCR) where one or two directional primers are immobilized on solid substrates could analyze multiplex genetic targets. However, conventional SP-PCR was subjected to restriction of application for lack of PCR efficiency and quantitative resolution. Here we introduce an advanced qPCR with primer-incorporated network (PIN). One directional primers are immobilized in the porous hydrogel particle by covalent bond and the other direction of primers are temporarily immobilized at so-called 'Supplimers'. Supplimers released the primers to aqueous phase in the hydrogel at the thermal cycling of PCR. It induced the high PCR efficiency over 92% with high reliability. It reduced the formation of primer dimers and improved the selectivity of qPCR thanks to the strategy of 'right primers supplied to right place only'. By conducting a six-plex qPCR of 30 minutes, we analyzed DNA samples originated from malaria patients and successfully identified malaria species in a single reaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Hydrogels / chemistry*
  • Multiplex Polymerase Chain Reaction / methods*
  • Plasmodium / classification*
  • Real-Time Polymerase Chain Reaction / methods*
  • Temperature

Substances

  • Hydrogels

Grants and funding

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant number: HI13C2262; URL: http://www.mohw.go.kr). This research was also supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) & funded by the Korean government (MSIP & MOHW) (No. 2016M3A9B6918639; URL: https://www.nrf.re.kr). Additionally, this work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean Government (NRF-2016-Fostering Core Leaders of the Future Basic Science Program / Global Ph.D. Fellowship Program, NRF- 2016H1A2A1908829; URL: https://www.nrf.re.kr). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.