Colorimetric polymerase chain reaction mediated by pH indicator: Rapid detection of drug-resistant nosocomial...

Polymerase chain reaction (PCR) is the gold standard for molecular diagnoses due to its high sensitivity and accuracy. However, conventional PCR exhibits limitations including extended analysis time and complicated sample treatment. To shorten the analysis time of standard three-step PCR, we employed a two-step PCR allowing for fast amplification of nucleic acid in a chip format. Moreover, to realize a fully-integrated and on-site visual discrimination of opportunistic and multidrug-resistant pathogens, namely, Enterococcus faecium and Acinetobacter baumannii. DNA was extracted using FTA card, and pH-dependent color changes of the PCR amplicons were monitored by using phenolphthalein as a pH indicator. The overall reaction took less than 35 min and exhibited high selectivity and sensitivity confirmed by the limit of detection of approximately 10³ CFU/mL for E. faecium. In addition, carbapenem-resistant A. baumannii was successfully detected demonstrating the clinical applicability of the introduced technique using a thermoplastic chip. E. faecium both in bacterial cell culture solution and a contaminated surface were also successfully detected proving the feasibility of the chip-based detection system and paving the way for a facile discrimination of opportunistic pathogens prevalent in hospitals and our environment.