Colorimetric detection of viable antibiotic resistant Enterococcus mediated by cordless operation of reverse transcription loop-mediated isothermal amplification

Abstract

In this study, we applied a tube-based reverse transcription loop-mediated isothermal amplification technique using preloaded amplification and detection reagents for simple screening of viable vancomycin-resistant Enterococcus in a cordless manner. We adopted an mRNA-based approach to detect live Enterococcus in vancomycin-treated cultures. We used agarose to preload and store all reagents for amplification and detection inside the tube, which could achieve on-site isothermal nucleic acid amplification and detection in less than 1 h without using sophisticated instruments. Moreover, the use of a portable insulated water tumbler eliminated the need for electricity, which is usually important in nucleic acid amplification-based assays. The water tumbler acted as a heat source to supply a stable heat required for the amplification reaction, which could last up to 45 min. In addition, colorimetric detection was realized using pH-based methods. The detection was triggered by shaking the tube so that the amplified solution was reacted with phenolphthalein embedded in the tube cap. The introduced one-pot strategy has many advantages such as easy and cordless operation, low cost, disposability, and less chance of contamination because the amplification and detection occur in a closed system. The system could have a great impact on nucleic acid analyses in instrument-free and low-resource areas.