An integrated one-step assay combining thermal lysis and loop-mediated isothermal DNA amplification (LAMP) in 30 min from E. coli and M. smegmatis cells on a paper substrate

摘要

Developing sensors for food safety, soil analysis, water quality monitoring and healthcare often requires distinguishing between different species of bacteria. The most rapid, sensitive and specific method to identify bacteria is to analyse their DNA sequence, which comprises of disinfection and lysis of bacterial cells, amplification of the isolated DNA and detection of the amplified sequence. Seamless integration of these steps on a paper substrate is a big challenge. This problem is even more complex for mycobacteria as its thick cell wall structure impedes lysis and the high GC-content of the genome requires careful optimization of enzymatic denaturation. Here we successfully combine thermal lysis and loop-mediated isothermal amplification (LAMP) into a single reaction step on paper. We demonstrate our integrated assay by amplifying DNA from 100 CFU/mL of Escherichia coli (MG1655) and Mycobacterium smegmatis (mc(2)155) cells in 30 min on a paper substrate. We also confirm that E. coli and M. smegmatis can be completely disinfected on paper by heating at 60 degrees C for 5 min and 15 min respectively, making this assay safe and suitable for incorporation into diverse paperfluidic sensors for field use.