An integrated one-step assay combining thermal lysis and loop-mediated isothermal DNA amplification (LAMP) in 30 min fromE. coliandM. smegmatiscells on a paper substrate

Abstract

Developing sensors in the domains of 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 by analysing 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 assays on a paper substrate remains a big challenge in paperfluidic nucleic acid analyis.Combining lysis and isothermal amplification in a single reaction step is difficult because the porosity of paper and the presence of cell debris following lysis reduces the efficiency of DNA amplification. On the other hand, extracting and purifying the DNA after lysis to improve the amplification efficiency involves addition of chemical reagents, one or more wash steps and manual intervention. 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 during isothermal amplification. Here we successfully combine thermal lysis and loop-mediated isothermal amplification (LAMP) into a single reaction step on paper without the need for any intermediate intervention. 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 o C for 5 min and 15 min respectively, making this assay safe and suitable for incorporation into diverse paperfluidic sensors for field use.

Development and comparison of Loop-Mediated Isothermal Amplification with Quantitative PCR for the specific detection ofSaprolegniaspp

“…It is noteworthy that our method can directly detect an organism from a sample without DNA purification, which refers to a one-step LAMP. This method has been reported in several studies such as detection of E. coli, Mycobacterium smegmatis [56] and Mycoplasma ovipneumoniae [57]. Similarly, detection by LAMP at femtogram level has been reported in numerous studies such as acute viral necrobiotic virus in scallop [58], classical swine flu virus [59] and…”

Development and comparison of loop-mediated isothermal amplification with quantitative PCR for the specific detection of Saprolegnia spp.

“…It is noteworthy that our method can directly detect an organism from a sample without DNA purification, which refers to a one-step LAMP. This method has been reported in several studies such as detection of E. coli, Mycobacterium smegmatis [56] and Mycoplasma ovipneumoniae [57]. Similarly, detection by LAMP at femtogram level has been reported in numerous studies such as acute viral necrobiotic virus in scallop [58], classical swine flu virus [59] and Enterococcus hirae [60].…”