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

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.

Advanced Signal-Amplification Strategies for Paper-Based Analytical Devices: A Comprehensive Review

“…Conventionally, polymerase chain reaction (PCR) is the technique extensively used to rapidly process millions to billions of DNA samples, but it requires precise temperature control for reactions, and hence, to date has been unsuccessfully applied to PADs [ 149 , 150 ]. Alternatively, various signal amplification isothermal methods have been tried for PAD development, including rolling circle amplification (RCA) [ 147 , 151 , 152 , 153 , 154 , 155 ], loop-mediated isothermal amplification (LAMP) [ 156 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 ], strand-displacement amplification (SDA) [ 167 ], recombinase polymerase amplification (RPA) [ 168 , 169 , 170 , 171 ], helicase dependent amplification (HDA) [ 172 , 173 , 174 ], and nucleic acid sequence-based amplification (NASBA) [ 175 ].…”

“…In particular, the LAMP technique provides enlarged specificity and sensitivity based on an exponential amplification capacity that can distinguish primers in one reaction, and hence, can simultaneously identify various target sequences [ 176 ]. To determine a mycobacterium’s DNA in a single reaction for the first time, Naik and coworkers successfully designed a novel LAMP assay on a paper substrate to detect E. coli and M. smegmatis ( Figure 8 A) [ 161 ]. This system can perform lysis and amplify DNA from only 100 CFU/mL with high sensitivity within 30 min through recorded fluorescence intensities, especially ensuring safety due to an effective ability to kill all bacteria in the samples.…”

“…Adapted with permission from Ref. [ 161 ]. ( B ) The process for human adenoviral detection was carried out by novel RPA vertical flow-paper microarray based on the visualization of anti-biotin antibody-conjugated AuNPs with positive spots.…”

Developing a Point-of-Care Molecular Test to Detect SARS-CoV-2

“…Finally, we demonstrated the integrated workflow on a paper substrate with Escherichia coli (MG1655) and Mycobacterium smegmatis (mc 2 155) bacterial cultures by incubating them with the reaction components at 60 °C for 15 min and 30 min, respectively (Naik et al 2019). We switched to a different DNA amplification technique called "loop-mediated isothermal amplification" (LAMP), while optimizing the integrated protocol on a paperfluidic substrate.…”

Mobile origami immunosensors for the rapid detection of urinary tract infections

“…14 They are also faster and easier to use than biosensors based on detecting nucleic acids. [22][23][24][25] Moreover, the biosensors provide more information about the type of pathogen causing the UTI compared to the traditional urine strip. While this information is limited to a single pathogen at the moment, it has the potential to be expanded into a multisensor design including multiple reservoirs with antibodydecorated nanoparticles against different types of pathogens.…”