Implementation of antimicrobial peptides for sample preparation prior to nucleic acid amplification in point-of-care settings

Abstract

These results show that AMPs treatment is a very efficient sample preparation technique that is suitable for application prior to nucleic acid amplification directly within biological samples. Furthermore, the entire process of AMPs treatment was performed at room temperature for 5 min thereby making it a good candidate for use in POC applications.

Chemical Trends in Sample Preparation for Nucleic Acid Amplification Testing (NAAT): A Review

“…Excessive surfactant use, however, may lead to bubble nucleation, which may cause practical challenges, including decreased solvent concentration, interrupted electrical and fluidic conductivity, and changes in hydraulic resistance [62,63]. Surfactants can also inhibit the amplification reaction by damaging amplification enzymes due to their denaturing properties effect [46].…”

“…Complete lysis of green fluorescence protein (GFP)-expressing cells was achieved within 1 min [58]. In contrast, when Escherichia coli (E. coli) were incubated in 1% Triton X-100 at room temperature for 5 min, only about 10-15% of viability was observed owing to the stronger bacterial walls and the E. coli cell permeability was enhanced to 30% with the aid of additional 1 mg/mL lysozyme [46]. Furthermore, a three-detergent method combining the anionic sodium dodecyl sulphate (SDS), Tween 20, and Triton X-100 (STT) was reported for lysis before RNA extraction from several Gram-negative bacteria, including Pseudomonas putida, Burkholderia cepacia, Agrobacterium tumefaciens, E. coli, and Edwardsiella tarda, and Gram-positive Bacillus subtills [59].…”

“…In enzymatic lysis, a biocatalyst is used to cleave and digest chemical bonds in the membranes. Enzymatic lysis is often combined with detergent for hard-to-lyse samples or samples in a complex matrix to improve the lysis efficiency as mentioned above [28,29,46]. During cell lysis, proteinase K promotes proteolysis to digest proteins and protects the NAs from DNase or RNase, but it requires thermal activation at 50-65 • C to optimize its activity [29,[64][65][66].…”

“…AL is faster than lysis using detergent or enzymes. Using AL with 400 mM KOH, 100 mM DTT, and 10 mM ETDA, 80% of E. coli cells were lysed after a 5 min incubation at room temperature, while 1% Triton X-100, 1 mg/mL lysozyme, and their mixture led to only ~30% lysis under same incubation conditions [46]. As speed is important for PONT, this makes AL an attractive option; however, the requirement for neutralisation before amplification may form an operational bottleneck in the development of ideal PONT devices.…”

“…A study of the use of AMPs for the lysis of hard-to-lyse bacteria, S. typhimurium and S. aureus, systematically correlated wall structure and AMP activity [85]. AMPs including melittin, magainin analogues (MSI), bombolitin, and cecropin were utilized for lysing bacteria cells in urine samples prior to LAMP [46]. As is shown in Figure 1 of ref.…”