Rapid sample preparation with Lyse-It® for Listeria monocytogenes and Vibrio cholerae

Abstract

Sample preparation is a leading bottleneck in rapid detection of pathogenic bacteria. Here, we use Lyse-It® for bacterial cellular lysis, genomic DNA fragmentation, and protein release and degradation for both Listeria monocytogenes and Vibrio cholerae. The concept of Lyse-It® employs a conventional microwave and Lyse-It® slides for intensely focused microwave irradiation onto the sample. High microwave power and a <60 second irradiation time allow for rapid cellular lysis and subsequent intracellular component release. The pathogenic bacteria are identified by quantitative polymerase chain reaction (qPCR), which subsequently demonstrates the viability of DNA for amplification post microwave-induced lysis. Intracellular component release, degradation, and detection of L. monocytogenes and V. cholerae has been performed and shown in this paper. These results demonstrate a rapid, low-cost, and efficient way for bacterial sample preparation on both food and water-borne Gram-positive and -negative organisms alike.

A comparison of Lyse-It to other cellular sample preparation, bacterial lysing, and DNA fragmentation technologies

“…Previously, it was shown that extracellular DNA was visible after making bacterial suspensions. [16] However, it was only until after days to a week that L . monocytogenes cells osmotically lyse and the genomic DNA began to degrade.…”

“…monocytogenes cells osmotically lyse and the genomic DNA began to degrade. [16] Following one month, the saver cards containing the sample were rehydrated with 1.25 mL DI water and mixed to aid in the release of bacterial cells. The mixed suspension was microwave irradiated with or without Lyse-It at 10%, 30%, or 50% power for 60 seconds.…”

Effects of Lyse-It on endonuclease fragmentation, function and activity

“…This data is concurrent with previous data that shows that bacterial DNA is also fragmented in to various sizes under a wide variety of conditions. [12–14] To aid in the fragment analysis, a mathematical weighted size () was calculated per trial as shown in Eqs 1→ 2, where n 0 is the first peak detected and n is the final peak reported from the Bioanalyzer data. For these equations, y i wa s the reported fragment size at peak n 0 ≤ i ≤ n , R i is the relative concentration reported for each fragment, and x i was the calculated weight factor where 1 = ∑ i x i .…”

“…Lyse-It has been reviewed in the literature as a highly efficient way to rapidly break open a wide variety of bacterial cells and subsequently fragment DNA and RNA and degrade proteins. [10–14] Lyse-It is a technology that utilizes highly focused microwave that increases the electromagnetic energy and subsequently temperature to the sample. By using gold equilateral triangles and a standard microwave, microwave power and time can be adjusted for tunable cellular lysis, intracellular component release, DNA fragmentation, and protein degradation.…”

“…By using gold equilateral triangles and a standard microwave, microwave power and time can be adjusted for tunable cellular lysis, intracellular component release, DNA fragmentation, and protein degradation. [12–14] We investigate the effects of conventional heating, standard microwave irradiation versus Lyse-It for their ability to fragment and inactivate three endonucleases. By utilizing Lyse-It, we can conclude that cellular lysis, DNA fragmentation, protein release and degradation [14] along with nuclease fragmentation, can occur at specific Lyse-It power and time settings, and be tuned to achieve over 98% inactivation of RNase A, RNase B, and DNase I.…”