Rapid Microbial Lysing and DNA Fragmentation by Microwave Focusing

Abstract

Bacterial infections are a major health problem worldwide. Identification of disease-causing organisms by culture-based approaches is time-consuming and often lacks sensitivity. Molecular approaches such as PCR and microwave-accelerated metal-enhanced fluorescence (MAMEF) assays1, are more sensitive and faster than traditional culture-based approaches, but require isolation of the target DNA. In order to determine the effect of both boiling and microwave irradiation on microbial lysing and DNA fragmentation, cultures of Neisseria gonorrhoeae and Listeria monocytogenes (108 CFU /mL) were either boiled (range 40° - 70°C) or lysed in a 900-watt microwave on isolator-mounted microscope slides, both with and without the assistance of disjointed antenna gold bow-tie structures. The temperatures of cultures were obtained prior to and after lysing and the resulting lysate cultured on selective agar plates. DNA isolation and fragmentation efficiency were determined by gel electrophoresis and PCR. N. gonorrhoeae lysed at a lower temperature (°C) than L. monocytogenes. Microbial lysing and DNA fragmentation was more effectively carried out in the presence dis-jointed gold triangle structures, but only when small sample volume were used. Standard boiling was successful for bacterial lysing and DNA fragmentation, but required higher temperatures and longer times than microwave focusing. PCR results suggest that low power microwave irradiation is ideal for PCR methods while higher microwave powers are required to generate DNA fragments ideal for MAMEF analysis. Microbial lysing and DNA fragmentation can be achieved by either boiling or microwave, but microwave lysing is more efficient for DNA fragmentation and is significantly faster. Microwave lysing is the recommended method when rapid isolation and DNA fragmentation is required.1Melendez, et at. (2013). Blind Evaluation of the Microwave-Accelerated Metal-Enhanced Fluorescence Ultrarapid and Sensitive Chlamydia Trachomatis test by use of Clinical Samples, Journal of Clinical Microbiology, 51(9), 2913-2920.