Rapid One-tube Lysis Method of Oral Samples for Quantitative PCR

Quantitation of Streptococcus mutans from oral samples is an important factor for caries risk assessment. Rapid and accurate quantitation of S. mutans in saliva and plaque would maximize sample accuracy and fully exploit the quantitative power of well designed quantitative PCR (Q-PCR) assays. Currently used traditional DNA isolation methods or commercially available DNA isolation kits produce template amounts that may not accurately reflect the total amount of DNA present in the oral sample. Objectives: This purpose of this study was use a Q-PCR assay to assess the effectiveness of modifications of a single-tube bead-beating lysis method using saliva and plaque samples over five second intervals. Methods: Seven hundred microliters of either enzyme-treated or non enzyme-treated human saliva and plaque samples were mixed with 500µl of 0.1mm silica beads in a microcentrifuge tube. Bead-beating was performed for 50 seconds with samples taken at five second intervals for plaque samples. Similarly, saliva samples were processed for 120 seconds. Twenty microliters of each time-interval aliquot was removed, and after centrifugation at 10,000 rpm for one minute, five microliters of the supernatant was used as template in a SYBR green Q-PCR assay. Results: Q-PCR data indicate that bead-beating is an efficient way to lyse S. mutans in enzyme pretreated and non pretreated saliva samples. The lowest Ct values occurred in the time interval between 35s to 50s and 60s to 120s for plaque and saliva samples, respectively. Conclusions: This one-tube bead-beating lysis method is an efficient technique to rapidly lyse S. mutans from oral samples regardless of sample pretreatment. This method may be better suited to be used as a pretreatment for an automated method of DNA isolation.

This study supported by grant #DE016684 from the NIDCR.