New methods for selective isolation of bacterial DNA from human clinical specimens.
Separation of bacterial DNA from human DNA in clinical samples may have an important impact on downstream applications, involving microbial diagnostic systems. We evaluated two commercially available reagents (MolYsis), Molzym GmbH & Co. KG, Bremen and Pureprove, SIRS-Lab GmbH, Jena, both Germany) for their potential to isolate and purify bacterial DNA from human DNA. We chose oral samples, which usually contain very high amounts of both human and bacterial cells. Three different DNA preparations each were made from eight caries and eight periodontal specimens using the two reagents above and a conventional DNA extraction strategy as reference. Based on target-specific real-time-quantitative PCR assays we compared the reduction of human DNA versus loss of bacterial DNA. Human DNA was monitored by targeting the beta-2-microglobulin gene, while bacteria were monitored by targeting 16S rDNA (total bacteria and Porphyromonas gingivalis) or the glycosyltransferase gene (Streptococcus mutans). We found that in most cases at least 90% of human DNA could successfully be removed, with complete removal in eight of 16 cases using MolYsis, and two (of 16) cases using Pureprove. Conversely, detection of bacterial DNA was possible in all cases with a recovery rate generally ranging from 35% to 50%. In conclusion, both strategies have the potential to reduce background interference from the host DNA which may be of remarkable value for nucleic-acid based microbial diagnostic systems.
Horz
Hans-Peter
HP
Division of Oral Microbiology and Immunology, Department of Operative and Preventive Dentistry & Periodontology, Medical Faculty, RWTH Aachen University Hospital, Pauwelsstrasse 30, D-52057 Aachen, Germany.
Scheer
Sebastian
S
Vianna
Morgana E
ME
Conrads
Georg
G
D000328:Adult; D001419:Bacteria / Q000235:genetics*; D001424:Bacterial Infections / Q000382:microbiology*; D001826:Body Fluids / Q000382:microbiology*; D004269:DNA, Bacterial / Q000302:isolation & purification*; D006801:Humans; D011933:Reagent Kits, Diagnostic; D012463:Saliva / Q000382:microbiology; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial; D011933:Reagent Kits, Diagnostic