Microbial food safety: Potential of DNA extraction methods for use in diagnostic metagenomics.
The efficiency of ten widely applied DNA extraction protocols was evaluated for suitability for diagnostic metagenomics. The protocols were selected based on a thorough literature study. Chicken fecal samples inoculated with about 1×10(3) and 1×10(6) CFU/g Campylobacter jejuni were used as a model. The evaluation was performed based on total DNA yield measured by fluorometry, and quality and quantity of C. jejuni DNA measured by real-time PCR. There was up to a 25-fold variance between the lowest (NucliSens miniMAG, BIOMÉRIEUX) and highest (PowerLyzer PowerSoil DNA Isolation Kit, MO BIO Laboratories) yielding protocols. The PowerLyzer PowerSoil DNA Isolation Kit performed significantly better than all other protocols tested. Selected protocols were modified, i.e., extended heating and homogenization, resulting in increased yields of total DNA. For QIAamp Fast DNA Stool Mini Kit (Qiagen) a 7-fold increase in total DNA was observed following the protocol for human DNA analysis and including a 5 min heating step at 70°C. For the PowerLyzer PowerSoil and the PowerFecal DNA Isolation Kit (MO BIO Laboratories) the total DNA fold increase was 1.6 to 1.8 when including an extra 10 min of bead-vortexing. There was no correlation between the yield of total DNA and the amount of PCR-amplifiable DNA from C. jejuni. The protocols resulting in the highest yield of total DNA did not show correspondingly increased levels of C. jejuni DNA as determined by PCR. In conclusion, substantial variation in the efficiency of the protocols to extract DNA was observed. The highest DNA yield was obtained with the PowerLyzer PowerSoil DNA Isolation Kit, whereas the FastDNA SPIN Kit for Feces (MP Biomedicals) resulted in the highest amount of PCR-amplifiable C. jejuni DNA.
Technical University of Denmark, National Food Institute, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark.
Mathilde H
MH
Josefsen
Technical University of Denmark, National Food Institute, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark.
Sandra C
SC
Andersen
Technical University of Denmark, National Food Institute, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark.
Julia
J
Christensen
Technical University of Denmark, National Food Institute, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark. Electronic address: Jhoo@food.dtu.dk.
Jeffrey
J
Hoorfar
D000818:Animals; D016123:Campylobacter jejuni / Q000235:genetics / Q000302:isolation & purification; D002645:Chickens; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology; D005470:Fluorometry; D005516:Food Microbiology / Q000379:methods*; D059022:Food Safety / Q000379:methods*; D056186:Metagenomics / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction
D004269:DNA, Bacterial
Chicken fecal samples; DNA extraction; DNA quality; DNA quantity; Diagnostic metagenomics