Novel cost-efficient real-time PCR assays for detection and quantitation of Listeria monocytogenes.
Listeriosis is a serious food-borne infection with mortality rates approaching 30%. Therefore, the rapid, cost-effective, and automated detection of Listeria monocytogenes throughout the food chain continues to be a major concern. Here we describe three novel quantitative real-time PCR assays for L. monocytogenes based on amplification of a target hlyA gene with SYBR Green I chemistry and hydrolysis probe (TaqMan MGB probe). In order to offer sensitive, rapid and robust tool of additional economical value the real-time PCR assays were designed and optimized to only 5 μl-reactions. All assays were evaluated by using different non-reference Listeria strains isolated from various food matrices. Results demonstrated specificity to L. monocytogenes with accurate quantification over a dynamic range of 5-6 log units with R² higher than 0.98 and amplification efficiencies reaching above 92%. The detection and quantification limits were as low as 165 genome equivalents. Comparison of novel assays to commercially available TaqMan® Listeria monocytogenes Detection Kit and previously published studies revealed similar specificity, sensitivity and efficiency, but greater robustness and especially cost-efficiency in the view of smaller reaction volumes and continuous increase in sample throughput.
Omega D.O.O., Dolinškova 8, Ljubljana, Slovenia. urban.traunsek@guest.arnes.si
Urban
U
Traunšek
Nataša
N
Toplak
Barbara
B
Jeršek
Aleš
A
Lapanje
Tamara
T
Majstorović
Minka
M
Kovač
D058491:Bacterial Load / Q000379:methods*; D001427:Bacterial Toxins / Q000235:genetics; D052160:Benzothiazoles; D003959:Diamines; D006360:Heat-Shock Proteins / Q000235:genetics; D006460:Hemolysin Proteins / Q000235:genetics; D006801:Humans; D008089:Listeria monocytogenes / Q000302:isolation & purification*; D008088:Listeriosis / Q000175:diagnosis* / Q000382:microbiology*; D015345:Oligonucleotide Probes / Q000235:genetics; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000191:economics* / Q000379:methods*; D011804:Quinolines; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
D001427:Bacterial Toxins; D052160:Benzothiazoles; D003959:Diamines; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D015345:Oligonucleotide Probes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; C038705:hlyA protein, Listeria monocytogenes