A validated PCR-based method to detect Listeria monocytogenes using raw milk as a food model--towards an international standard.
A PCR assay with an internal amplification control was developed for Listeria monocytogenes. The assay has a 99% detection probability of seven cells per reaction. When tested against 38 L. monocytogenes strains and 52 nontarget strains, the PCR assay was 100% inclusive (positive signal from target) and 100% exclusive (no positive signal from nontarget). The assay was then evaluated in a collaborative trial involving 12 European laboratories, where it was tested against an additional 14 target and 14 nontarget strains. In that trial, the inclusivity was 100% and the exclusivity was 99.4%, and both the accordance (repeatability) and the concordance (reproducibility) were 99.4%. The assay was incorporated within a method for the detection of L. monocytogenes in raw milk, which involves 24 h of enrichment in half-Fraser broth followed by 16 h of enrichment in a medium that can be added directly into the PCR. The performance characteristics of the PCR-based method were evaluated in a collaborative trial involving 13 European laboratories. In that trial, a specificity value (percentage of correct identification of blank samples) of 81.8% was obtained; the accordance was 87.9%, and the concordance was 68.1%. The sensitivity (correct identification of milk samples inoculated with 20 to 200 L. monocytogenes cells per 25 ml) was 89.4%, the accordance was 81.2%, and the concordance was 80.7%. This method provides a basis for the application of routine PCR-based analysis to dairy products and other foodstuffs and should be appropriate for international standardization.
D'Agostino
M
M
DEFRA Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK.
Wagner
M
M
Vazquez-Boland
J A
JA
Kuchta
T
T
Karpiskova
R
R
Hoorfar
J
J
Novella
S
S
Scortti
M
M
Ellison
J
J
Murray
A
A
Fernandes
I
I
Kuhn
M
M
Pazlarova
J
J
Heuvelink
A
A
Cook
N
N
D000818:Animals; D005188:False Negative Reactions; D005189:False Positive Reactions; D005516:Food Microbiology; D007391:International Cooperation; D008089:Listeria monocytogenes / Q000302:isolation & purification*; D008892:Milk / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity