Quantitative detection of Listeria monocytogenes and Listeria innocua by real-time PCR: assessment of hly, iap, and lin02483 targets and AmpliFluor technology

Abstract

We developed and assessed real-time PCR (RTi-PCR) assays for the detection and quantification of the food-borne pathogen Listeria monocytogenes and the closely related nonpathogenic species L. innocua. The target genes were hly and iap for L. monocytogenes and lin02483 for L. innocua. The assays were 100% specific, as determined with 100 Listeria strains and 45 non-Listeria strains, and highly sensitive, with detection limits of one target molecule in 11 to 56% of the reactions with purified DNA and 3 CFU in 56 to 89% of the reactions with bacterial suspensions. Quantification was possible over a 5-log dynamic range, with a limit of 15 target molecules and R(2) values of >0.996. There was an excellent correspondence between the predicted and the actual numbers of CFU in the samples (deviations of <23%). The hly-based assay accurately quantified L. monocytogenes in all of the samples tested. The iap-based assay, in contrast, was unsuitable for quantification purposes, underestimating the bacterial counts by 3 to 4 log units in a significant proportion of the samples due to serovar-related target sequence variability. The combination of the two assays enabled us to classify L. monocytogenes isolates into one of the two major phylogenetic divisions of the species, I and II. We also assessed the new AmpliFluor technology for the quantitative detection of L. monocytogenes by RTi-PCR. The performance of this system was similar to that of the TaqMan system, although the former system was slightly less sensitive (detection limit of 15 molecules in 45% of the reactions) and had a higher quantification limit (60 molecules).

FIG. 1.

RTi-PCR detection and amplification of the hly, iap, and lin02483 sequences. Representative amplification plots are shown. Serial dilutions of L. monocytogenes or L. innocua genomic DNA, equivalent to 3 × 10⁵ (▪), 3 × 10⁴ (□), 3 × 10³ (•), 3 × 10² (○), 60 (▴), 30 (▵), 15 (★), and 8 (⋆) target molecules per reaction, were used. Note that the AmpliFluor reactions could not detect 15 and 8 target molecules. Insets show representative standard curves generated from the amplification data. ΔR_n, normalized reporter value (with ROX). ΔR, reporter value (without ROX).

FIG. 2.

Effect of target gene sequence polymorphisms on the performance of the iap RTi-PCR assay. (A) Normalized C_T values (iap/hly; see the text) obtained with 40 L. monocytogenes strains of different serovars (indicated below the graphs). The results for each strain (mean of four replicates) are represented by bars. Note that the strains can be ascribed to two statistically significant groups (P < 0.05) which coincide with serovar-related phylogenetic division I or III (serovars 1/2b, 3b, 4a, 4b, 4c, 4d, and 7) and division II (serovars 1/2a, 1/2c, 3a, and 3c) of L. monocytogenes delimited by an iap/hly C_T value of 3. Note also that two strains of serovar 1/2a have higher iap/hly C_T values (indicated by an asterisk); these strains harbor a mismatch in the target sequence of the probe (see panel B, sequence 2, black shading). (B) Nucleotide sequences of hly and iap fragments targeted by PCR primers and probes (boxed, with arrows indicating the 5′→3′ orientation) (Table 3). The bottom panel shows strains CECT 911, CECT 938, UdG 1010, and UdG 1011 (sequence 1); strains CECT 932 and CECT 4031 (sequence 2); strains CECT 935, CECT 936, CECT 937, CECT 940, CECT 4032, and UdG 1034 (sequence 3); and strain CECT 934 (sequence 4) (Table 1). Mismatches are represented by black shading.