A Novel Real-Time PCR for Listeria monocytogenes That Monitors Analytical Performance via an Internal Amplification Control

Abstract

We describe a novel quantitative real-time (Q)-PCR assay for Listeria monocytogenes based on the coamplification of a target hly gene fragment and an internal amplification control (IAC). The IAC is a chimeric double-stranded DNA containing a fragment of the rapeseed BnACCg8 gene flanked by the hly-specific target sequences. This IAC is detected using a second TaqMan probe labeled with a different fluorophore, enabling the simultaneous monitoring of the hly and IAC signals. The hly-IAC assay had a specificity and sensitivity of 100%, as assessed using 49 L. monocytogenes isolates of different serotypes and 96 strains of nontarget bacteria, including 51 Listeria isolates. The detection and quantification limits were 8 and 30 genome equivalents, and the coefficients for PCR linearity (R 2 ) and efficiency (E) were 0.997 and 0.80, respectively. We tested the performance of the hly-IAC Q-PCR assay using various broth media and food matrices. Fraser and half-Fraser media, raw pork, and raw or cold-smoked salmon were strongly PCR-inhibitory. This Q-PCR assay for L. monocytogenes, the first incorporating an IAC to be described for quantitative detection of a food-borne pathogen, is a simple and robust tool facilitating the identification of false negatives or underestimations of contamination loads due to PCR failure.

Internally Controlled Real-Time PCR Method for Quantitative Species-Specific Detection and vapA Genotyping of Rhodococcus equi

“…M24199), which we previously validated as a Q-PCR probe target (24), flanked by the R. equi-specific choE gene sequences targeted by reqF and reqR primers (Table 1). This chimeric DNA molecule was generated by two rounds of PCR as previously described (26). The first PCR used 1 ng L. monocytogenes DNA template and primers riacF and riacR (Table 1), which contained the corresponding hly target sequences plus a 5Ј tail with the reqF or reqR primer sequence.…”

“…The second PCR used the purified first-round PCR product (diluted 1:1,000) as a template and the reqF and reqR primers. PCR conditions were as previously described (26). The IAC PCR product was purified using the QIAquick gel extraction kit (QIAGEN, Hilden, Germany), quantified, and diluted to the appropriate concentration in 10 mM Tris-HCl, pH 8.0, in the presence of 500 ng/ml of acetylated bovine serum albumin as a blocking agent to minimize binding of the negatively charged IAC DNA to the plastic microtubes.…”

Quantitative Detection of Clostridium tyrobutyricum in Milk by Real-Time PCR

“…When a negative signal is obtained for the target fla signal, the absence of a positive IAC signal indicates that amplification has failed (19). The IAC was constructed as previously described (22). The IAC consisted of a 121-bp chimeric DNA containing a portion (nucleotide positions 421 to 490) of the Listeria monocytogenes positive regulatory factor A (prfA) gene (14), flanked by the C. tyrobutyricum-specific fla gene sequences targeted by the CTflaF and CTflaR primers.…”

Molecular methodology in Food Microbiology diagnostics: trends and current challenges

“…Therefore, adequate control of the efficiency of the reaction is a fundamental aspect in such assays Cook, 2003, Hoorfar et al, 2004). An internal amplification control or IAC is a non-target nucleic acid sequence which is co-amplified simultaneously with the target sequence (Cone et al, 1992;Rodríguez-Lázaro et al, 2004;2005). In a reaction without an IAC, a negative response (no signal) can mean that there was no target sequence present in the reaction.…”