Demonstration of the effective performance of a combined enrichment /real‐time PCR method targeting the prfA gene of Listeria monocytogenes by testing fresh naturally contaminated acid curd cheese

Aims: A rapid real‐time PCR‐based method for the detection of Listeria monocytogenes was applied to the examination of 44 Quargel cheese samples from a recent outbreak in Austria and compared to the standard method according to ISO‐16140. Methods and Results: The combined enrichment/real‐time PCR method amplifying the prfA locus was performed according to [Rossmanith et al. (2006) Res Microbiol, 157, 763–771]. Qualitative and quantitative examination of the samples was performed according to the standard method ISO‐11290. Comparison of the combined enrichment/real‐time PCR method with ISO‐11290 resulted in 100% relative accuracy, 100% relative sensitivity and 100% relative specificity. Conclusions: A previously published study describing the validation of the method, including samples after storage at ?80°C, resulted in lower performance values. In contrast, the samples were stored at +4°C in this study. The results of this study indicate an effect of storage, thus masking the true performance of the method. Significance and Impact of the Study: The results of this study are discussed together with the previously published data to demonstrate the excellent qualities of this rapid (≤30 h) method when applied to fresh specimens stored at +4°C.     

A real‐time PCR assay for detection and quantification of Lactococcus garvieae

Aims: To develop a rapid, sensitive, specific tool for the detection and quantification of Lactococcus garvieae in food and environmental samples. Methods and Results: A real‐time quantitative PCR (qPCR) assay with primers for CAU12F and CAU12R based on the 16S rRNA gene of L. garvieae was successfully established. The limit of detection for L. garvieae genomic DNA was 1 ng DNA in conventional PCR and 32 fg with a mean C_T value of 36·75 in qPCR. Quantification of L. garvieae vegetative cells was linear (R² = 0·99) over a 7‐log‐unit dynamic range down to ten L. garvieae cells. Conclusions: This method is highly specific, sensitive and reproducible for the detection of L. garvieae compared to gel‐based conventional PCR assays, thus providing precise quantification of L. garvieae in food and natural environments. Significance and Impact of the Study: This work provides efficient diagnostic and monitoring tools for the rapid identification of L. garvieae, an emerging pathogen in aquaculture and an occasional human pathogen from other members of the genus Lactobacillus.     

smcL as a novel diagnostic marker for quantitative detection of Listeria ivanovii in biological samples

Aims: To develop a novel molecular tool for the quantitative detection of the ruminant pathogen Listeria ivanovii in different biological matrices. Methods and Results: A real‐time PCR (RTi‐PCR) for the quantitative and species‐specific identification of L. ivanovii was designed to target the region of the smcL gene. The assay includes an internal amplification control (IAC) to avoid false‐negative results. The smcL‐IAC RTi‐PCR assay was 100% selective and allowed the detection of as little as one genome equivalent in 45% of reactions. The quantification accuracy was excellent, as demonstrated by its high linearity (R² > 0·9989) and PCR efficiency (E > 0·984) over a 6‐log dynamic range, down to 10 genome equivalents. Finally, the applicability of this assay was evaluated with artificially contaminated biological matrices implicated in the transmission of this bacterium such as sheep raw milk, blood and amniotic fluid. The smcL‐IAC RTi‐PCR assay allowed the detection of as few as 50 colony forming unit numbers (CFUs) per 25 ml of raw milk, 43 CFUs per 1 ml of blood or 50 CFUs per 1 ml of amniotic fluid. Conclusions: This method can be an adequate alternative for the identification of L. ivanovii and for complete diagnosis of animal and human listeriosis. Significance and Impact of the Study: We present an alternative for the detection of another pathogenic member of Listeria genus, which can help to distinguish from Listeria monocytogenes and therefore facilitates the establishment of preventive and prophylactic measures in food and farm environments.     

Development and field testing of a real-time PCR assay for cylindrospermopsin-producing cyanobacteria

Aims: To develop and test a real-time PCR assay to detect and quantify genes specific to Cylindrospermopsis sp. and cylindrospermopsin-producing cyanobacteria. Method and Results: A duplex real-time PCR assay was developed that targets a cylindrospermopsin-specific and Cylindrospermopsis raciborskii-specific DNA sequence. The C. raciborskii-specific sequence was based on the rpoC1 DNA-dependent RNA polymerase gene, whilst the cylindrospermopsin-specific sequence was selected by surveying an extensive number of potential cylindrospermopsin-producing cyanobacterial strains for genes implicated in toxin production, aoaA, aoaB and aoaC. In toxic strains, sequences of each of these three genes were always present; whilst in nontoxic strains the distribution of these sequences was patchy, resulting in what are likely to be natural deletion mutants. The real-time assay was optimized on a fixed and portable device, with results indicating that the reliable limit of detection for the assay was 100 copies per reaction or 1000 cells ml⁻¹ for both target sequences on both devices. In routine environmental samples enumerated by microscopy, the assay results were positive for all samples where C. raciborskii cells were observed at >1000 cells ml⁻¹ and negative in 15 samples where no C. raciborskii cells were observed. In field samples, the number of copies of the rpoC1 sequence more closely approximated the number of cells enumerated by microscopy, the number of copies of the pks sequence and detection of the toxin-specific sequence matched the results of toxin testing. Conclusions: The duplex real-time PCR assay was a sensitive and rapid method for detecting potential cylindrospermopsin-producing cyanobacteria in the laboratory or in the field. The observation of probable natural deletion mutants provides further evidence that the aoaA, aoaB and aoaC genes are involved in toxin production. Significance and Impact of the Study: This assay provides a new monitoring capability for tracking cylindrospermopsin-producing cyanobacteria that are an emerging threat to water quality.     

Development of Streptococcus gordonii‐specific quantitative real‐time polymerase chain reaction primers based on the nucleotide sequence of rpoB

In this study, Streptococcus gordonii‐specific quantitative real‐time polymerase chain reaction (qPCR) primers, RTSgo‐F2/RTSgo‐R2, were developed based on the nucleotide sequences of RNA polymerase β‐subunit gene (rpoB). The specificity of the RTSgo‐F2/RTSgo‐R2 primers was assessed by conventional PCR on 99 strains comprising 63 oral bacterial species, including the type strain and eight clinical isolates of S. gordonii. PCR products were amplified from the genomic DNAs of only S. gordonii strains. The qPCR primers were able to detect as little as 40 fg of S. gordonii genomic DNA at a cycle threshold value of 33. These findings suggest that these qPCR primers detect S. gordonii with high specificity and sensitivity.     

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

Targeted species‐specific and community‐wide molecular diagnostics tools are being used with increasing frequency to detect invasive or rare species. Few studies have compared the sensitivity and specificity of these approaches. In the present study environmental DNA from 90 filtered seawater and 120 biofouling samples was analyzed with quantitative PCR (qPCR), droplet digital PCR (ddPCR) and metabarcoding targeting the cytochrome c oxidase I (COI) and 18S rRNA genes for the Mediterranean fanworm Sabella spallanzanii. The qPCR analyses detected S. spallanzanii in 53% of water and 85% of biofouling samples. Using ddPCR S. spallanzanii was detected in 61% of water of water and 95% of biofouling samples. There were strong relationships between COI copy numbers determined via qPCR and ddPCR (water R² = 0.81, p < .001, biofouling R² = 0.68, p < .001); however, qPCR copy numbers were on average 125‐fold lower than those measured using ddPCR. Using metabarcoding there was higher detection in water samples when targeting the COI (40%) compared to 18S rRNA (5.4%). The difference was less pronounced in biofouling samples (25% COI, 29% 18S rRNA). Occupancy modelling showed that although the occupancy estimate was higher for biofouling samples (ψ = 1.0), higher probabilities of detection were derived for water samples. Detection probabilities of ddPCR (1.0) and qPCR (0.93) were nearly double metabarcoding (0.57 to 0.27 marker dependent). Studies that aim to detect specific invasive or rare species in environmental samples should consider using targeted approaches until a detailed understanding of how community and matrix complexity, and primer biases affect metabarcoding data.     

Evaluation of reference genes for real‐time PCR quantification of gene expression in the Australian sheep blowfly,Lucilia cuprina

The Australian sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae), is an important pest of sheep in Australia and other parts of the world. However, the paucity of information on many fundamental molecular aspects of this species limits the ability to exploit functional genomics techniques for the discovery of new drug targets for its control. The present study aimed to facilitate gene expression studies in this species by identifying the most suitable reference genes for normalization of mRNA expression data. Quantitative real‐time polymerase chain reaction (PCR) was performed with 11 genes across RNA samples from eggs, L1, L3, pupae and adult life stages, and two normalization programs, Normfinder and geNorm, were then applied to the data. The results showed an ideal set of genes (18S rRNA, 28S rRNA, GST1, β‐tubulin and RPLPO) for data normalization across all life stages. The most suitable reference genes for studies within specific life stages were also identified. GAPDH was shown to be a poor reference gene. The reference gene recommendations in this study will be of use to laboratories investigating gene expression in L. cuprina and related blowfly species     

A decade with nucleic acid‐based microbiological methods in safety control of foods

In the last decade, nucleic acid‐based methods gradually started to replace or complement the culture‐based methods and immunochemical assays in routine laboratories involved in food control. In particular, real‐time polymerase chain reaction (PCR) was technically developed to the stage of good speed, sensitivity and reproducibility, at minimized risk of carry‐over contamination. Basic advantages provided by nucleic acid‐based methods are higher speed and added information, such as subspecies identification, information on the presence of genes important for virulence or antibiotic resistance. Nucleic acid‐based methods are attractive also to detect important foodborne pathogens for which no classical counterparts are available, namely foodborne pathogenic viruses. This review briefly summarizes currently available or developing molecular technologies that may be candidates for involvement in microbiological molecular methods in the next decade. Potential of nonamplification as well as amplification methods is discussed, including fluorescent in situ hybridization, alternative PCR chemistries, alternative amplification technologies, digital PCR and nanotechnologies.     

Evaluation of Bacillus anthracis and Yersinia pestis sample collection from nonporous surfaces by quantitative real‐time PCR

Aim: We will validate sample collection methods for recovery of microbial evidence in the event of accidental or intentional release of biological agents into the environment. Methods and Results: We evaluated the sample recovery efficiencies of two collection methods – swabs and wipes – for both nonvirulent and virulent strains of Bacillus anthracis and Yersinia pestis from four types of nonporous surfaces: two hydrophilic surfaces, stainless steel and glass, and two hydrophobic surfaces, vinyl and plastic. Sample recovery was quantified using real‐time qPCR to assay for intact DNA signatures. We found no consistent difference in collection efficiency between swabs or wipes. Furthermore, collection efficiency was more surface‐dependent for virulent strains than nonvirulent strains. For the two nonvirulent strains, collection efficiency was similar between all four surfaces, albeit B. anthracis Sterne exhibited higher levels of recovery compared to Y. pestis A1122. In contrast, recovery of B. anthracis Ames spores and Y. pestis CO92 from the hydrophilic glass or stainless steel surfaces was generally more efficient compared to collection from the hydrophobic vinyl and plastic surfaces. Conclusions: Our results suggest that surface hydrophobicity may play a role in the strength of pathogen adhesion. The surface‐dependent collection efficiencies observed with the virulent strains may arise from strain‐specific expression of capsular material or other cell surface receptors that alter cell adhesion to specific surfaces. Significance and Impact of the Study: These findings contribute to the validation of standard bioforensics procedures and emphasize the importance of specific strain and surface interactions in pathogen detection.     

Quantification of Campylobacter spp. in chicken carcass rinse by real‐time PCR

Aims: In this study, a real‐time quantitative polymerase chain reaction (PCR) method was examined for its ability to quantify Campylobacter spp. in chicken carcass rinses and compared with bacteriological culturing. Methods and Results: The linearity of the real‐time PCR quantification protocol was assessed on pure DNA. The amplification efficiency was 100% and the square regression coefficient (R²) was 0·998. Quantification was linear over at least 7 log units. Using a crude cell lysate gave the highest sensitivity and the detection limit of the method was 3·3 log CFU per carcass. The statistical correlation between the bacteriological enumeration and the real‐time quantitative (Q)‐PCR determined using chicken carcasses sampled at the end of the slaughter line was 0·733. The difference in detection levels was probably because of the detection of stressed, dead or viable but not culturable cells by Q‐PCR. Conclusion: The real‐time Q‐PCR method described in this study is a powerful tool for determining the number of Campylobacter cells on carcasses. Significance and Impact of the Study: The real‐time Q‐PCR method is available to quantify the Campylobacter contamination at the slaughterhouse level and could be used to evaluate primary production.     

Triplex real‐time polymerase chain reaction assay for detection and quantification of norovirus (GI and GII) and sapovirus

To improve detection of norovirus (NoVGI, NoVGII) and sapovirus (SaV), a simultaneous quantitative RT‐PCR method was established. This triplex real‐time PCR method was evaluated using a combination of optimized specific primers and probes. The performance of the developed PCR assay was equivalent to that of monoplex real‐time PCR across a broad dynamic range of 10²–10⁷ copies/assay using plasmid DNA standards. The limit of detection was 10² copies/assay. The quantitative value was comparable with that of monoplex real‐time PCR of stool samples. Our triplex real‐time PCR is useful for detection of NoV and SaV infections.     

Serum miR‐122 and miR‐192 as biomarkers of intrinsic and idiosyncratic acute hepatotoxicity: A quantitative real‐time polymerase chain reaction study in adult albino rats

miR‐122 and miR‐192 were investigated as indicators of toxic liver injury caused by acetaminophen, but their role in idiosyncratic toxic liver injury remains controversial. So, this work aimed to assess and compare the expressions of miR‐122 and miR‐192 in two different types of toxic liver injury (intrinsic [acetaminophen] and idiosyncratic [diclofenac]). Forty male adult Wistar albino rats were divided into equal five groups, in which serum liver enzymes; microRNAs (miRNAs) expressions (miR‐122 and miR‐192) and histopathological findings were studied. The present study showed that (1) miR‐122 and miR‐192 are good serum biomarkers of toxic liver injury whatever its etiology, as their serum levels exhibited a significantly earlier increase and earlier return to normal baseline levels as compared to serum aminotransferase levels; (2) miR‐122 is more specific than miR‐192; and (3) both serum levels of miR‐122 and miR‐192 showed non‐significant differences in relation to the type of toxic liver injury.