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.     

Competitor internal standards for quantitative detection of mycoplasma DNA

Abstract Homologous internal controls were used as competitor DNA in the polymerase chain reaction for the quantitative detection of mycoplasma DNA. PCR primer sets were designed on the basis of the most conserved nucleotide sequences of the 16S rRNA gene of mycoplasma species. Amplification of this gene was examined in five different mycoplasma species: Mycoplasma orale, M. hyorhinus, M. synoviae, M. gallisepticum and M. pneumonias . To evaluate the primers, a number of different cell lines were assayed for the detection of mycoplasma infections. All positive cell lines showed a distinct product on agarose gels while uninfected cells showed no DNA amplification. Neither bacterial nor eukaryotic DNA produced any cross-reaction with the primers used, thus confirming their specificity. Internal control DNA to be used for quantitation was constructed by modifying the sizes of the wild-type amplified products and cloning them in plasmid vectors. These controls used the same primer binding sites as the wild-type and the amplified products were differentiated by a size difference. The detection limits for all the mycoplasma species by competitive quantitative PCR were estimated to range from 4 to 60 genome copies per assay as determined by ethidium bromide-stained agarose gels. These internal standards also serve as positive controls in PCR-based detection of mycoplasma DNA, and therefore accidental contamination of test samples with wild-type positive controls can be eliminated. The quantitative PCR method developed will be useful in monitoring the progression and significance of mycoplasma in the disease process.     

Collaborative study report: Evaluation of the ATCC experimental mycoplasma reference strains panel prepared for comparison of NAT-based and conventional mycoplasma detection methods

The main goal of this collaborative study was to evaluate the experimental panel of cryopreserved mycoplasma reference strains recently prepared by the American Type Culture Collection (ATCC^®) in order to assess the viability and dispersion of cells in the mycoplasma stocks by measuring the ratio between the number of genomic copies (GC) and the number of colony forming units (CFU) in the reference preparations. The employment of microbial reference cultures with low GC/CFU ratios is critical for unbiased and reliable comparison of mycoplasma testing methods based on different methodological approaches, i.e., Nucleic Acid Testing (NAT) and compendial culture-based techniques. The experimental panel included ten different mycoplasma species known to represent potential human and animal pathogens as well as common contaminants of mammalian and avian cell substrates used in research, development, and manufacture of biological products. Fifteen laboratories with expertise in field of mycoplasma titration and quantification of mycoplasmal genomic DNA participated in the study conducted from February to October of 2012. The results of this study demonstrated the feasibility of preparing highly viable and dispersed (possessing low GC/CFU ratios) frozen stocks of mycoplasma reference materials, required for reliable comparison of NAT-based and conventional mycoplasma detection methods.     

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.     

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.     

Statistical assessment of DNA extraction reagent lot variability in real‐time quantitative PCR

Aims: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real‐time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. Methods and Results: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. Conclusions: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot‐to‐lot variation is accounted for in data interpretation. Significance and Impact of the Study: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms.     

Application of PCR for detection and identification of mycoplasma contamination in virus stocks

Summary A nested polymerase chain reaction (PCR) was used to detect and identify mycoplasma contaminants in viral stocks. The results of the PCR assay proved to be a sensitive and accurate indicator of the true status of the stock tested. Those samples positive by agar culture or Hoechst stain were also positive by PCR. Those samples that were inconclusive by Hoechst stain (10.05%) could be clearly determined to be mycoplasma positive or negative by PCR. The PCR assay also detected those fastidious species of mycoplasma that gave false negative results by the direct culture method. In many respects the PCR-based mycoplasma detection method described is superior to the agar culture and Hoechst staining detection methods. In this study, the PCR assay detected substantially more mycoplasma-positive viral stocks than did the agar culture assay. Due to its speed, sensitivity, and reliability, the PCR assay is of particular value in monitoring the process of removing mycoplasma from contaminated stocks. Furthermore, the PCR amplification products can be analyzed by restriction analysis to rapidly identify the species of the mycoplasma contaminating the stock tested.     

抗生素抗性基因检测方法发展历程及应用现状

耐药性的传播已引起全球广泛关注,抗生素抗性基因(antibiotic resistance genes, ARGs)检测技术的开发是研究ARGs在环境-动植物-人群中迁移传播的关键。本文梳理了现有核酸检测技术的发展历程及首次应用于ARGs检测的时间节点,并从检测原理、应用优缺点、开发潜力等方面对各技术进行分类综述。在此基础上提出以等温扩增结合CRISPR/Cas技术为核心的ARGs原位快速检测技术的开发及应用前景展望。本综述旨在回顾各技术发展历程的基础上,为ARGs新检测技术的开发及应用提供参考,为耐药性传播的研究及控制提供技术支撑。     

A single-tube nested real-time polymerase chain reaction for sensitive contained detection of Cryptosporidium parvum

Aim:  A new real-time polymerase chain reaction (PCR) was developed for sensitive contained detection of Cryptosporidium parvum .
Methods and Results:  The method is a nested PCR targeting a specific region of rDNA of C. parvum , which takes place in one tube, using different annealing temperatures to control the first and the second rounds of PCR, with real-time fluorogenic probe-based detection of the second round of PCR. The DNA-based detection limit of the method was 2 fg, which corresponds to approx. one genome per reaction. The detection level determined using diluted samples of C. parvum oocysts was ten oocysts per millilitre.
Conclusions:  The method facilitates sensitive detection of C. parvum thanks to the nested format, while reducing the risk of laboratory contamination thanks to the single-tube, real-time fluorimetric format.
Significance and Impact of the Study:  The developed method may be useful for sensitive contained detection of C. parvum in environmental and food samples, after appropriate separation of oocysts.     

Correlation between the distribution pattern of virulence genes and virulence of Aeromonas hydrophila strains

Nine strains of Aeromonas hydrophila isolated from diseased fish or soft-shelled tortoise were tested for the presence of three virulence genes including the genes encoding aerolysin,hemolysin,and extracellular serine protease (i.e.,aerA,hlyA,and ahpA,respectively).These genes were investigated using polymerase chain reaction (PCR)with specific primers for each gene.And the pathogenicities to Carrassius auratus ibebio of these strains were also assayed.PCR results demonstrated that the distribution patterns of aerA,hlyA,and ahpA were different in these strains.6/9 of A.hydrophila strains were aer A positive,8/9 of strains hly A positive,7/9 of strains ahp A positive,respectively.However,the assay for pathogenesis showed that two strains (A.hydrophila XS91-4-1 and C2)were strong virulent,two strains (A.hydrophila ST78-3-3 and 58-20-9)avirulent and the rest middle virulent was to the fish.In conclusion,there are significant correlation between the distribution pattern of the three virulence genes and the pathogenicity to Carrassius auratus ibebio.All strong virulent A.hydrophila strains were aerA+hlyA+ahpA+genotype,and all aerA+hlyA+ahpA+strains were virulent.Strains with the genotype of aerA-hlyA-ahpA+have middle pathogenicity.In the present study,we found for the first time that all A.hydrophila isolated from the ahpA positive were virulent to Carrassius auratus ibebio.Additionally,there was a positive correlation between the virulence of A.hydrophila and the presence of aerA and ahpA.     

Detection of virulent Escherichia coli O157 strains using multiplex PCR and single base sequencing for SNP characterization

Aims: To compare 167 Norwegian human and nonhuman Escherichia coli O157:H7/NM (nonmotile) isolates with respect to an A/T single nucleotide polymorphism (SNP) in the tir gene and to detect specific SNPs that differentiate STEC O157 into distinct virulence clades (1–3 and 8). Methods and Results: We developed a multiplex PCR followed by single base sequencing for detection of the SNPs, and examined the association among SNP genotype, virulence profile (stx and eae status), multilocus variable number of tandem repeats analysis (MLVA) profile and clinical outcome. We found an over‐representation of the T allele among human strains compared to nonhuman strains, including 5/6 haemolytic‐uraemic syndrome cases. Fourteen strains belonged to clade 8, followed by two clade 2 strains. No clade 1 nor 3 isolates were observed. stx1 in combination with either stx2_EDL933 or stx2c were frequently observed among human strains, whereas stx2c was dominating in nonhuman strains. MLVA indicated that only single cases or small outbreaks with E. coli O157 have been observed in Norway through the years 1993–2008. Conclusion: We observed that the tir‐255 A/T SNP and the stx status were different between human and nonhuman O157 strains. No major outbreaks were observed, and only a few strains were differentiated into the virulence clades 2 and 8. Significance and Impact of the Study: The detection of virulence clade‐specific SNPs enables the rapid designation of virulent E. coli O157 strains, especially in outbreak situations.     

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.