Molecular beacon-based real-time PCR detection of primary isolates of Salmonella Typhimurium and Salmonella Enteritidis in environmental and clinical samples

Background  

Medium density DNA microchips that carry a collection of probes for a broad spectrum of pathogens, have the potential to be powerful tools for simultaneous species identification, detection of virulence factors and antimicrobial resistance determinants. However, their widespread use in microbiological diagnostics is limited by the problem of low pathogen numbers in clinical specimens revealing relatively low amounts of pathogen DNA.     

A capillary polymerase chain reaction for Salmonella detection from poultry meat

AIMS: In this study, a capillary polymerase chain reaction (cPCR) was applied for Salmonella detection from poultry meat. METHODS AND RESULTS: Salmonella detection limits of the optimized cPCR were determined with DNA templates from the samples of tetrathionate broth (TTB), Rappaport Vassiliadis broth (RVB) and selenite cystine broth (SCB) artificially contaminated with 10-fold dilutions of 6 x 10(8) CFU ml(-1) of pure Salmonella enterica ssp. enterica serovar Enteritidis 64K stock culture. Detection limits of cPCR from TTB, RVB and SCB were found as 6, 6 x 10(1) and 6 x 10(4) CFU ml(-1), respectively. In addition, detection limits of bacteriology were also determined as 6 CFU ml(-1) with TTB and SCB, and 6 x 10(1) CFU ml(-1) with RVB. A total of 200 samples, consisting of 100 chicken and 100 turkey meat samples, were tested with optimized cPCR and bacteriology. Eight and six per cent of the chicken meat samples were found to harbour Salmonella by cPCR and standard bacteriology, respectively. Of six Salmonella isolates, four belonged to serogroup D, two to serogroup B. CONCLUSIONS: The TTB cultures of both artificially and naturally contaminated samples were found to be superior to those of RVB and SCB cultures in their cPCR results. This cPCR, utilizing template from 18-h TTB primary enrichment broth culture, takes approximately 40 min in the successful detection of Salmonella from poultry meat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that cPCR from TTB enrichment culture of poultry meat would enable rapid detection of Salmonella in laboratories with low sample throughput and limited budget.     

Diagnostic real-time PCR for detection of Salmonella in food

A robust 5' nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 10(3) CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 10(4) CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method.     

A novel multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces

AIMS: To develop a multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces. METHODS AND RESULTS: A total of 54 Salmonella strains representing 19 serovars and non-Salmonella strains representing 11 different genera were used. Five primer pairs were employed in the assay. Three of them targeted to the genes hilA, spvA and invA that encode virulence-associated factors. A fourth primer pair amplified a fragment of a unique sequence within S. enterica serovar Enteritidis genomes. An internal amplification control (a fragment of a conservative sequence within the 16S rRNA genes) was targeted by a fifth primer pair. The assay produced two or three amplicons from the invA, hilA and 16S rRNA genes for 19 Salmonella serovars. All Salmonella and non-Salmonella strains yielded a band of an internal amplification control. For S. enterica serovar Typhimurium, four products (the fourth from the spvA gene), and for S. enterica serovar Enteritidis five amplicons (the fifth from the sdf gene) were observed. S. enterica serovar Enteritidis was cultured from three of 71 rectal swabs from diarrhoeal patients. Five specific amplicons were generated with the multiplex PCR assay only from culture-positive faecal samples. CONCLUSION: The multiplex PCR assay specifically detects S. enterica serovar Enteritidis. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a novel multiplex PCR assay, which contains an internal amplification control and enables concurrent survey for Salmonella virulence genes.     

Salmonella Enteritidis bacteriophage candidates for phage therapy of poultry

Aims: Salmonella is a worldwide foodborne pathogen causing acute enteric infections in humans. In the recent years, the use of bacteriophages has been suggested as a possible tool to combat this zoonotic pathogen in poultry farms. This work aims to isolate and perform comparative studies of a group of phages active against a collection of specific Salmonella Enteritidis strains from Portugal and England. Also, suitable phage candidates for therapy of poultry will be selected. Methods and Results: The Salm. Enteritidis strains studied were shown to have a significantly high occurrence of defective (cryptic) prophages; however, no live phages were found in the strains. Bacteriophages isolated from different environments lysed all except one of the tested Salm. Enteritidis strains. The bacteriophages studied were divided into different groups according to their genetic homology, RFLP profiles and phenotypic features, and most of them showed no DNA homology with the bacterial hosts. The bacteriophage lytic efficacy proved to be highly dependent on the propagation host strain. Conclusions: Despite the evidences shown in this work that the Salm. Enteritidis strains used did not produce viable phages, we have confirmed that some phages, when grown on particular hosts, behaved as complexes of phages. This is most likely because of the presence of inactive phage‐related genomes (or their parts) in the bacterial strains which are capable of being reactivated or which can recombine with lytic phages. Furthermore, changes of the bacterial hosts used for maintenance of phages must be avoided as these can drastically modify the parameters of the phage preparations, including host range and lytic activity. Significance and Impact of the Study: This work shows that the optimal host and growth conditions must be carefully studied and selected for the production of each bacteriophage candidate for animal therapy.     

A TaqMan-based real-time PCR assay for the specific detection and quantification of Leuconostoc mesenteroides in meat products

A new real-time PCR procedure was developed for the specific detection and quantification of Leuconostoc mesenteroides in meat products. It is a TaqMan assay based on 23S rRNA gene targeted primers and probe. Specificity was evaluated using purified DNA from 132 strains: 102 lactic acid bacteria (LAB), including 57 reference strains and 46 food isolates, belonging to genus Leuconostoc and related genera, and 30 non-LAB strains. Quantification was linear over at least 5 log units using both serial dilutions of purified DNA and calibrated cell suspensions from Leuconostoc mesenteroides ssp. dextranicum CECT 912T. This assay was able to detect at least five genomic equivalents, using purified DNA or 59 CFU per reaction when using calibrated cell suspensions. It performed successfully when tested on an artificially inoculated meat product, with a minimum threshold of 10(4) CFU g(-1) for the accurate quantification of Leuconostoc mesenteroides.     

Comparison of primers for the detection of Salmonella enterica serovars using real-time PCR

AIMS: To evaluate the specificity and sensitivity of PCR primers for the detection of Salmonella enterica in a real-time PCR assay using pure cultures. METHODS AND RESULTS: Unenriched whole cells in sterile water were used as template for each PCR. SYBR Green dye was used for the nonspecific detection of dsDNA. The real-time PCR detection limits of five previously published primer sets used in conventional PCR applications were not below 3 x 10(3) CFU per reaction (rxn). A new primer set, Sen, was designed, which detected Salm. enterica Newport down to 6 CFU rxn(-1) in one case, and gave an average detection limit of 35 CFU rxn(-1) over three separate runs. CONCLUSIONS: Primers originally designed for end-point PCR did not have adequate specificity or sensitivity compared with those specifically designed for real-time PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: This study emphasizes the importance of evaluating real-time PCR primer sets in pure cultures prior to testing in field samples. This study will benefit other researchers in selecting an appropriate primer set for real-time PCR detection of Salm. enterica.     

Detection of Salmonella Enteritidis in asymptomatic carrier animals: comparison of quantitative real-time PCR and bacteriological culture methods

Quantification of Salmonella in asymptomatic carrier animals can be used to assess microbial risk and monitor the level of contamination in domestic animals used for food production. We examined the sensitivity, specificity and accuracy of real-time qPCR, without pre-enrichment or selective enrichment stages, for the quantification of S. enterica serovar Enteritidis in resistant mice, as a model of asymptomatic carrier animal. The results were compared with those obtained by traditional bacteriological culture methods, the gold standard test. Two hundred and forty-three samples, including spleen, liver, mesenteric lymph nodes, portions of intestine, intestinal content of the ileocecal portion, and feces, were collected from a group of 27 C57BL/6 mice, that had been intragastrically inoculated with high doses of S. enterica serovar Enteritidis. The real-time qPCR assay presented a consistent linearity of the standard curve (r(2) = 0.999), with very low differences between melting temperatures, and low coefficients of variation in intra- (< 1%) and interassay (< 2%) comparisons. The primers were highly specific; there was no amplification with other Salmonella serovars or with DNA from uninfected tissues and feces from mice. The detection limit of the technique was defined as 32 copies of S. enterica serovar Enteritidis. A sensitivity of 90%, a specificity of 77% and an accuracy of 79% were obtained. The higher sensitivity of PCR was reflected in a kappa coefficient of 0.41, showing moderate agreement between tests. We conclude that real-time qPCR is a good alternative for diagnostic scanning in asymptomatic carrier animals, due to its high sensitivity and rapidity.     

Nested real-time PCR for hepatitis A detection

Aims:  The hepatitis A virus (HAV) is one of the most important human foodborne pathogens causing a number of worldwide outbreaks each year. The detection of HAV in food samples remains a complex issue, because commonly used detection tools, such as conventional or even real-time PCR assays, are often unable to detect HAV with sufficient sensitivity. The aims of this study were to develop highly sensitive and specific nested real-time PCR (NRT-PCR)-based method for HAV detection in food and to compare it with currently available methods.
Methods and Results:  By combining conventional PCR, nested PCR and real-time PCR techniques, we have developed a specific NRT-PCR assay for the detection of HAV. The procedure involves two consecutive PCRs, the first of which is performed as a conventional RT-PCR using primers specific for HAV 5' noncoding region. The second reaction involves a real-time PCR using a nested primer pair specific for the first PCR product and a TaqMan probe.
Conclusions:  We have developed a novel NRT-PCR method capable of detecting as little as 0·2 PFU of HAV, which is significantly more sensitive than any other PCR technique tested in our system.
Significance and Impact of the Study:  NRT-PCR provides a potentially useful method for detecting HAV at extremely low levels, as frequently found in food samples, and can be potentially adopted as a regulatory method to ensure food safety.     

Rapid quantitative detection of Listeria monocytogenes in meat products by real-time PCR

We describe a quick and simple method for the quantitative detection of Listeria monocytogenes in meat products. This method is based on filtration, Chelex-100-based DNA purification, and real-time PCR. It can detect as few as 100 CFU/g and quantify as few as 1,000 CFU/g, with excellent accuracy compared to that of the plate count method. Therefore, it is a promising alternative for the detection of L. monocytogenes in meat products.     

Development of RNA aptamers for detection of Salmonella Enteritidis

We developed and evaluated RNA aptamers to analyze their potential for use in detecting Salmonella Enteritidis. The selected aptamer was observed to specifically bind to Salmonella Enteritidis without any cross-reactivity to other Salmonella serovars. Thus, this study suggests that aptamers specific to Salmonella Enteritidis have a high potential for use in presumptive presumptive screening methods or alternative serotyping methods.     

Adding a selective enrichment step to the iQ-Check real-time PCR improves the detection of Salmonella in naturally contaminated retail turkey meat products

AIMS: The aim of this study was to compare the real-time iQ-Check Salmonella kit (Bio-Rad) with the immunocapture assay RapidCheck Salmonella method, and a conventional culture method (FSIS, USDA) in detecting Salmonella in naturally contaminated turkey meat products. This study was also designed to determine if a selective enrichment step might improve the real-time detection of Salmonella. METHODS AND RESULTS: Using the culture method, Salmonella was recovered from 49 out of 99 retail turkey meat samples collected. RapidCheck failed to detect 11 Salmonella samples that were positive by the culture method. The iQ-Check real-time PCR also failed to detect three samples that were positive by the culture method. However, when carried out after a selective enrichment step, the iQ-Check real-time PCR detected all 49 Salmonella samples recovered by the culture method. The iQ-Check real-time PCR detected the presence of Salmonella in some samples that were not recovered by the culture method. CONCLUSIONS: Adding a selective enrichment step to the iQ-Check real-time PCR improves the detection of Salmonella in naturally contaminated turkey meat samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The iQ-Check Salmonella real-time PCR can be used as a rapid method to monitor Salmonella in turkey meat, together with conventional culture methodology.     

Application of real-time PCR for quantitative detection of Campylobacter jejuni in poultry, milk and environmental water

Campylobacter jejuni is a leading human food-borne pathogen. The rapid and sensitive detection of C. jejuni is necessary for the maintenance of a safe food/water supply. In this article, we present a real-time polymerase chain reaction (PCR) assay for quantitative detection of C. jejuni in naturally contaminated poultry, milk and environmental samples without an enrichment step. The whole assay can be completed in 60 min with a detection limit of approximately 1 CFU. The standard curve correlation coefficient for the threshold cycle versus the copy number of initial C. jejuni cells was 0.988. To test the PCR system, a set of 300 frozen chicken meat samples, 300 milk samples and 300 water samples were screened for the presence of C. jejuni. 30.6% (92/300) of chicken meat samples, 27.3% (82/300) of milk samples, and 13.6% (41/300) of water samples tested positive for C. jejuni. This result indicated that the real-time PCR assay provides a specific, sensitive and rapid method for quantitative detection of C. jejuni. Moreover, it is concluded that retail chicken meat, raw milk and environmental water are commonly contaminated with C. jejuni and could serve as a potential risk for consumers in eastern China, especially if proper hygienic and cooking conditions are not maintained.     

Rapid quantitative detection of Lactobacillus sakei in meat and fermented sausages by real-time PCR

A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages.     

A MIQE-compliant real-time PCR assay for Aspergillus detection

The polymerase chain reaction (PCR) is widely used as a diagnostic tool in clinical laboratories and is particularly effective for detecting and identifying infectious agents for which routine culture and microscopy methods are inadequate. Invasive fungal disease (IFD) is a major cause of morbidity and mortality in immunosuppressed patients, and optimal diagnostic criteria are contentious. Although PCR-based methods have long been used for the diagnosis of invasive aspergillosis (IA), variable performance in clinical practice has limited their value. This shortcoming is a consequence of differing sample selection, collection and preparation protocols coupled with a lack of standardisation of the PCR itself. Furthermore, it has become clear that the performance of PCR-based assays in general is compromised by the inadequacy of experimental controls, insufficient optimisation of assay performance as well as lack of transparency in reporting experimental details. The recently published "Minimum Information for the publication of real-time Quantitative PCR Experiments" (MIQE) guidelines provide a blueprint for good PCR assay design and unambiguous reporting of experimental detail and results. We report the first real-time quantitative PCR (qPCR) assay targeting Aspergillus species that has been designed, optimised and validated in strict compliance with the MIQE guidelines. The hydrolysis probe-based assay, designed to target the 18S rRNA DNA sequence of Aspergillus species, has an efficiency of 100% (range 95-107%), a dynamic range of at least six orders of magnitude and limits of quantification and detection of 6 and 0.6 Aspergillus fumigatus genomes, respectively. It does not amplify Candida, Scedosporium, Fusarium or Rhizopus species and its clinical sensitivity is demonstrated in histological material from proven IA cases, as well as concordant PCR and galactomannan data in matched broncho-alveolar lavage and blood samples. The robustness, specificity and sensitivity of this assay make it an ideal molecular diagnostic tool for clinical use.     

Development of a surface adhesion immunofluorescent technique for the rapid detection of Salmonella spp. from meat and poultry

A rapid method based on bacterial adhesion was developed for the detection of Salmonella in an enriched meat system. Minced beef samples inoculated with Salm. enteritidis (10 cfu g-1) were incubated overnight (18 h) at 37 degrees C in buffered peptone water. Salmonella enteritidis cells were isolated from the enriched meat sample by surface adhesion onto a polycarbonate membrane attached to a glass slide. The organisms attached to this polycarbonate membrane were subsequently visualized using immunofluorescent microscopy. The technique had a detection level of log10 3.5 Salmonella ml-1. The surface adhesion immunofluorescent technique correlated well with Salmonella plate counts (r2 = 0.99). Application of the rapid method to retail beef and poultry samples (n = 100) confirmed the correlation between this technique and traditional microbiological procedures. Thirty-one retail samples were reported positive for Salmonella species. No false positives or negatives were recorded for the rapid method.     

Direct detection of Shigella flexneri and Salmonella typhimurium in human feces by real-time PCR

We have established a SYBR Green-based realtime PCR method using AnyDirect solution, which enhances PCR from whole blood, for direct amplification of the virA gene of Shigella flexneri and the invA gene of Salmonella typhimurium from human feces without prior DNA purification. When we compared the efficiency of conventional or realtime PCR amplification of the virA and invA genes from the supernatant of boiled feces supplemented with S. flexneri and S. typhimurium in the presence or absence of AnyDirect solution, amplification products were detected only in reactions to which AnyDirect solution had been added. The detection limit of real-time PCR was 1 x 10(4) CFU/g feces for S. flexneri and 2 x 10(4) CFU/g feces for S. typhimurium this sensitivity level was comparable to other studies. Our real-time PCR assay with AnyDirect solution is simple, rapid, sensitive, and specific, and allows simultaneous detection of S. flexneri and S. typhimurium directly from fecal samples without prior DNA purification.     

Fluorescent bio-barcode DNA assay for the detection of Salmonella enterica serovar Enteritidis

Salmonella enterica serovar Enteritidis is one of the most frequently reported causes of foodborne illness. It is a major threat to the food safety chain and public health. A highly amplified bio-barcode DNA assay for the rapid detection of the insertion element (Iel) gene of Salmonella Enteritidis is reported in this paper. The biosensor transducer is composed of two nanoparticles: gold nanoparticles (Au-NPs) and magnetic nanoparticles (MNPs). The Au-NPs are coated with the target-specific DNA probe which can recognize the target gene, and fluorescein-labeled barcode DNA in a 1:100 probe-to-barcode ratio. The MNPs are coated with the 2nd target-specific DNA probe. After mixing the nanoparticles with the 1st target DNA, the sandwich structure (MNPs-2nd DNA probe/Target DNA/1st DNA probe-Au-NPs-barcode DNA) is formed. A magnetic field is applied to separate the sandwich from the unreacted materials. Then the bio-barcode DNA is released from the Au-NPs. Because the Au-NPs have a large number of barcode DNA per DNA probe binding event, there is substantial amplification. The released barcode DNA is measured by fluorescence. Using this technique, the detection limit of this bio-barcode DNA assay is as low as 2.15 x 10(-16)mol (or 1 ng/mL).