Rapid detection of Salmonella in field samples by nested polymerase chain reaction

A simple and universal protocol for the rapid detection of Salmonella spp. in various samples using nested polymerase chain reaction (PCR) was developed. The protocol takes advantage of the rapid purification and concentration of Salmonella by centrifugation onto a layer of 60% sucrose solution. DNA was released by treatment with proteinase K and Triton X-100. Even without pre-enrichment, the detection limit for the nested PCR was 10(5) CFU g-1 of faeces. After pre-enrichment, it was possible to detect Salmonella in faeces where their original concentration was approximately 10(2) CFU g-1 of faeces and in meat samples, it was possible to detect Salmonella in samples where their original concentration was less than 10 cells g-1 of minced meat.     

Use of the polymerase chain reaction for Salmonella detection

J. KWANG, E.T. LITTLEDIKE AND J.E. KEEN. 1996. A primer set of oligonucleotides (S18 and S19) from the _omp C gene of Salmonella has been evaluated for specific detection of Salmonella by polymerase chain reaction (PCR). This primer set successfully amplified 40 Salmonella serovars (60 isolates), but not 24 non-Salmonella bacteria (42 isolates) that have been tested so far. The uniqueness of these primer sequences was also confirmed. The sensitivity of PCR detection in extracted chromosomal DNA for Salm. typhimurium was 1 pg. The sensitivity for boiled whole bacteria was 400 cells. The detection of Salm. typhimurium in ground beef samples required 4–6 h enrichment with an initial inocula of 100 bacteria.     

Development of species-specific polymerase chain reaction primers for detection of Fusobacterium periodonticum

The purpose of this study was to develop species-specific PCR primers for detection of Fusobacterium periodonticum. The specificity data showed that two sets of PCR primers, Fp-F3/Fp-R2 and Fp-F1/Fp-R2 PCR, produced amplicons from all the F. periodonticum, but not from the other species tested, which included 12 Fusobacterium species or subspecies and representative oral bacteria. The sensitivity of the primer sets was 4 or 40 pg of the chromosomal DNA from F. periodonticum ATCC 33693(T) . These results suggest that these two sets of PCR primers are quite sensitive in detection of F. periodonticum in molecular epidemiological studies of periodontitis.     

Rapid polymerase chain reaction method for detection of Vibrio cholerae in foods.

The polymerase chain reaction was used to selectively amplify sequences within the cholera toxin operon from Vibrio cholerae O1. Oysters, crabmeat, shrimp, and lettuce were seeded with V. cholerae and then homogenized or washed with alkaline peptone water, followed by short-term (6- to 8-h) enrichment. A detection limit of as few as 1 V. cholerae CFU per 10 g of food was obtained with amplification reactions from crude bacterial lysates. The method is extremely rapid and obviates the need for DNA isolation from a variety of complex food matrices.     

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.     

Comparison of different primers for rapid detection of Vibrio parahaemolyticus using the polymerase chain reaction

AIMS: The aim of this study was to compare different primers for rapid and effective detection of Vibrio parahaemolyticus by polymerase chain reaction (PCR). METHODS AND RESULTS: A total of four pairs of primers, three previously published and one based on a newly developed V. parahaemolyticus metalloprotease (vpm) gene, have been assayed for PCR detection of V. parahaemolyticus. They have been tested for specificity and sensitivity on a total of 101 strains including reference and environment isolates belonging to V. parahaemolyticus and other species in Vibrio. Of the four sets of primers tested, the one designed on the basis of the metalloprotease gene (675 bp) gave optimal results with bacterial strains examined as they only amplified the specific fragment in strains that had been genetically and biochemically assessed as V. parahaemolyticus and the limit of detection was 4 pg of purified target DNA. CONCLUSIONS: The primers designed on the metalloprotease gene gave optimal results for specific, sensitive and rapid detection of V. parahaemolyticus by PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR amplification with the optimal primer set VPM1/VPM2 could facilitate the rapid diagnosis and surveillance of potentially pathogenic strains of V. parahaemolyticus and reduce food-borne illness.     

Specific detection of soybean residues in processed foods by the polymerase chain reaction

A sensitive qualitative detection method for soybeans in foods by using the polymerase chain reaction (PCR) was developed. For specific detection of soybeans with high specificity, the primer pair of Gym 81/Gym 82 was designed on the gene encoding the Glycine max repetitive sequence. The trace amount of soybeans in commercial food products could be qualitatively detected by this method.     

Antemortem detection of mouse parvovirus and mice minute virus by polymerase chain reaction (PCR) of faecal samples

Parvoviruses remain one of the most common viral infections seen in laboratory mouse colonies. The purpose of this study was to develop an antemortem polymerase chain reaction (PCR) assay to detect mice infected with mouse parvovirus-1 (MPV) and mice minute virus (MMV) using faecal samples. The MMV PCR assay consistently detected as few as 100 plasmid copies of MMV in faecal samples, while the MPV PCR assay detected as few as 10 plasmid copies of MPV. Faecal pellets from infected mice held at room temperature from 1 to 7 days tested positive by MMV and MPV PCR, respectively. This demonstrates that parvovirus DNA is stable in faecal samples kept at room temperature. PCR assays were also used to follow the length of MMV and MPV shedding in faeces from SENCAR mice, which were endemically infected with multiple agents. MMV faecal shedding was detected in 60-70% of the mice 5-7 weeks old, and by 13 weeks of age, faecal samples from all mice were negative for MMV. MPV faecal shedding was detected in 90-100% of the mice 5-11 weeks old; however, by 19 weeks of age, faecal samples from all mice were negative for MPV. These findings confirm that faecal shedding occurs for a limited time and suggest that 5-9-week-old mice are the most appropriate age group in endemically infected mice for faecal testing by MMV and MPV PCR.     

Development of a polymerase chain reaction assay for the detection of Listeria monocytogenes in foods

N.S. BANSAL. 1996. Species-specific oligonucleotide primers were selected from the coding region of the listeriolysin O gene of Listeria monocytogenes and were used in conjunction with genus-specific primers and an internal control fragment for polymerase chain reaction amplification. The specificity of the primers was confirmed by testing 40 isolates of L. monocytogenes , other Listeria species and other micro-organisms which are ubiquitous in the environment. The reliability of these primers was further tested in parallel with standard cultural methods. In a preliminary study, over 250 different food samples were examined and PCR results were in complete agreement with those obtained from standard cultural procedures.     

Specific detection of wheat residues in processed foods by polymerase chain reaction

A sensitive qualitative detection method for wheat in foods using polymerase chain reaction (PCR) was developed. Trace amounts of wheat in commercial food products could be qualitatively detected by this method. The sensitivity of the proposed PCR method appears to be similar to that of ELISA. The present method should be very useful for detecting wheat residues in processed foods.     

Designing primers from multiple sequences using matchup program to improve detection of hepatitis B virus by polymerase chain reaction

Traditionally primers for PCR detection of viruses have been selected from genomic sequence of single or representative viral strain. However, high mutation rate of viral genomes often results in failure in detecting viruses in clinical and environmental samples. Thus, it seems necessary to consider primers designed from multiple viral sequences in order to improve detection of viral variants. Matchup is a program intended to select universal primers from multiple sequences. We designed using Matchup program primer pairs for HBV detection from 691 full genomic HBV DNA sequences available from NCBI GenBank database. Thousands of primer candidates were initially extracted and these were sequentially filtered down to 5 primer pairs. These primer pairs were tested by PCR using 5 HBV Korean HBsAg(+) patient sera, and eventually one universal primer pair was selected and named MUW (multiple-universal-worldwide). This primer pair, 3 HBV reference primer pairs reported by others and 1 commercial primer pair were compared using 86 HBV HBsAg(+) sera from Korean and Vietnamese patients. The detection rate for MUW primer pair was 72.1%, much greater than those obtained by reference and commercial primers (32.5 to 40.7%). The superiority of MUW primer pair appeared to be correlated with the conserved sequences of the forward primer binding sites and primer quality score. These results suggest that the universal primers designed by the Matchup program from multiple sequences could be useful in detecting viruses from clinical samples.     

Chemically modified primers for improved multiplex polymerase chain reaction

Multiplex polymerase chain reaction (PCR), the amplification of multiple targets in a single reaction, presents a new set of challenges that further complicate more traditional PCR setups. These complications include a greater probability for nonspecific amplicon formation and for imbalanced amplification of different targets, each of which can compromise quantification and detection of multiple targets. Despite these difficulties, multiplex PCR is frequently used in applications such as pathogen detection, RNA quantification, mutation analysis, and (recently) next generation DNA sequencing. Here we investigated the utility of primers with one or two thermolabile 4-oxo-1-pentyl phosphotriester modifications in improving multiplex PCR performance. Initial endpoint and real-time analyses revealed a decrease in off-target amplification and a subsequent increase in amplicon yield. Furthermore, the use of modified primers in multiplex setups revealed a greater limit of detection and more uniform amplification of each target as compared with unmodified primers. Overall, the thermolabile modified primers present a novel and exciting avenue for improving multiplex PCR performance.     

Specific detection of buckwheat residues in processed foods by polymerase chain reaction

A specific and qualitative detection method for buckwheat in foods using the polymerase chain reaction (PCR) was developed. Trace amounts of buckwheat in commercial food products were qualitatively detected by this method. It should be reliable for detecting buckwheat residues in processed foods and practical for monitoring the labeling system for allergenic food materials.     

A new set of primers for the detection of Toxoplasma gondii in amniotic fluid using polymerase chain reaction

Abstract A new PCR system including a pair of primers, a probe and an internal control were designed from the B1 gene of Toxoplasma gondii . The system described allowed the detection of less than 10 tachyzoites of the RH strain of T. gondii . Among 21 amniotic fluid samples, this system diagnosed the cases of congenital toxoplasmosis which were simultaneously diagnosed using mice inoculation, in vitro culture, and serology from both amniotic fluid and fetal blood. These results show that these new primers allow for a highly sensitive detection of T. gondii DNA.     

Application of polymerase chain reaction (PCR) and TaqMan PCR techniques to the detection and identification of Rhodococcus coprophilus in faecal samples.

Rhodococcus coprophilus, a natural inhabitant of herbivore faeces, has been suggested as a good indicator of animal (as opposed to human) faecal contamination of aquatic environments. However, conventional detection methods limit its use for this as they require up to 21 days to obtain a result. In this paper an optimised method for extracting R. coprophilus DNA from faecal samples is described. PCR and 5'-nuclease (TaqMan) PCR methods were developed to allow the detection and enumeration of R. coprophilus in faecal samples within 2-3 days. Both PCR methods targeted the 16S rRNA gene, producing an amplicon of 443 bp which was specific for R. coprophilus. Sixty cells were required to produce an amplification product by conventional PCR, while as little as one cell was required for the TaqMan PCR method. The latter approach gave a linear quantitative response over at least four log units with both bacterial cells and DNA. Successful amplification by PCR was achieved using DNA extracted from cow, sheep, horse and deer faeces but was negative for samples from humans, pig, possum, duck and rabbit. These PCR methods enhance the feasibility of using R. coprophilus to distinguish faecal pollution of farmed herbivores from human pollution.     

Specific detection of Salmonella spp. by multiplex polymerase chain reaction.

Three sets of oligonucleotide primers were used in the polymerase chain reaction (PCR) assay to detect Salmonella species. phoP primers specific to the phoP/phoQ loci of coliform pathogenic bacteria such as Salmonella, Shigella, Escherichia coli, and Citrobacter species served as presumptive indicators of enteric bacteria. In addition to the phoP primers, the Hin and the H-1i primers, which targeted a 236-bp region of hin/H2 and a 173-bp region of the H-1i flagellin gene, respectively, were used. Both Hin and H-1i primers are specific to motile Salmonella species and are not present in Shigella, E. coli, or Citrobacter species. Thus, by multiplex PCR amplification, Salmonella species including Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi A, and Salmonella enteritidis can be specifically detected. Optimal reaction conditions have been described to demonstrate this specific, sensitive detection of Salmonella species. By using agarose gel electrophoresis for detection of the PCR-amplified products, the sensitivity of detection was 10(2) CFU after 25 cycles of PCR and 1 (10(0)) CFU after a 50-cycle double PCR. The efficacy of these primers was demonstrated on environmental isolates which had previously been confirmed as Salmonella species by the use of conventional cultural techniques. In addition, positive amplifications resulted from Salmonella species in environmental samples including soil and water.     

Comparison of polymerase chain reaction and conventional culture for the detection of legionellas in hospital water samples

A detection system for Legionella spp. based on the polymerase chain reaction (PCR) was used to assess the diagnostic value of PCR for the surveillance of contamination of man-made water systems by legionellas. A previously-published primer system was chosen to amplify a fragment of the 5S-ribosomal gene of Legionella spp. A total of 78 water samples from various sources were examined by PCR and culture on MWY Legionella selective agar. Fifty-seven of 78 water samples were positive by both test systems (73%), nine were positive by PCR only (11.5%), another nine were positive by culture but negative by PCR (11.5%), and three were negative by both techniques (3.8%). The PCR was inhibited when large amounts of rust were present in the samples. Culture failed to detect legionellas in samples that contained large numbers of other bacteria capable of overgrowing the legionellas. These results show that PCR is a rapid and sensitive technique for the detection of legionella contamination in water samples and that PCR and culture complement each other in monitoring of environmental water samples.     

Multiplex polymerase chain reaction and ligation detection reaction/universal array technology for the traceability of genetically modified organisms in foods

A multiplex polymerase chain reaction (PCR) system was developed for the simultaneous detection of target sequences in genetically modified soybean (Roundup Ready) and maize (MON810, Bt176, Bt11, and GA21). Primer pairs were designed to amplify the junction regions of the transgenic constructs analyzed and the endogenous genes of soybean (lectin) and maize (zein) were included as internal control targets to assess the efficiency of all reactions. This multiplex PCR has constituted the basis for an efficient platform for genetically modified organism traceability based on microarray technology. In particular, the ligation detection reaction combined to a universal array approach, using the multiplex PCR as target, was applied. High specificity and sensitivity were obtained.     

Development of a combined selective enrichment method and polymerase chain reaction (PCR) assay for sensitive detection of Salmonella in food samples

PCR assays were formatted using primer pairs homologous to phoE and invA genes. The amplification conditions were optimized with pure cultures and reactions were carried out to define selectivity, specificity and sensitivity of both primer pairs. The performance of the invA primer pair was better than that of the phoE pair, making the specific detection of Salmonella serovars and strains isolated from different food samples possible. Using the invA primer pair, the combined selective enrichment method with the polymerase chain reaction assay was established and used to detect Salmonella from artificially multi-contaminated food samples. The complete procedure detected as few as three cells of Salmonella (3 c.f.u.) from milk and meat samples.     

An interlaboratory comparison for the detection of Mycoplasma pneumoniae in respiratory samples by the polymerase chain reaction

A panel of 78 respiratory samples collected from 43 patients was analyzed in three different Centers for the presence of Mycoplasma pneumoniae DNA by polymerase chain reaction (PCR). One Center collected the samples and extracted the DNA by two different methods. DNA extracted according to the first method was amplified using primers targetting the 16 S rRNA gene. DNA extracted according to the second method was amplified using the same primers in a semi-nested format and was sent to the two other Centers. The latter Centers both used the same primers targetting the P1 gene but with a different detection format. Thirty-nine samples (50%) from 19 patients were positive by at least two PCR assays. None of the laboratories were free of false positive or false negative PCR results. Calculated specificities of the individual PCR assays ranged from 97.4% to 87.2% and sensitivities ranged from 97.4% to 89.2%. Complement fixation was done on sera of 33 patients. The calculated specificity and sensitivity of serology was 100% and 58.8%, respectively. Several aspects concerning false positive and false negative results with PCR are discussed.