Use of Polymerase Chain Reaction and Restriction Enzyme Analysis to directly detect and identify Salmonella typhimurium in food

A primer set of oligonucleotides (Salm 3 and Salm 4) from the inv A gene of Salmonellae has been evaluated for the specific detection of Salmonella spp. by the polymerase chain reaction (PCR). This primer set amplified 33 Salmonella serovars but did not amplify 16 non- Salmonella bacteria. Moreover, after PCR amplification, it was possible to identify Salm. typhimurium by restriction enzyme analysis. The PCR-RE method developed could represent a helpful tool for detecting Salmonella spp., and for directly and rapidly identifying Salm. typhimurium in food.     

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.     

Evaluation of selective and non-selective enrichment PCR procedures for Salmonella detection

AIMS: To compare PCR combined with enrichment media with the standard microbiological techniques (SMT) and to determine the most sensitive method for the detection of Salmonella and the identification of Salm. typhimurium (ST), Salm. enteritidis (SE), Salm. gallinarum (SG) and Salm. pullorum (SP). METHODS AND RESULTS: We analysed 87 samples from poultry using PCR and SMT, PCR being performed from non-selective (NS) and Rappaport-Vassiliadis (RV) media. PCR-NS was less sensitive than PCR-RV and SMT for the detection and identification of Salmonella. PCR-RV detected more positive samples of Salmonella sp. than SMT but both these methods showed similar sensitivity regarding the identification of Salmonella serovars. CONCLUSIONS: PCR-RV was more sensitive and decreased the time necessary to detect and identify Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR-RV is a powerful tool for the rapid and accurate detection and identification of Salmonella and can be implemented in diagnostic and food analysis laboratories.     

Evaluation of four template preparation methods for polymerase chain reaction-based detection of Salmonella in ground beef and chicken

AIMS: To compare procedures for recovering template DNA from ground beef or chicken for polymerase chain reaction (PCR)-based detection of Salmonella. METHODS AND RESULTS: The primer set of ST11 and ST15 was utilized to amplify a 429-bp product from Salmonella serotype Typhimurium. Boiling and three commercial kits were evaluated for extracting DNA from pure suspensions and artificially contaminated ground beef and chicken. The detection sensitivity of the PCR assay for pure cultures was independent of the template preparation method (P=0.946). Boiling and GeneReleaser failed to detect Salm. Typhimurium at 4 x 106 cfu g(-1) in ground chicken. PrepMan Ultra and the high pure PCR template preparation kit facilitated reliable and sensitive detection of Salm. Typhimurium in two types of food. The sensitivities were approx. 4 x 103 cfu g(-1). When spiked samples were enriched in peptone water for 6 h, an initial inoculum of 1 cfu g(-1) was detectable. CONCLUSIONS: Four template DNA preparation methods differed in performance with respect to the type of samples tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Template DNA for the PCR detection of pathogenic bacteria, such as Salmonella in meat and poultry, could be effectively obtained using a simple rapid method such as the commercially available PrepMan Ultra kit.     

Development of a multiplex PCR detection of Salmonella spp. and Shigella spp. in mussels

Multiplex PCR amplification of invA and virA genes was developed enabling simultaneous detection in mussels of Salmonella spp. and Shigella spp., respectively. Simultaneous amplification of products of 215 and 275 bp was obtained either by using mixtures of individual strains of Sh. dysenteriae and Salm. typhimurium or spiked contaminated mussels with both bacteria. In the case of the mussels, 10-100 cells of Salmonella spp. and Shigella per millilitre of homogenate were detected by the multiplex PCR following a pre-enrichment step to increase sensitivity and to ensure that detection was based on the presence of cultivable bacteria. Also, the sensitivity and specificity of this method was evaluated. Multiplex PCR amplification was shown to be an effective, sensitive and rapid method for the simultaneous detection of pathogens in mussels.     

禽大肠杆菌、肠炎、鼠伤寒、鸡白痢及鸡伤寒沙门菌多重PCR方法的建立及应用

【背景】大肠杆菌病和沙门菌病是最常见的家禽细菌性疾病,给养禽业造成严重经济损失。另外,禽大肠杆菌和沙门菌也是重要的人畜共患病原菌,可通过禽类及其产品传播给人类,对人类健康造成严重威胁。加强禽大肠杆菌和沙门菌的快速鉴别检测,对养禽业和公共卫生都具有重要意义。【目的】建立禽大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌的多重PCR检测方法。【方法】通过比较分析确定禽致病性大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌的特异靶标基因,设计5对特异性引物,通过条件优化建立多重PCR方法,分析该多重PCR方法的特异性、敏感性及可靠性。【结果】该方法能特异性地鉴定禽致病性大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌,每个PCR反应的最低检出限分别为103 CFU细菌和100 pg基因组DNA。临床分离菌株检测显示,多重PCR与传统血清学方法结果一致。【结论】建立的多重PCR方法能够快速鉴别禽致病性大肠杆菌和不同血清型沙门菌,对禽大肠杆菌病和沙门菌病的流行病学调查及临床检测具有重要意义。     

Antagonistic effects of intestinal Lactobacillus isolates on pathogens of chicken

L.Z. JIN, Y.W. HO, N. ABDULLAH, M.A. ALI AND S. JALALUDIN. 1996. Twelve Lactobacillus strains isolated from chicken intestine, which demonstrated a strong and moderate capacity to adhere to the ileal epithelial cells in vitro , were used to investigate their inhibitory ability against five strains of salmonella, i.e. Salmonella enteritidis 935/79, Salm. pullorum, Salm. typhimurium, Salm. blockley and Salm. enteritidis 94/448, and three serotypes of Escherichia coli , viz. E. coli O1 : K1, O2 : K1 and O78 : K80. The results showed that all the 12 Lactobacillus isolates were able to inhibit the growth of the five strains of salmonella, and the three strains of E. coli in varying degrees. Generally, they were more effective in inhibiting the growth of salmonella than E. coli . Inhibition of the pathogenic bacteria was probably due to the production of organic acids by the Lactobacillus isolates.     

The detection of Salmonella using a combined immunomagnetic separation and ELISA end-detection procedure

The aim of this study was to develop a rapid immunoassay to detect Salmonella bacteria. Skimmed milk powder (SMP) in buffered peptone water was inoculated with six Salmonella strains (Salm. typhimurium, Salm. virchow, Salm. enteritidis, Salm. give, Salm. ealing and Salm. arizonae) at three inoculum levels (about 2-200 cfu 25 g(-1) SMP) and incubated (37 degrees C) overnight. Heat-treated salmonella cells were immobilized on paramagnetic particles and detected within 3 h using the Salmonella genus-specific monoclonal antibody M105 in a microtitre plate based assay. The rapid Salmonella detection method combining immunomagnetic separation and ELISA had a total isolation and detection time of less than 24 h, which is significantly shorter than the conventional techniques requiring 72-96 h. The technique had a sensitivity limit of 10(5)-10(6) cfu ml(-1).     

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.     

Detection of Salmonella typhi by polymerase chain reaction

A rapid and sensitive method for detection of Salmonella typhi would help in preventing the spread of outbreaks and in clinical diagnosis. In order to develop unique PCR primers to detect Salm. typhi , ribosomal RNA genes from Salm. typhi (Rawlings) were cloned in pUC18. The resulting clone was confirmed by sequencing. The cloned DNA fragment contained the 5S, part of the 23S rRNA genes and the 5S-23S spacer region (EMBL/GenBank accession No. U04734).
It was expected that the 5S-23S spacer region is divergent unlike the highly conserved 23S+5S genes. This was confirmed by comparison with the rRNA gene sequences in the EMBL/GenBank database. A pair of PCR primers specific for Salm. typhi was obtained, based on this spacer region sequence. The specificity of this pair of primers was tested with 54 Salm. typhi strains (of 27 different phage types). All these Salm. typhi strains showed the positive 300 bp PCR product with this pair of primers. Six other Salmonella species as well as six other non- Salmonella bacteria were tested and none showed the 300 bp PCR product. The sensitivity of the detection level was 0·1 pg of pure Salm. typhi genomic DNA, or approximately 40 Salm. typhi cells in a spiked food sample. This pair of primers therefore has the potential for development into a diagnostic tool for the rapid diagnosis of typhoid fever.     

Detection of low numbers of Salmonella in environmental water, sewage and food samples by a nested polymerase chain reaction assay

A polymerase chain reaction (PCR) assay with two nested pairs of primers selected from conserved sequences within a 2.3 kb randomly cloned DNA fragment from the Salmonella typhimurium chromosome was developed. The nested PCR assay correctly identified 128 of a total of 129 Salmonella strains belonging to subspecies I, II, IIIb and IV. One strain of Salm. arizona (ssp. IIIa) tested negative. No PCR products were obtained from any of the 31 non-Salmonella strains examined. The sensitivity of the assay was 2 cfu, as determined by analysis of proteinase K-treated boiled lysates of Salm. typhimurium. The performance of the assay was evaluated for environmental water, sewage and food samples spiked with Salm. typhimurium. Water and sewage samples were filtered and filters were enriched overnight in a non-selective medium. Prior to PCR, the broth cultures were subjected to a rapid and simple preparation procedure consisting of centrifugation, proteinase K treatment and boiling. This assay enabled detection of 10 cfu 100 ml(-1) water with background levels of up to 8700 heterotrophic organisms ml(-1) and 10000 cfu of coliform organisms 100 ml(-1) water. Spiked food samples were analysed with and without overnight enrichment in a non-selective medium using the same assay as above. Nested PCR performed on enriched broths enabled detection of <10 cfu g(-1) food. Variable results were obtained for food samples examined without prior enrichment and most results were negative. This rapid and simple assay provides a sensitive and specific means of screening drinking water or environmental water samples, as well as food samples, for the presence of Salmonella spp.     

Application of Random Amplified Polymorphic DNA analysis to differentiate strains of Salmonella typhi and other Salmonella species

A Random Amplified Polymorphic DNA (RAPD) fingerprinting method was developed to differentiate isolates of Salmonella serotype typhi ( S. typhi ) and other Salmonella isolates. A panel of five primers was used to examine 63 isolates of Salm. typhi , including 56 strains isolated in Taiwan and seven strains obtained abroad. Twenty-one RAPD types were revealed using the RAPD fingerprinting method. An RAPD with primer 6032 yielded a polymorphism in a 350 bp fragment that differentiated the attenuated vaccine strain Salm. typhi Ty21a from the rest of the Salm. typhi strains. Strains of Salm. typhi were divided into five types with primer D14307. Primer D14307 also proved capable of discrimination among 65 other Salmonella isolates representing 42 different serotypes. The bacterial DNA used in this RAPD protocol was obtained using a commercially available DNA extraction kit (GeneReleaser). The DNA of various strains of Salmonella from this simple extraction procedure could be discriminated within a few hours using the RAPD technique.     

Effect of Saccharomyces boulardii against experimental oral infection with Salmonella typhimurium and Shigella flexneri in conventional and gnotobiotic mice

A.C.P. RODRIGUES, R.M. NARDI, E.A. BAMBIRRA, E.C. VIEIRA AND J.R. NICOLI. 1996. Saccharomyces boulardii was shown to be capable of inhibiting multiplication of enteropathogenic bacteria in vitro and is currently used for its anti-diarrhoea properties. We studied the capacity of this yeast to antagonize Salmonella typhimurium and Shigella flexneri in the intestinal tract of conventional or gnotobiotic NMRI mice. Conventional animals were given daily 10 mg doses of S. boulardii , whereas germ-free animals were given a single 10 mg dose. Both groups were challenged orally 5 d later with the pathogenic bacteria (10⁸ or 10² viable cells, respectively). Control groups were treated with saline instead of S. boulardii. Mortality and/or histopathological data showed a protective effect against the pathogenic bacteria in yeast-treated mice. Saccharomyces boulardii colonized the digestive tract of gnotobiotic mice and the number of viable cells ranged around 10¹⁰ g^-1 of faeces. In experimental and control gnotobiotic animals, Salm. typhimurium and Sh. flexneri became rapidly established at a level of about 10¹⁰ viable cells g^-1 of faeces and remained at high levels until the animals died or were sacrificed. The protection against Salm. typhimurium and Sh. flexneri obtained in conventional and/or gnotobiotic mice previously associated with S. boulardii is not due to the reduction of the bacterial populations in the intestines.     

Modulation of Salmonella infection by the lectins of Canavalia ensiformis (Con A) and Galanthus nivalis (GNA) in a rat model in vivo

The plant lectins, Concanavalin A (Con A) and Galanthus nivalis agglutinin (GNA) have been prefed to rats for 3 d pre- and 6 d postinfection with Salmonella typhimurium S986 or Salm. enteritidis 857. Con A significantly increased numbers of Salm. typhimurium S986 in the large intestine and in faeces, and severely impaired growth of the rats, more severely than is the case of infection with Salmonella typhimurium alone. Con A had much less effect on rats infected with Salm. enteritidis 857 only showing a significant increase in numbers in the colon, accompanied by intermittent increases of Salmonella in the faeces during the study. GNA significantly reduced pathogen numbers in the lower part of the small bowel and the large intestine of rats infected with Salm. typhimurium S986 and significantly improved rat growth. GNA had little effect on infection by Salm. enteritidis 857 with slight decreases in Salmonella numbers in the small intestine and large intestine and transient increases in the faeces.     

Real-time PCR method for Salmonella spp. targeting the stn gene

AIM: To develop a real-time PCR assay for Salmonella spp. targeting the stn gene. METHODS AND RESULTS: The presence of stn in the Salmonella bongori genome was found by a BLAST with Salmonella enterica stn sequence. Manual alignment of stn sequences showed that Salm. bongori had 88% sequence identity with Salm. enterica. Two primers (stnL-433 and stnR-561) and a probe (stnP-452) were designed to target conserved regions in stn and meet the requirements of a 5'-nuclease assay. The primers and probe were evaluated against 353 isolates, including 255 Salm. enterica representing 158 serotypes, 14 Salm. bongori representing 12 serotypes and 84 non-Salmonella representing 56 species from 31 genera. All isolates were correctly identified, with the exception of three isolates of Citrobacter amalonaticus, which gave false positives. The limit of detection with cultured Salmonella was 3 CFU per reaction. CONCLUSIONS: The stn real-time PCR method had 100% inclusivity, 96.4% exclusivity and a level of detection of 3 CFU per reaction for cultured Salmonella spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The study showed that stn is present in Salm. bongori and is a valid target for both species of Salmonella. The Salmonella s tn real-time PCR is a useful method for identifying Salmonella spp.     

Multicenter validation of the analytical accuracy of Salmonella PCR: towards an international standard

As part of a major international project for the validation and standardization of PCR for detection of five major food-borne pathogens, four primer sets specific for Salmonella species were evaluated in-house for their analytical accuracy (selectivity and detection limit) in identifying 43 Salmonella spp. and 47 non-Salmonella strains. The most selective primer set was found to be 139-141 (K. Rahn, S. A. De Grandis, R. C. Clarke, S. A. McEwen, J. E. Galán, C. Ginocchio, R. Curtiss III, and C. L. Gyles, Mol. Cell. Probes 6:271-279, 1992), which targets the invA gene. An extended determination of selectivity by using 364 strains showed that the inclusivity was 99.6% and the exclusivity was 100% for the invA primer set. To indicate possible PCR inhibitors derived from the sample DNA, an internal amplification control (IAC), which was coamplified with the invA target gene, was constructed. In the presence of 300 DNA copies of the IAC, the detection probability for primer set 139-141 was found to be 100% when a cell suspension containing 10(4) CFU/ml was used as the template in the PCR (50 CFU per reaction). The primer set was further validated in an international collaborative study that included 16 participating laboratories. Analysis with 28 coded ("blind") DNA samples revealed an analytical accuracy of 98%. Thus, a simple PCR assay that is specific for Salmonella spp. and amplifies a chromosomal DNA fragment detected by gel electrophoresis was established through extensive validation and is proposed as an international standard. This study addresses the increasing demand of quality assurance laboratories for standard diagnostic methods and presents findings that can facilitate the international comparison and exchange of epidemiological data.     

Serotyping, PCR, phage-typing and antibiotic sensitivity testing of Salmonella serovars isolated from urban drinking water supply systems of Nepal

AIMS: To study the occurrence and diversity of Salmonella serovars in urban water supply systems of Nepal. METHODS AND RESULTS: Occurrence of Salmonella was detected in 42 out of 300 water samples by enrichment culture technique in selenite F broth followed by plating on Salmonella Shigella agar. A total of 54 isolates identified to genus level by standard tests were subsequently confirmed by serotyping, phage typing and PCR detection of virulence genes (inv A and spv C). The predominant serotype was Salmonella Typhimurium, followed by Salm. Typhi, Salm. Paratyphi A and Salmonella Enteritidis. Most of the Salm. Typhi isolates were E1 phage type followed by UVS4, A and UVS1. All isolates of Salm. Paratyphi A and Salm. Enteritidis were an untypable (UT) phage type. The majority of isolates were multi-drug resistant as revealed by Kirby-Bauer disc diffusion technique. Ceftriaxone resistant isolates of Salm. Enteritidis indicated the presence of one of the ESBL genes, blaSHV, whereas the genes blaTEM and blaCTX were absent. CONCLUSIONS: The microbiological quality of the urban water supply is poor and indicates possibility of fatal outbreaks of enteric fever and related infections in Nepal. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study will be useful in water borne disease control and prevention strategy formulation in Nepal and in the global context.     

Variation in Salmonella core lipopolysaccharide as detected by the monoclonal antibody M105

L.P. MANSFIELD, E. BILLETT, E. OLSEN AND S.J. FORSYTHE. 1996. The lipopolysaccharide antigenicity of 22 Salmonella strains (representing nine serogroups) and four non-salmonellae Enterobacteriaceae to the Salmonella genus specific monoclonal antibody M105 was analysed. The monoclonal antibody M105 reacted with all 22 Salmonella strains. Probing SDS-PAGE separated LPS molecules with MAb M105 revealed that the antibody reacted with the core region of all Salmonella serovars. However, no reaction was obtained to the long-chain LPS of serovars O ( Salm. adelaide and Salm. ealing ), C₁ ( Salm. infantis, Salm. livingstone and Salm. virchow ) or Salm. arizonae . It is plausible that the presence of a second core antigenic type results in the lack of reaction between long-chain LPS and the Salmonella genus specific monoclonal antibody M105.     

Evaluation of the specificity of Salmonella PCR primers using various intestinal bacterial species

AIMS: Nine sets of PCR primers targeting Salmonella were evaluated for their specificity with pure cultures of intestinal-associated bacteria prior to their application to Salmonella detection in faecal samples. METHODS AND RESULTS: Gene targets of PCR primers included: 16S rDNA, a Salmonella pathogenicity island I virulence gene, Salmonella enterotoxin gene (stn), invA gene, Fur-regulated gene, histidine transport operon, junction between SipB and SipC virulence genes, Salmonella-specific repetitive DNA fragment, and multiplex targeting invA gene and spvC gene of the virulence plasmid. Fifty-two Salmonella strains were used to determine sensitivity; five strains from related genera and 45 intestinal bacteria were used to evaluate specificity. All primers amplified DNA from Salmonella strains, although two primer sets failed to amplify Salmonella DNA from either Salmonella bongori (hilA) or subgroups VI or VII (16S rDNA). There was no detected amplification of DNA from related bacterial genera with any of nine PCR assays. Six of the PCR assays amplified DNA for some intestinal bacteria. CONCLUSIONS: Only three primer pairs were determined to be suitable for application of PCR amplification of Salmonella in faecal samples - 16S rDNA, stn and histidine transport operon. We are currently evaluating their sensitivity of detection of Salmonella in faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the importance of internal lab validation of PCR primers prior to application to the type of samples of interest. Information from this evaluation can be applied in other labs to facilitate choosing Salmonella PCR primers.     

Detection of coliform bacteria in water by polymerase chain reaction and gene probes

Polymerase chain reaction (PCR) amplification and gene probe detection of regions of two genes, lacZ and lamB, were tested for their abilities to detect coliform bacteria. Amplification of a segment of the coding region of Escherichia coli lacZ by using a PCR primer annealing temperature of 50 degrees C detected E. coli and other coliform bacteria (including Shigella spp.) but not Salmonella spp. and noncoliform bacteria. Amplification of a region of E. coli lamB by using a primer annealing temperature of 50 degrees C selectively detected E. coli and Salmonella and Shigella spp. PCR amplification and radiolabeled gene probes detected as little as 1 to 10 fg of genomic E. coli DNA and as a few as 1 to 5 viable E. coli cells in 100 ml of water. PCR amplification of lacZ and lamB provides a basis for a method to detect indicators of fecal contamination of water, and amplification of lamB in particular permits detection of E. coli and enteric pathogens (Salmonella and Shigella spp.) with the necessary specificity and sensitivity for monitoring the bacteriological quality of water so as to ensure the safety of water supplies.