Analysis of major band of Enterobacter sakazakii by ERIC-PCR and development of a species-specific PCR for detection of Ent. sakazakii in dry food samples

ERIC (Enterobacterial Repetitive Intergenic Consensus)-PCR was employed to generate stable and reproductive ERIC-PCR fingerprints of Ent. sakazakii ATCC51329. Moreover, this study also cloned and sequenced a major band of Ent. sakazakii (ATCC51329) ERIC-PCR fingerprints. The major band was amplified with primer ERIC2 and sequences extending primer ERIC 2 showed poor similarity with ERIC elements. A comparison of the nucleotide acid with other sequences available in the GenBank revealed 90% of identity with Ent. sakazakii ATCC BAA-894, and 73%-74% of identity with oligopeptiase gene or protease gene of some species from the Enterobacteriaceae family. Two primers were synthesized to develop and optimize an Enterobacter sakazakii-specific PCR based on regions of major band unique to Ent. sakazakii. The expected fragment was amplified from all of Ent. sakazkaii but not from the negative controls. As few as 10(2) CFU/ml of Ent. sakazakii of PCR were directly detected in the infant formulas. This was the case even in the presence of other bacteria. A comparison of traditional methods and new developed PCR in commercial foods suggested that without using API20-E test, the DFI chromogenic medium and FDA method showed 46.15% and 50% false positive respectively. Moreover, one false negative was observed with FDA method. In contrast, PCR was highly sensitive and specific to Ent. sakazakii. A high heterogeneity between Ent. sakazakii and the other microorganisms was found on expected fragment sequence. In addition, Ent. sakazakii ATCC51329 formed a separate branch with >5% divergence from the type strain ATCC BAA-894 and major strains.     

Characterization of the zinc-containing metalloprotease encoded by zpx and development of a species-specific detection method for Enterobacter sakazakii

Enterobacter sakazakii causes a severe form of neonatal meningitis that occurs as sporadic cases as well as outbreaks. The disease has been epidemiologically associated with consumption of reconstituted, dried infant formulas. Very little information is available regarding pathogenicity of the organism and production of virulence factors. Clinical and environmental strains were screened for production of factors which have activity against Chinese hamster ovary (CHO) cells in tissue culture. Polymyxin B lysate and sonicate preparations but not culture supernatants from the strains caused "rounding" of CHO cells. Subsequent studies showed that the CHO cell-rounding factor is a proteolytic enzyme that has activity against azocasein. The cell-bound protease was isolated by using a combination of polymyxin B lysis, followed by sonication of cells harvested from tryptone broth. The protease was purified to homogeneity by sequential ammonium sulfate precipitation, gel filtration chromatography with Sephadex G-100, hydrophobic interaction chromatography with phenyl-Sepharose CL-4B, and a second gel filtration with Sephadex G-100. In addition to activity against azocasein, the purified protease also exhibits activity against azocoll and insoluble casein but not elastin. The protease has a molecular weight of 38,000 and an isoelectric point of 4.4. It is heat labile and for maximal activity against azocasein has an optimum temperature of 37 degrees C and a pH range of 5 to 7. Proteolytic activity is inhibited by ortho-phenanthroline and Zincov but is not affected by phenylmethylsulfonyl fluoride, N-ethylmaleimide, and trypsin inhibitors, which demonstrates that the protease is a zinc-containing metalloprotease. The metalloprotease does not hemagglutinate chicken or sheep erythrocytes. Twenty-three to 27 of the first 42 N-terminal amino acid residues of the metalloprotease are identical to proteases produced by Serratia proteamaculans, Pectobacterium carotovorum, and Anabaena sp. PCR analysis using primers designed from a consensus nucleotide sequence showed that 135 E. sakazakii strains possessed the metalloprotease gene, zpx, and 25 non-E. sakazakii strains did not. The cloned zpx gene of strain 29544 consists of 1,026 nucleotides, and the deduced amino acid sequence of the metalloprotease has 341 amino acid residues, which corresponds to a theoretical protein size of 37,782 with a theoretical pI of 5.23. The sequence possesses three well-characterized zinc-binding and active-site motifs present in other bacterial zinc metalloproteases.     

Cloning and sequencing of the ompA gene of Enterobacter sakazakii and development of an ompA-targeted PCR for rapid detection of Enterobacter sakazakii in infant formula

Enterobacter sakazakii is an emerging, infant formula-borne pathogen that causes severe meningitis, meningoencephalitis, sepsis, and necrotizing enterocolitis in neonates and infants, with a high fatality rate. Traditional detection methods take up to 7 days to identify E. sakazakii. The outer membrane protein A gene (ompA), along with its flanking sequences from E. sakazakii (ATCC 51329), was cloned in the pGEM-T Easy vector and sequenced. Comparison of the nucleotide and deduced amino acid sequences of the ompA gene with other sequences available in the GenBank database revealed a high degree of homology with ompA genes of other gram-negative bacteria belonging to the Enterobacteriaceae. Based on regions of the ompA gene unique to E. sakazakii, two primers were synthesized to develop and optimize an E. sakazakii-specific PCR. The PCR amplified a 469-bp DNA product from all E. sakazakii strains tested but not from other bacteria. Experiments to determine the sensitivity of the PCR indicated that it could detect as few as 10(3) CFU/ml of E. sakazakii bacteria in infant formula directly and 10(-1) CFU/ml after an 8-h enrichment step. We conclude that this PCR, combined with enrichment culturing, has the potential to be used as a rapid tool for detecting the presence of E. sakazakii in infant formula.     

Real time PCR using TaqMan and SYBR Green for detection of Enterobacter sakazakii in infant formula

Enterobacter sakazakii is an emerging pathogen that causes meningitis, bacteremia, sepsis, and necrotizing enterocolitis in neonates and children. Powdered milk-based infant formulas have been associated with the E. sakazakii-related outbreaks in premature or other immunocompromised infants. In this study, we developed two real time PCR assays using TaqMan and SYBR Green to identify the pathogen after selective enrichment in mLST and BHI. The accuracy of two detections was tested by 35 strains of E. sakazakii and 88 non-E. sakazakii bacterial strains. The results showed that all of these E. sakazakii strains were positive reaction to the detections and all of the non-E. sakazakii strains were negative. The newly developed assays enable us to detect 1.1 CFU/100 g infant formula. And both of the assays can be accomplished within 2 business days. Compared to the traditional detection, the real time PCR procedures are quicker and simpler. In this study, we also developed a new method to design the primers, which can support multiple real time PCR with one pair of primers in SYBR Green detection. The detection methods are more sensitive and effective based on Two-Tm-Value of PCR.     

A robotic DNA purification protocol and real-time PCR for the detection of Enterobacter sakazakii in powdered infant formulae

Background  

Enterobacter sakazakii is the causative agent of rare but severe food-borne infections associated with meningitis, necrotizing enterocolitis and sepsis in infants. Rehydrated powdered infant formulae have been implicated as the source of infection in several outbreaks and sporadic cases. In this work, a real time fluorescence resonance energy transfer PCR assay incorporating an internal amplification control (IAC) was developed for the specific detection of E. sakazakii in foods. Performance of the assay, coupled to an automated DNA extraction system and the E. sakazakii ISO-IDF (TS 22964/RM 210) enrichment procedure, was evaluated on infant formulae and samples from production environment.     

Cloning and Sequencing of the ompA Gene of Enterobacter sakazakii and Development of an ompA-Targeted PCR for Rapid Detection of Enterobacter sakazakii in Infant Formula

Enterobacter sakazakii is an emerging, infant formula-borne pathogen that causes severe meningitis, meningoencephalitis, sepsis, and necrotizing enterocolitis in neonates and infants, with a high fatality rate. Traditional detection methods take up to 7 days to identify E. sakazakii. The outer membrane protein A gene (ompA), along with its flanking sequences from E. sakazakii (ATCC 51329), was cloned in the pGEM-T Easy vector and sequenced. Comparison of the nucleotide and deduced amino acid sequences of the ompA gene with other sequences available in the GenBank database revealed a high degree of homology with ompA genes of other gram-negative bacteria belonging to the Enterobacteriaceae. Based on regions of the ompA gene unique to E. sakazakii, two primers were synthesized to develop and optimize an E. sakazakii-specific PCR. The PCR amplified a 469-bp DNA product from all E. sakazakii strains tested but not from other bacteria. Experiments to determine the sensitivity of the PCR indicated that it could detect as few as 10³ CFU/ml of E. sakazakii bacteria in infant formula directly and 10⁻¹ CFU/ml after an 8-h enrichment step. We conclude that this PCR, combined with enrichment culturing, has the potential to be used as a rapid tool for detecting the presence of E. sakazakii in infant formula.     

食品中克罗诺杆菌属双重PCR检测试剂盒的评价

【背景】在食品安全领域,克罗诺杆菌属于需要重点监测的致病菌,当前随着分子检测相关技术的不断发展,研制有关食品中克罗诺杆菌简便、高效的检测产品至关重要。【目的】研制克罗诺杆菌检测的双重PCR检测试剂盒并评价其用于食品中克罗诺杆菌检测的实效性。【方法】优化双重PCR反应体系,反应试剂采用冻干工艺,确立了试剂盒组成,并评价其特异性、灵敏度、重复性、保质期等性能指标。【结果】克罗诺杆菌标准菌株和分离菌株均在目标位置出现两条明显条带,非克罗诺杆菌标准菌株和分离菌株均检测为阴性,纯基因组DNA检测灵敏度为2.3×10-1 ng/μL,纯培养菌检验限为3.2×104CFU/mL;对65份食品样品进行克罗诺杆菌检测,该试剂盒检测结果与标准方法检测结果一致性较高;批内、批间检测重复率均为100%,可在42°C环境放置120 h且其检测效力不受影响,4°C保质期可长达12个月。【结论】该试剂盒性能好,检测结果稳定、可靠,适用于食品中克罗诺杆菌的快速检测。     

Rapid detection and identification of Streptococcus ratti by a species-specific PCR method

To establish a rapid and species-specific detection and identification method of Streptococcus ratti by polymerase chain reaction, two PCR primer pairs specific to S. ratti were designed on the basis of the nucleotide sequence of the dextranase gene (dex) of S. ratti ATCC19645(T). The primer pairs specifically detected S. ratti, but none of the other mutans streptococci (16 strains of 6 species). The PCR procedure was capable of detecting 1 pg of genomic DNA purified from S. ratti ATCC19645. We developed the Streptococcus mutans-, Streptococcus sobrinus-, Streptococcus downei- and Streptococcus salivarius-specific PCR methods (the dex PCR methods) with the primer pairs specific for a portion of the dex gene of each species. The mixture of these primer pairs including S. ratti (this study) successfully differentiated the five species of mutans streptococci by species-specific amplicons of different lengths. These results suggest that the present PCR method is suitable for the specific detection and identification of S. ratti, and that the mixture of primer pairs for the dex PCR methods is useful for species-specific detection and rapid discrimination of each species in mutans streptococci.     

改良环介导等温扩增技术快速检测婴儿配方奶粉中的阪崎肠杆菌

[目的]采用改良环介导等温扩增(LAMP)技术,快速检测婴儿配方奶粉中的阪崎肠杆菌.[方法]以阪崎肠杆菌(ATCC29544)的16S-23S rRNA间区序列作为靶序列,设计内、外引物和环引物,通过肉眼观察白色沉淀,判断检测结果.[结果]LAMP检测阪崎肠杆菌的灵敏度为0.101 CFU/mL,人工污染阪崎肠杆菌的婴儿配方奶粉的检出限为1.1 CFU/g.采用试剂盒提取DNA,从样品处理到报告结果,耗时1 h.而对照,PCR检测阪崎肠杆菌的灵敏度为101 CFU/mL,人工污染阪崎肠杆菌的婴儿配方奶粉的检出限为1100 CFU/g.采用同样方法提取DNA,从样品处理到报告结果,耗时3 h.[结论]因此,LAMP检测婴儿配方奶粉中的阪崎肠杆菌灵敏度高,耗时短,方法简便.     

Detection of Legionella pneumophila in water samples by species-specific real-time and nested PCR assays

AIMS: Legionella pneumophila is a contaminant of man-made water systems, including potable water, cooling towers, water systems of large buildings, etc. It is the most common causative agent of legionellosis, a respiratory infection, which may give rise to restricted outbreaks. To survey environmental water samples from hospitals and private habitations in Bologna, we developed a species-specific nested and a TaqMan real-time PCR for the detection of L. pneumophila. We compared the two assays and both to cultural isolation. METHODS AND RESULTS: The targeted gene was macrophage infectivity potentiator (mip), conserved in L. pneumophila, and divergent in other legionellae. One assay was based on a nested PCR and the other on a TaqMan real-time PCR protocol. Their sensitivities were 14 % or 5% higher than that of cultural isolation respectively. The detection limits were 1-2 genome equivalents per 50-microl reaction. Specificity was assessed using DNA from nine target and 20 nontarget organisms. CONCLUSIONS: When applied to water samples, both assays detected L. pneumophila at 80% or higher frequency. SIGNIFICANCE AND IMPACT OF THE STUDY: The species-specific molecular diagnosis of L. pneumophila by means of nested PCR does not require a specific instrumentation, exhibits a high sensitivity, and is advantageous over the cultural isolation and real-time PCR detection. It allows to quickly monitor water samples for the risk assessment of environmental contaminations.     

Application of groEL gene for the species-specific detection of Vibrio parahaemolyticus by PCR

Aims: Vibrio parahaemolyticus is a significant cause of human gastrointestinal disorders and is transmitted through ingestion of raw or undercooked contaminated seafood. We used the groEL gene for the species‐specific detection of V. parahaemolyticus from artificially inoculated shellfish, fish and seawater. Methods and Results: The nucleotide sequences of 24 Vibrio and seven non‐Vibrio spp. were compared, and less conserved regions were selected for the designing of primer sets. To detect V. parahaemolyticus specifically, PCR conditions were standardized and tested to evaluate the specificity of primers. A 510‐bp band was appeared only from V. parahaemolyticus by PCR. Notably, the detection was shown to be functional at high annealing temperature above 68°C. The groEL primers detected 100 pg and 1 ng of DNA purified from V. parahaemolyticus culture and artificially infected oyster tissue, respectively. Conclusions: The groEL gene is a potential marker for the species‐specific detection of V. parahaemolyticus and could be used to detect this bacterium in contaminated food by PCR. Significance and Impact of the Study: PCR using primers designed from groEL gene provide an efficient method for the accurate identification of V. parahaemolyticus from contaminated samples.     

Genus- and species-specific PCR primers for the detection and identification of bifidobacteria

16SrDNA-targeted genus- and species-specific PCR primers have been developed and used for the identification and detection of bifidobacteria. These primers cover all of the described species that inhabit the human gut, or occur in dairy products. Identification of cultured bifidobacteria using PCR primer pairs is rapid and accurate, being based on nucleic acid sequences. Detection of bifidobacteria can be achieved using DNA extracted from human faeces as template in PCR reactions. We have found that, in adult faeces, the Bifidobacterium catenulatum group was the most commonly detected species, followed by Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium bifidum. In breastfed infants, Bifidobacterium breve was the most frequently detected species, followed by Bifidobacterium infantis, B. longum and B. bifidum. It was notable that the B. catenulatum group was detected with the highest frequency in adults, although it has often been reported that B. adolescentis is the most common species. Real-time, quantitative PCR using primers targeting 16S rDNA shows promise in the enumeration of bifidobacteria in faecal samples. The approach to detect the target bacteria with quantitative PCR described in this review will contribute to future studies of the composition and dynamics of the intestinal microflora.     

Comparison of methods for DNA isolation from food samples for detection of Shiga toxin-producing Escherichia coli by real-time PCR

In this study, food samples were intentionally contaminated with Escherichia coli O157:H7, and then DNA was isolated by using four commercial kits. The isolated DNA samples were compared by using real-time PCR detection of the Shiga toxin genes. The four kits tested worked similarly.     

Development of a PCR protocol for sensitive detection of Cryptosporidium oocysts in water samples.

The development of a reliable method of using PCR for detection of Cryptosporidium oocysts in environmental samples with oligonucleotide primers which amplify a portion of the sequence encoding the small (18S) subunit of rRNA producing a 435-bp product was demonstrated. The PCR assay was found to provide highly genus-specific detection of Cryptosporidium spp. after release of nucleic acids from oocysts by a simple freeze-thaw procedure. The assay routinely detected 1 to 10 oocysts in purified oocyst preparations, as shown by direct microscopic counts and by an immunofluorescence assay. The sensitivity of the PCR assay in some seeded environmental water samples was up to 1,000-fold lower. However, this interference was eliminated by either flow cytometry or magnetic-antibody capture. Sensitivity was also improved 10- to 1,000-fold by probing of the PCR product on dot blots with an oligonucleotide probe detected by chemiluminescence. Confirmation of the presence of Cryptosporidium oocysts in water samples from the outbreak in Milwaukee, Wis., was obtained with this technique, and PCR was found to be as sensitive as immunofluorescence for detection of oocysts in wastewater concentrates.     

Development of species-specific primers for detection of Streptococcus mutans in mixed bacterial samples

Streptococcus mutans is the major microbial pathogen associated with dental caries in children. The objectives of this study were to design and evaluate species-specific primers for the identification of S. mutans. Validation of the best primer set, Sm479F/R, was performed using seven S. mutans reference strains, 48 ATCC non-S. mutans strains, 92 S. mutans clinical isolates, DNA samples of S. mutans-Streptococcus sobrinus or S. mutans-Streptococcus sanguinis, and mixed bacterial DNA of saliva samples from 33 18-month-old children. All of the S. mutans samples tested positive, and no PCR products were amplified from members of the other streptococci or nonstreptococci strains examined. The lowest detection level for PCR was 10(-2) ng of S. mutans DNA (c. 4.6 x 10(3) copies) in the test samples. The results of this study suggest that the Sm479F/R primer pair is highly specific and sensitive for identification of S. mutans in either purified or mixed DNA samples.     

Comparison of two chromogenic media and evaluation of two molecular based identification systems for Enterobacter sakazakii detection

Background  

Enterobacter sakazakii is a foodborne pathogen that has been associated with sporadic cases and outbreaks causing meningitis, necrotizing enterocolitis and sepsis especially in neonates. The current FDA detection method includes two enrichment steps, the subculturing of the second enrichment broth on a selective agar (VRBG), a further subculturing of selected grown colonies on TSA and the subsequent biochemical identification of yellow-pigmented colonies by API20E. However, there is a strong need for simplified methods for isolation and identification of E. sakazakii. In this study, two chromogenic media, which allow to indicate presumptive E. sakazakii colonies by the alpha glucosidase activity, as well as a newly developed 1,6-alpha-glucosidase based conventional PCR assay and a rRNA oligonucleotide probe based commercial test system for identification of presumptive E. sakazakii were evaluated on 98 target and non-target strains. The methods were compared with respect to specifiCity aspects.     

食品中阪崎肠杆菌快速检测鉴定方法的研究

通过设计特异性的引物,采用SYBR Green I实时荧光PCR,经引物的优化筛选、特异性和重现性试验,以及模拟污染样品检验,建立了食品中阪崎肠杆菌的快速检测和鉴定方法。     

Comprehensive approaches to molecular biomarker discovery for detection and identification of Cronobacter spp. (Enterobacter sakazakii) and Salmonella spp

Cronobacter spp. (formerly Enterobacter sakazakii) and Salmonella spp. are increasingly implicated internationally as important microbiological contaminants in low-moisture food products, including powdered infant formula. Estimates indicate that 40 to 80% of infants infected with Cronobacter sakazakii and/or Salmonella in the United States may not survive the illness. A systematic approach, combining literature-based data mining, comparative genome analysis, and the direct sequencing of PCR products of specific biomarker genes, was used to construct an initial collection of genes to be targeted. These targeted genes, particularly genes encoding virulence factors and genes responsible for unique phenotypes, have the potential to function as biomarker genes for the identification and differentiation of Cronobacter spp. and Salmonella from other food-borne pathogens in low-moisture food products. In this paper, a total of 58 unique Salmonella gene clusters and 126 unique potential Cronobacter biomarkers and putative virulence factors were identified. A chitinase gene, a well-studied virulence factor in fungi, plants, and bacteria, was used to confirm this approach. We found that the chitinase gene has very low sequence variability and/or polymorphism among Cronobacter, Citrobacter, and Salmonella, while differing significantly in other food-borne pathogens, either by sequence blasting or experimental testing, including PCR amplification and direct sequencing. This computational analysis for Cronobacter and Salmonella biomarker identification and the preliminary laboratory studies are only a starting point; thus, PCR and array-based biomarker verification studies of these and other food-borne pathogens are currently being conducted.