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.     

Amperometric DNA sensor using the pyrroquinoline quinone glucose dehydrogenase-avidin conjugate

A new amperometric DNA sensor was constructed using a pyrroquinoline quinone glucose dehydrogenase ((PQQ)GDH) conjugated with avidin. Our aim was to specifically detect the DNA sequence of the invA virulence gene from the pathogenic bacterium Salmonella. Probe DNA with a sequence complementary to that of a specific fragment of the invA gene was immobilized onto a carbon paste electrode. After hybridization with biotinylated target DNA, (PQQ)GDH-avidin conjugate was added and the resulting electric current was measured. The electric current is generated from glucose oxidization catalyzed by (PQQ)GDH via 1-methoxyphenazine methosulphate (m-PMS) electron mediator. The sensor response increased with the addition of glucose and in the presence of 6.3 mM glucose the response increased with increasing DNA in the range 5.0x10(-8)-1.0x10(-5) M.     

Quantification of mRNA in Salmonella sp. seeded soil and chicken manure using magnetic capture hybridization RT-PCR

Direct quantification of mRNA from Salmonella sp. seeded for 1 h to soil and chicken manure was accomplished using magnetic capture hybridization as a purification technique. This detection strategy targeted the invA gene present in Salmonella sp. After cell lysis, phenol/chloroform purification and isopropanol precipitation, the RNA extract was combined with the hybridization probe conjugated to paramagnetic beads. After hybridization, the captured nucleic acids were released by denaturation and purified of contaminating DNA using DNase. The resulting RNA was of high purity and there was no need for dilution of the samples prior to RT-PCR. The developed procedure was reproducibly used to quantify Salmonella sp. in high organic agricultural soil. The detection limit for mRNA using ordinary quantitative PCR (employing SYBRgreen-based detection) was 5 x 10(4)Salmonella sp. cells per gram of soil. Chicken manure amended into soil (1:4 w/w) did not reduce the ability to quantify Salmonella sp. mRNA in soil. Pasteurization (65 degrees C, 30 min) of chicken manure containing Salmonella sp. dramatically reduced the detection of invA mRNA (requiring 42 qPCR cycles for detection versus 26 cycles in unpasteurized manure), presumably due to degradation of the invA mRNA in Salmonella sp. cells killed by pasteurization. By contrast, DNA-based qPCR still detected Salmonella sp. in the pasteurized manure. Thus, in this case using samples seeded with fresh Salmonella sp. the mRNA-based detection appears to be superior to minimizing false-positive detection which was prevalent with DNA-based qPCR.     

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.     

Electrochemical genosensor for specific detection of the food-borne pathogen, <Emphasis Type="Italic">Vibrio cholerae</Emphasis>

A disposable horseradish peroxidase (HRP)-based electrochemical genosensor was developed for chronoamperometric detection of single-stranded asymmetric lolB gene PCR amplicon (118 bp in length) of the food-borne pathogen, Vibrio cholerae. A two-step sandwich-type hybridization strategy using two specific probes was employed for specific detection of the target single-stranded DNA (ssDNA). The analytical performances of the detection platform have been evaluated using a synthetic ssDNA (ST3) which was identical to the target single-stranded amplicon and a total of 19 bacterial strains. Under optimal condition, ST3 was calibrated with a dynamic range of 0.4883–15.6250 nM. By coupling asymmetric PCR amplification, the probe-based electrochemical genosensor was highly specific to the target organism (100% specificity) and able to detect as little as 0.85 ng/μl of V. cholerae genomic DNA.     

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.     

多重PCR检测无公害畜禽肉和水产品中4种致病菌

建立无公害畜禽肉和水产品中肠出血性大肠杆菌(EHEC)、沙门氏菌、副溶血性弧菌(VP)和单核细胞增生性李斯特氏菌(LM)的多重PCR检测方法,为这些致病菌的快速诊断提供实验依据。选择分别针对EHEC溶血素基因hlyAB、副溶血性弧菌属保守序列toxR基因、沙门氏菌侵袭基因invA和LM的iap基因特异的4对引物,先分别进行单重PCR扩增,再同时加入4对引物进行多重PCR扩增,扩增产物经测序验证。建立的多重PCR方法可简便、快速、灵敏地实现对EHEC、LM、沙门氏菌和VP的同时检测,在畜禽肉和水产品中的检测灵敏度达到10^3cfu/mL。     

A real-time PCR for the detection of Salmonella Enteritidis in poultry meat and consumption eggs

A robust duplex 5' nuclease (TaqMan) real-time PCR was developed and in-house validated for the specific detection of Salmonella enterica subspecies enterica serovar Enteritidis in whole chicken carcass rinses and consumption eggs. The assay uses specifically designed primers and a TaqMan probe to target the Prot6e gene located on the S. Enteritidis specific 60-kb virulence plasmid. As an internal amplification control to monitor Salmonella DNA in the sample, a second primer/TaqMan probe set detects simultaneously the Salmonella specific invA gene. The assay identified correctly 95% of the 79 Salmonella Enteritidis strains tested comprising 19 different phage types. None of the 119 non-Enteritidis strains comprising 54 serovars was positive for the Prot6e gene. The assay detection probability was for 10(2) or more genome equivalents 100% and for 10 equivalents 83%. A pre-PCR sample preparation protocol including a pre-enrichment step in buffered peptone water, followed by DNA extraction was applied on low levels of artificially contaminated whole chicken carcass rinses and eggs from hens as well as 25 potentially naturally contaminated chickens. The detection limit was less than three CFU per 50 ml carcass rinse or 10 ml egg. The sensitivity and specificity compared to the traditional culture-based detection method and serotyping were both 100%. Twenty-five potentially naturally contaminated chickens were compared by the real-time PCR and the traditional cultural isolation method resulting in four Salmonella positive samples of which two were positive for the Prot6e gene and serotyped as S. Enteritidis. We show also that Salmonella isolates which have a rough lipopolysaccharide structure could be assigned to the serovar Enteritidis by the real-time PCR. This methodology can contribute to meet the need of fast identification and detection methods for use in monitoring and control measures programmes.     

Eight-hour PCR-based procedure for the detection of Salmonella in raw oysters

The aim of this study was to evaluate the ability of a nested PCR system to detect Salmonella senftenberg in raw oysters. The specific primers of the PCR were derived from the invA gene sequence, essential for Salmonella invasiveness into epithelial cells. First, for the extraction of DNA, four methods (guanidine isothiocyanate, E.Z.N.A. Mollusc Kit, Chelex-100, and lysis with detergents) were compared. A nested PCR method combined with 3.5 h pre-enrichment in buffered peptone water (BPW) and DNA extraction by the resin Chelex-100 is proposed for the detection of S. senftenberg in oyster samples. The detection limit of the method is less than 0.1 CFU/ml (<1 CFU/g of oyster). This procedure is shown to be an excellent tool for the sensitive detection of S. senftenberg from naturally contaminated oysters, with results being obtained within 8 h.     

Detection of Salmonella enterica serovar Typhi (S. Typhi) by selective amplification of invA, viaB, fliC-d and prt genes by polymerase chain reaction in mutiplex format

AIMS: Development of a PCR assay that can target multiple genes for rapid detection of Salmonella enterica serovar Typhi (S. Typhi) from water and food samples. METHODS AND RESULTS: PCR primers for invasion, O, H and Vi antigen genes, invA, prt, fliC-d and viaB were designed and used for the rapid detection of S. Typhi by multiplex PCR. Internal amplification control, which co-amplified with prt primers, was also included in the assay. The results showed that all cultures of Salmonella were accurately identified by the assay with no nonspecific amplification in other cultures. The assay had 100% detection probability when a cell suspension of 10(4) CFU ml(-1) (500 CFU per reaction) was used. Salmonella Typhi bacteria were artificially inoculated in the water and food (milk and meat rinse) samples and detected by mPCR after overnight pre-enrichment in buffered peptone water. No Salmonella bacteria could be detected from water samples collected from the field by mPCR or standard culture method. CONCLUSIONS: The developed mPCR assay provides specific detection of S. Typhi. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid methods for detection of S. Typhi from complex environmental matrices are almost nonexistent. The mPCR assay reported in this study can be useful to identify S. Typhi bacteria in field environmental samples.     

A nanoparticle amplification based quartz crystal microbalance DNA sensor for detection of Escherichia coli O157:H7

A quartz crystal microbalance (QCM) DNA sensor, based on the nanoparticle amplification method, was developed for detection of Escherichia coli O157:H7. A thiolated single-stranded DNA (ssDNA) probe specific to E. coli O157:H7 eaeA gene was immobilized onto the QCM sensor surface through self-assembly. The hybridization was induced by exposing the ssDNA probe to the complementary target DNA, and resulted in the mass change and therefore frequency change of the QCM. Streptavidin conjugated Fe(3)O(4) nanoparticles (average diameter=145 nm) were used as "mass enhancers" to amplify the frequency change. Synthesized biotinylated oligonucleotides as well as E. coli O157:H7 eaeA gene fragments (151 bases) amplified using asymmetric PCR with biotin labeled primers were tested. As low as 10(-12)M synthesized oligonucleotides and 2.67 x 10(2) colony forming unit (CFU)/ml E. coli O157:H7 cells can be detected by the sensor. Linear correlation between frequency change and logarithmic number of bacterial cell concentration was found for E. coli O157:H7 from 2.67 x 10(2) to 2.67 x 10(6)CFU/ml.     

Electrochemical DNA biosensor for detecting cancer biomarker related to glutathione S-transferase P1 (GSTP1) hypermethylation in real samples

An electrochemical genosensor for the detection of hypermethylation of the glutathione S-transferase P1 (GSTP1) gene, a specific marker of prostate cancer, was reported. This new sensor was used in combination with a single-use carbon graphite working electrode and differential pulse voltammetry, with the results of sample analysis based on the guanine oxidation signals obtained at +1.0 V before and after hybridization between probe and synthetic target or denatured PCR samples. The detected DNA hybridization was also characterized by electrochemical impedance spectroscopy with potassium ferri/ferrocyanide as a redox probe. The protocol consisted of 2 different modes: (i) capture probes selective for methylation-specific and unmethylated GSTP1 sequences were immobilized onto the sensor directly, and hybridization was formed on the electrode surface; (ii) probe/target or probe/noncomplementary target couples were mixed in solution phase, and the transducer was modified through simple adsorption. The limit of detection (S/N=3) was calculated as 2.92 pmol of target sequence in a 100-μl reaction volume. The optimum analytical detection parameters for the biosensor, as well as its future prospects, were also presented.     

电化学方法在李斯特氏菌毒力基因检测中的应用

单核李斯特氏菌(Listeria monocytogenes, LM)是食源性李斯特氏病的病源菌, 该病可引起败血病、脑膜炎、流产等。李斯特氏菌的毒力因子listeriolysin O (LLO)是引发李斯特氏病的主要原因。文章使用一种特殊的电化学方法从样品中检测编码LLO的hlyA基因。该方法以化合物Nhydroxysulfosuccinimide (NHS) 和 N-(3-dimethylamion) propyl- N'-ethyl carbodiimidehydrochloride (EDC) 作为激活剂, 使单链DNA探针结合到金电极表面组成工作电极, 以[Co(phen)₃](ClO₄)₃ 作为指示剂来检测循环伏安曲线(Cyclic voltammetry , CV), 通过CV峰值的变化来估算hlyA基因的含量, 从而确定LM的污染情况。这种新颖的电化学方法用于免标记的目标DNA的杂交检测, 具有快速和方便的特点。     

Giant magnetoresistive biochip for DNA detection and HPV genotyping

A giant magnetoresistive (GMR) biochip based on spin valve sensor array and magnetic nanoparticle labels was developed for inexpensive, sensitive and reliable DNA detection. The DNA targets detected in this experiment were PCR products amplified from Human Papillomavirus (HPV) plasmids. The concentrations of the target DNA after PCR were around 10nM in most cases, but concentrations of 10pM were also detectable, which is demonstrated by experiments with synthetic DNA samples. A mild but highly specific surface chemistry was used for probe oligonucleotide immobilization. Double modulation technique was used for signal detection in order to reduce the 1/f noise in the sensor. Twelve assays were performed with an accuracy of approximately 90%. Magnetic signals were consistent with particle coverage data measured with Scanning Electron Microscopy (SEM). More recent research on microfluidics showed the potential of reducing the assay time below one hour. This is the first demonstration of magnetic DNA detection using plasmid-derived samples. This study provides a direct proof that GMR sensors can be used for biomedical applications.     

Salmonella DNA persistence in natural seawaters using PCR analysis

The risks of false-positive responses were examined when using the polymerase chain reaction (PCR) method for the detection of Salmonella in the marine environment (water and shellfish). The degradation rates of DNA, both free and from dead Salmonella , were evaluated in natural seawaters maintained at 10° and 20°C, using PCR with Vir and invA primers. The DNA of dead Salmonella was detected up to 55 d in seawater collected in winter and stored at 10°C. But in summer, the persistence was shorter: 10 d or even 2 d for a smaller inoculum (3 × 10³ Salmonella ml⁻¹). The role of the planktonic organisms present in spring and summer was pinpointed. For free DNA, the persistence times were shorter: from 2 to 4 d at 20°C, and from 3 to 8 d at 10°C showing that the nuclease activity of marine organisms is higher at warm temperatures. These data led us to recommend careful interpretations of direct PCR results, especially during cold periods and for samples collected close to terrestrial discharges of high concentrations of live, dead or lysed Salmonella . PCR is a rapid, specific and sensitive method, but should be applied with care to marine samples, in order to avoid false-positive responses.     

Dramatic increase in the signal and sensitivity of detection <Emphasis Type="Italic">via</Emphasis> self-assembly of branched DNA

In molecular testing using PCR, the target DNA is amplified via PCR and the sequence of interest is investigated via hybridization with short oligonucleotide capture probes that are either in a solution or immobilized on solid supports such as beads or glass slides. In this report, we report the discovery of assembly of DNA complex(es) between a capture probe and multiple strands of the PCR product. The DNA complex most likely has branched structure. The assembly of branched DNA was facilitated by the product of asymmetric PCR. The amount of branched DNA assembled was increased five fold when the asymmetric PCR product was denatured and hybridized with a capture probe all in the same PCR reaction mixture. The major branched DNA species appeared to contain three reverse strands (the strand complementary to the capture probe) and two forward strands. The DNA was sensitive to S1 nuclease suggesting that it had single-stranded gaps. Branched DNA also appeared to be assembled with the capture probes immobilized on the surface of solid support when the product of asymmetric PCR was hybridized. Assembly of the branched DNA was also increased when hybridization was performed in complete PCR reaction mixture suggesting the requirement of DNA synthesis. Integration of asymmetric PCR, heat denaturation and hybridization in the same PCR reaction mixture with the capture probes immobilized on the surface of solid support achieved dramatic increase in the signal and sensitivity of detection of DNA. Such a system should be advantageously applied for development of automated process for detection of DNA.     

Rapid detection of Campylobacter coli,C. jejuni,and Salmonella enterica on poultry carcasses by using PCR-enzyme-linked immunosorbent assay

Contamination of retail poultry by Campylobacter spp. and Salmonella enterica is a significant source of human diarrheal disease. Isolation and identification of these microorganisms require a series of biochemical and serological tests. In this study, Campylobacter ceuE and Salmonella invA genes were used to design probes in PCR-enzyme-linked immunosorbent assay (ELISA), as an alternative to conventional bacteriological methodology, for the rapid detection of Campylobacter jejuni, Campylobacter coli, and S. enterica from poultry samples. With PCR-ELISA (40 cycles), the detection limits for Salmonella and Campylobacter were 2 x 10(2) and 4 x 10(1) CFU/ml, respectively. ELISA increased the sensitivity of the conventional PCR method by 100- to 1,000-fold. DNA was extracted from carcass rinses and tetrathionate enrichments and used in PCR-ELISA for the detection of Campylobacter and S. enterica, respectively. With PCR-ELISA, Salmonella was detected in 20 of 120 (17%) chicken carcass rinses examined, without the inclusion of an enrichment step. Significant correlation was observed between PCR-ELISA and cultural methods (kappa = 0.83; chi-square test, P < 0.001) with only one false negative (1.67%) and four false positives (6.67%) when PCR-ELISA was used to screen 60 tetrathionate enrichment cultures for SALMONELLA: With PCR-ELISA, we observed a positive correlation between the ELISA absorbance (optical density at 405 nm) and the campylobacter cell number in carcass rinse, as determined by standard culture methods. Overall, PCR-ELISA is a rapid and cost-effective approach for the detection and enumeration of Salmonella and Campylobacter bacteria on poultry.     

重组酶介导等温核酸扩增方法快速检测土壤沙门氏菌

【背景】沙门氏菌(Salmonella)是一种可以引起人畜患病的致病菌,也是最主要的食源性细菌之一。土壤中的沙门氏菌可通过蔬菜等植物进入人体,引发食物中毒。但由于土壤性质及其他微生物的干扰,如何快速甄别土壤是否受到沙门氏菌的污染仍是一个难题。【目的】建立一种快速、灵敏检测土壤沙门氏菌的实时重组酶介导等温核酸扩增(Real-Time Recombinase Aided Amplification,RT-RAA)方法。【方法】针对沙门氏菌invA基因序列设计特异性引物和探针,构建含有invA基因待检片段的重组质粒,评价RT-RAA方法的灵敏度;分别以肠炎沙门氏菌、大肠杆菌、福氏志贺氏菌和金黄色葡萄球菌的基因组DNA为模板,评价RT-RAA方法的特异性;RT-RAA方法用于番茄、生姜土壤中沙门氏菌的检测,同时用平板培养法进行验证。【结果】RT-RAA方法可用于重组质粒中invA基因片段的检测,在39℃条件下,20 min内即可获得检测结果,最低检测质粒拷贝数为10拷贝/反应,而且与大肠杆菌、福氏志贺氏菌和金黄色葡萄球菌无交叉反应。土壤样品DNA的RT-RAA检测结果显示,供试番茄土已被沙门氏菌污染,而生姜土则没有,与平板培养结果一致。【结论】RT-RAA方法具有灵敏度高和特异性强的特点,可用于土壤沙门氏菌污染的快速检测。     

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.     

Detection and modelling of DNA hybridization by EIS measurements. Mention of a polythiophene matrix suitable for electrochemically controlled gene delivery

A conducting polymer sensor for direct label-free DNA detection based on a polythiophene bearing an electroactive linker group is investigated. DNA hybridization is studied by electrochemical impedance spectroscopy (EIS) and quartz crystal microbalance (QCM) techniques. Modelling of DNA hybridization by EIS measurements exhibits the contribution of nucleic acid to a superficial p-doping process. A 675-mer single-stranded DNA is produced using asymmetric PCR from a DNA sequence of a transposable element mariner and hybridized to the previously immobilized probe. Electrochemical stimulus leads to the release "on demand" of DNA fragments and the amount delivery permits to do PCR amplification.