Development and evaluation of multiplex PCR for detection of T-2 and zearalenone producing Fusarium spp.

The study aimed to develop and evaluate a multiplex polymerase chain reaction assay (mPCR) for the concurrent detection of three major mycotoxin metabolic pathway genes, namely tri8 (T-2 toxin), tri6 (trichothecene) and pks4 (zearalenone), along with competitive internal amplification control. Specific primers for each of the aforementioned genes were optimized and validated using 14 reference strains and 10 pure culture isolates. The optimized mPCR assay detected the three metabolic pathway genes in artificially contaminated maize samples with a sensitivity of 2 × 103  CFU per g for tri6 and pks4 positive Fusarium strains, whereas 2 × 104  CFU per g for tri8 positive Fusarium strains. Application of the developed mPCR assay to 30 cereal and 20 feed samples revealed 24% (12 of 50) contamination with either one or more mycotoxins. The results of mPCR assay were further evaluated with high performance liquid chromatography (HPLC), and both methods provided unequivocal results. This mPCR assay might be a supplementary tool to conventional mycotoxin analytical techniques like thin-layer chromatography, HPLC, etc. The current mPCR assay is a rapid and reliable tool for simultaneous, sensitive and specific detection of T-2, zearalenone and trichothecene producing Fusarium spp. from naturally contaminated foods and to monitor them during the processing steps of food and feed commodities.

A novel multiplex PCR for the simultaneous detection of Salmonella enterica and Shigella species.

Salmonella enterica and Shigella species are commonly associated with food and water borne infections leading to gastrointestinal diseases. The present work was undertaken to develop a sensitive and reliable PCR based detection system for simultaneous detection of Salmonella enterica and Shigella at species level. For this the conserved regions of specific genes namely ipaH1, ipaH, wbgZ, wzy and invA were targeted for detection of Shigella genus, S. flexneri, S. sonnei, S. boydii and Salmonella enterica respectively along with an internal amplification control (IAC). The results showed that twenty Salmonella and eleven Shigella spp., were accurately identified by the assay without showing non-specificity against closely related other Enterobacteriaceae organisms and also against other pathogens. Further evaluation of multiplex PCR was undertaken on 50 natural samples of chicken, eggs and poultry litter and results compared with conventional culture isolation and identification procedure. The multiplex PCR identified the presence of Salmonella and Shigella strains with a short pre-enrichment step of 5 h in peptone water and the same samples were processed by conventional procedures for comparison. Therefore, this reported multiplex PCR can serve as an alternative to the tedious time-consuming procedure of culture and identification in food safety laboratories.

Development and validation of an immunochromatographic assay for rapid detection of fumonisin B1 from cereal samples.

Fumonisins are one of the most agriculturally significant environmental toxins produced by Fusarium and Aspergillus species that grow on agricultural commodities in the field or during storage. Cereals contaminated with fumonisins causes serious loss to agricultural produce leads to health problems in humans and other farm animals. In the present study, polyclonal hyperimmune sera was raised against FB1 in rabbits immunized with FB1-keyhole limpet haemocyanin (KLH). Purified antibodies were used to establish a sensitive gold nanoparticle based immunochromatographic strip (ICG) for detecting FB1 levels in cereal grains. Effective on-site detection of FB1 was achieved by developing a rapid and sensitive pAb based ICG strip. This strip had a detection limit of 5 ng mL(-1) for FB1 in cereal samples and it could be completed within 3 min. Close examination of 150 cereal samples by ICG strip method revealed that 77 were fumonisin-positive. Results obtained by the developed method was further validated with well standardized HPLC method and results of strip method was correlated well with those obtained by HPLC method. In conclusion, the developed method was a better alternative for onsite detection of FB1 in cereal samples intended for human consumption to reduce risk of humans and other farm animals. The high level of FB1 concentrations recorded in present study warrants the need to develop an awareness creation programme to the farmers of India for safe handling of cereal grains at the time of harvesting and storage of grains.

Application of r-PFE hyperimmune sera for concurrent detection of Bacillus anthracis, Yersinia pestis and staphylococcal enterotoxin B.

AIM: To evaluate the potential of an intergeneric multidomain recombinant chimeric protein for the simultaneous detection of Bacillus anthracis, Yersinia pestis and Staphylococcal enterotoxin B. METHODS AND RESULTS: Truncated portions of protective antigen (pag) of B. anthracis, fraction 1 capsular antigen (F1) of Y. pestis and enterotoxin B (entB) of Staphylococcus aureus were PCR amplified and linked each other using ligation-dependent cloning. The fusion gene was codon-optimized for expression in Escherichia coli and encoded a 55 kDa recombinant PFE protein (rPFE). Hyperimmune antiserum raised against rPFE specifically reacted individually with the native PA of B. anthracis, F1 antigen of Y. pestis and SEB of S. aureus on Western blot analysis as well as in enzyme-linked immunosorbent assay (ELISA). For simultaneous detection of these three antigens from culture supernatants, common media consisting of BHI broth supplemented with 0·2% xylose were used. To assess the detection capability, a known number of these organisms (10(8) -10(2) CFU ml(-1)) were experimentally spiked on to the meat and blood samples, the polyclonal antibodies were again clearly able to identify all three target proteins up to a dilution of 10(5) CFU ml(-1). CONCLUSIONS: This recombinant chimeric protein-based immunodetection approach may eventually provide advantages over PCR formats during onsite investigations of biological emergencies or even during routine testing by laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: The trivalent recombinant PFE protein could be a novel intervention for possible diagnosis/detection of potential biological agents simultaneously in environmental and clinical samples to reduce the responding time and minimize the impact of the bioattack.

A novel multiplex PCR for the simultaneous detection of Salmonella enterica and Shigella species

Salmonella enterica and Shigella species are commonly associated with food and water borne infections leading to gastrointestinal diseases. The present work was undertaken to develop a sensitive and reliable PCR based detection system for simultaneous detection of Salmonella enterica and Shigella at species level. For this the conserved regions of specific genes namely ipaH1, ipaH, wbgZ, wzy and invA were targeted for detection of Shigella genus, S. flexneri, S. sonnei, S. boydii and Salmonella enterica respectively along with an internal amplification control (IAC). The results showed that twenty Salmonella and eleven Shigella spp., were accurately identified by the assay without showing non-specificity against closely related other Enterobacteriaceae organisms and also against other pathogens. Further evaluation of multiplex PCR was undertaken on 50 natural samples of chicken, eggs and poultry litter and results compared with conventional culture isolation and identification procedure. The multiplex PCR identified the presence of Salmonella and Shigella strains with a short pre-enrichment step of 5 h in peptone water and the same samples were processed by conventional procedures for comparison. Therefore, this reported multiplex PCR can serve as an alternative to the tedious time-consuming procedure of culture and identification in food safety laboratories.

Zearalenone induced toxicity in SHSY-5Y cells: The role of oxidative stress evidenced by N-acetyl cysteine.

Zearalenone (ZEN) is a mycotoxin from Fusarium species commonly found in many food commodities and are known to cause reproductive disorders, genotoxic and immunosuppressive effects. Although many studies have demonstrated the cytotoxic effects of ZEN, the mechanisms by which ZEN mediates its cytotoxic effects appear to differ according to cell type and route of exposure. Meantime, the available information on the neurotoxic effects of ZEN is very much limited. In the present study we evaluated the role of oxidative stress in ZEN mediated neurotoxicity in SH-SY5Y cells and investigated the possible underlying mechanism. ZEN induced ROS formation and elevated levels of MDA, loss of mitochondrial membrane potential (MMP) and increase in DNA damage in a dose dependent manner as assessed by COMET assay and agarose gel electrophoresis. However, there was no DNA damage by plasmid breakage assay at 6, 12 and 24h time points. DAPI staining showed apoptotic nuclei at 12 and 24h. Further, ZEN treated SH-SY5Y cells showed a marked suppressive effect on the neuronal gene expression. Use of an antioxidant N-acetylcysteine (NAC) reversed the toxin-induced generation of ROS and also attenuated loss of MMP. Collectively, these results suggest that ROS is the main upstream signal leading to increased ZEN mediated neurotoxicity in SH-SY5Y cells.

Development of ISSR-derived SCAR marker-targeted PCR for identification of Aspergillus section Flavi members.

UNLABELLED: Aspergillus section Flavi is a heterogeneous fungal cluster including some of the most economically important Aspergillus species. The section is comprised of toxigenic and nontoxigenic aspergilli that are phenotypically undistinguishable. The aim of this study was to develop a genetic marker specific to Aspergillus section Flavi on the whole. Based on inter-simple sequence repeat (ISSR) fingerprinting profiles of major Aspergillus section Flavi members, a sequence-characterized amplified region (SCAR) marker was identified. Primers were designed in the conserved regions of the SCAR marker and were utilized in a PCR for concurrent identification of major members of the section. The detection level of the SCAR-PCR was found to be 0·1 ng purified DNA, and when applied to 45 naturally contaminated food samples, 28 samples were found infected with Aspergillus section Flavi members. The present SCAR-PCR is rapid and less cumbersome unlike conventional identification techniques. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of Aspergillus section Flavi members is important owing to their impact on human health and economy. The ISSR-based SCAR-PCR developed in this study is superior over the other existing Aspergillus section Flavi detection systems due to its simplicity and minimal requirement of sample handling. This PCR could be a supplementary strategy to time-consuming and rather ambiguous conventional polyphasic detection techniques and a reliable tool for high-throughput sample analysis.

Incidence and multiplex PCR based detection of trichothecene chemotypes of Fusarium culmorum isolates collected from freshly harvested Maize kernels in Southern India.

Hundred Fusarium culmorum strains, isolated from freshly harvested maize grain samples from Southern parts of India, were incubated in czapek-dox medium and analyzed for trichothecene (DON/NIV) production. The mPCR assay was standardized targeting trichothecene metabolic pathway genes viz., Tri6, Tri7, Tri13 for detection of trichothecene (DON/NIV) chemotypes and rDNA gene for specific detection of F. culmorum species. Primers for targeted genes were designed and used to predict whether these isolates could produce deoxynivalenol/nivalenol, 94 isolates were able to produce DON/NIV by mPCR assay. Chemical analysis of DON/NIV was carried out for mPCR positive isolates by high performance-thin layer chromatography (HPTLC). To check the practical usefulness of developed mPCR assay, 150 field samples of maize were evaluated and results were compared with conventional HPTLC method. Out of 150 samples, 34% samples stayed as a positive for NIV contamination whereas 44% were found to have deoxynivalenol contamination. Moreover, mPCR results are equivocally matched with the HPTLC chemical analysis for field samples. Chemotyping of F. culmorum isolates were reported for the first time from India, and highlights the important potential of F. culmorum to contaminate maize with DON/NIV.

Evaluation of a multiplex PCR assay for concurrent detection of four major mycotoxigenic fungi from foods.

AIM: To develop and evaluate a multiplex polymerase chain reaction assay (mPCR) for the concurrent detection of four major mycotoxin metabolic pathway genes, viz. nor1 (aflatoxin), Tri6 (trichothecene), FUM13 (fumonisin) and otanps (ochratoxin A). METHODS AND RESULTS: A mPCR assay with competitive internal amplification control, employing specific primers for each of the aforementioned four genes, was optimized and validated using 10 reference strains and 60 pure culture isolates. The standardized mPCR assay detected all four mycotoxin metabolic genes in artificially contaminated maize samples with a sensitivity of 2 × 10(3) CFU g(-1) for nor1-positive Aspergillus strains, Tri6 and FUM13-positive Fusarium strains and 2 × 10(4) CFU g(-1) for otanps-positive Penicillium strains. When the developed mPCR assay was applied to 40 natural foods, 35% (14 of 40) of the samples were contaminated with either one or more mycotoxins. The mPCR results were further evaluated with high-performance liquid chromatography (HPLC), and in general, both the methods provided unequivocal results. CONCLUSION: The current mPCR assay is a rapid and reliable tool for simultaneous specific and sensitive detection of aflatoxigenic Aspergillus strains, trichothecene- and fumonisin-producing Fusarium strains, and ochratoxigenic Penicillium species from naturally contaminated foods. SIGNIFICANCE AND IMPACT OF THE STUDY: This mPCR assay could be a supplementary strategy to current conventional mycotoxin analytical techniques such as thin-layer chromatography (TLC), high performance thin layer chromatography, HPLC, etc., and a reliable tool for high-throughput monitoring of major mycotoxin-producing fungi during the processing steps of food and feed commodities.

Incidence and multiplex PCR based detection of trichothecene chemotypes of Fusarium culmorum isolates collected from freshly harvested Maize kernels in Southern India

Hundred Fusarium culmorum strains, isolated from freshly harvested maize grain samples from Southern parts of India, were incubated in czapek-dox medium and analyzed for trichothecene (DON/NIV) production. The mPCR assay was standardized targeting trichothecene metabolic pathway genes viz., Tri6, Tri7, Tri13 for detection of trichothecene (DON/NIV) chemotypes and rDNA gene for specific detection of F. culmorum species. Primers for targeted genes were designed and used to predict whether these isolates could produce deoxynivalenol/nivalenol, 94 isolates were able to produce DON/NIV by mPCR assay. Chemical analysis of DON/NIV was carried out for mPCR positive isolates by high performance-thin layer chromatography (HPTLC). To check the practical usefulness of developed mPCR assay, 150 field samples of maize were evaluated and results were compared with conventional HPTLC method. Out of 150 samples, 34% samples stayed as a positive for NIV contamination whereas 44% were found to have deoxynivalenol contamination. Moreover, mPCR results are equivocally matched with the HPTLC chemical analysis for field samples. Chemotyping of F. culmorum isolates were reported for the first time from India, and highlights the important potential of F. culmorum to contaminate maize with DON/NIV.

Specific identification of pathogenic Yersinia enterocolitica by monoclonal antibodies generated against recombinant attachment invasion locus (rAil) protein.

Classical pathogenic strains of Yersinia enterocolitica produce a 17 kDa outer membrane protein, Ail (attachment-invasion locus), which mediates bacterial attachment to some cultures epithelial cell lines and invasion of others. In the present study, hybridomas were developed for the production of monoclonal antibodies (MAbs) against Ail protein of Y. enterocolitica. A set of five stabilized hybridoma cell lines were generated, of which, two MAbs, YEA 302 and YEA 303, exhibited specific reaction to the native Ail protein (17 kDa) present in whole cell lysate of Y. enterocolitica strains beside having reaction with rAil. The other three MAbs, YEA 5, 17 and 32, had some cross reactions with proteins other than Ail. Two out of five MAbs were IgG1, two were IgG2b and one in IgM in nature. MAbs (YEA 302 and YEA 303) did not show any cross-reaction with whole cell lysate of Brucella abortus, Vibrio cholerae, Salmonella typhimurium and Escherichia coli and other species of Enterobacteriaceae including Y. pestis in ELISA and Western blot analysis. The presence of Ail protein among the strains recovered from pork and milk samples was evaluated by these sets of MAbs and the results were compared with the duplex PCR. Collectively, the data suggest that these MAbs may have the potential for their use in the detection of pathogenic Y. enterocolitica reliably, rapidly and at a relatively low cost.

Generation and characterization of an inter-generic bivalent alpha domain fusion protein αCS from Clostridium perfringens and Staphylococcus aureus for concurrent diagnosis and therapeutic applications.

AIM: To evaluate an inter-generic recombinant alpha domain fusion protein for simultaneous detection and neutralization of Clostridium perfringens and Staphylococcus aureus alpha toxins. METHODS AND RESULTS: Truncated portions of clostridial and staphylococcal alpha haemolysin genes were PCR amplified and linked to each other through a hydrophilic flexible Glycine linker sequence using overlap-extension PCR to form a chimeric gene αCS. The recombinant αCS fusion protein was expressed and characterized for its toxicity, cell binding capacity and haemolysis inhibition properties. The fusion protein was nontoxic and effectively retarded staphylococcal alpha haemolysis, probably by competitively interacting with putative staphylococcal alpha haemolysin receptors on erythrocytes. Murine hyperimmune polysera raised against r-αCS specifically detected 42-kDa and 33-kDa proteins when culture supernatants of Cl. perfringens (clostridial alpha toxin) and Staph. aureus (staphylococcal alpha toxin), respectively, were analysed in Western blot. The polyclonal antisera effectively diminished the haemolytic action of both the wild-type toxins in vitro. CONCLUSIONS: The r-αCS fusion protein was nontoxic competitive inhibitor of staphylococcal alpha haemolysin. The protein elicited specific immune response against Cl. perfringens and Staph. aureus alpha toxins. The antisera also neutralized the toxicities of both the native wild-type toxins in vitro. SIGNIFICANCE OF THE STUDY: The bivalent recombinant αCS protein could be a novel intervention in the field of diagnostics and therapeutics against Cl. perfringens and Staph. aureus infections, particularly, in case of co-infections like gangrenous ischaemia, gangrenous mastitis, etc.

Application of a Chimeric Protein Construct having Enterotoxin B and Toxic Shock Syndrome Toxin Domains of S. aureus in Immunodiagnostics.

Staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 are the super antigens responsible for diseases such as staphylococcal food poisoning and toxic shock syndrome. At low serum concentrations, SEB can trigger toxic shock, profound hypotension and multi organ failure and hence is recognized as biowarfare molecule. In this study, a multidomain fusion protein (r-TE) was generated with specificity for SEB and toxic shock syndrome toxin (Tsst-1). The fusion gene comprising the conserved regions of seb and the tsst genes was codon-optimized for expression in Escherichia coli and encoded a 26 kDa recombinant multidomain chimeric protein (r-TE). Hyperimmune antiserum raised against r-TE specifically reacted with SEB (~28 kDa) and Tsst-1 (~22 kDa) components during Western blot analysis and by plate ELISA in confirmed toxin producing strains of S. aureus. The antigenicity of the SEB component of the r-TE protein was also confirmed using TECRA kit. The described procedure of creating a single protein molecule carrying components of two different toxins whilst still retaining the original antigenic determinants of individual toxins proved highly advantageous in the development of rapid, reliable and cost effective immunoassays and may also have the potential to serve as candidate molecule for vaccine studies.

Detection of toxigenic strains of Aeromonas species in foods by a multiplex PCR assay.

Aeromonas hydrophila and other aeromonads are ubiquitous organisms found in meat, vegetables, drinking water and various other food items. They cause diarrhea and extra-intestinal infections in normal and immunocompromised patients. The aim of the study was to develop a multiplex PCR assay for the detection of virulence-associated genes of Aeromonas including hemolysin (hlyA), aerolysin (aerA), glycerophospholipid-cholesterol acyl transferase (GCAT) alongwith a 16S rRNA gene. Internal amplification control (IAC), which was coamplified with aerA primers, was also included in this study. The results showed that all cultures of Aeromonas were accurately identified by the assay without showing non-specificity. A. hydrophila could be detected at a range of 10-50 CFU ml(-1) from experimentally spiked fish, chicken and milk samples following overnight enrichment in alkaline peptone water supplemented with 10 µg/ml ampicillin (APW-A) by this multiplex PCR (mPCR). When evaluated on a total of 74 naturally occurring food samples, four samples were identified to contain Aeromonas by mPCR. All these results were compared to the conventional culture, isolation and biochemical identification procedures. The high throughput and cost-effective mPCR method developed in this study could provide a powerful tool for detection of pathogenic Aeromonas spp. from food and environmental samples and in addition, the method has advantages in terms of specificity, sensitivity and ease of use compared to other reported PCR methods and DNA hybridization assays.

Multiplex PCR assay for the detection of enterotoxic Bacillus cereus group strains and its application in food matrices.

Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis are the major concerns for the food safety in terms of frequency and/or seriousness of the disease. Being members of the same group and sharing DNA homology to a larger extent, they do create problems when their specific detection/identification is attempted from different food and environmental sources. Numerous individual polymerase chain reaction (PCR) and few multiplex PCR (mPCR) methods have been employed to detect these organisms by targeting toxin genes but with lack of internal amplification control (IAC). Therefore, we attempted a mPCR with IAC for the detection of enterotoxic B. cereus group strains by selecting hbl A, nhe A and cyt K genes from B. cereus, indicative of the diarrheal potential and cry I A and pag genes, the plasmid borne phenotypic markers specific to B. thuringiensis and B. anthracis strains, respectively. Multiplex PCR assay validation was performed by simultaneous comparison with the results of single-target PCR assays and correlated to the classical conventional and biochemical identification of the organisms. The mPCR was able to detect as low as 10(1)-10(2) organisms per ml following overnight enrichment of spiked food samples (vegetable biriyani and milk) in buffered peptone water (BPW). The presence of these organisms could also be detected by mPCR in naturally contaminated samples of rice based dishes and milk. The high throughput and cost-effective mPCR method described could provide a powerful tool for simultaneous, rapid and reliable detection of enterotoxic B. cereus group organisms.

Molecular characterization of lactic acid bacteria recovered from natural fermentation of beet root and carrot Kanji.

The lactic acid bacteria (LAB) play an important role in the fermentation of vegetables to improve nutritive value, palatability, acceptability, microbial quality and shelf life of the fermented produce. The LAB associated with beetroot and carrot fermentation were identified and characterized using different molecular tools. Amplified ribosomal DNA restriction analysis (ARDRA) provided similar DNA profile for the 16 LAB strains isolated from beetroot and carrot fermentation while repetitive extragenic palindromic PCR (rep-PCR) genotyping could differentiate the LAB strains into eight genotypes. Thirteen strains represented by five genotypes could be clustered in five distinct groups while three LAB strains exhibiting distinct genotypes remained ungrouped. These genotypes could be identified to be belonging to L. plantarum group by 16S rDNA sequencing. The recAnested multiplex PCR employing species-specific primers for the L. plantarum group members identified the LAB strains of six genotypes to be L. paraplantarum and the other two genotypes to be L. pentosus. Three genotypes of L. paraplantarum were consistently found on the third and sixth day of beetroot fermentation whereas a distinct genotype of L. paraplantarum and L. pentosus appeared predominant on the tenth day. From carrot Kanji two distinct genotypes of L. paraplantarum and one genotype of L. pentosus were identified. REP-PCR DNA fingerprinting coupled with 16S rDNA sequencing and recA-nested multiplex PCR could clearly identify as well as differentiate the diverse L. plantarum group strains involved in the fermentation.

Cloning, expression and characterization of attachment-invasion locus protein (Ail) of Yersinia enterocolitica and its utilization in rapid detection by immunoassays.

AIMS: Rapid detection of pathogenic Yersinia enterocolitica isolates by using antisera raised against recombinant attachment-invasion locus (Ail) protein. METHODS AND RESULTS: The complete gene (471 bp) encoding for the Ail protein was amplified by PCR and cloned in pQE 30 UA vector. The recombinant clones were selected by polymerase chain reaction (PCR). Recombinant protein was expressed using induction with 1 mmol l(-1) final concentration of isopropylthiogalactoside (IPTG). Polyclonal antibodies were raised in mice against this purified recombinant protein. An indirect plate ELISA was standardized based on rAil protein for the detection of Y. enterocolitica. Western blot analysis with the sera raised against recombinant Ail protein exhibited reaction at 17 kDa region of the native Ail protein present in pathogenic Y. enterocolitica standard strains and strains isolated from pork samples suggesting that the antigenicity of recombinant Ail protein was similar to that of native Ail protein. Nonpathogenic Y. enterocolitica and the other species of Yersinia, namely, Y. pseudotuberculosis, Y. intermedia, Y. kristenseni, Y. fredrickseni and also the Enterobacteriaceae organisms tested were not found reacting to polyclonal antisera against this recombinant Ail protein. CONCLUSION: The antibodies raised against recombinant Ail protein could specifically identify pathogenic Y. enterocolitica strains both by indirect plate ELISA and Western blot immunoassay. SIGNIFICANCE AND IMPACT OF THE STUDY: The method developed in this study may find application in the detection of pathogenic Y. enterocolitica not only from food and environmental samples but also from clinical samples.

Construction of a recombinant intergenus multidomain chimeric protein for simultaneous expression of haemolysin BL of Bacillus cereus, listeriolysin O of Listeria monocytogenes and enterotoxin B of Staphylococcus aureus.

Haemolysin BL (HBL) of Bacillus cereus, listeriolysin O (LLO) of Listeria monocytogenes and enterotoxin B (SEB) of Staphylococcus aureus are among the major toxin components contributing to the pathogenicity of these organisms in foodborne illnesses. In this study, an intergenus non-toxic multidomain fusion protein (r-HLE) was generated with specificity for HBL, LLO and SEB. The fusion gene (r-hle) comprising the conserved regions of hblD and the hly and entB genes was codon-optimized for expression in Escherichia coli and encoded a 50 kDa recombinant multidomain chimeric protein (r-HLE). Hyperimmune antiserum raised against r-HLE specifically reacted with the L1 (38 kDa) component of the HBL complex of B. cereus, LLO (58 kDa) of L. monocytogenes and SEB (28 kDa) of S. aureus during Western blot analysis when tested on standard strains. During testing on isolates, the antiserum again identified the appropriate toxin molecules and was highly specific to the relevant bacterial species. The antigenicity of the SEB component of the r-HLE protein was also confirmed using a commercially available TECRA kit. The described procedure of creating a single antigenic molecule carrying components of three different toxins whilst still retaining the original antigenic determinants of individual toxins will be highly advantageous in the development of rapid, reliable and cost-effective immunoassays.

Simultaneous detection of pathogenic B. cereus, S. aureus and L. monocytogenes by multiplex PCR.

Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus are of major concern for food safety in terms of frequency and seriousness of the disease. The occurrence these three important pathogens and their coexistence in food matrices are predominant. Moreover, symptoms associated with B. cereus and S. aureus food poisoning not only closely resembles each other but can also be overlapping with other foodborne infections. In this context, detection of these three pathogens simultaneously in food samples by a single multiplex PCR (mPCR) would have advantages in terms of rapidity and cost saving, when compared with single organism specific PCRs. mPCR has been standardized by targeting three major diarrheal enterotoxin genes hbl A, cyt K and nhe A of B. cereus, virulence associated nuc and Ent B genes of S. aureus and virulence associated hly and iap genes of L. monocytogenes along with internal amplification control (IAC). The results showed that mPCR accurately identified all the three organisms individually or in combination without non-specificity. The mPCR was able to detect as low as 10 to 100 organisms per ml of growth following overnight enrichment of spiked food samples (vegetable biriyani and milk) and their presence in naturally contaminated samples also. The high throughput and cost effective multiplex PCR method developed in this study could provide a powerful tool for simultaneous, rapid and reliable detection of B. cereus, S. aureus and L. monocytogenes in food samples.

Construction of a non toxic chimeric protein (L1-L2-B) of Haemolysin BL from Bacillus cereus and its application in HBL toxin detection.

Among the many potential virulence factors of B. cereus, Haemolysin BL is a unique and potent three component pore forming toxin composed of a binding component, B, and two lytic components, L(1) and L(2). Heterogeneity in nucleic acid and protein sequences of HBL components and problems during expression of L(1) and L(2) proteins in recombinant host due to their toxicity causes problems for development of specific detection systems based on PCR and Immunoassay, respectively. Commercially available kit (BCET RPLA, Oxoid) is useful for detection of L(2) component of HBL, but detection of only one component is insufficient to give comprehensive view on HBL toxin producing strains as some strains produced only one or two of the three HBL components. To address above mentioned problems, in this study, we cloned conserved domains of B, L(1) and L(2) components together as single fusion gene and expressed as recombinant multidomain chimeric protein in E. coli. The resultant protein having L(1), B and L(2) components in the form of single protein had no toxicity towards E. coli as we followed truncated protein approach. The hyperimmune antisera raised in mice against r-chimeric protein reacted with all the three components of HBL toxin of B. cereus (ATCC 14579) and provided three reaction bands at ~40 kDa to ~50 kDa regions during Western blot analysis. The hyperimmune sera of r-chimeric protein also notably neutralized the hemolytic activity of native HBL toxin. These results demonstrated that the obtained chimeric protein is correct and retained the antigenicity of native HBL toxin components. Therefore, it has better application in the development of a comprehensive HBL detection immunoassay and may also be a potential candidate molecule for vaccine studies.