Gold-FISH: A new approach for the in situ detection of single microbial cells combining fluorescence and scanning electron microscopy

A novel fluorescence in situ hybridisation (FISH) method is presented that allows the combination of epifluorescence and scanning electron microscopy (SEM) to identify single microbial cells. First, the rRNA of whole cells is hybridised with horseradish peroxidase-labelled oligonucleotide probes and this is followed by catalysed reporter deposition (CARD) of biotinylated tyramides. This facilitates an amplification of binding sites for streptavidin conjugates covalently labelled with both fluorophores and nanogold particles. The deposition of Alexa Fluor 488 fluoro-nanogold–streptavidin conjugates was confirmed via epifluorescence microscopy and cells could be quantified in a similar way to standard CARD–FISH approaches. To detect cells by SEM, an autometallographic enhancement of the nanogold particles was essential, and allowed the in situ localisation of the target organisms at resolutions beyond light microscopy. Energy dispersive X-ray spectroscopy (EDS) was used to verify the effects of CARD and autometallography on gold deposition in target cells.     

Quantification methods for Bacillus cereus vegetative cells and spores in the gastrointestinal environment

There is an interest to understand the fate and behaviour of the food-borne pathogen Bacillus cereus in the gut, a challenging environment with a high bacterial background. We evaluated the current detection methods to select an appropriate strategy for B. cereus monitoring during gastrointestinal experiments. Application of quantitative real-time PCR (qPCR) in a gastrointestinal matrix required careful selection of the qPCR reaction and elaborate optimization of the DNA extraction protocol. Primer competition and depletion problems associated with qPCR reactions targeting general 16S rRNA gene can be avoided by the selection of a target sequence that is unique for and widespread among the target bacteria, such as the toxin gene nheB in the case of pathogenic B. cereus. Enumeration of B. cereus during the ileum phase was impossible by plating due to overgrowth by intestinal bacteria, while a carefully optimized qPCR enabled specific detection and quantification of B. cereus. On the other hand, plating allowed the distinction of viable, injured and dead bacteria and the germination of spores, which was not possible with qPCR. In conclusion, both plating and qPCR were necessary to yield the maximal information regarding the viability and physiology of the B. cereus population in various gastrointestinal compartments.     

Peptides panned from a phage-displayed random peptide library are useful for the detection of Bacillus anthracis surrogates B. cereus 4342 and B. anthracis Sterne

Recent use of Bacillus anthracis as a bioweapon has highlighted the need for a sensitive monitoring system. Current bacterial detection tests use antibodies as bio-molecular recognition elements which have limitations with regard to time, specificity and sensitivity, creating the need for new and improved cost-effective high-affinity detection probes. In this study, we screened a commercially available bacteriophage-displayed random peptide library using Bacillus cereus 4342 cells as bait to identify peptides that could be used for detection of Bacillus. The method enabled us to identify two 12-amino acid consensus peptide sequences that specifically bind to B. cereus 4342 and B. anthracis Sterne, the nonpathogenic surrogates of B. anthracis strain. The two Bacillus-binding peptides (named BBP-1 and BBP-2) were synthesized with biotin tag to confirm their binding by four independent detection assays. Dot-blot analysis revealed that the peptides bind specifically to B. cereus 4342 and B. anthracis Sterne. Quantitative analysis of this interaction by ELISA and fluorometry demonstrated a detection sensitivity of 10² colony forming U/ml (CFU/ml) by both assays. When the peptides were used in combination with Qdots, the sensitivity was enhanced further by enabling detection of even a single bacterium by fluorescence microscopy. Immunoblot analysis and protein sequencing showed that BBP-1 and BBP-2 bound to the S-layer protein of B. anthracis Sterne. Overall, our findings validate the usefulness of synthetic versions of phage-derived peptides in combination with Qdot-liquid nanocrystals as high sensitivity bioprobes for various microbial detection platforms.     

Molecular analysis of SMN1, SMN2, NAIP, GTF2H2, and H4F5 genes in 157 Chinese patients with spinal muscular atrophy

Spinal muscular atrophy (SMA) is a common and lethal autosomal recessive neurodegenerative disorder, which is caused by mutations of the survival motor neuron 1 (SMN1) gene. Additionally, the phenotype is modified by several genes nearby SMN1 in the 5q13 region. In this study, we analyzed mutations in SMN1 and quantified the modifying genes, including SMN2, NAIP, GTF2H2, and H4F5 by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), multiplex ligation-dependent probe amplification (MLPA), TA cloning, allele-specific long-range PCR, and Sanger sequencing in 157 SMA patients. Most SMA patients (94.90%) possessed a homozygous SMN1 deletion, while 10 patients demonstrated only the absence of exon 7, but the presence of exon 8. Two missense mutations (c.689 C > T and c.844 C > T) were identified in 2 patients who both carried a single copy of SMN1. We found inverse correlations between SMN2, the NAIP copy number, and the clinical severity of the disease. Furthermore, 7 severe type I patients possessed large-scale deletions, including SMN1, NAIP, and GTF2H2. We conclude that SMN1 gene conversion, SMN1 subtle mutations, SMN2 copy number, and the extent of deletion in the 5q13 region should all be considered in the genotype–phenotype analysis of SMA.     

Conjugal transfer between Bacillus thuringiensis and Bacillus cereus strains is not directly correlated with growth of recipient strains

Bacillus thuringiensis and Bacillus cereus belong to the B. cereus species group. The two species share substantial chromosomal similarity and differ mostly in their plasmid content. The phylogenetic relationship between these species remains a matter of debate. There is genetic exchange both within and between these species, and current evidence indicates that insects are a particularly suitable environment for the growth of and genetic exchange between these species. We investigated the conjugation efficiency of B. thuringiensis var. kurstaki KT0 (pHT73-Em^R) as a donor and a B. thuringiensis and several B. cereus strains as recipients; we used one-recipient and two-recipient conjugal transfer systems in vitro (broth and filter) and in Bombyx mori larvae, and assessed multiplication following conjugation between Bacillus strains. The B. thuringiensis KT0 strain did not show preference for genetic exchange with the B. thuringiensis recipient strain over that with the B. cereus recipient strains. However, B. thuringiensis strains germinated and multiplied more efficiently than B. cereus strains in insect larvae and only B. thuringiensis maintained complete spore germination for at least 24 h in B. mori larvae. These findings show that there is no positive association between bacterial multiplication efficiency and conjugation ability in infected insects for the used strains.     

Fluorescence in situ hybridization for intracellular localization of nifH mRNA

Few reports on in situ mRNA detection in bacteria have been published, even though a major aim in environmental microbiology is to link function/activity to the identity of the organisms. This study reports a reliable approach for the in situ detection of nifH mRNA using fluorescence hybridization based on a previously described protocol for pmoA. nifH codes for a dinitrogenase reductase, a key enzyme in dinitrogen fixation. nifH mRNA was hybridized with a digoxigenin-labelled polynucleotide probe. The hybrid was detected with an anti-DIG-antibody labelled with horseradish peroxidase. Subsequently, the signal was amplified by catalyzed reporter deposition (CARD) with fluorochrome-labelled tyramides. Furthermore, the imaged organisms were identified using standard fluorescence in situ hybridization of rRNA. Thus, the approach enabled us specifically to link in situ the information from the dinitrogen fixation activity of an organism to its identity. Unexpectedly, the signals derived from nifH mRNA hybridization showed a distinct uneven pattern within the cells. This indicated that the method used could even give insights about the localization of the detected mRNA within the cell, which is a potential use of mRNA fluorescence in situ hybridization (FISH) that has not been reported up to now for bacterial cells.     

Intraspecific variation of the crotamine and crotasin genes in Crotalus durissus rattlesnakes

Crotamine is a small basic myotoxin peptide of Crotalus durissus venom, with β-defensin scafold and variable concentration in individual venoms. The crotamine gene was mapped to the end of chromosome 2 and the signal intensity differed significantly between the two homologues. In contrast to crotamine, the paralogous crotasin gene is scarcely expressed in the venom glands. In this study, we analyzed the crotamine concentrations in the venoms of a total of 23 rattlesnakes from diverse Brazilian localities by ELISA as well as the copy number of both crotamine and crotasin genes by real-time PCR. Crotamine was found to constitute 5–29% of venom proteins varying greatly among individual animals. The crotamine gene exists from 1 to 32 copies per haploid genome, whereas the crotasin gene is present from 1 to 7 copies. Furthermore, we observed that the crotamine concentration and crotamine gene copy number are positively correlated (r² = 0.68), implying the variation of crotamine in venom results from the variation of the gene copy number. Sequencing of 50 independent copies of crotamine and crotasin genes from four different rattlesnakes revealed the presence of six crotasin isoforms with a single amino acid difference from the original crotasin sequence, whereas only two additional crotamine isoforms were observed. Taken together, our results suggested that after duplication from a common ancestor gene, crotamine and crotasin may have diverged in such a way that the crotamine gene underwent repetitive duplication to increase its copy number, whereas the crotasin gene diversified its sequence.     

Phaseolus vulgaris seed-borne endophytic community with novel bacterial species such as Rhizobium endophyticum sp. nov.

The bacterial endophytic community present in different Phaseolus vulgaris (bean) cultivars was analyzed by 16S ribosomal RNA gene sequences of cultured isolates derived from surface disinfected roots and immature seeds. Isolated endophytes from tissue-macerates belonged to over 50 species in 24 different genera and some isolates from Acinetobacter, Bacillus, Enterococcus, Nocardioides, Paracoccus, Phyllobacterium, and Sphingomonas seem to correspond to new lineages. Phytate solubilizing bacteria were identified among Acinetobacter, Bacillus and Streptomyces bean isolates, phytate is the most abundant reserve of phosphorus in bean and in other seeds. Endophytic rhizobia were not capable of forming nodules. A novel rhizobial species Rhizobium endophyticum was recognized on the basis of DNA–DNA hybridization, sequence of 16S rRNA, recA, rpoB, atpD, dnaK genes, plasmid profiles, and phenotypic characteristics. R. endophyticum is capable of solubilizing phytate, the type strain is CCGE2052 (ATCC BAA-2116; HAMBI 3153) that became fully symbiotic by acquiring the R. tropici CFN299 symbiotic plasmid.     

Nocardiosis in Mediterranean bivalves: First detection of Nocardia crassostreae in a new host Mytilus galloprovincialis and in Ostrea edulis from the Gulf of Naples (Italy)

In this work M. galloprovincialis and O. edulis specimens were surveyed for a pathological study in the Gulf of Naples (Mediterranean sea, Campania Region, southern Italy). Clusters of Nocardia sp.-like cells were observed in histological slides. PCR amplification, sequencing and in situ hybridization were carried out in order to corroborate Nocardia species identification for both hosts. Blast results showed a 99% of maximum identity with Nocardia crassostreae sequences in Genbank. This is the first report of N. crassostreae in the new host M. galloprovincialis and, in a new area, the Mediterranean Sea.     

A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera)

Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R² = 0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available.     

Genetic detection and quantification of Nosema apis and N. ceranae in the honey bee

The incidence of nosemosis has increased in recent years due to an emerging infestation of Nosema ceranae in managed honey bee populations in much of the world. A real-time PCR assay was developed to facilitate detection and quantification of both Nosema apis and N. ceranae in both single bee and pooled samples. The assay is a multiplexed reaction in which both species are detected and quantified in a single reaction. The assay is highly sensitive and can detect single copies of the target sequence. Real-time PCR results were calibrated to spore counts generated by standard microscopy procedures. The assay was used to assess bees from commercial apiaries sampled in November 2008 and March 2009. Bees from each colony were pooled. A large amount of variation among colonies was evident, signifying the need to examine large numbers of colonies. Due to sampling constraints, a subset of colonies (from five apiaries) was sampled in both seasons. In November, N. apis levels were 1212 ± 148 spores/bee and N. ceranae levels were 51,073 ± 31,155 spores/bee. In March, no N. apis was detected, N. ceranae levels were 11,824 ± 6304 spores/bee. Changes in N. ceranae levels were evident among apiaries, some increasing and other decreasing. This demonstrates the need for thorough sampling of apiaries and the need for a rapid test for both detection and quantification of both Nosema spp. This assay provides the opportunity for detailed study of disease resistance, infection kinetics, and improvement of disease management practices for honey bees.     

Construction and characterization of a copy number-inducible fosmid library of Xanthomonas oryzae pathovar oryzae MAFF311018

A fosmid library of Xanthomonas oryzae pathovar oryzae MAFF311018 (T7174), the causative agent of bacterial blight on rice, was constructed and characterized. The average fosmid library insert size was > 34 kb, and 967 clones were uniquely positioned on its sequenced genome. The entire Xoo MAFF311018 genome was covered by end-sequenced clones with at least 5 kb of overlap. The fosmid vector contains both the single-copy Escherichia coli fertility factor origin, which enhances fosmid stability, and the multi-copy IncPα origin, allowing amplification of copy number upon induction with l-arabinose. Real-time quantitative PCR on 12 randomly picked fosmid library clones determined that fosmid copy number increased 8- to 58-fold after 5 hour induction. This library provides a new resource for complementation experiments and systematic functional studies in Xoo and related species.     

rDNA-targeted PCR primers and FISH probe in the detection of Ophiocordyceps sinensis hyphae and conidia

Ophiocordyceps sinensis (Berk.) Sung, Sung, Hywel-Jones & Spatafora (syn. Cordyceps sinensis) one of the entomopathogenic fungi, is a rare Traditional Chinese Medicine (TCM) found in the Qinghai-Tibetan Plateau. Polymerase Chain Reaction (PCR) and Fluorescence in situ hybridization (FISH) methods are necessary to identify the mycelia or spores of O. sinensis from its habitat and to monitor its dispersal, colonization and infectivity. To develop both primers and probe specific to O. sinensis, ribosomal DNA (rDNA) amplified with universal primers from O. sinensis genomic DNA and seven closely related fungi were sequenced. According to these sequences, the upper and lower primers (OsT-F and OsT-R) were designed within internal transcribed spacer region 1 (ITS1) and ITS2 and flanked by universal primers ITS5 and ITS4, respectively. The designed primers were used for general PCR, touchdown PCR, or both together with the universal primers for nested-touchdown PCR. The results showed that only the extracted DNA of O. sinensis was specifically amplified. The sensitivity of nested-touchdown PCR with extracted DNA of O. sinensis is as low as 10⁻¹⁴ g (10 fg) and at least 1000 times higher than the other PCR methods. In addition, Cy5-labeled probe (OsLSU) for cytoplasmic LSU rRNA was hybridized with the ascospores of O. sinensis. It showed a strong red fluorescence throughout the whole cell but did not cross-react with other entomopathogenic fungi. Taken together, these methods were useful for studying the biology and ecology of O. sinensis.     

Identification of a Bacillus anthracis specific indel in the yeaC gene and development of a rapid pyrosequencing assay for distinguishing B. anthracis from the B. cereus group

Bacillus anthracis, the causative agent of anthrax, is a potential source of bioterrorism. The existing assays for its identification lack specificity due to the close genetic relationship it exhibits to other members of the B. cereus group. Our comparative analyses of protein sequences from Bacillus species have identified a 24 amino acid deletion in a conserved region of the YeaC protein that is uniquely present in B. anthracis. PCR primers based on conserved regions flanking this indel in the Bacillus cereus group of species (viz. Bacillus cereus, B. anthracis, B. thuringiensis, B. mycoides, B. weihenstephnensis and B. pseudomycoides) specifically amplified a 282 bp fragment from all six reference B. anthracis strains, whereas a 354 bp fragment was amplified from 15 other B. cereus group of species/strains. These fragments, due to large size difference, are readily distinguished by means of agarose gel electrophoresis. In contrast to the B. cereus group, no PCR amplification was observed with any of the non-B. cereus group of species/strains. This indel was also used for developing a rapid pyrosequencing assay for the identification of B. anthracis. Its performance was evaluated by examining the presence or absence of this indel in a panel of 81 B. cereus-like isolates from various sources that included 39 B. anthracis strains. Based upon the sequence data from the pyrograms, the yeaC indel was found to be a distinctive characteristic of various B. anthracis strains tested and not found in any other species/strains from these samples. Therefore, this B. anthracis specific indel provides a robust and highly-specific chromosomal marker for the identification of this high-risk pathogen from other members of the B. cereus group independent of a strain's virulence. The pyrosequencing platform also allows for the rapid and simultaneous screening of multiple samples for the presence of this B. anthracis-specific marker.     

Comparative analysis of the virulence of invertebrate and mammalian pathogenic bacteria in the oral insect infection model Galleria mellonella

Infection of Galleria mellonella by feeding a mixture of Bacillus thuringiensis spores or vegetative bacteria in association with the toxin Cry1C results in high levels of larval mortality. Under these conditions the toxin or bacteria have minimal effects on the larva when inoculated separately. In order to evaluate whether G. mellonella can function as an oral infection model for human and entomo-bacterial pathogens, we tested strains of Bacillus cereus, Bacillus anthracis, Enterococcus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa and a Drosophila targeting Pseudomonas entomophila strain. Six B. cereus strains (5 diarrheal, 1 environmental isolate) were first screened in 2nd instar G. mellonella larvae by free ingestion and four of them were analyzed by force-feeding 5th instar larvae. The virulence of these B. cereus strains did not differ from the B. thuringiensis virulent reference strain 407Cry⁻ with the exception of strain D19 (NVH391/98) that showed a lower virulence. Following force-feeding, 5th instar G. mellonella larvae survived infection with B. anthracis, L. monocytogenes, E. faecalis and P. aeruginosa strains in contrast to the P. entomophila strain which led to high mortality even without Cry1C toxin co-ingestion. Thus, specific virulence factors adapted to the insect intestine might exist in B. thuringiensis/B. cereus and P. entomophila. This suggests a co-evolution between host and pathogens and supports the close links between B. thuringiensis and B. cereus and more distant links to their relative B. anthracis.     

Comparison of multiplex PCR, enzyme immunoassay and cell culture methods for the detection of enterotoxinogenic Bacillus cereus

Fast and reliable methods are needed for the detection of pathogenic Bacillus cereus which should provide consistent results. Therefore, we tested a panel of 176 strains, including B. cereus strains, B. cereus group strains and other Bacillus spp. with polymerase chain reaction, immunoassays and cytotoxicity tests and assessed the consistency of the results. A screening multiplex PCR for the detection of hbl, nhe, ces and cytK1 as well as two multiplex PCRs for the differentiation of Hbl genes (hblC, hblD, hblA) and Nhe genes (nheA, nheB, nheC) was applied. All PCRs included an internal amplification control. Component specific antibody based immunoassays were used for the detection of the three components of Hbl and Nhe and the overall cytotoxicity to Vero cells and HEp-2 cells was checked. An overall excellent correlation was obtained for the results of the three, methodically independent assays and no false-negative PCR results were seen for any of the strains tested positive in immunoassays and cytotoxicity tests. The three multiplex PCRs proved to be a facile method for the identification of enterotoxinogenic B. cereus isolates.     

Combined deletion of DAZ2 and DAZ4 copies of Y chromosome DAZ gene is associated with male infertility in Tunisian men

The relationship between male infertility and AZFc micro-deletions that remove multiple genes of the Y chromosome varies among countries and populations. The purpose of this study was to analyze the prevalence and the characteristics of different Deleted in azoospermia (DAZ) gene copy deletions and their association with spermatogenic failure and male infertility in Tunisian men. 241 infertile men (30.7% azoospermic (n = 74), 31.5% oligozoospermic (n = 76) and 37.7% normozoospermic (n = 91)) and 115 fertile healthy males who fathered at least one child were included in the study. Three DAZ-specific single nucleotide variant loci and six bi-allelic DAZ-SNVs (I–VI) were analyzed using polymerase chain reaction (PCR)–restriction fragment length polymorphism and PCR. Our findings showed high frequencies of infertile men (73.85%) and controls (78.26%) having only three DAZ gene copies (DAZ1/DAZ2/DAZ3 or DAZ1/DAZ3/DAZ4 variants); so deletion of DAZ2 or DAZ4 were frequent both in infertile (36.5% and 37.3%, respectively) and fertile groups (33.9% and 44.3%, respectively) and removing DAZ4 copy was significantly more frequent in oligospermic than in normospermic men (p = 0.04) in infertile group. We also report for the first time that simultaneous deletion of both DAZ2 and DAZ4 copies was significantly more common in infertile men (12.4%) than in fertile men (4.3%) (p = 0.01). However, deletions of DAZ1/DAZ2 and DAZ3/DAZ4 clusters were very rare. Analysis of DAZ gene copies in Tunisian population, suggested that the simultaneous deletion of DAZ2 and DAZ4 gene copies is associated with male infertility, and that oligospermia seems to be promoted by removing DAZ4 copy.     

Development of rRNA and rDNA-targeted probes for fluorescence in situ hybridization to detect Heterosigma akashiwo (Raphidophyceae)

Heterosigma akashiwo (Hada) is a fragile, fish-killing alga. Efforts to understand and prevent blooms due to this harmful species to mitigate the impact on aquaculture require the development of methods for rapid and precise identification and quantification, so that adequate warning of a harmful algal bloom may be given. Here, we report the development and application of rRNA and rDNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection and enumeration of H. akashiwo. The designed probes were species specific, showing no cross-reactivity with four common HAB causative species: Prorocentrum micans Ehrenberg, P. minimum (Pavillard) Schiller, Alexandrium tarmarense (Lebour) Balech, and Skeletonema costatum (Greville) Cleve, or with four other microalgae, including Gymnodinium sp. Stein, Platy-monas cordiformis (Karter) Korsch, Skeletonema sp.1 Greville and Skeletonema sp.2. The rRNA-targeted probe hybridized to cytoplasmic rRNA, showing strong green fluorescence throughout the whole cell, while cells labeled by rDNA-targeted probe exhibited exclusively fluorescent nucleus. The detection protocols were optimized and could be completed within an hour. For rRNA and rDNA probes, about a corresponding 80% and 70% of targeted cells could be identified and quantified during the whole growth circle, despite the inapparent variability in the average probe reactivity. The established FISH was proved promising for specific, rapid, precise, and quantitative detection of H. akashiwo.     

Multiplex PCR assay for the detection of enterotoxic <Emphasis Type="Italic">Bacillus cereus</Emphasis> 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 10¹–10² 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.