The ARK Assay Is a Sensitive and Versatile Method for the Global Detection of DNA-Protein Crosslinks

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Multiplex TaqMan qPCR Assay for Detection,Identification, and Quantification of Three Sclerotinia Species

White mold (or Sclerotinia stem rot), caused by Sclerotinia species, is a major air, soil, or seed-transmitted disease affecting numerous crops and wild plants. Microscopic or culture-based methods currently available for their detection and identification are time-consuming, laborious, and often erroneous. Therefore, we developed a multiplex quantitative PCR (qPCR) assay for the discrimination, detection, and quantification of DNA collected from each of the three economically relevant Sclerotinia species, namely, S. sclerotiorum, S. minor, and S. nivalis. TaqMan primer/probe combinations specific for each Sclerotinia species were designed based on the gene sequences encoding aspartyl protease. High specificity and sensitivity of each probe were confirmed for sclerotium and soil samples, as well as pure cultures, using simplex and multiplex qPCRs. This multiplex assay could be helpful in detecting and quantifying specific species of Sclerotinia, and therefore, may be valuable for disease diagnosis, forecasting, and management.     

A Novel Immunological Assay for Hepcidin Quantification in Human Serum

Background

Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes a specific and non-operator demanding immunoassay for hepcidin quantification in human sera.

Methods and Findings

An ELISA assay was developed for measuring hepcidin serum concentration using a recombinant hepcidin25-His peptide and a polyclonal antibody against this peptide, which was able to identify native hepcidin. The ELISA assay had a detection range of 10–1500 µg/L and a detection limit of 5.4 µg/L. The intra- and interassay coefficients of variance ranged from 8–15% and 5–16%, respectively. Mean linearity and recovery were 101% and 107%, respectively. Mean hepcidin levels were significantly lower in 7 patients with juvenile hemochromatosis (12.8 µg/L) and 10 patients with iron deficiency anemia (15.7 µg/L) and higher in 7 patients with Hodgkin lymphoma (116.7 µg/L) compared to 32 age-matched healthy controls (42.7 µg/L).

Conclusions

We describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility.     

Detection and Quantification of Botulinum Neurotoxin Type A by a Novel Rapid In Vitro Fluorimetric Assay

Botulinum neurotoxin type A (BoNT/A), the most poisonous substance known to humans, is a potential bioterrorism agent. The light-chain protein induces a flaccid paralysis through cleavage of the 25-kDa synaptosome-associated protein (SNAP-25), involved in acetylcholine release at the neuromuscular junction. BoNT/A is widely used as a therapeutic agent and to reduce wrinkles. The toxin is used at very low doses, which have to be accurately quantified. With this aim, internally quenched fluorescent substrates containing the fluorophore/repressor pair pyrenylalanine (Pya)/4-nitrophenylalanine (Nop) were developed. Nop and Pya were, respectively, introduced at positions 197 and 200 of the cleavable fragment (amino acids 187 to 203) of SNAP-25 (with norleucine at position 202 [Nle²⁰²]), which is acetylated at its N terminus and amidated at its C terminus. Cleavage of this peptide occurred between positions 197 and 198, as in SNAP-25, and was easily quantified by the strong fluorescence emission of the metabolite. To increase the assay sensitivity, the peptide sequence of the previous substrate was lengthened to account for exosite binding to BoNT/A. We synthesized the peptide PL50 (SNAP-25-NH₂ acetylated at positions 156 to 203 [Nop¹⁹⁷, Pya²⁰⁰, Nle²⁰²]) and its analogue PL51, in which all methionines were replaced by nonoxidizable Nle. Consistent with a large increase in affinity for BoNT/A, PL50 and PL51 exhibit catalytic efficiencies of 2.6 × 10⁶ M⁻¹ s⁻¹ and 8.85 × 10⁶ M⁻¹ s⁻¹, respectively, and behave as the best fluorigenic substrates of BoNT/A reported to date. Under optimized assay conditions, they allow simple quantification of as little as 100 and 60 pg of BoNT/A, respectively, within 2 h with a classical fluorimeter. Calibration of the method against the mouse 50% lethal dose assay unequivocally validates the enzymatic assay.The botulinum neurotoxin (BoNT) family consists of seven antigenically distinct serotypes, BoNT/A to BoNT/G, which act on the peripheral nervous system (19). Of these toxins, serotypes A, B, E, and F cause botulism in humans, a disease characterized by flaccid muscular paralysis. The neurotoxins are produced as single inactive polypeptides of 150 kDa, which are subsequently processed by proteolytic cleavage into biologically active di-chains (19). These forms consist of an approximately 50-kDa light chain (LC) linked by a disulfide bridge to a 100-kDa heavy chain (HC) that contains two domains, designated the binding and translocation domains. The neurotoxins reach their intracellular targets by translocating the LC into the cytosol after endocytosis via interaction of the HC with a high-affinity membrane-bound receptor complex (9, 20). The LC, which possesses a highly specific zinc-endopeptidase activity (29), then blocks the fusion of synaptic vesicles with the presynaptic membrane by selectively cleaving one of the three polypeptides involved in neuroexocytosis. BoNT/A, for instance, cleaves the 206-amino-acid, 25-kDa synaptosome-associated protein (SNAP-25) exclusively between the Q¹⁹⁷ and R¹⁹⁸ residues, thus inhibiting neurotransmitter release at the neuromuscular junction (37, 38).BoNT/A is recognized as the most toxic serotype; its oral 50% lethal dose (LD₅₀) for humans is estimated at 1 μg/kg of body weight (2). Because of this extreme toxicity and prolonged effect, BoNTs are classified by the Centers for Disease Control and Prevention (CDC) as one of the six highest-risk threat agents for bioterrorism in “category A” (27). In spite of this, BoNT/A and -B are widely used as therapeutic agents for the treatment of muscular and nerve disorders, as well as in the treatment of neurological diseases (14, 15, 28). There is also an increasing use of BoNT/A in esthetics for wrinkle reduction (4). Because of their high toxicity, BoNTs are used at very low concentrations, and procedures to be used for their detection and quantification in toxin preparations for medical applications or in the event of malevolent bioterrorist acts have to be highly sensitive, rapid, and easy to use; the use of all lengthy in vivo assays is excluded (2, 11). The advantage of the currently used pharmacotoxicological mouse LD₅₀ (MLD₅₀) assay, considered the gold standard assay, is that it provides the in vivo toxicity of a given botulinum toxin sample, whatever the nature of the infected medium. However, this assay is time-consuming, requires the use of a large number of animals, and has poor repeatability due to many fluctuant parameters involved in this method (22). Several in vitro assays have been reported for the detection of BoNT/A, relying either on mass spectrometry (3, 16), immunological detection (10, 25), or BoNT/A''s endopeptidase activity (12, 30). The advantage of the endopeptidase assay is that it measures and quantifies the “active” part of the toxin, which is directly responsible for neurotransmission inhibition. Various methods have been developed to quantify the BoNT/A proteolytic activity (12, 23, 32-33). Although some of these assays are very sensitive (11), they cannot be used for the field detection of BoNT/A, as they require a multistep procedure, and they are also not easily amenable to quantification of toxin preparations used for medical applications.In this paper, we have designed novel, specific, high-affinity, mimetic peptide substrates for BoNT/A using the internal-collision-induced fluorescence-quenching technique (13). This technique, the use of which has previously been successful in the design of peptide substrates for other Zn-metallopeptidases, e.g., ECE-1 (18) and BoNT/B (1, 26), involves the introduction of a fluorophore/repressor pair, here the highly fluorescent pyrenylalanine (Pya) along with a nitro-phenylalanine (Nop) repressor residue on each side of the cleavage site. Once the better positions of the fluorophore/repressor pair Pya/Nop were determined using a fragment of the SNAP-25 sequence from amino acids 187 to 203 [(187-203) SNAP-25] (30), the kinetic parameters of the peptide substrate were optimized and the stability of the final substrate, acetylated SNAP-25 from positions 156 to 203 [(Ac-156-203) SNAP-25] (Nop¹⁹⁷, Pya²⁰⁰, Nle²⁰²), also called PL50, was finally improved in PL51 by replacing the oxidizable methionine residues within the sequence with norleucines. Thus, the specificity constants (catalytic constant [k_cat]/Michaelis constant [K_m]) of PL50 and of its analogue PL51 were 2.6 × 10⁶ M⁻¹ s⁻¹ and 8.85 × 10⁶ M⁻¹ s⁻¹, respectively. The use of these novel high-affinity substrates provides a simple, one-step, specific, robust, and rapid enzymatic assay, thus fulfilling all the requirements for BoNT/A field detection and for BoNT/A''s quantification in preparations for medical applications.     

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).Download video file.^{(44M, mov)}     

Species-Specific Detection and Quantification of Toxic Marine Dinoflagellates Alexandrium tamarense and A. catenella byReal-Time PCR Assay

A Real-time polymerase chain reaction (PCR) assay was designed and evaluated for rapid detection and quantification of the toxic dinoflagellates Alexandrium catenella and A. tamarense, which cause paralytic shellfish poisoning. Two sets of PCR primers and fluorogenic probes targeting these two species were derived from the sequence of 28S ribosomal DNA. PCR specificity was examined in closely related Alexandrium spp. and many other microalgae. A. catenella–specific primers and probe detected the PCR amplification only from A. catenella strains, and nonspecific signals were not detected from any microalgae. Also, A. tamarense–specific primers and probe also detected the targeted species, suggesting the strict species specificity of each PCR. This assay could detect one cell of each species, showing its high sensitivity. Moreover, using the developed standard curves, A. tamarense and A. catenella could be quantified in agreement with the quantification by optical microscopy. The performance characteristics of species specificity, sensitivity, and rapidity suggest that this method is applicable to the monitoring of the toxic A. tamarense and A. catenella.     

Double-Layer Plaque Assay for Quantification of Enteroviruses

We describe here a double-layer plaque assay for the quantification of enteroviruses, combining a monolayer plaque assay and a suspended-cell plaque assay. The double-layer assay provides significantly greater counts than other methods of virus quantification of both suspensions of pure culture viruses and naturally occurring viruses. The counts obtained by this method are approximately one order of magnitude greater than those obtained with the more commonly used method, the monolayer plaque assay. We conclude that the methods available for quantifying viruses rank in efficiency as follows: double-layer plaque assay ≥ suspended-cell plaque assay > counting cytopathogenic virus adsorbed to cellulose nitrate membrane filters ≥ most probable number of cytopathogenic units > monolayer plaque assay. Moreover, the double-layer plaque assay allows the use of two different cell lines in the two layers. Using the human colonic carcinoma cell line CaCo2 facilitates the recovery of a greater number and diversity of naturally occurring enteroviruses in water than the monolayer agar method. In addition, the pretreatment of cells with 5-iodo-2′-deoxyuridine (IDU) prior to the quantification of enteroviruses by the double-layer plaque assay provides significantly higher recoveries than the use of IDU does with the other methods of quantification.     

Enzyme-Linked Immunosorbent Assay for Detection of Ochratoxin A

A simple microtest plate enzyme-linked immunosorbent assay was developed for the detection of ochratoxin A at levels as low as 25 pg per assay. The relative cross-reactivities of the antibody in this system with ochratoxin A (OA), OB, OC, and Oα were found to be 1.0, 0.14, 0.44, and 0.01, respectively.     

A Versatile Simple Capture Assay for Assessing the Structural Integrity of MHC Multimer Reagents

Antigen-specific T cell responses can be visualized using MHC:peptide multimers. In cases where robust T cell controls are not readily available to assess the integrity of multimer reagents prior to analyzing limited sample, the ability to assess the structural integrity of MHC multimers before their use in critical experiments would be useful. We present a method to probe the structural integrity of MHC multimers using antibodies specific for conformational determinants. Beads coated with anti-mouse Ig are incubated with conformation-specific mouse monoclonal antibody and then with fluorescently tagged MHC multimer. The ability of the bead to capture the labeled multimer can be measured semi-quantitatively by flow cytometry. In this manner, the correct folding of MHC multimers can be visualized and batches of multimer can be compared for quality control. Because there are multiple conformational epitopes formed by various molecular interactions among heavy chain, peptide, and β₂M, this capture assay can assess the fidelity of each aspect of multimer structure, depending on the availability of antibodies. The described approach could be particularly useful for studies using irreplaceable samples, including patient samples collected in clinical trials.     

A Direct and Versatile Assay Measuring Membrane Penetration of Adenovirus in Single Cells

Endocytosis is the most prevalent entry port for viruses into cells, but viruses must escape from the lumen of endosomes to ensure that viral genomes reach a site for replication and progeny formation. Endosomal escape also helps viruses bypass endolysosomal degradation and presentation to certain Toll-like intrinsic immunity receptors. The mechanisms for cytosolic delivery of nonenveloped viruses or nucleocapsids from enveloped viruses are poorly understood, in part because no quantitative assays are readily available which directly measure the penetration of viruses into the cytosol. Following uptake by clathrin-mediated endocytosis or macropinocytosis, the nonenveloped adenoviruses penetrate from endosomes to the cytosol, and they traffic with cellular motors on microtubules to the nucleus for replication. In this report, we present a novel single-cell imaging assay which quantitatively measures individual cytosolic viruses and distinguishes them from endosomal viruses or viruses at the plasma membrane. Using this assay, we showed that the penetration of human adenoviruses of the species C and B occurs rapidly after virus uptake. Efficient penetration does not require acidic pH in endosomes. This assay is versatile and can be adapted to other adenoviruses and members of other nonenveloped and enveloped virus families.     

A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins

Staphylococcal and streptococcal exotoxins, also known as superantigens, mediate a range of diseases including toxic shock syndrome, and they exacerbate skin, pulmonary and systemic infections caused by these organisms. When present in food sources they can cause enteric effects commonly known as food poisoning. A rapid, sensitive assay for the toxins would enable testing of clinical samples and improve surveillance of food sources. Here we developed a bead-based, two-color flow cytometry assay using single protein domains of the beta chain of T cell receptors engineered for high-affinity for staphylococcal (SEA, SEB and TSST-1) and streptococcal (SpeA and SpeC) toxins. Site-directed biotinylated forms of these high-affinity agents were used together with commercial, polyclonal, anti-toxin reagents to enable specific and sensitive detection with SD₅₀ values of 400 pg/ml (SEA), 3 pg/ml (SEB), 25 pg/ml (TSST-1), 6 ng/ml (SpeA), and 100 pg/ml (SpeC). These sensitivities were in the range of 4- to 80-fold higher than achieved with standard ELISAs using the same reagents. A multiplex format of the assay showed reduced sensitivity due to higher noise associated with the use of multiple polyclonal agents, but the sensitivities were still well within the range necessary for detection in food sources or for rapid detection of toxins in culture supernatants. For example, the assay specifically detected toxins in supernatants derived from cultures of Staphylococcus aureus. Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of Staphylococcus aureus and Streptococcus pyogenes.     

Development of a Real-Time PCR Assay for Rapid Detection and Quantification of Alexandrium minutum (a Dinoflagellate)

The marine dinoflagellate genus Alexandrium includes a number of species which produce neurotoxins responsible for paralytic shellfish poisoning (PSP), which in humans may cause muscular paralysis, neurological symptoms, and, in extreme cases, death. A. minutum is the most widespread toxic PSP species in the western Mediterranean basin. The monitoring of coastal waters for the presence of harmful algae also normally involves microscopic examinations of phytoplankton populations. These procedures are time consuming and require a great deal of taxonomic experience, thus limiting the number of specimens that can be analyzed. Because of the genetic diversity of different genera and species, molecular tools may also help to detect the presence of target microorganisms in marine field samples. In this study, we developed a real-time PCR-based assay for rapid detection of all toxic species of the Alexandrium genus in both fixative-preserved environmental samples and cultures. Moreover, we developed a real-time quantitative PCR assay for the quantification of A. minutum cells in seawater samples. Alexandrium genus-specific primers were designed on the 5.8S rDNA region. Primer specificity was confirmed by using BLAST and by amplification of a representative sample of the DNA of other dinoflagellates and diatoms. Using a standard curve constructed with a plasmid containing the ITS1-5.8S-ITS2 A. minutum sequence and cultured A. minutum cells, we determined the absolute number of 5.8S rDNA copies per cell. Consequently, after quantification of 5.8S rDNA copies in samples containing A. minutum cells, we were also able to estimate the number of cells. Several fixed A. minutum bloom sea samples from Arenys Harbor (Catalan Coast, Spain) were analyzed using this method, and quantification results were compared with standard microscopy counting methods. The two methods gave comparable results, confirming that real-time PCR could be a valid, fast alternative procedure for the detection and quantification of target phytoplankton species during coastal water monitoring.     

TaqMan荧光定量RT-PCR检测水稻RAcl基因mRNA方法的建立

构建包含RAcl基因cDNA片段的质粒,作为水稻肌动蛋白基因RAcl之mRNA定量检测的标准品,建立检测方法,为水稻其他基因的定量建立内参。从水稻叶总RNA中逆转录扩增总cDNA,PCR扩增RAcl基因中设计的目的片段,将纯化的目的片段与pMD19-T Simple载体进行连接,转化宿主菌JM-109,提取重组质粒DNA,PCR鉴定并测序分析。纯化质粒并检测260nm吸光值,确定重组质粒原液的拷贝浓度并以此制备荧光定量PCR梯度浓度标准品,进行实时荧光定量PCR实验。建立了RAcl基因mRNA表达实时荧光定量PCR检测方法,特异性好,检测灵敏度达102拷贝,线性范围为102—1护拷贝,阈值循环数（Ct）与PCR体系中起始模板量的对数值之间有着良好的线性关系（r=1．000）,扩增效率高（E=98．2％）。建立了基因RAcl实时定量PCR的质粒标准品。     

Characterization of Egg Yolk Antibodies for Detection and Quantification of Selenomonas ruminantium by Using an Enzyme-Linked Immunosorbent Assay

The specificity of polyclonal antibodies prepared against strains of Selenomonas ruminantium, the effect of assay conditions, and quantification of individual strains in mixed-cell suspensions of selenomonad strains were examined in this study. Whole-cell suspensions were prepared with pure cultures of S. ruminantium PC18, HD₄, GA192, and D. Each cell suspension was injected into a Leghorn laying hen, and polyclonal antibodies were harvested from eggs laid in week 3 or 7 following initial immunization. Antibodies made to the S. ruminantium strains readily discerned the homologous strain from the heterologous strains. Cross-reactivity among antibodies and the heterologous S. ruminantium strains ranged from 5 to 26%. Among non-S. ruminantium species, cross-reactivity of S. ruminantium antibodies was greatest with Selenomonas sputigena (3 to 34%) and Succinivibrio dextrinosolvens (0 to 37%). Antibodies made to strains GA192 and D were used to quantify a mixture of the two strains. Both antibodies responded to graded concentrations of the homologous antigen in the biculture mixtures in accord with the change in the direct cell counts for each strain (strain D, R² = 0.92; strain GA192, R² = 0.90). This enzyme-linked immunosorbent assay enabled concurrent and accurate quantification of two strains of S. ruminantium subsp. ruminantium in a mixed-cell suspension with a precision of much less than 1 order of magnitude.     

新型鸭呼肠孤病毒RT-LAMP检测方法的建立

建立适于基层实验室快速检测新型鸭呼肠孤病毒(Novel duck reovirus,NDRV)的一步反转录环介导等温扩增(RT-LAMP)方法。基于新型鸭呼肠孤病毒S3基因的6个保守区域设计了4条LAMP引物,利用Bst DNA聚合酶在63℃恒温保持45 min即可完成反转录和扩增反应,由此建立了RT-LAMP检测方法。该方法具有良好的特异性,除NDRV外对其他6种常见鸭病的检测结果均为阴性。该方法对病毒RNA的最低检出量为0.1 pg,是常规RT-PCR方法的100倍。临床应用结果表明,该方法与病毒分离鉴定方法的符合率为98%,而且对仪器的要求低,适于基层实验室和现场检测。     

Isolation and Quantification of MicroRNAs from Urinary Exosomes/Microvesicles for Biomarker Discovery

Recent studies indicate that microRNA (miRNA) is contained within exosome. Here we sought to optimize the methodologies for the isolation and quantification of urinary exosomal microRNA as a prelude to biomarker discovery studies. Exosomes were isolated through ultracentrifugation and characterized by immunoelectron microscopy. To determine the RNA was confined inside exosomes, the pellet was treated with RNase before RNA isolation. The minimum urine volume, storage conditions for exosomes and exosomal miRNA was evaluated. The presence of miRNAs in patients with various kidney diseases was validated with real-time PCR. The result shows that miRNAs extracted from the exosomal fraction were resistant to RNase digestion and with high quality confirmed by agarose electrophoresis. 16ml of urine was sufficient for miRNA isolation by absolute quantification with 4.15×10⁵ copies/ul for miR-200c. Exosomes was stable at 4℃ 24h for shipping before stored at -80℃ and was stable in urine when stored at -80°C for 12months. Exosomal miRNA was detectable despite 5 repeat freeze-thaw cycles. The detection of miRNA by quantitative PCR showed high reproducibility (>94% for intra-assay and >76% for inter-assay), high sensitivity (positive call 100% for CKD patients), broad dynamic range (8-log wide) and good linearity for quantification (R²>0.99). miR-29c and miR-200c showed different expression in different types of kidney disease. In summary, the presence of urinary exosomal miRNA was confirmed for patients with a diversity of chronic kidney disease. The conditions of urine collection, storage and miRNA detection determined in this study may be useful for future biomarker discovery efforts.     

TaqMan荧光定量RT-PCR检测水稻RAc1基因mRNA方法的建立

构建包含RAc1基因cDNA片段的质粒,作为水稻肌动蛋白基因RAc1之mRNA定量检测的标准品,建立检测方法,为水稻其他基因的定量建立内参.从水稻叶总RNA中逆转录扩增总cDNA,PCR扩增RAc1基因中设计的目的片段,将纯化的目的片段与pMD19-T Simple载体进行连接,转化宿主菌JM-109,提取重组质粒DNA,PCR鉴定并测序分析.纯化质粒并检测260nm吸光值,确定重组质粒原液的拷贝浓度并以此制备荧光定量PCR梯度浓度标准品,进行实时荧光定量PCR实验.建立了RAc1基因mRNA表达实时荧光定量PCR检测方法,特异性好,检测灵敏度达102拷贝,线性范围为102～107拷贝,阈值循环数(Ct)与PCR体系中起始模板量的对数值之间有着良好的线性关系(r=1.000),扩增效率高(E=98.2%).建立了基因RAc1实时定量PCR的质粒标准品.     

Pentaplexed Quantitative Real-Time PCR Assay for the Simultaneous Detection and Quantification of Botulinum Neurotoxin-Producing Clostridia in Food and Clinical Samples

Botulinum neurotoxins are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven distinct serotypes (A to G) known to cause botulism in animals and humans. In this study, a multiplexed quantitative real-time PCR assay for the simultaneous detection of the human pathogenic C. botulinum serotypes A, B, E, and F was developed. Based on the TaqMan chemistry, we used five individual primer-probe sets within one PCR, combining both minor groove binder- and locked nucleic acid-containing probes. Each hydrolysis probe was individually labeled with distinguishable fluorochromes, thus enabling discrimination between the serotypes A, B, E, and F. To avoid false-negative results, we designed an internal amplification control, which was simultaneously amplified with the four target genes, thus yielding a pentaplexed PCR approach with 95% detection probabilities between 7 and 287 genome equivalents per PCR. In addition, we developed six individual singleplex real-time PCR assays based on the TaqMan chemistry for the detection of the C. botulinum serotypes A, B, C, D, E, and F. Upon analysis of 42 C. botulinum and 57 non-C. botulinum strains, the singleplex and multiplex PCR assays showed an excellent specificity. Using spiked food samples we were able to detect between 10³ and 10⁵ CFU/ml, respectively. Furthermore, we were able to detect C. botulinum in samples from several cases of botulism in Germany. Overall, the pentaplexed assay showed high sensitivity and specificity and allowed for the simultaneous screening and differentiation of specimens for C. botulinum A, B, E, and F.Botulinum neurotoxins (BoNTs), the causative agents of botulism, are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven serotypes, A to G. While the botulinum neurotoxins BoNT/A, BoNT/B, BoNT/E, and BoNT/F are known to cause botulism in humans, BoNT/C and BoNT/D are frequently associated with botulism in cattle and birds. Despite its toxicity, BoNT/G has not yet been linked to naturally occurring botulism (26).Botulism is a life-threatening illness caused by food contaminated with BoNT (food-borne botulism), by the uptake and growth of C. botulinum in wounds (wound botulism), or by colonization of the intestinal tract (infant botulism) (14). In addition, C. botulinum and the botulinum neurotoxins are regarded as potential biological warfare agents (8).The gold standard for the detection of BoNTs from food or clinical samples is still the mouse lethality assay, which is highly sensitive but rather time-consuming. In addition to various immunological assays for BoNT detection, several conventional and real-time PCR-based assays for the individual detection of bont genes have been reported (2, 9-12, 15, 20, 23, 27-30). A major improvement is the simultaneous detection of more than one serotype, which results in a reduction of effort and in the materials used. In recent years, both conventional and real-time PCR-based multiplex assays have been developed for the simultaneous detection of C. botulinum serotypes (1, 6, 22, 24). To date, however, no internally controlled multiplex real-time PCR assay for the simultaneous detection and differentiation of all four serotypes relevant for humans has been reported.We describe here a highly specific and sensitive multiplex real-time PCR assay based on the 5′-nuclease TaqMan chemistry (17) for the simultaneous detection of the C. botulinum types A, B, E, and F, including an internal amplification control (IAC). Furthermore, we developed six different singleplex assays based on the TaqMan chemistry for the detection of C. botulinum serotypes A to F. Assays were validated on 42 C. botulinum strains, 57 non-C. botulinum strains, on spiked food samples, and on real samples from cases of botulism in Germany.