Comparison of real-time and nested PCR assays for detection of herpes simplex virus DNA

We performed a real-time PCR assay to detect herpes simplex virus (HSV) DNA, and compared it prospectively with a nested PCR assay in 164 clinical samples (109 cerebrospinal fluid and 55 sera) from patients suspected of having neonatal HSV infection or HSV encephalitis. In 25 of 164 samples, HSV DNA was detected by the nested PCR assay. All samples positive for HSV DNA in the nested PCR assay were also positive in the real-time PCR assay, and all but two samples negative for HSV DNA in the nested assay were negative in the real-time assay. The real-time PCR assay thus had a sensitivity of 100% and a specificity of 99%, when compared with the nested assay. Sequential assays in a case of disseminated HSV showed that a decrease in HSV DNA paralleled clinical improvement. Quantification of HSV DNA by real-time PCR was useful for diagnosing and monitoring patients with HSV encephalitis and neonatal HSV infection.     

Characterization of six type A strains of Clostridium botulinum that contain type B toxin gene sequences

Six Clostridium botulinum isolates exhibiting type A toxicity as measured by the mouse bioassay were found to contain both type A and type B neurotoxin DNA sequences. The six strains were divided into three groups based on the DNA sequence of the type B neurotoxin gene. Members of each group exhibited 100% sequence identity over the 3876 bp type B toxin open reading frame. The type B toxin sequence of all groups differed at more than 60 positions when compared to the BGB control strain.     

Simple Method for Detection of Clostridium botulinum Type A to F Neurotoxin Genes by Ploymerase Chain Reaction

A polymerase chain reaction (PCR)-based method was established to detect each type of neurotoxin genes of Clostridium botulinum types A to F by employing the oligonucleotide primer sets corresponding to special regions of the light chains of the neurotoxins. In this procedure, the PCR products were easily confirmed by restriction enzyme digestion profiles, and as little as 2.5 pg of template DNAs from toxigenic strains could be detected. The specific PCR products were obtained from toxigenic C. botulinum types A to F, a type E toxin-producing C. butyricum strain, and a type F toxin-producing C. baratii strain, but no PCR product was detected in nontoxigenic strains of C. botulinum and other clostridial species. The neurotoxin genes were also detected in food products of a seasoned dry salmon and a fermented fish (Izushi) which had caused type E outbreaks of botulism. Therefore, it is concluded that this PCR-based detection method can be used for the rapid diagnosis of botulism.     

Immunological Characterization of the Neurotoxin Produced by Clostridium botulinum Type A Associated with Infant Botulism in Japan

The neurotoxin associated with type A infant botulism in Japan shows different antigenic properties from those produced by authentic strains. The monoclonal antibodies recognizing the light chain reacted to both neurotoxins, whereas half the antibodies recognizing the heavy chain reacted specifically to the respective neurotoxin. Each neurotoxin showed its own manner of binding to brain synaptosomes. These results indicate that the distinguishable characteristics are ascribable to the heavy chain but not to the light chain. In both neurotoxins, an epitope recognized by the monoclonal antibody that reacts to the light chain and neutralizes the toxin was found to be very close to the amino-terminal half (H-1 fragment) of the heavy chain. This may support the hypothesis that the H-1 fragment functions in the transport of the light chain in the target cell.     

Production of monoclonal antibodies against Clostridium botulinum type E derivative toxin

Abstract Five monoclonal antibodies (MCA; E–8–2, 9–1, 11–2, 12–4, and 13–1) against Clostridium botulinum type E derivative toxin were prepared. Their ELISA titers were higher than or equivalent to that of conventional polyclonal antibody. Three of them (E-8–2, 12–4, and 13–1) possessed the neutralizing activity comparable to that of polyclonal antibody. The results of binding-competition experiments indicated that the monoclonal antibodies bound to different sites on the type E toxin molecule. Immunoblotting analyses demonstrated that E-8–2, 9–1, and 11–2 react to fragment I (heavy chain) of the toxin. By use of these monoclonal antibodies, it may be possible to scrutinize the structure-function relationship of botulinum toxins and cross reactions between type E and F toxins.     

Sensitive and rapid quantitative detection of anthrax spores isolated from soil samples by real-time PCR

Quantitative analysis of anthrax spores from environmental samples is essential for accurate detection and risk assessment since Bacillus anthracis spores have been shown to be one of the most effective biological weapons. Using TaqMan real-time PCR, specific primers and probes were designed for the identification of pathogenic B. anthracis strains from pag gene and cap gene on two plasmids, pXO1 and pXO2, as well as a sap gene encoded on the S-layer. To select the appropriate lysis method of anthrax spore from environmental samples, several heat treatments and germination methods were evaluated with multiplex-PCR. Among them, heat treatment of samples suspended with sucrose plus non-ionic detergent was considered an effective spore disruption method because it detected up to 10(5) spores/g soil by multiplex-PCR. Serial dilutions of B. anthracis DNA and spore were detected up to a level of 0.1 ng/ microliters and 10 spores/ml, respectively, at the correlation coefficient of 0.99 by real-time PCR. Quantitative analysis of anthrax spore could be obtained from the comparison between C(T) value and serial dilutions of soil sample at the correlation coefficient of 0.99. Additionally, spores added to soil samples were detected up to 10(4) spores/g soil within 3 hr by real-time PCR. As a consequence, we established a rapid and accurate detection system for environmental anthrax spores using real-time PCR, avoiding time and labor-consuming preparation steps such as enrichment culturing and DNA preparation.     

C terminal half fragment (50 kDa) of heavy chain components of Clostridium botulinum type C and D neurotoxins can be used as an effective vaccine

Recombinant whole heavy chains (H, 100 kDa) and their N-terminal (Hn, 50 kDa) and C-terminal (Hc, 50 kDa) half fragments of Clostridium botulinum type C and D neurotoxins were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. GST eliminated-preparations of H (10 microg), Hn (5 microg), Hc (5 microg), or a mixture of Hn (5 microg) and Hc (5 microg) of types C and D were mixed with an equal volume of adjuvant, and then were twice injected into mice subcutaneously. After immunization, the mice were challenged with up to 10(6) the minimum lethal doses (MLD)/0.5 ml of C or D toxin, the type of which was same as that of the immunogens. All of the mice immunized with antigens except for Hn survived against 10(5) to 10(6) MLD/0.5 ml of the toxins, but the mice immunized with Hn were killed by 100 MLD/0.5 ml. The mice immunized with a mixture of C-Hc and D-Hc, each 5 microg, also showed a high level of resistance against both C and D toxins. Antibody levels immunized with GST fused-or GST eliminatedpreparation were quite similar. These results indicate that recombinant GST-fused Hc can be used as a safe and effective vaccine for type C and D botulism in animals. It also became clear that one time inoculation with a large amount of C-Hc or D-Hc, 100 microg, is useful for vaccine trials in mice.     

A novel type A2 neurotoxin gene cluster in Clostridium botulinum strain Mascarpone

The partial nucleotide sequence ( approximately 10 kb) of the cluster of genes encoding the botulinum neurotoxin complex in Clostridium botulinum type A strain Mascarpone was determined. The analysis revealed six ORFs (orfs), which were organized as in the type A2 and type A3 botulinum neurotoxin gene clusters of strains Kyoto-F and NCTC 2916, respectively. While the orfs at the proximal and distal ends of the sequence (orfX2 and bont/A genes) shared a high level of similarity with the corresponding sequences of strain Kyoto-F, the segment encompassing the orfX1 and botR/A genes within the sequence exhibited a higher degree of homology to the related region in strain NCTC 2916. The mosaic structure of the Mascarpone neurotoxin gene cluster suggests recombinational exchanges.     

Cloning of a Clostridium botulinum type B toxin gene fragment encoding the N-terminus of the heavy chain

Two lambda gt11 clones of the toxin gene of Clostridium botulinum type B were identified by the monoclonal antibody specific to the heavy chain of type B toxin. Neither of the expressed fusion proteins from the lysates of lysogenic E. coli Y1089 showed any botulinal toxic activity. One of the clones hybridized to the oligonucleotide probe which was synthesized according to the amino acid sequence of N-terminus of heavy chain. The sequence analysis revealed that highly homologous regions in N-terminus of heavy chain exist among botulinum neurotoxins (type A, B) and tetanus toxin on the amino acid sequence level.     

Difficulties of molecular dissociation of Clostridium botulinum type G progenitor toxin

Clostridium botulinum type G progenitor toxin was chromatographed on DEAE-Sephadex and Q-Sepharose equilibrated with 0.05 M Tris-HCl buffer, pH 8.0, containing 0.2 M urea. The toxin was eluted in a single protein peak from DEAE-Sephadex, but it was eluted in four protein peaks from Q-Sepharose; the third peak was toxic and the others were nontoxic. The third peak, appearing to be the toxic component, had a molecular mass of 150,000. In SDS-polyacrylamide gel electrophoresis, purified type G progenitor toxin migrated in six bands, with molecular masses of 150,000, 140,000, 58,000, 10,800, 10,600, and 10,400. Type G progenitor toxin may be composed of a toxin component with a molecular mass of 150,000 and a nontoxic component in a manner similar to progenitor toxins of other types. Type G toxic component, whether it was reduced or not, migrated in a single band to the same relative positions in SDS-PAGE; type A toxic component reduced with 2-mercaptoethanol migrated in two bands.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum toxin type A.

The enzyme linked immunosorbent assay using the so-called "double-sandwich technique" has been applied to determine botulinum toxin type A. By this assay, 50-100 mouse ip LD50 of toxin type A can be detected. No cross-reaction occurs with botulinum toxins of other types tested. In all probability this is due to the high specificity of the antiserum prepared against the toxic component of type A toxin.     

Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producing Clostridium botulinum, Clostridium baratii and Clostridium butyricum

Aims:  To develop real-time PCR assays for tracking and tracing clostridia responsible for human botulism.
Methods and Results:  Real-time PCR assays based on the detection of the genes ntnh encoding the nontoxin-nonhaemagglutinin (NTNH) proteins or the most homologous regions of the botulinum neurotoxin ( bont ) genes have been developed together with four real-time PCR assays, each being specific of the genes bont/A , bont/B , bont/E , bont/F and enables a toxin type-specific identification. The specificity of the assays was demonstrated using a panel of botulinum toxin producing clostridia (29 strains), nonbotulinum toxin producing clostridia (21 strains) and various other bacterial strains. The toxin type-specific assays had a sensitivity of 100 fg–1000 fg of total DNA in the PCR tube (25–250 genome equivalents) which correspond to 10³ to 10⁴ cells ml⁻¹. After a 48 h enrichment in anaerobic conditions, these PCR assays allowed the detection of Clostridium botulinum type A in a naturally contaminated sample of 'foie gras' suspected in a C. botulinum outbreak.
Conclusion:  These PCR tests are specific and reliable for detection of heterogeneous BoNT producing clostridia responsible for human botulism.
Significance and Impact of the Study:  Adoption of these PCR assays is a step forward a reliable and rapid detection of these clostridia in food samples.     

A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth

Aim: To develop a real‐time PCR‐based strategy for the detection of Paenibacillus larvae vegetative cells and spores to improve the diagnosis and the screening of American foulbrood (AFB), the most harmful pathology of honeybee brood. Methods and Results: A real‐time PCR that allowed selective identification and quantification of P. larvae 16S rRNA sequence was developed. Using standard samples quantified by flow cytometry, detection limits of 37·5 vegetative cells ml^?1 and 10 spores ml^?1 were determined. Compared to spread plate method, this real‐time PCR‐based strategy allowed, in only 2 h, the detection of P. larvae in contaminated honeys. No false‐positive results were obtained. Moreover, its detection limit was 100 times lower than that of the culture method (2 vs 200 spores g^?1 of honey). Conclusion: A rapid, selective, with low detection limit, sensitive and specific method to detect and quantify vegetative cells and spores of P. larvae is now available. Significance and Impact of Study: In addition to honey samples, this real‐time PCR‐based strategy may be also applied to confirm AFB diagnosis in honeybee brood and to screen other apiary supplies and products (bees, pollen, wax), thus broadening the control of AFB spreading.     

A quantitative TaqMan PCR assay for the detection of Mycoplasma suis

Aim: To develop a TaqMan probe‐based, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of Mycoplasma suis in the blood of pigs. Methods and Results: Primers and probes specific to Myc. suis 16S rRNA gene were designed. The qPCR assay’s specificity, detection limit, intra‐ and inter‐assay variability were evaluated and its performance was compared with a Myc. suis conventional PCR assay (cPCR). Blood of two experimentally infected pigs, 40 Indiana pigs, 40 Brazilian sows and 28 peccaries were tested. The assay detected as few as ten copies of Myc. suis plasmids and was 100‐fold more sensitive than the cPCR. No cross‐reactivity with nontarget pig mycoplasmas was observed. An average of 1·62 × 10¹¹ and 2·75 × 10⁸ target copies ml^?1 of blood were detected in the acutely and chronically infected pigs, respectively. Three (7·5%) pigs and 32 (80·0%) sows were positive while all peccaries were negative for Myc. suis. Conclusion: The developed qPCR assay is highly sensitive and specific for Myc. suis detection and quantification. Significance and Impact of the Study: TaqMan qPCR is an accurate and quick test for detection of Myc. suis infected pigs, which can be used on varied instrumentation platforms.     

Sequence of the gene encoding type F neurotoxin of Clostridium botulinum

Primers designed to conserved regions of botulinum and tetanus clostridial toxins were used to amplify DNA fragments from non-proteolytic Clostridium botulinum type F (202F) DNA using polymerase chain reaction technology. The fragments were cloned and the complete nucleotide sequence of the gene encoding type F toxin determined. Analysis of the nucleotide sequence demonstrated the presence of an open frame encoding a protein of 1274 amino acids, similar to other botulinum neurotoxins. Upstream of the toxin gene is the end of an open reading frame which encodes the C-terminus of a protein with homology to non-toxic-non-hemagglutinin component of type C progenitor toxin.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type E toxin.

The enzyme-linked immunosorbent assay using the "double-sandwich" technique was utilized to determine Clostridium botulinum type E toxin. With this technique, about 80 mouse intraperitoneal 50% lethal doses of toxin could be detected. Cross-reaction was hardly observed with C. botulinum type A and B toxins. No cross-reaction was observed with culture supernatants of C. botulinum type C or other Clostridium strains. In all probability this was due to the high specificity of the antiserum prepared aginst the toxic component of type E toxin.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type E toxin.

The enzyme-linked immunosorbent assay using the "double-sandwich" technique was utilized to determine Clostridium botulinum type E toxin. With this technique, about 80 mouse intraperitoneal 50% lethal doses of toxin could be detected. Cross-reaction was hardly observed with C. botulinum type A and B toxins. No cross-reaction was observed with culture supernatants of C. botulinum type C or other Clostridium strains. In all probability this was due to the high specificity of the antiserum prepared aginst the toxic component of type E toxin.