Yoshihito Fujinami

Rapid discrimination of Legionella by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Molecular typing is an important tool in the surveillance and investigation of human Legionella infection outbreaks. In this study, two molecular typing methods, pulsed-field gel electrophoresis (PFGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), were used to discriminate 23 Legionella pneumophila strains. The usefulness of MALDI-TOF-MS was demonstrated. The MALDI-TOF-MS fingerprinting with filtered small acid-soluble molecules gave different molecular profiles among strains, and the clustal analysis with MALDI-TOF-MS showed a high discrimination of strains the same as that with PFGE. In addition, MALDI-TOF-MS data could be generated within a few hours after the initial culture, although PFGE analyses took several days to complete. Thus, MALDI-TOF-MS offers a simple and rapid discrimination technique that could aid in the tracking of fast-spreading outbreaks of Legionella.

Sensitive Detection of Bacillus anthracis Using a Binding Protein Originating from γ-Phage

Detection of biological weapons is a primary concern in force protection, treaty verification, and safeguarding civilian populations against domestic terrorism. One great concern is the detection of Bacillus anthracis, the causative agent of anthrax. Therefore, there is a pressing need to develop novel methods for rapid, simple, and precise detection of B. anthracis. Here, we report that the C‐terminal region of γ‐phage lysin protein (PlyG) binds specifically to the cell wall of B. anthracis and the recombinant protein corresponding to this region (positions, 156–233), PlyGB, is available as a bioprobe for detection of B. anthracis. Our detection method, based on a membrane direct blot assay using recombinant PlyGB, was more rapid and sensitive than the γ‐phage test and was simpler and more inexpensive than genetic methods such as PCR, or immunological methods using specific antibodies. Furthermore, its specificity was comparable to the γ‐phage test. PlyGB is applicable in conventional methods instead of antibodies and could be a potent tool for detection of B. anthracis.

Expression of statherin mRNA and protein in nasal and vaginal secretions.

Nasal secretion has been regarded as one of the most difficult body fluids to identify and is especially difficult to discriminate from vaginal secretions and saliva. At present, few specific markers are known for nasal secretions. The aim of this study is to find a new approach for the identification of nasal secretions. We examined expression levels of statherin and histatin, peptides which are commonly found in saliva, in nasal and vaginal secretions by real-time RT-PCR and ELISA assays. Statherin mRNA was highly expressed in all nasal samples (dCt value=-1.49±1.10, n=8) and was detected even in 1-day-old 0.1-μL stains. However, the stability of mRNA in nasal stains was significantly (P<0.01) lower than in saliva. Low levels of statherin mRNA were detected in 4 of the 17 vaginal samples (dCt value=11.65-14.72). Histatin mRNA was not detected in any nasal or vaginal samples, although it was highly expressed in all saliva samples. ELISA assays with anti-statherin goat polyclonal antibody showed that statherin peptide was detected in all nasal and saliva samples even after dilution of more than 1000-fold. The statherin peptide was not detected in any vaginal samples, including samples that expressed low levels of statherin mRNA. The amount of statherin peptide in vaginal samples might be less than the limit of detection of this assay. In the present study, statherin was highly expressed in nasal secretions, but histatin was not. These markers may be useful for discriminating nasal secretions from vaginal secretions and saliva. However, the usefulness of these markers in practical forensic case samples has not yet been examined. Therefore, further research is required to establish the utility of these assays for identification of nasal secretions.

Applicability of ELISA detection of statherin for forensic identification of saliva

Statherin is a low molecular-weight phosphoprotein secreted from the parotid gland. Statherin mRNA was previously reported to be a useful marker for mRNA-based saliva identification. In this study, applicability of ELISA detection of statherin for forensic identification of saliva was investigated. The specificity and sensitivity of ELISA for detection of statherin were compared with those of ELISA for α-amylase and the Phadebas® amylase test. Statherin was specifically detected in saliva but not in other body fluids. In addition, statherin was successfully detected in aged saliva stains, mixed body fluids–saliva stains, and simulated casework samples. On the other hand, although ELISA for α-amylase showed higher sensitivity than ELISA for statherin, it was not specific enough to identify saliva. The Phadebas® amylase test also showed positive results in other body fluids that are known to have α-amylase activity; however, it is easy to use for screening forensic casework samples. In conclusion, ELISA for detection of statherin developed in this study could be an effective tool for the forensic identification of saliva because of its specificity for saliva among other body fluids. Forensic casework samples should be tested by ELISA detection or mRNA-based analysis for statherin, depending on the condition of the sample, to supplement presumptive tests for α-amylase, such as the Phadebas® amylase test.

Rapid and simple detection of Clostridium botulinum types A and B by loop-mediated isothermal amplification

Aims:  To develop a convenient and rapid detection method for toxigenic Clostridium botulinum types A and B using a loop‐mediated isothermal amplification (LAMP) method.

Evaluation of tools for environmental sampling of Bacillus anthracis spores.

This study describes the validation of sampling techniques used to detect biological warfare agents used in terror attacks. For this purpose, we tested the efficiencies of different sampling media and extraction solutions for the recovery of bacterial pathogens. We first used Bacillus cereus ATCC 4342 spores as a surrogate for highly pathogenic B. anthracis to compare recovery efficiencies of spores from four different surfaces. We used three different types of sampling swabs and four different solutions to extract spores from the swabs. The most effective sampling method employed rayon swabs moistened with water. The efficencies of the four extraction solutions did not differ significantly, although yields were highest using phosphate-buffered saline containing Tween 80 (PBS-T). Using rayon swabs and sterile water, we recovered B. cereus ATCC 4342 and B. anthracis spores with equivalent efficiencies. These findings indicate that because of its reduced pathogenicity and relative ease in handling (Biosafety Level 1), use of B. cereus ATCC 4342 will facilitate further optimization of techniques to detect B. anthracis.

Evaluation of the Universal Viral Transport system for long-term storage of virus specimens for microbial forensics

Forensic microbial specimens, including bacteria and viruses, are collected at biocrime and bioterrorism scenes. Although it is preferable that the pathogens in these samples are alive and kept in a steady state, the samples may be stored for prolonged periods before analysis. Therefore, it is important to understand the effects of storage conditions on the pathogens contained within such samples. To evaluate the capacity to preserve viable virus and the viral genome, influenza virus was added to the transport medium of the Universal Viral Transport system and stored for over 3 months at various temperatures, after which virus titrations and quantitative analysis of the influenza hemagglutinin gene were performed. Although viable viruses became undetectable 29 days after the medium was stored at room temperature, viruses in the medium stored at 4°C were viable even after 99 days. A quantitative PCR analysis indicated that the hemagglutinin gene was maintained for 99 days at both 4°C and room temperature. Therefore, long-term storage at 4°C has little effect on viable virus and viral genes, so the Universal Viral Transport system can be useful for microbial forensics. This study provides important information for the handling of forensic virus specimens.