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Background: The aim of this study was to measure the changes in the bacterial bioburden in orthokeratology (OK) lens storage cases using the DNA dot hybridization assay (DHA) after forewarning patients about their bacterial contamination severity. Methods: Thirty-one OK lens wearers were prospectively enrolled in this study. Dot hybridization assay was used for serial measurements of bacterial bioburden in OK storage cases after lenses had been soaked for approximately 6 hr. After the first assessment, the lens wearers were informed of the extent of case contamination and the possible risk of microbial keratitis (MK), and best practices for lens care and lens case hygiene were reviewed and reinforced. A second assessment by the same DHA method was performed after approximately 6 months. Results: Two universal bacterial probes confirmed a significant decrease in bacterial bioburden at the second assessment (P

A sandwich enzyme-immunoassay performed on plastic sticks was developed to specifically identify Escherichia coli O157. Colonies of test bacteria grown on tryptic soy agar were suspended in phosphate-buffered saline, heated in a 100°C water bath for 15 min, and incubated with the plastic sticks coated with a commercial preparation of anti-E. coli O157 antibodies at 37°C for 1.5 h. After incubation, the same antibodies labeled with peroxidase were used to produce the signal of antigen-antibody reaction. For 35 strains of E. coli O157 (among them 34 were E. coli O157:H7) tested, all produced strong reactions by the immunoassay. For 162 strains of E. coli with somatic antigens other than O157 and 38 strains of other genera tested, only one strain (Salmonella bietri) produced a false-positive reaction. The specificity and sensitivity of the immunostick assay were 100% (35/35) and 99.5% (199/200), respectively. The detection limit of the assay for E. coli O157:H7 (CCRC 15991) was about 105 CFU/ml. The method, ...

The coagulase test is routinely used for the confirmation of suspect Staphylococcus aureus on Baird-Parker agar (BPA). Overnight (18 to 24 h) preincubation of suspect colonies in brain heart infusion (BHI) broth is generally practiced prior to the coagulase test. In order to shorten the test protocol, different preincubation times of S. aureus in BHI were evaluated. Stock cultures (257 strains) of S. aureus were subcultured on BPA, and single colonies of each strain on BPA were analyzed for coagulase activities by the conventional procedure except that the preincubation times in BHI were varied (0, 4, and 24 h). The formation of a clot was examined at 2-h intervals over a 6-h period and at 24 h, and any degree of clot formation (1+ to 4+) was considered a positive reaction. Low sensitivities were found for tests without preincubation in BHI prior to the enzyme test. However, for 4- and 24-h preincubation, there was no difference in the degree of clot formation and in the sensitivities of the coagulase tests with incubation periods from 6 (98.1%) to 24 h (99.6%). Compared with the conventional procedures which may need two days, the modified protocol (4-h preincubation in BHI) can identify a large majority (97.7-98.1 %) of suspect S. aureus on BPA within one working day.

The activities of coagulase and thermostable nuclease (TNase) and the production of protein A were studied in 338 bacterial strains. These included 213 isolates of Staphylococcus aureus to determine which characteristic was most specific for the identification of S. aureus. The evaluation of different protocols for interpretation of coagulase results was also undertaken. Protein A was analyzed by a sandwich enzyme-linked immunosorbent assay using microtiter plates coated with anti-protein A antibodies. Coagulase activities were determined according to the criteria recommended by Association of Official Analytical Chemists (AOAC ; any degree of clot formation is a positive reaction), American Public Health Association (APHA ; coagulase activities ≥3+ are positive reactions), and the Bacteriological Analytical Manual (BAM ; only 4+ reaction is positive). It was found that the AOAC protocol, which had a test sensitivity of 97.7% and a specificity of 95.1% and could be completed within six hours, was more practical than the methods used by APHA and BAM. Compared with coagulase and TNase, protein A was a better marker of S. aureus ; a high sensitivity (100%) and specificity (96.8%) were obtained by using protein A for the identification of S. aureus.

A latex agglutination test for the rapid identification of Vibrio parahaemolyticus has been developed. Two bacterial outer membrane proteins, with molecular weights of 36,000 and 34,000, were obtained by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and were subsequently used as antigens for producing antibodies in rabbits. Immunoelectron microscopy demonstrated that the two proteins were present in the outer membrane of the microorganism. Latex particles sensitized with the affinity-purified antibodies were used as a reagent for the rapid identification of the bacterium. Of 173 strains (including 94 isolates of V. parahaemolyticus , 40 isolates of other vibrios and 39 strains of other bacteria) tested, the false-negative and false-positive rates were 1.4 and 3.1%, respectively. Some strains of three vibrios ( V. alginolyticus , V. harveyi , and V. mimicus ) produced false-positive results. However, no cross-reactions were observed with the latex reagent among the 39 strains (33 species) of other bacteria. It is proposed that suspicious colonies (green or blue) of V. parahaemolyticus on thiosulfate-citrate-bile salts-sucrose agar (TCBS) be subcultured to tryptic soy agar with 3% NaCl for overnight incubation. Cultures grown on tryptic soyagar-3% NaCl may be used for latex agglutination and galactosidase assays. V. parahaemolyticus will be latex positive and galactosidase negative. Under this condition, V. parahaemolyticus can be identified in only one day after suspect colonies were observed on TCBS agar.

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the rapid detection of Staphylococcus aureus in foods. The assay was based on the detection of protein A which is a specific protein secreted by S. aureus . Following a 24-h incubation in a staphylococcal selective broth containing mannitol as the carbon source, the culture supematant was added to the microtiter plate coated with anti-protein A immunoglobulin G (IgG). After incubation, peroxidase-labeled anti-protein A IgG was used to produce the signal of antigen-antibody reaction. The sensitivity of the assay for protein A was 0.1 ng/ml. For 37 strains of S. aureus studied, all produced protein A, and the amount (13-1,100 ng/ml) of protein A secreted by different strains varied to a large degree. For another 57 strains (including 19 Staphylococcus spp.) of bacteria tested, two strains (5. capilis subsp. capitis CCRC 12161 and S. lentus CCRC 12926) produced very low amounts of protein A (0.6-1 ng/ml) after 24-h incubation. Staphylococcus aureus was detected by the ELISA in all of six samples of precooked foods naturally contaminated with the bacterium. Twenty-two processed foods artificially inoculated with S. aureus at levels of < 2 CFU/g and 10 to 20 CFU/g, respectively, were all positive by the ELISA. As compared to the conventional culture methods which take 5 to 6 days to complete, the ELISA can detect low numbers of S. aureus in processed foods with a total analytical time of only 28 h.

Purpose Developing a DNA dot hybridization model for diagnosing parasitic keratitis. Methods Newly designed oligonucleotide probes for detecting Acanthamoeba and microsporidia were tested with target reference strains of Acanthamoeba (n = 20) and microsporidia (n = 3), and non-target microorganisms, including bacteria (n = 20) and fungi (n = 20). These probes, which had passed the preliminary tests, were then assembled as a parasite dot hybridization (PDH) model for assessing 33 clinical samples from patients with clinically suspected Acanthamoeba and microsporidia keratitis, including eight positives for Acanthamoeba, 13 positives for microsporidia, and 12 negatives for both pathogens. Results Two probes for detecting Acanthamoeba and two for detecting microsporidia passed the tests using target and non-target strains and then were assembled in the PDH model. For clinical samples, one Acanthamoeba-positive sample (proved with pathology) was falsely negative according to the PDH assay. The sensitivity and specificity of the PDH assay for diagnosing Acanthamoeba keratitis were 87.5% and 100%, respectively, while the sensitivity and specificity for diagnosing microsporidia keratitis were 100%. The infectious agent of all clinical samples of microsporidia keratitis was identified as Vittaforma corneae with DNA sequencing, while those of Acanthamoeba keratitis were caused by four species of Acanthamoeba, with Acanthamoeba castellanii found in four samples (50%, 4/8). Conclusions The PDH model has the potential to be a molecular assay for diagnosing Acanthamoeba and microsporidia keratitis. However, a prospective clinical study might be needed before the model is adopted in routine clinical practice.

PURPOSE The aim of this study was to evaluate a DNA dot hybridization assay (DHA) for assessing bacterial bioburden in orthokeratology lens (OK) storage cases. METHODS Forty-one OK wearers participated in this study. The dot hybridization assay was used to assess the bacterial bioburden of OK after removal and 6-hour soaking in a storage case. Signals of the DHA were standardized after gray image transformation. The correlations between the hybridization intensities of three universal bacteria probes (BP1, BP2, and BP3) and bacterial bioburden determined by culture (colony forming units per milliliter) was analyzed by Pearson's correlation coefficient and receiver operating characteristic plots. In addition, three genus-specific probes for Pseudomonas, Acinetobacter, and Klebsiella were used to detect potentially hazardous bacterial contamination regardless of bacterial viability status. RESULTS Among the three universal probes, there were good correlations between probe BP2 (r2 = 0.31, P = 9.5 × 10(-5)) and probe BP3 (r2 = 0.35, P = 3.1 × 10(-5)) with bacterial bioburden, but no correlation was found between probe BP1 and bacterial bioburden (r2 = 0.04, P = 0.11). In 41 samples, one was Pseudomonas-positive by both DHA and culture, while 10 were Pseudomonas-positive by DHA but negative by culture. Furthermore, nine samples tested positive for Acinetobacter (n = 7) and Klebsiella (n = 2) by DHA only. CONCLUSIONS The dot hybridization assay provides a novel way to assess the bacterial bioburden of OK storage cases. Lens care quality can be assessed with universal bacteria probes, while potentially hazardous bacterial contamination can be traced with genus-specific probes.

PURPOSE We verified a multiplex dot hybridization (MDH) assay for the rapid detection and differentiation of Acanthamoeba keratitis (AK) and herpes simplex keratitis (HSK). METHODS Molecular detection of Acanthamoeba and herpes simplex virus in corneal scrapes was performed with the MDH assay and standard diagnostic methods. The experimental group included corneal scrapes (n = 33) from patients with culture- or pathology-confirmed AK (n = 15) and real-time PCR-confirmed HSK (n = 16). The control group included 50 samples from cases of bacterial keratitis (n = 15), fungal keratitis (n = 15), and initially presumed AK (n = 5) or HSK (n = 17) which finally were excluded by culture for Acanthamoeba or by real-time PCR for herpes simplex virus, respectively. Discrepant results between methods were resolved by DNA sequencing of the PCR amplicons. RESULTS After discrepant analysis, the sensitivity for the diagnosis of AK and HSK was both 93.3% by the MDH assay, while the specificity was 100% for the two types of keratitis. The turnaround time of MDH assay was within a working day using an already prepared array. Two false-negatives (one AK case and one HSK case) were obtained by the MDH assay. CONCLUSIONS The MDH assay could effectively prevent missed or delayed diagnosis of AK and HSK and has a potential to be adopted in routine clinical practice if the test is commercialized.

More than 20 species of Legionella have been identified in relation to human infections. Rapid detection and identification of Legionella isolates is clinically useful to differentiate between infection and contamination and to determine treatment regimens. We explored the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) Biotyper system (Bruker Daltonik GmbH, Bremen, Germany) for the identification of Legionella species. The MALDI MS spectra were generated and compared with the Biotyper database, which includes 25 Legionella strains covering 22 species and four Legionella pneumophila serogroups. A total of 83 blind-coded Legionella strains, consisting of 54 reference and 29 clinical strains, were analyzed in the study. Overall, the Biotyper system correctly identified 51 (61.4%) of all strains and isolates to the species level. For species included in the Biotyper database, the method identified 51 (86.4%) strains out of 59 Legionella strains to the correct species level, including 24 (100%) L. pneumophila and 27 (77.1%) non-L. pneumophila strains. The remaining 24 Legionella strains, belonging to species not covered by the Biotyper database, were either identified to the Legionella genus level or had no reliable identification. The Biotyper system produces constant and reproducible MALDI MS spectra for Legionella strains and can be used for rapid and accurate Legionella identification. More Legionella strains, especially the non-L. pneumophila strains, need to be included in the current Biotyper database to cover varieties of Legionella species and to increase identification accuracy.

Summary Objectives Bacteremia due to Acinetobacter baumannii complex (ABC), which composed of four genomic species (gen. sp.), is a serious and potentially fatal condition. The epidemiology and outcome of such infections due to individual gen. sp. remain undefined. Methods A retrospective study of patients with monomicrobial ABC bacteremia over six years was conducted at a medical center to determine the association of gen. sp. with clinical outcome. Results Included were 291 patients with monomicrobial ABC bacteremia. Of them, 222 (76.3%) patients had bacteremia caused by gen. sp. 2, i.e. A. baumannii . The presence of multidrug-resistant phenotype was the only independent predictor of Acinetobacter gen. sp. 2 bacteremia (adjusted odd ratio, 7.5; 95% confidence interval, 3.8–14.7; P Acinetobacter gen. sp. 2 bacteremia had a higher sepsis-related ( P = 0.006) and 30 day ( P = 0.028) mortality rates than the non- Acinetobacter gen. sp. 2 group. The fatal outcome was independently associated with high SPAS II scores ( P = 0.002), rapidly fatal underlying diseases ( P = 0.002), bacteremia caused by Acinetobacter gen. sp. 2 ( P = 0.01), inappropriate definitive antimicrobial therapy ( P P Conclusion Acinetobacter gen. sp. 2 bacteremia heralded a worse clinical outcome, and therefore the gen. sp. identification of ABC bacteremic isolates is justified.

The crystal structures of 2-chlorobenzophenone (1) and 2,5-dichlorobenzophenone (2) were obtained by single-crystal X-ray diffraction. Crystallization of 1 occurs in the centrosymmetric monoclinic space group P21/c (No. 14) with a = 8.2812(8), b = 16.5933(1), c = 7.7599(7); β = 98.488(5)° and Z = 4. Crystallization of 2 occurs in the centrosymmetric orthorhombic space group Pbca (No. 61) with a = 16.414(3), b = 7.7896(1), and c = 17.754(4); and Z = 8. Details of the structures and spectroscopic results are presented and discussed. Torsion angles about the carbonyl functionality were found to be outside the previously observed values for unsubstituted benzophenones by greater than 20°.

The effects of gallium additions on microstructures and thermal and mechanical properties of the Sn-9Zn solder alloys are investigated in this study. The results show that the melting temperature of the alloys decreases with the increase in the Ga concentration, while the pasty ranges of the alloys are simultaneously enlarged. By adding a 0.25–0.5 wt.% Ga element, the Sn-matrix region is slightly increased and the Zn-rich phase becomes slightly coarser; however, the overall microstructure is still very similar to that of the Sn-9Zn alloy. It is found that, when the Ga concentration is less than 0.50 wt.%, the ultimate tensile strength and elongation are maintained at the same values. The addition of a 0.25–0.50 wt.% Ga to the Sn-9Zn alloy also leads to small cup and cone fracture surfaces which exhibit near-complete ductile fracturing. With the addition being increased to 0.75 wt.%, larger cup and cone fractures are observed. The 1.00 wt.% Ga alloy has lower strength and ductility due to the coarser and nonuniform microstructures. However, the fracture surfaces of the 1.00 wt.% Ga alloy show partial cleavage and a partially dimpled fracture.

The purpose of this study was to compare the IMViC (indole, methyl red, Voges-Proskauer and citrate utilization) tests with the beta-glucuronidase (GUD) assay for the identification of suspect Escherichia coli on Levine's eosin-methylene blue (EMB) agar. After testing 258 suspect E. coli colonies from raw meat and meat products, 163 and 44 were found to be E. coli and non-E. coli, respectively, by both methods. Nine isolates were IMViC positive (i.e., + + - - or - + - -) but GUD negative; among these isolates, six were confirmed to be E. coli by API 20E (bioMerieux, Marcy-I'Etoile, France) with the remaining three being non-E. coli. There were 42 isolates that were IMViC negative but GUD positive; among these isolates, seven were pure E. coli cultures, 33 were mixed cultures containing E. coli, and the remaining two were Proteus spp. The sensitivities for the identification of E. coli on EMB were 80.9% (169/209) and 97.1% (203/209), respectively, by the IMViC tests and GUD assay; whereas the specificities were 93.9% (46/49) and 95.9% (47/49), respectively, by the IMViC tests and GUD assay. It is proposed that the GUD assay can be an effective alternative to the conventional IMViC tests for the identification of suspect E. coli on EMB.

Conventional procedures for the detection of Listeria monocytogenes require selective enrichment and isolation, followed by several potentially lengthy identification steps. To facilitate the identification of the bacterium, polyclonal antibodies against a 68-kDa cell-surface antigen of L. monocytogenes were used to develop a sandwich enzyme-linked immunosorbent assay (ELISA). Of 51 strains of L. monocytogenes tested, 50 (98%) produced positive results using the ELISA method, whereas 17 non- Listeria strains yielded negative reactions. However, the polyclonal antibodies were found to cross-react with L. innocua and some strains of L. welshimeri . Further purification of the antibodies by affinity chromatography was impractical, since the increase in sensitivity was accompanied with decreased specificity. It is proposed that a combination of the ELISA with a single biochemical test, such as phosphoinositol-specific phospholipase C (PI-PLC), arylamidase, or hemolytic activity could differentiate L. monocytogenes (PI-PLC [+], hemolysin [+], arylamidase [-]) from L. innocua and L. welshimeri (PI-PLC [-], hemolysin [-], arylamidase [+]) , thus considerably reducing the number of steps required and the length of time necessary for the identification of L. monocytogenes .

Fungal keratitis (FK) is an important cause of ocular morbidity, especially for people living in the agricultural communities of the developing world. Current diagnostic methods may lack sensitivity (direct microscopy) or are time consuming (culture). The aim of this study was to develop a dot hybridization assay for sensitive and rapid diagnosis of FK.Evaluation of diagnostic test or technology.Fifty corneal scrapes (49 patients) from consecutive cases of clinically suspected microbial keratitis were analyzed prospectively.Molecular detection of fungi in the scrapes was performed by amplification of the internal transcribed spacer region (ITS) that contained the target gene (5.8S rRNA gene) by polymerase chain reaction (PCR), followed by hybridization of the PCR product to a fungus-specific oligonucleotide probe immobilized on a nylon membrane. The results were compared with those obtained by gram-stain microscopy, culture, and gel electrophoresis of the PCR products. Discrepant results were resolved by cloning and resequencing of the amplified ITS fragments.Performance of the dot hybridization assay, including sensitivity, specificity, and positive and negative predictive values, was evaluated.Ten scrapes demonstrated positive results by both the dot hybridization assay and culture. However, 11 scrapes demonstrated positive results by the dot hybridization assay, but demonstrated negative results by culture, and 10 of the 11 samples were considered to be positive for FK by cloning and resequencing of the amplified ITS fragment and by a pathologic examination or clinical course review. The sensitivities for diagnosis of FK by the dot hybridization assay and culture were 100% and 50%, respectively, whereas the specificities were 96.7% and 100%, respectively.The dot hybridization assay is a highly sensitive and specific diagnostic tool for FK. The method provides a much higher sensitivity than that of culture (100% vs. 50%; P0.001). The hybridization procedure can be finished within a working day. It is expected that the method can have an impact on the diagnosis and treatment of FK in the future.The author(s) have no proprietary or commercial interest in any materials discussed in this article.

More than 20 species of Legionella have been identified in relation to human infections. Rapid detection and identification of Legionella isolates is clinically useful to differentiate between infection and contamination and to determine treatment regimens. We explored the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) Biotyper system (Bruker Daltonik GmbH, Bremen, Germany) for the identification of Legionella species. The MALDI MS spectra were generated and compared with the Biotyper database, which includes 25 Legionella strains covering 22 species and four Legionella pneumophila serogroups. A total of 83 blind-coded Legionella strains, consisting of 54 reference and 29 clinical strains, were analyzed in the study. Overall, the Biotyper system correctly identified 51 (61.4%) of all strains and isolates to the species level. For species included in the Biotyper database, the method identified 51 (86.4%) strains out of 59 Legionella strains to the correct species level, including 24 (100%) L. pneumophila and 27 (77.1%) non-L. pneumophila strains. The remaining 24 Legionella strains, belonging to species not covered by the Biotyper database, were either identified to the Legionella genus level or had no reliable identification. The Biotyper system produces constant and reproducible MALDI MS spectra for Legionella strains and can be used for rapid and accurate Legionella identification. More Legionella strains, especially the non-L. pneumophila strains, need to be included in the current Biotyper database to cover varieties of Legionella species and to increase identification accuracy.

A latex agglutination kit (AUREUS TEST™) for the rapid identification of Staphylococcus aureus was evaluated in this study. The reagent consists of polystyrene latex particles sensitized with rabbit anti-protein A immunoglobulin G and fibrinogen. Due to the respective binding of bacterial protein A with immunoglobulin G and coagulase with fibrinogen, an agglutination reaction occurs within 1 min when the latex is mixed with a suspension of S. aureus . Of 157 S. aureus isolates (138 from foods) and 110 non- S. aureus isolates (58 species belonging to 19 genera), the sensitivity and specificity of the latex test were 100 and 94.4-100%, respectively. The results were comparable to the conventional coagulase test. Therefore, it is proposed that suspicious colonies of S. aureus on Baird-Parker agar medium be subcultured to tryptic soy agar for overnight incubation. Cultures grown on tryptic soy agar are used for latex agglutination test. The latex agglutination test can be finished within minutes and could be used as an alternative rapid procedure of the coagulase test, which needs several hours or even overnight incubation for completion. In addition, all S. aureus isolates grown on Baird-Parker agar also could be correctly identified by the latex reagent.

Delayed and incorrect diagnoses are potential risk factors leading to high mortality of invasive aspergillosis (IA). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to acquire a wide mass spectral range and characterize the early process of asexual sporulation of lethal IA pathogens recovered on agar plates. Proteins were extracted using trifluoroacetic acid and soft ionized using an ultraviolet laser with the assistance of ferulic acid. At the second stage of sporulation with various differentiated structures, there are more specific peaks that can be used to discriminate different Aspergillus species than at the first stage, which features vegetative hyphae. Certain specific peaks are found in different strains of the same species, Aspergillus fumigatus. In addition, the relative standard deviations of the m/z ratios are much smaller than those of the relative intensities in these peaks. Therefore, common lethal Aspergillus species can be identified after short-term cultivation by matching species-specific m/z values.

Background and aim: We have characterized a membrane current associated with a decrease in pHi, which can be induced by either elevating intracellular calcium or extracellular application of methylmercury (a potent agent in elevating intracellular Ca2+ concentration) in the alveolar macrophages bathing in the impermeant bilateral cesium aspartate solution.Results: Decreasing pHi and elevating [Ca2+]i profoundly enhanced, but H-7 (a broad-spectrum kinase inhibitor), W-7 (a selective calmodulin antagonist) and KN-93 (a calmodulin kinase II inhibitor) inhibited the currents.Conclusion: These results indicate that rat alveolar macrophages possess a calcium-activated and pHi-sensitive proton channel which can be phosphorylated and activated by calmodulin kinase II.

This report describes the detection of Staphylococcus aureus in buffer and in several kinds of food by flow cytometry. Fluorescein isothiocyanate conjugated anti-protein A antibodies were used in a 4-h procedure to label cells, 105-106 cells/mL are needed. the use of single parameter, green fluorescence, enabled specific differentiation of S. aureus from other bacteria including 11 Staphylococcus species. the flow cytometric method can detect S. aureus in food samples after 48-h enrichment in trypticase soy broth with 10% NaCl. As low as 2 S. aureus cells present in 10 mL enrichment broth could grow to a population density detectable by the flow cytometric method after enrichment. This method was faster and less laborious than the conventional BAM (Bacteriological Analytical Manual) or AOAC (Association of Official Analytical Chemists) methods, and could be automated for analysis of S. aureus in food.

We report a rare case of hepatosplenic abscesses caused by Verticillium spp. in a 40-year-old female leukemic patient. Identification of the microorganism was based on amplification of the D1-D2 region of the large-subunit RNA gene from DNA extracted from paraffin sections of liver tissue, followed by sequencing of the amplified polymerase chain reaction product.

A modified immuno-polymerase chain reaction (immuno-PCR) for the detection of E. coli beta-glucuronidase (GUD) is described. Flexible polycarbonate microtiter plates (Biozyme, Landgraaf) with 96 V-bottomed wells were used throughout all steps including the antigen-antibody reaction and polymerase chain reaction. The plates were coated with anti-GUD antibodies to capture the antigen, which was then detected using biotinylated anti-GUD antibodies. Following this, avidin was used to bridge the biotinylated antibodies and biotinylated lamda phage DNA, which was amplified by PCR to produce a product of 500 nucleotides. Following optimization, the detection limit of the immuno-PCR for GUD was 1 x 10(-17) g/ml (or 5 x 10(-19) g/well); this is equivalent to two GUD molecules in a sample solution of 50 microliters. The method was used to detect GUD in a cell extract of E. coli, and it was found that the enzyme released from a single E. coli cell in a solution of 10 l could be detected. So far, this is the most sensitive method ever published for the detection of an antigen. In addition to high sensitivity, the present protocol is capable of automation.

A method of indirect immunofluorescence microscopy was developed for the rapid detection of Vibrio parahaemolyticus in oysters. Affinity-purified antibodies against two outer membrane proteins (molecular mass 36 and 34 kDa, respectively) of V. parahaemolyticus were used as the primary antibodies to label the vibrio, and fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G was used to reveal the antigen-antibody reaction. Of 85 strains of V. parahaemolyticus tested, all produced strong fluorescence under the fluorescence microscope. However, for 63 strains (including 27 vibrios) of other bacteria tested, 6 produced weak to moderate fluorescence. The sensitivity and specificity of the immunostaining technique were 100 and 90.5%, respectively. In the inoculation experiments, as low as 1.7 CFU/g of V. parahaemolyticus spiked in oysters could be detected by the immunostaining method after 18-h enrichment in alkaline peptone water containing 3% NaCl. The immunofluorescence microscopy is recommended for rapid screening of V. parahaemolyticus in oysters; a presumptive positive result could be obtained within 24 h.

The reaction of α,α,4-tri­chloro-α-phenyl-o-tolyl di­chloro­phosphinate with phenol leads to the selective substitution of the phospho­rus-bound chlorines by OPh groups. The product, 4-chloro-(α,α-di­chloro­benzyl)­phenyl di­phenyl phosphate, C25H18Cl3O4P, has a CCl2 group that remains intact.