Development of a monoclonal antibody for specific detection of Vibrio parahaemolyticus and analysis of its antigen.

Vibrio parahaemolyticus is a major foodborne pathogen worldwide. Contamination of V. parahaemolyticus in foods must be detected as quickly as possible because raw seafood, a major source of V. parahaemolyticus infection, is shipped immediately after production due to its short expiration date. In this study, we generated monoclonal antibodies (mAbs) against V. parahaemolyticus to develop a rapid and specific detection assay. Obtained mAbs were categorized into four groups according to their specificity. Of the groups, Group 1 (mAb VP7, VP11, and VP24) reacted to O1-O12 of V. parahaemolyticus without cross-reaction with human pathogenic Vibrio spp. (V. alginolyticus, V. cholerae, V. fluvialis, V. furnissii, V. mimicus, and V. vulnificus). We developed an immunochromatographic (IC) strip for the rapid detection of V. parahaemolyticus in the field using VP7 as a membrane-immobilized antibody and VP24 as a colloidal gold-conjugated antibody. The IC strip detected any and all serogroups (O1 to O12) or isolates (clinical, food, and environmental strains) of V. parahaemolyticus, regardless of the presence of virulence factors thermostable direct hemolysin (TDH) or TDH-related hemolysin (TRH). It did not cross-react with any other non-V. parahaemolyticus strains tested. To elucidate the target of the IC strip, we analyzed the antigen recognized by these mAbs. Group 1 mAbs showed two specific bands at molecular masses of approximately 11 and 16 kDa by western blotting analysis. Nano liquid chromatography mass spectrometry (LC-MS)/MS analysis revealed that the candidate antigen recognized by these mAbs was outer membrane (OM) lipoprotein Q87G48. We verified that mAb VP7 detected His-tagged OM lipoprotein synthesized by reconstituted cell-free protein synthesis reagent. Reactivity to an N-terminus deletion form and protease digestion form of the OM lipoprotein showed that the extent of epitope recognized by VP mAbs was 22nd-41st amino acids (AAs) from N-terminus of the OM lipoprotein, with the sequence "22SDDAATANAAKLDEL36." This region was also confirmed to be a V. parahaemolyticus-specific sequence by comparing putative orthologs of OM lipoprotein among Vibrio spp. The C-terminus deletion form (1st-39th AAs) including the sequence primarily recognized by VP mAbs (22nd-36th AAs) showed poor reactivity, indicating that the sequence after 40 residues of OM lipoprotein is also important for recognition by VP mAbs and VP mAbs recognize a conformational epitope. Bioinformatics research demonstrated that the OM lipoprotein is an ortholog of the lpp protein conserved throughout many bacteria. Lpp is an abundant and constitutively expressed protein and exists on the bacterial surface, suggesting it may be a good target for detection of V. parahaemolyticus.

Development of a Rapid and Versatile Method of Enzyme-Linked Immunoassay Combined with Immunoaffinity Column for Aflatoxin Analysis.

We combined immunoaffinity column (IAC) and enzyme-linked immunosorbent assay (ELISA) methods to develop a rapid method to analyze total aflatoxin (AF) in foods, using a large number of samples. Using newly developed monoclonal antibodies, with high cross-reactivity and high organic solvent tolerance, we developed the IAC-ELISA method. Our IAC-ELISA method showed a good correlation with the high-performance liquid chromatography method for corn samples spiked with total AF. Certain food samples, such as chili powder, chocolate, green coffee beans, and roasted coffee beans, are difficult to measure owing to their matrices, which affect ELISA adversely. Our IAC-ELISA method clearly improved the recovery rates (79 to 109%) compared with the ELISA method (97 to 164%) for four food samples. Our method is simple and quick; thus, it may be ideal for routine inspections.

Development of a rapid immunochromatographic assay to detect contamination of raw oysters with enteropathogenic Vibrio parahaemolyticus.

Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are major virulence factors of enteropathogenic Vibrio parahaemolyticus. TDH and TRH are bacterial exotoxins, and their presence in culture medium serves as a specific marker for detecting this significant pathogen. Here, we developed and evaluated an immunochromatographic assay (TDH/TRH-ICA) to simultaneously or individually detect TDH and TRH. The TDH/TRH-ICA detected TDH in all broth cultures of 47 V. parahaemolyticus strains carrying tdh. The genes encoding TRH are classified as variants trh1 and trh2, and TRH was detected in all broth cultures of 25 V. parahaemolyticus strains carrying trh1 and certain proportion (5/31) of broth cultures of V. parahaemolyticus strains carrying trh2. In contrast, TDH and TRH were not detected in broth cultures of 12 non-enteropathogenic V. parahaemolyticus strains without tdh and trh. It was difficult to detect TRH2 using the TDH/TRH-ICA. However, TRH2 may not serve as a suitable marker for detecting enteropathogenic V. parahaemolyticus, and evidence indicates that TRH2 may not contribute to enteropathogenesis. Further, a screening method using a combination of TDH/TRH-ICA and SPP medium supplemented with 1.5% NaCl (modified-SPP medium) detected oyster samples artificially spiked with 1.1-22 colony-forming units of enteropathogenic V. parahaemolyticus per 25g of oysters within approximately 8.5h, including the enrichment culture. The assay may serve as a method that facilitates the rapid and easy detection of raw oysters contaminated with enteropathogenic V. parahaemolyticus.

Evaluation of an immunochromatographic assay for direct identification of thermostable direct hemolysin-producing Vibrio parahaemolyticus colonies on selective agar plates.

We evaluated the utility of an immunochromatographic assay (NH IC TDH) in identifying thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus colonies on selective agar plates. The sensitivity of the NH IC TDH assay was 100% (189 samples) and its specificity was 100% (41 samples) compared with the presence of tdh.

Simple, rapid, and reliable detection of Escherichia coli O26 using immunochromatography.

Shiga toxin-producing Escherichia coli (STEC) O26 has been increasingly associated with diarrheal disease all over the world. We developed an immunochromatographic (ic) strip for the rapid detection of E. coli O26 in food samples. To determine the specificity of the IC strip, pure cultures of 67 E. coli and 22 non-E. coli strains were tested with the IC strip. The IC strip could detect all (18 of 18) E. coli O26 strains tested and did not react with strains of any other E. coli serogroup or non-E. coli strains tested (0 of 71). The minimum detection limits for E. coli O26 were 2.2 × 10(3) to 1.0 × 10(5) cfu/ml. To evaluate the ability of the IC strip to detect E. coli O26 in food, 25-g food samples (ground beef, beef liver, ground chicken, alfalfa sprout, radish sprout, spinach, natural cheese, and apple juice) were spiked with E. coli O26. The IC strip was able to detect E. coli O26 at very low levels (approximately 1 cfu/25 g of food samples) after an 18-h enrichment, and the IC strip results were in 100% agreement with the results of the culture method and pcr assay. When 115 meat samples purchased from supermarkets were tested, 5 were positive for E. coli O26 with the IC strip; these results were confirmed with a pcr assay. These results suggest that the IC strip is a useful tool for detecting E. coli O26 in food samples.

Potential rapid and simple lateral flow assay for Escherichia coli O111.

We developed and evaluated a lateral flow assay (LFA) as a simple and rapid method for direct detection of Escherichia coli O111 in food after enrichment. When cell suspensions of 8 E. coli O111 strains and 77 non-E. coli O111 strains were tested with the LFA, the former all yielded positive results and the latter all yielded negative results. The minimum detection limits for the E. coli O111 strains were 1.8 × 10(3) to 5.6 × 10(5) CFU/ml of cell suspension, and the LFA was able to detect live cultures or those killed by autoclaving at nearly the same level of sensitivity. To evaluate the ability of LFA to detect its target in food, enrichment cultures of meat samples inoculated with 10-fold serial dilutions of E. coli O111 were tested with the LFA and PCR. Even when there were very few E. coli O111 cells in the meat samples (1.6 × 10(0) to 1.6 × 10(1) CFU/25 g of food), when they were cultured in modified E. coli broth with novobiocin for 22 h at 42°C, the LFA yielded positive results that corresponded to the PCR results. Although the LFA requires further evaluation and field study, these results suggest that this assay has sufficient sensitivity and specificity. This procedure can be completed with a one-step incubation after the test strip has been inserted into the sample after 22 h of culture, whereas the standard culture method requires multiple cultures, skilled personnel, a well-equipped laboratory, and 4 or 5 days. The speed and simplicity of this LFA make it suitable for use as part of routine screening assays in the food industry.

Development of a novel multiplex lateral flow assay using an antimicrobial peptide for the detection of Shiga toxin-producing Escherichia coli.

The binding capacity of peptides with broad antimicrobial activity, or antimicrobial peptides (AMPs), to microbes has recently been applied to the specific detection of bacteria and viruses. We established a novel lateral flow assay (LFA) that combines AMPs labeled with colloidal gold and a target-specific antibody immobilized on a nitrocellulose membrane. α-Helical AMPs, especially cecropin P1 (CP1), magainin 2 (MG2), and ceratotoxin A (CtxA), were shown to have optimal properties as probes in LFA. We also established a multiplex LFA for the simultaneous detection and identification of three serogroups of Shiga toxin-producing Escherichia coli (STEC) using the CP1 probe with polyclonal antibodies anti-O157, anti-O26, and anti-O111. Each serogroup of E. coli could easily and rapidly be detected by multiplex LFA using CP1 and each was clearly visualized in a different position on the LFA strip. The multiplex LFA could detect all tested E. coli strains from serogroups O157 (22/22), O26 (17/17), and O111 (7/7), and the detection limit was 10(4)CFU/mL. No other serogroups of E. coli, including STEC O45, O91, O103, O121, and O145, or non-E. coli strains, reacted. The multiplex LFA could detect E. coli O157, O26, and O111 in food samples at very low levels (6.3, 2.9, and 5.6 CFU per 25 g of ground beef, respectively) after 18-h enrichment, and these results were in accordance with the results of the culture method, immunochromatography (IC) strip, and PCR. Given the broad binding capacity, AMP probes in combination with specific antibodies in the novel multiplex LFA may have the potential to detect various microbes simultaneously with identification on a single strip.

Genotypic change of porcine circovirus type 2 on Japanese pig farms as revealed by restriction fragment length polymorphism analysis.

Porcine circovirus type 2 (PCV2) has been recognized as the causal agent of postweaning multisystemic wasting syndrome and can be divided into two major genotypic groups. We developed a method of restriction fragment length polymorphism (RFLP) analysis of PCV2 open reading frame 2 for easy discrimination between the two major groups. Genotyping of PCV2 isolates from 10 Japanese commercial pig farms was performed, and the analysis revealed that both PCV2 groups and at least five RFLP types of PCV2 are prevalent in Japan. On two farms, the genotypes of the PCV2 isolates in the spring of 2007 were different from those in the autumn of 2006. One genotype may have become dominant within only six months on these farms.