Enrichment-ELISA for Detection of Salmonella typhi From Food and Water Samples

Objective Development of monoclonal antibody based sandwich enzyme linked immunosorbant assay （sELISA） for rapid detection of Salmonella enterica serovar typhi （S. typhi） from food and water samples and optimization of enrichment procedures for use with the developed sELISA to increase the detection sensitivity of the assay. Methods Spleen cells from BALB/c mice immunized with flagellin （H=d） antigen of S. typhi were fused with Sp2/0 myeloma cells. The hybridoma cell line specific to H=d antigen was established, characterized and ascites raised against one of these clones. The hyperimmune serum to flagellin antigen was raised in New Zealand White rabbits. An sELISA was developed using polyclonal antibody as capture and monoclonal antibody as detection antibody. To design the efficient culture strategies for use with the sELISA, different pre-enrichment and enrichment broths were evaluated. The media included buffered peptone water （BPW） and brain heart infusion broth for pre-enrichment and selenite F broth and Rappaport-Vassiliadis broth as enrichment broths. The developed sELISA with preceding enrichment step in BPW （Enrichment-ELISA） was evaluated in various food samples artificially inoculated with S. typhi bacteria. Various food （30） and water （35） samples collected from field were also tested by Enrichment-ELISA and culture method. Results Out of four specific clones to H=d antigen, one clone （# 2/56, IgG2a isotype） was used in sELISA. The sELISA had the detection limit of 10^4-10^5 cfu of S. typhi. Of the various broths used with sELISA, BPW was found to yield maximum ELISA values. Enrichment-ELISA, when tested in artificially inoculated food samples, generally, could detect 10^2 S. typhi cfu/mL within 10 h from various food rinses （meat, vegetable） and milk samples. After overnight enrichment in BPW, as less as 2 bacteria per 10 mL of milk, meat rinse, and chicken rinse could be detected. Only one of the field samples （water） gave false positive result by Enrichment-ELISA. Conclusion In comparison to culture, the Enrichment-ELISA is a rapid, sensitive, and specific method for detection of S. typhi from food or water samples. This method may be used as rapid screening procedure for environmental monitoring during outbreak situation.

A Novel Non Radioactive PCR-DNA Probe for the Detection of Aflatoxin Producing <i>Aspergillus</i>Species from Major Food Crops Grown in India

In the present study, a novel non radioactive digoxigenen labelled PCR-DNA probe was developed targeting nor-1 gene to assess the contamination of aflatoxigenic Aspergillus species in food grain samples from Southern parts of India. The sensitivity of developed PCR-DNA probe was determined to be 10 pg of genomic DNA and 1 pg of purified PCR product. The specificity of the DNA probe was validated by testing against an array of Aspergillus, Fusarium and Penicillium spp. A total of 89 Aspergillus isolates were recovered from 152 grain samples of maize, paddy, and groundnut. Among them, maize had the highest (90%) incidence of toxigenic Aspergillus species. When developed PCR-DNA probe was evaluated onto pure cultures of toxigenic and nontoxigenic Aspergillus species, 60 samples were positive for the nor-1 gene probe. DNA probe results unequivocally matched with the HPLC analysis. In conclusion, the novel PCR-DNA probe developed in this study may find its application in rapid detection of aflatoxin-producing Aspergillus isolates from contaminated cereal grains.