SF6039 - Improvement and Validation of a Novel Protein Microarray Assay for Salmonella Serotyping

| Food Safety Research Programs 2004 Project Summaries Index Page |

Researcher:

Dr. Shu Chen, Laboratory Services Division, University of Guelph

Objectives:

To improve and validate and existing protein microarray-based assay for rapid, accurate and cost-effective Salmonella serotyping.

Specific Objectives:

1. To improve an existing protein microarray method for enhanced assay robustness and data quality for rapid, simple, accurate and cost-effective serotyping of 20 commonly isolated and clinically important Salmonella serovars.
2. To develop a computational method and create a customized software program to standardize and streamline data analysis, interpretation and reporting for the serotyping assay.
3. To validate the method under applied conditions for its future implementation in diagnostic laboratories.

Expected Benefits:

1. Improve and validate the system to make it operational by diagnostic labs. Assay conditions will be further optimized to enhance data quality and assay robustness.
2. A customized software program will be created to facilitate data analysis, interpretation and reporting.
3. A rapid, simple, accurate and cost-effective protein chip based-test and associated software that can be used for Salmonella serotyping by diagnostic labs. The availability of such a test will allow diagnostic labs to provide faster and cost-effective results to food industries and provide an avenue to enhance the safety of Ontario's food supplies.

Summary of Research Results:

Salmonella is one of the most important agents of human food-borne illness. Salmonella and Campylobacter account for over 90% of all reported cases of bacteria-related food poisoning. The annual cost of salmonellosis was estimated at more than $800 million for Canada. Salmonella isolates need to be characterized to accurately diagnose their infection, to understand the epidemiology of infection, to investigate outbreaks and to effectively prevent and minimize their transmission through the food chain and other routes. Traditionally, Salmonella isolates are characterized by serotyping, an immunological method that determines two surface structures (O and H antigens) of Salmonella cells. Currently, the method employs more than 250 O and H antibodies for the characterization of over 2,500 Salmonella serovars. To determine the O and H antigens that a Salmonella strain carries, the cells are tested against selected antibodies, one at a time. A positive reaction occurs if a correct antibody is chosen. A minimum of three antibody-antigen reactions is needed for recognition of a particular Salmonella serovar. The current serotyping method requires well-experienced technologists to perform, and takes at least 3 days to determine a serovar. We previously developed a prototype antibody microarray method that allowed rapid and simultaneous detection of multiple antibody-antigen reactions. The purpose of this research was to improve and evaluate the method using a large number (over 1000) of Salmonella strains under applied conditions.

In this research, we have improved method for Salmonella serotyping based on the microarray technology that allows detection of many antibody-antigen reactions at a time on a single glass slide. The method involved attachment of Salmonella antibodies onto microscopic slides, staining Salmonella cells with a fluorescent dye, capturing the fluorescent cells by the slides, and detection of the fluorescent signals using a microarray scanner. A serotyping antibody microarray consisting of 92 antibodies was constructed for identification of top 20 commonly identified and clinically important Salmonella serovars, representing 80-90% Salmonella isolated in Canada. The system was evaluated side-by-side with the standard slide agglutination method over a period of ten months using 582 target strains, belonging to the 20 common Salmonella serovars, as well as 422 strains of other 123 Salmonella serovars. The microarray profiles allowed complete or partial serovar identification for 96.5% of the target strains tested, and also allowed exclusion of all the non-target strains from the target serovars. The detection sensitivity and specificity were 84-100% and 85-100% respectively based on the minimum number of antibody-antigen reactions involved in the 20 common serovars. Because of its speed, accuracy, simplicity and ability to identify many antigens simultaneously, the antibody microarray-based assay has further demonstrated its potential as an alternative for Salmonella serotyping to the conventional immunological method. The method is now being used to characterize Salmonella strains isolated from Ontario poultry samples. The method will allow diagnostic laboratories to provide faster and cost-effective results to food industries and animal heath system. The methodology platform can probably be extended to perform detection and serotyping of other bacteria.