Background

Current methods for detecting harmful, and sometimes deadly, bacteria are often time consuming and lack the required specificity to be considered reliable. This is particularly an issue for Salmonella enterica serovar Enteritidis (SE), a pathogenic subspecies of Salmonella enterica. It, like other types of pathogenic bacteria, shares high similarity to its associated non-pathogenic relatives. Serological tests employing antibodies typically cross-react between pathogenic and non-pathogenic species due to high surface structure similarity. PCR and plasmid-borne sequence-based detection methods also have difficulty distinguishing between different strains of SE due to their similarity. A method designed to overcome the obstacle of species similarity would allow for the specific detection of SE among close relatives.

Description

LLNL scientists have invented a method that is able to identify SE specific sequences that can be utilized as diagnostics markers. The method, called suppression subtractive hybridization (SSH), is a PCR-based technique that identifies restriction fragments that are present in the target strain but not other strains. A set of restriction enzymes specific to the target species isolates their associated restriction fragments which are then amplified via PCR. These PCR amplicons are then validated for specificity by testing with primer pairs from several types of phages for the target pathogen and its close relatives. In this validation test, only the specific target pathogen would pair with its known phage primers, isolating it with high specificity even among its close relatives.

Advantages
  • Accurate identification of SE specific sequences
  • Limited number of steps required for SE identification simplifies the process
Potential Applications
  • Detection of SE pathogen in infected eggs or chicken coops to reduce risk of food-borne illness
  • Rapid screening of production areas, plants, or food distribution centers to lower risk of spread of infection
Development Status

LLNL currently holds patent 7,442,517 "Method for the detection of Salmonella enterica serovar Enteritidis" for this method (LLNL internal # IL-10943).

Reference: Identification by subtractive hybridization of sequences specific for Salmonella enterica serovar Enteritidis

Reference Number
43019