Rapid method for detection of pathogen
The present specification discloses methods of detecting a pathogen of interest and components useful in carrying out these methods, including a pre-enrichment media, and enrichment media and a detection solution.
1. A method of detecting Salmonella in a sample, the method consisting essentially of:
a) incubating the sample in a liquid pre-enrichment medium consisting essentially of a peptone low growth nutrient component, a malachite green growth inhibiting agent, and a nonylphenol ethoxylate surfactant with bacteriostatic or bactericidal action, wherein the incubation is at about 35° C. to about 39° C. for about 14 to 15 hours, and wherein the sample is a food sample, a feces sample or a raw livestock sample:
b) incubating an aliquot of the pre-enrichment medium from step (a) in a separate liquid enrichment medium, the enrichment medium consisting essentially of a peptone high growth nutrient component and ferric ammonium citrate, wherein the incubation is at about 39° C. to about 43° C. for about 5-7 hours, wherein the aliquot of the pre-enrichment medium is about 1/50 to about 1/1000 a volume of the enrichment medium;
c) purifying the liquid enrichment medium to increase concentration of the Salmonella and/or decrease contaminants using an immuno-precipitation method employing antibodies for the Salmonella linked to magnetic particles; and
d) detecting the presence or absence of the Salmonella by analyzing an aliquot of the purified enrichment medium from step (c), using an electrochemical sensor-based detection method.
2. The method according to claim 1 , wherein the nonylphenol ethoxylate surfactant is selected from the group consisting of Nonoxynol-4 (NP-4), Nonoxynol-6 (NP-6), Nonoxynol-7 (NP-7), Nonoxynol-8 (NP-8), Nonoxynol-9 (NP-9), Nonoxynol-10 (NP-10), Nonoxynol-11 (NP-11), Nonoxynol-12 (NP-12), Nonoxynol-13 (NP-13), Nonoxynol-15 (NP-15), Nonoxynol-30 (NP-30), Nonoxynol-40 (NP-40), Nonoxynol-50 (NP-50), Nonoxynol-55 (NP-55), and Nonoxynol-70 (NP-70).
3. The method according to claim 1 , wherein the malachite green growth inhibiting agent is selected from the group consisting of malachite green dye and brilliant green dye.
4. The method according to claim 1 , wherein the aliquot of pre-enrichment medium in step (b) is about 1/50 to about 1/500 of the volume of the enrichment medium used in step (b).
5. The method according to claim 1 , wherein the ammonium ferric citrate is at a concentration of about 0.1 mg/m L to about 5.0 mg/m L.
6. The method according to claim 1 , wherein the electrochemical sensor-based detection method is a biosensor-based detection method.
7. The method according to claim 1 , wherein the Salmonella is Salmonella typhimurium or Salmonella typhi.
8. The method according to claim 1 , wherein the malachite green growth inhibiting agent is at a concentration of about 0.5% (v/v) to about 3% (v/v).
9. The method according to claim 1 , wherein the surfactant is at a concentration of about 0.5% (v/v) to about 5% (v/v).