Research On Gold Nanopariticle Lateral Flow Immunoassay For Detecting E. Coli O157:H7 In Pork

E. coli O157:H7 is a kind of food-borne pathogen which easily infects humans and animals through contaminated food. E. coli O157:H7 causes the greatest number of food poisoning all around the world. It often leads to acute diarrhea, vomiting, fever and septicemia. Thus, a method for detecting E. coli O157:H7 in a fast and accurate way is important. Traditional detection methods of E. coli O157:H7 would cost several days. It couses time consuming and laborious. China is the biggest pork consumptive country in the world. So it is important to control the E. coli O157:H7 contaminated for pork. A gold nanoparticle(AuNP) lateral flow immunoassay(LFIA) was studied to detect E. coli O157:H7 in pork.In this study, Au-LFIA was optimized for stability, sensitivity, and specitivity. Then, it was used for detecting E. coli O157:H7 in pork. False positive results were frequently appeared both in Au-LFIA by our lab and DuPontTM. A strain of Citrobacter freundii was isolated and identified from some pork’s samples. The Citrobacter freundii had cross-reactivity with these Au-LFIAs. An enrichment protocol instead of Chinese standard protocol(mEC) was optimized for eliminating this false positive. Modified MacConkey Broth(mCT-SMC) was optimized for enrichment of E. coli O157:H7 and inhibition of Citrobacter freundii at the optimized concentration of antibotics(5mg/L potassium tellurite and 0.10mg/L cefixime). This mCT-SMC was combined with Au-LFIAs for accurately detecting of E. coli O157:H7 in pork. E. coli O157:H7 was detected in 100% of pork contaminated at 2 CFU/g by this method. And it eliminated false positive by Citrobacter freundii. This method was apprepriate for detecting E. coli O157:H7 in pork.A novel AuNP-LFIAs was developed in this study for enhancing detection sensitivity of E. coli O157:H7. In the signal amplification direct LFIA method, anti-mouse secondary antibody-linked AuNP(anti-mouse-Ab-AuNP) was mixed with sample solution in an ELISA well, after which it was added to LFIA, which already contained anti-E. coli O157:H7 monoclonal antibody-AuNP(anti-E. coli O157:H7-mAb-AuNP) dispersed in the conjugate pad. Polyclonal antibody was the test line, and anti-mouse secondary antibody was the control line in nitrocellulose(NC) membrane. In the signal amplification indirect LFIA method, anti-mouse-Ab-Au NP was mixed with sample solution and anti-E. coli O157:H7-mAb-AuNP complex in ELISA well, creating a dual AuNP complex. This complex was added to LFIA, which had a polyclonal antibody as the test line and secondary antibody as the control line in NC membrane. The detection sensitivity of both LFIAs improved 20-fold and reached 1.14×103 CFU/mL. The 28 nm and 45 nm AuNPs were demonstrated to be the optimal dual AuNP pairs. Signal amplification LFIA was perfectly applied to the detection of milk samples with E. coli O157:H7 via naked eye observation.