Immuno Colloidal Gold Test Strips For Listeria Monocytogenes, Listeriolysin O And Three Staphylococcal Enterotoxins In Food

Listeria monocytogenes, belonging to Listeria genus, is a kind of pathogenic bacteria whichcan cause human and animal diseases such as meningitis, encephalitis, sepicaemia, endocaritis,abortion, abscess and topical purulent damage, and bring the pregnant woman abortion, stillbirthand etc. The patients' death rate may reach as high as 30～40%. In recent years, the fact that manycountries have already reported Listeria monocytogenes polluted food toxicosis events paid moreand more serious attention to this bacterium, made a lot of new food security codes and enforcedlegal inspection on Listeria monocytogenes in food. The staphylococcal enterotoxins (SEs) arerectories for the pathogenesis of human and animal illnesses. These toxins are responsible forfood poisoning outbreaks and toxigenic syndrome in humen. The enterotoxins are classifiedserologically into SEA, SEB, SEC, SED, SEE, SEG, SHE, SEI and SEJ, and SEC can be furthercategorized into SEC₁, SEC₂, and SEC₃.Based on the sequence of hly gene in NCBI database under Accession No. gi: 134116121,oligonucleotide primers with mutual overlaps were synthesized based on the optimizedsequences. A 1320 bp gene fragment encoding hly was gained by PCR of two steps, confirmedby sequencing, and subcloned into the expression vector pET32a (+) to construct recombinantexpression vector pET32a (+)-hly. The resulting expression vector pET32a (+)-hly wastransformed into E.coli BL21 (DE3) competent cells and induced at 30℃for 3.5 hours. Aspecific expression band with a relative molecular mass 6kDa was detected by SDS-PAGE ininclusion body form and the protein accounted for 65.48% of total cell protein. The expressedprotein was purified to homogeneity with 96.21% purity using Ni-NTA affinity chromatographymethod under denatured condition, with a yield of 2mg/L of induced culture.In order to develop diagnosis antibody and establish colloidal gold-basedimmunochromatographic assay for rapid detection of L. monocytogenes, Staphylococalenterotoxin and Listeriolysin O (LLO), the L.monocytogenes, SE and rLLO protein were used toimmunize rabbit for producing the polyclonal antibody against L. monocytogenes, SE or rLLOand then were purified to 98% by caprylic acid and ammonium sulfate precipitation and proteinA affinity chromatography method. The ELISA showed the titter was up to 1:10⁸. The WBshowed the antigen's immunity was good. Then we have generated rabbit antibodies usingL. monocytogenes or rLLO protein as immunogens. The specificity and titer of the antibodieswere determined by ELISA using L.monocytogenes,SE or rLLO directly coated to the sameantigens. Then anti- L. monocytogenes, anti-SE or anti-LLO antibody was immobilized to a defineddetection zone (as captured antibody) on a porous nitrocellulose membrane, while the anti- L.monocytogenes, anti-SE or anti-LLO antibody was conjugated to colloidal gold particles whichserved as a detection reagent. The L. monocytogenes, SE or rLLO containing sample was addedto the membrane and allowed to react with antibody coated particles. The mixture was thenpassed along the porous membrane by capillary action reacted past the antibody in the detectionzone, which allow binding particles that had antibodies of L. monocytogenes,SE or rLLO boundto their surfaces, giving a red color within this detection zone with an intensity in proportion to L.monocytogenes, SE or LLO concentration. In the absence of L. monocytogenes,SE or LLO, noimmunogold parties were bound to the solid phase antibody. With visual observation, the lowestdetection limit was found to be 10⁶CFU/ml of L. monocytogenes, 150ng/mL of LLO, 10ng/mL ofSEA, 1ng/mL of SEB and 10ng/mL of SEC in less than 10 minutes respectively. These colloidalgold strips no cross-reaction with other bacteria and toxins. The accelerate trail with 37℃in 15days showed the stability of these strips were good and the conservation time would last oneyear.The colloidal gold detection method of L. monocytogenes and SE established in thisexperiment has high sensitivity and specificity. It is quick and accurate. The detection systemestablished in this experiment can be further used in the department of detection and quarantineto do L. monocytogenes and Staphylococcus aureus detection work in the imports and exportsfood. After the method is established, the classical and common method can be completed, andinspection of L. monocytogenes and Staphylococcus aureus can be taken at the molecular biologylevel. This method will have a wide use in the department of inspection and quarantine, foodindustry and sanitation supervision. It will bring a lot of benefits to economy and society.Meanwhile, it may lay the scientific foundation for the revision or supplement of theinternational detection method of food microbiology.