The Immunomagnetic Preenrichment Of Listeria Monocytogenes In 10 ML Separation System

Listeria monocytogenes has been identified as one of the most significant human and animal foodborne pathogens,which can be spread by food,water and animal's faeces,and can be alive even at 4 ?.The L.monocytogenes can cause severe listeriosis with a significant mortality rate of 30%,and even a higher mortality rate of70% in infants and immunocompromised population.Therefore,it is required to develop a rapid,simple,and reliable method for sensitive detection of L.monocytogenes.Immunomagnetic separation is a sample preenrichment method which can effectively improve the sensitivity of foodborne pathogenic detection.But,up to date,most of research works of immunomagnetic separation for L.monocytogenes preenrichment were based on 1 m L of separation system.In this study,a large-volume(10 m L)immunomagnetic separation(IMS)combined with m PCR was reported for rapid and sensitive detection of Listeria monocytogenes in lettuce without further enrichment process,in which the IMS has performed based on one-step or two-step immunomagnetic separation process.The main contents are described as follows:In the first chapter,the research advances of L.monocytogenes andimmunomagnetic separation technologies were reviewed.In the second chapter,a large-volume(10 m L)IMS method was studied for preenrichment of L.monocytogenes based on one-step and two-step immuomagnetic process,respectively.In one-step method,the Listeria-antibody coated magnetic beads were prepared by conjugation of the streptavidin coupled magnetic beads with biotin labelled anti-Listeria-antibody,and then were utilized for separation and purification of L.monocytogenes in a 10 m L volume.Various parameters were systematically investigated,and the optimized parameters were described as follows:1)the amounts of streptavidin for conjugation of 1 mg of magnetic beads were 80 ?g,2)the amounts of the biotin-m Abs for 1 mg of streptavidin coated magnetic beads were 100 ?g,3)700 ?g of immuomagnetic beads were suggested to use for 10 m L magnetic separation volume,4)the optimum immuoreaction time betweenimmunomagnetic beads and L.monocytogenes was 90 min,5)the immuomagnetic seperation time was 5 min.Under the optimum conditions,the large volume IMS method only took less than 2 h with a high capture efficiency(> 90.9% ± 6%)for L.monocytogenes spiked samples(the bacterial concentration less than 106 CFU/m L).The above results showed that one-step IMS method in 10 m L separation volume can reach a high capture efficiency for L.monocytogenes separation and can significantly shorten the preenrichment time.But,the consumption of antibodies of each immunomagnetic separation in 10 m L volume is too large for practical application.In order to reduce the amounts of antibodies used in large volume,a novel IMS method based on two-step immuomagnetic process was proposed,in which biotin labelled anti-L.monocytogene antibodies were pre-mixed with L.monocytogene first in 10 m L volume,and then the streptavidin coated magnetic beasds were used for capture of biotin labelled antibodies coated L.monocytogene.The optimum parameters of two-step method are described as follows: 1)50 ?g of biotin-m Ab were pre-mixed with L.monocytogene in a 10 m L of reaction volume,2)the amounts of streptavidin coated magnetic were 500 ?g in 10 m L volume,3)the immuoreaction time between biotin-m Ab and L.monocytogene was 60 min,4)immuomagnetic seperation time was 3 min.Under this optimum condition,the capture efficiency of two-step method was more than 85.4% ± 4.8% when the concentration of L.monocytogene was lower than 105CFU/m L,and the amounts of biotin-m Ab used for magnetic separation in two-step method were reduced 14 times compared with that of on-step method.In the third chapter,a m PCR method coupled with a large volume IMS was developed for simultaneous and sensitive detection of the L.monocytogenes and Listeria ivanovii in real lettuce sample without further enrichment process.The optimum experimental parameters for m PCR are as following: The concentrations of the primers in m PCR were 0.75 m M(act A gene of L.monocytogenes),0.05 m M(iact A gene of Listeria ivanovii),0.08 m M(prs gene of Listeria),and 0.05 m M(16S r RNA gene as IAC),respectively.The optimal annealing temperature was for co-amplification was set at 52? with the 35 amplification cycles.The detection limit of m PCR coupled with a large volume IMS for L.monocytogenes reached as low as10 CFU/g in real lettuce samples,and the total assay time took less than 7 h.Insummary,the proposed large volume IMS combined with m PCR method can be used for rapid and sensitive detection of L.monocytogenes and Listeria ivanovii.