| Listeria is primarily a soil-borne microorganism dispersed throughout the environment. Listeria monocytogenes(L.monocytogenes)has been found in such sources as water,agricultural products and in animals.Recognized as a human pattogen for more than 50 years.L. monocytogenes has been identified as the etiologic agent of listeriosis,causing meningitis, encephalitis,septicemia,endocarditis,abortion,abscesses and local purulent lesions in humans. Four major outbreaks of listeriosis within the last decade have been connected to consumption of contaminated food.Pregnant women,neonates,immunocompromized patients and the elderly are at greatest risk of acquiring listeriosis.Since the prevalence of Listeria monocytogenes in foods and its implication as the causative agent of food-borne listeriosis have been recognized, substantial efforts have been made to develop selective media and methods to isolate and enumerate this pathogen rapidly.Listeria monocytogenes and its relative diseases have been paid close attention by public health and food bureaus the world over.The traditional detection of Listeria monocytogenes is common enrichment culture and biochemistry identity and the process can be lasted for 4 to 10 days.The immunization is faster but the antibody preparation of the monoclonal antibody is very hard.Itealth safety.Recent advances of Listeria monocytogenes detection methods was reviewed.Thus,a foundation of rapid and accurate detecting methods of the bacteria were laid.The study established the methods of nucleic acid hybridization to detect the Listeria monocytogenes.The methods simplified inspection flow-sheet,enhanced detective efficiency and can be applied in practical sample's detection.HlyA gene with high homology was selected to designed primers by using the software of Primer 5.0,O1igo 6 and Lasergene.The length of the nucleic acid probe is 21bp.This method utilized a chemiluminescent acridinium ester to label an oligonucleotide probe that was designed to hybridize with a target oligonucleotide sequence.And then,they generated DNA-RNA hybridity.After hybridization,hybridized probe and unhybirdized probe can be identified without separation,but by differential hydrolysis,that was,when adding mild alkaline,unhybrodizied probe lost luminous character(the half-life periods less than 1 minute),but the solution hybridization of the probe with the target sequence protected the acridinium ester from mild alkaline hydrolysis.Because the acridinium ester with plane structure was set in double-helical interior and protected by stereo specific blockade,the speed of hybridized probe hydrolysis was lowed down(the half-life period is more than 10 minute),so that the acridinium ester still had luminous efficiency.Therefore,when adding alkaline hydrogen peroxide,only the chemiluminescence's of the hybridized probe can be detected in illuminometer.30 minutes could be used during the process.This method is named nucleic acid hybridization protection assay(HPA),but it was applied less in China now.The pure culture's colonies of Listeria monocytogenes type strain were detected by HPA. Listeria monocytogenes can be detected specifically by HPA at screening and optimizing reactive condition,but the Escherichia coli,Salmonella,Campylobacter jejuni,Pseudomonas aeruginosa the bacteria that were not Listeria monocytogenes were negative by HPA.The different pieces of the positive colonies were picked to carry out sensitivity assay,the lowest detectable limit is two colony(10cfu/mL).100 samples of chicken meat were detected by established HPA.During the process of isolating culture,the pure culture colonies were detected by HPA,and 15%(15/100)of them were positive.The applying result showed that the study had established the methods of HPA to detect Listeria monocytogenes.The method was convenient,fast,exact and reliable,.They could be used to quarantine chicken meat for Listeria monocytogenes in the future. |
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