无公害畜产品微生物快速检测方法的研究
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摘要
随着人们生活水平的不断提高,畜产品在人们饮食结构中所占的比例越来越大,畜产品质量的安全问题已经成为人们广泛关注的重大问题。食源性致病菌是影响畜产品安全的主要原因之一,检测食源性致病菌是保障畜产品质量安全的关键环节,因此研究畜产品中微生物的检测方法即成为极其重要的一个课题。就全球而言,沙门氏菌和大肠菌群是引起细菌性食物中毒的主要原因,而传统的沙门氏菌和大肠菌群检测,操作繁琐,费时费力,难以应对食物中毒的快速诊断和对畜产品安全的实时监控。本论文对畜产品沙门氏菌和大肠菌群的快速检测方法做了一些研究,全文共分为四个部分。
第一部分综述了当前我国畜产品的质量安全现状,以及当前沙门氏菌及大肠菌群检测方法的研究进展。
第二部分对无公害畜产品中大肠菌群的检测方法进行了研究,对测试片法检测大肠菌群进行了特异性和重复性试验,并将国标法及测试片法用于大肠菌群的检测进行了比较,试验结果表明PetrifilmTM大肠菌群测试片用于测定无公害畜产品中大肠菌群可省却繁重的准备、收尾工作,操作简单,快速省时,检测结果与国标法相符,可用于无公害畜产品中大肠菌群的测定。
第三部分对沙门氏菌快速检测试剂盒的应用效果进行了评价,用多种沙门氏菌株考察了试剂盒的准确度、检测限、特异性,并通过对90份生鲜鸡肉和90份生鲜猪肉试剂盒法与国标法检验比较考察了试剂盒的应用效果,结果表明对沙门氏菌检测的检测限可达到1 cfu/25g样品,所考察的非沙门氏菌检测沙门氏菌快速检测试剂盒的特异度为100%,通过共计180份样品用国标法和试剂盒法进行比对实验,分析得出该试剂盒的灵敏度为99.3%。
第四部分建立了沙门氏菌PCR快速检测体系。研究了5种增菌液对PCR反应的影响,确定了SC增菌液为培养沙门氏菌的最佳增菌液；确定了特异性强的上下游引物对；对沙门氏菌PCR检测方法的特异性、灵敏度及重复性进行了研究,试验结果表明建立的沙门氏菌PCR检测简便快速、特异性强、灵敏度高、重复性好,本研究确定的沙门氏菌PCR检测方法将会有广泛的应用。
With the development of People's living standards, the proportion of livestock and poultry products in people's diet are changing ceaselessly,so the safety of quality in livestock and poultry products is causing serious problems in people's great attention. The quality of livestock and poultry products is bringing great impact to people's health and life, Food-borne pathogen is one of the main causes of food safety, Detection of food-borne pathogens is the key link in food-borne diseases prevention and control. So the microorganism inspection method of livestock and poultry products becomes a very important topic. In the world, salmonella and bacterium are main causes in bacterial food poisoning, the traditional detection is operating trival, time-consuming, It can not match the fast food poisoning diagnosis and the real-time monitoring of food safety.This article is about some researchs in rapid detection methods of salmonella and bacterium. It is divided into four chapters altogether.
The first chapter is about the summmarizing of the current situation of livestock and poultry products in our country, the quality and safety of livestock and poultry products. and the progress of salmonella and coliforms detection methods.
In the second chapter, the rapid detection method of coliforms was illustrated in the green livestock and poultry products, the specificity and repeatability were tested by the coliforms PetrifilmTM test pieces, compared with the traditional method, the experimental results showed that PetrifilmTM coliforms test pieces for determination of green livestock and poultry products can dispense with a lot of preparations and ending works. The operation is simple and fast, the testing results had no difference with the GB, It can be used in the test of coliforms in green livestock and poultry products.
The third chapter evaluated the ELISA kit for Salmonella rapid detection.The detection of Samonella by ELISA kit and the standard method of China were compared.The results showed that the veracity of the kit was 100%.The sensitivity was 1 cfu/25g, and the specificity for non-salmonella strains was 100%, too. By tests of 90 poultry meat and 90 porks, it showed that the sensitivity rate and the specificity rate were 99.3% and 100%, respectively.
The fourth chapter found the method of rapid detection of salmonella by polymerase chain reaction (PCR). Five kinds of enrichment medium were compared, and the selenite-cystine (SC medium) was selected as the best one for enrichment and PCR methods. Primers were designed, by lots of Salmonella and non-salmonella isolations, the primers had high specificity. The specificity, sensitivity, anti-interference and reproducibility were evaluated for the detection of samonella, too. The experiments showed that the PCR method was specific, sensitive, and reproducible, so it would have more application in the field of the detection of samonella.
第一部分综述了当前我国畜产品的质量安全现状,以及当前沙门氏菌及大肠菌群检测方法的研究进展。
第二部分对无公害畜产品中大肠菌群的检测方法进行了研究,对测试片法检测大肠菌群进行了特异性和重复性试验,并将国标法及测试片法用于大肠菌群的检测进行了比较,试验结果表明PetrifilmTM大肠菌群测试片用于测定无公害畜产品中大肠菌群可省却繁重的准备、收尾工作,操作简单,快速省时,检测结果与国标法相符,可用于无公害畜产品中大肠菌群的测定。
第三部分对沙门氏菌快速检测试剂盒的应用效果进行了评价,用多种沙门氏菌株考察了试剂盒的准确度、检测限、特异性,并通过对90份生鲜鸡肉和90份生鲜猪肉试剂盒法与国标法检验比较考察了试剂盒的应用效果,结果表明对沙门氏菌检测的检测限可达到1 cfu/25g样品,所考察的非沙门氏菌检测沙门氏菌快速检测试剂盒的特异度为100%,通过共计180份样品用国标法和试剂盒法进行比对实验,分析得出该试剂盒的灵敏度为99.3%。
第四部分建立了沙门氏菌PCR快速检测体系。研究了5种增菌液对PCR反应的影响,确定了SC增菌液为培养沙门氏菌的最佳增菌液；确定了特异性强的上下游引物对；对沙门氏菌PCR检测方法的特异性、灵敏度及重复性进行了研究,试验结果表明建立的沙门氏菌PCR检测简便快速、特异性强、灵敏度高、重复性好,本研究确定的沙门氏菌PCR检测方法将会有广泛的应用。
With the development of People's living standards, the proportion of livestock and poultry products in people's diet are changing ceaselessly,so the safety of quality in livestock and poultry products is causing serious problems in people's great attention. The quality of livestock and poultry products is bringing great impact to people's health and life, Food-borne pathogen is one of the main causes of food safety, Detection of food-borne pathogens is the key link in food-borne diseases prevention and control. So the microorganism inspection method of livestock and poultry products becomes a very important topic. In the world, salmonella and bacterium are main causes in bacterial food poisoning, the traditional detection is operating trival, time-consuming, It can not match the fast food poisoning diagnosis and the real-time monitoring of food safety.This article is about some researchs in rapid detection methods of salmonella and bacterium. It is divided into four chapters altogether.
The first chapter is about the summmarizing of the current situation of livestock and poultry products in our country, the quality and safety of livestock and poultry products. and the progress of salmonella and coliforms detection methods.
In the second chapter, the rapid detection method of coliforms was illustrated in the green livestock and poultry products, the specificity and repeatability were tested by the coliforms PetrifilmTM test pieces, compared with the traditional method, the experimental results showed that PetrifilmTM coliforms test pieces for determination of green livestock and poultry products can dispense with a lot of preparations and ending works. The operation is simple and fast, the testing results had no difference with the GB, It can be used in the test of coliforms in green livestock and poultry products.
The third chapter evaluated the ELISA kit for Salmonella rapid detection.The detection of Samonella by ELISA kit and the standard method of China were compared.The results showed that the veracity of the kit was 100%.The sensitivity was 1 cfu/25g, and the specificity for non-salmonella strains was 100%, too. By tests of 90 poultry meat and 90 porks, it showed that the sensitivity rate and the specificity rate were 99.3% and 100%, respectively.
The fourth chapter found the method of rapid detection of salmonella by polymerase chain reaction (PCR). Five kinds of enrichment medium were compared, and the selenite-cystine (SC medium) was selected as the best one for enrichment and PCR methods. Primers were designed, by lots of Salmonella and non-salmonella isolations, the primers had high specificity. The specificity, sensitivity, anti-interference and reproducibility were evaluated for the detection of samonella, too. The experiments showed that the PCR method was specific, sensitive, and reproducible, so it would have more application in the field of the detection of samonella.
引文
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[2]Present"s council on food safety.19 Janunry,2001.129-134
[3]郑克新.猪胴体沙门氏菌污染的调查[J],中国兽医杂志,2007,43(1)；51-52
[4]李秀桂,郭云昌,吕素玲,等,2005年广西食源性沙门氏菌污染监测分析J],应用预防医学,2008,14(1)：149-152
[5]刘建军,魏晓燕,靳烨,等.浅议微生物与畜产品安全及控制[J],农产品加工,2007,7(7)：41～44
[6]李莉,蒋作明.PCR技术在食品沙门氏菌检测中的应用[J],食品科技,2002,(4)： 156-158
[7]李志峰,聂军,陈义忠,等.一种快速检测副溶血弧菌的PCR方法[J]；解放军预防医学杂志;2004：(6)：109-111
[8]李云玖.实时定量PCR检测血中大肠杆菌的方法学及在外科的初步应用,中国协和医科大学,中国医学科学院,[D].
[9][9]Morb Mortal Wkly Rep.Center for disease Control and Prevention Preliminary food net data on the incidence of infection with pathogens transmitted commonly through food-10 states[J] 2006,(55):392-395
[10]汪月霞,赵会杰,赵一丹,等.PCR方法检测食品中沙门氏菌研究进展[J],安徽农业科学,2009,37(24)：11435-11436,11445
[11]吴永宁.现代食品安全科学.见吴永宁主编.北京：化学工业出版社,2003.(7)：336-337
[12]句立言,杨立秋,王世平,等.大肠菌群检测技术研究进展[J],中国初级卫生保健, Chinese Primary Health Care[J],2008 (5):136-140.
[13]刘亚军.水体中大肠杆菌快速检测方法和仪器的研究,华东师范大学硕士学位论文,2009,[D]
[14]李洪,粪涛,达永淑,等.探讨影响大肠菌群快速纸片法的因素[J],职业卫生与防治,2006,17(3)：198-199
[15]Robert S S.Amplication of nucleic acid sequences:the choices multiply[J] NIH Res,1991,3(2):81-94
[16]谢明勇,万益群,王远兴.食品安全与卫生实用手册[M],南昌：江西科学技术出版社,2005(12)：161
[17]刘建军,魏晓燕,靳烨,等.浅议微生物与畜产品安全及控制措施[J],农产品加工,学刊,2007(7)：41-44
[18]Fung, D, Y, C.. Rapid methods and automation in food microbiology[J], VCH publishere,inc.,New York:3-22
[19]刘亚军.水体中大肠杆菌快速检测方法和仪器的研究,华东师范大学硕士学位论文,2009,[D]
[20]Kariyazono,H.,Ohno,T.Ihara,K.et al. Rapid detection of the 22q11.2 deletion with quantitative real-time PCR. Mol Cell Probes,2001,15(2):71～73
[21]杨小龙,陈朝琼.食品微生物快速检测技术研究进展[J],河北农业科学,2008,12(12)： 51-53
[22]文利平,唐伟松,蒋朱明,等.外科手术病人手术前后血中细菌DNA检出率的研究,中国临床营养杂志,1999,7：171-173
[23]刘华伟,郭蔼光,马立农,等.PCR技术在沙门氏菌快速检测中的应用,动物医学进展,2004,25(6)：55-58
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