粪便中沙门氏菌检测论文

PCR技术是一种体外酶促合成、扩增特定DNA片段的方法。下面是我整理的关于pcr技术论文，希望你能从中得到感悟!

技术的研究进展

摘要 PCR技术是一种体外酶促合成、扩增特定DNA片段的方法。因其高强的特异性和灵敏度以及检测速度快、准确性好等优点，已被广泛地应用于水产、微生物检测等许多领域。该文从PCR技术的原理及应用方面进行了综述，并对其发展做出了展望。

关键词 PCR技术;研究进展;应用

中图分类号 Q819 文献标识码 A 文章编号 1007-5739(2012)10-0047-02

PCR(polymerase chain reaction，PCR)即聚合酶链式反应，它是一种体外酶促合成，扩增特定DNA片段的方法。1985年，美国Karray等学者首创了PCR技术，并由美国Cetus公司开发研制[1]。随着科学技术的发展和突破，PCR技术已在多个领域得到广泛地应用，如微生物检测、兽医学、水产养殖等方面。由于该技术具有较强的灵敏度、准确度和特异性，又能快速进行检测，因而其应用领域也在不断延伸[2-3]。随着PCR技术的不断发展，在常规PCR技术的基础上又衍生出了许多技术，如多重PCR(mutiplex PCR)技术[4]、实时荧光定量PCR(real-time fluorescent quantitative PCR，FQ-PCR)技术[5]、单分子PCR技术[6]。

1 PCR技术原理

PCR技术是根据待扩增的已知DNA片段序列、人工合成与该DNA 2条链末端互补的2段寡核苷酸引物，在体外将待检DNA序列(模板)在酶促作用下进行扩增。PCR的整个技术过程经若干个循环组成，一个循环包括连续的3个步骤：第1步是高温条件下的DNA模板变性，即模板DNA在93~94 ℃的条件下变性解链;第2步是退火，即人工合成的2个寡核苷酸引物与模板DNA链3’端经降温至55 ℃退火;第3步是延伸，即在4种dNTP底物同时存在的情况下，借助TaqDNA聚合酶的作用，引物链将沿着5’-3’方向延伸与模板互补的新链[7]。经过这个循环后，合成了新链，可将其作为DNA模板继续反应，由此循环进行。循环进程中，扩增产物的量以指数级方式增加，一般单一拷贝的基因循环25~30次，DNA可扩增l00万~200万倍[1]。PCR反应的步骤很简单，但是具体的操作是复杂的，如退火温度的确定、延伸时间的长短以及循环数等。因此，不同的反应体系应该确定适当的反应条件，以避免假阴性或假阳性等情况的产生。

2 PCR技术的分类

在传统PCR技术的基础上，根据人们的需要以及各个领域的应用要求，又衍生出很多种类的PCR技术。新技术在各领域广泛应用并逐渐改进，为进一步的研究提供了基础。

实时荧光定量PCR技术

1996年，学者经过研究，在传统PCR技术的基础上，首创了实时荧光定量PCR技术，新技术已经应用至医学领域、分子生物学和其他基础研究领域。实时荧光定量PCR技术基于传统技术的优势，还具有实时性、准确性、无污染，实现了自动化操作和多重反应，是PCR技术研究史上从定性到定量的飞跃[8]。

荧光定量PCR技术最大的特点是能将荧光基团加入到PCR反应体系中，借助于荧光信号，累积实时监测整个PCR进程，最后通过标准曲线对未知模板进行定量分析[9]。实时监测这一特点是常规PCR技术所不具有的，因为其对扩增反应不能进行随时的检测。常规PCR技术的扩增终产物需要在凝胶电泳等条件下才能进行，无法对起始模板进行准确的定量，而荧光定量PCR技术的反应进程可以根据荧光信号的变化做出准确的判断[10-11]。一个PCR循环反应结束之后，定量PCR仪可以收集1个荧光强度信号，荧光信号强度的变化可以反映产物量的变化情况，这样就可以得到1条荧光扩增曲线[12]。荧光信号在指数扩增阶段，PCR产物荧光信号的对数值与起始模板量之间存在线性对应关系，然后进行定量分析[13]。

多重PCR技术

多重PCR(mutiplex PCR)技术是PCR技术的一种，为同一管中加入多对特异性引物，与PCR管内的多个模板反应，在一个PCR管中同时检测多个目标DNA分子。多重PCR技术可以扩增一个物种的一个片段，也可以同时扩增多个物种的不同片段[14]。

在同一反应体系中，多重PCR技术进行多个位点的特异性扩增时，引物间的配对、引物间的竞争性扩增等会对扩增效果产生重要影响。一方面，如果能选择适宜的反应体系和反应条件，可极大地提高多重PCR的扩增效果[15]。主要包括退火温度、退火及延伸时间、PCR缓冲液成分、dNTP的用量、引物及模板的量等。另一方面，DNA的抽提质量也影响多重PCR扩增效率，如DNA抽提不干净或降解都将影响PCR扩增效果[16]。

单分子PCR技术(SM-PCR)

单分子PCR技术是在传统PCR技术的基础上发展的，基本循环过程相同，但在反应条件、模板数量、DNA 聚合酶选择、引物设计方面具有不同点。该技术是以少量或单个DNA分子为模板进行的PCR[17]。

单分子PCR技术反应中，DNA 模板浓度极低，这就要求模板有较高的质量。因为这是试验成败的决定性因素。在设计引物时，应该严格控制GC的含量和Tm值，同时尽量避免引物间存在可配对序列。在反应混合物模板数极低的情况下，若引物之间存在少量配对序列，扩增时极易形成二聚体，使反应无法进行，得不到所需要的产物[18]。由于单分子PCR技术反应的变性温度(96~98 ℃)大多比常规PCR技术(94 ℃)略高，因而对DNA 聚合酶热稳定性的要求也更加严格，需要有较好的热稳定性，以防止温度过高而使其失活。其变性时间(5~15 s)、退火时间及延伸时间也短于常规PCR技术[17]。

3 PCR技术的应用

PCR技术在水产上的应用

基因表达是检测某个基因在不同发育期或不同组织中的表达量变化，或受到某种试验处理过程中的影响而出现表达量变化的情况。有学者应用real-time PCR技术研究碳水化合物含量对翘嘴红鲴糖代谢酶G6Pase、GK以及PEPCK表达量的影响[19-21]，研究结果可为翘嘴红鲴饲料配方中的最合适糖含量提供理论依据。孙淑娜等[22]研究叶酸拮抗剂对斑马鱼心脏发育相关基因BMP2b及HAS2表达的影响，表明叶酸拮抗剂对早期胚胎的心脏发育影响较大，可导致斑马鱼心脏发育延迟及心脏形态异常，并下调斑马鱼心脏发育相关基因BMP2b及HAS2的表达，这可能是叶酸生物学活性受抑后导致心脏发育异常的机制之一。Sawyer et al[23]以斑马鱼的未受精卵、胚胎、仔鱼和成鱼为研究材料，采用实时荧光定量PCR技术，检测了P450aromA和P450aromB在不同组织的表达量，表明在各组织中均有2种基因的表达，但表达量显著不同，呈现组织特异性。

PCR技术在微生物检测上的应用

1990年，Bej et al[24]在利用多重PCR的方法检测了Leg-ionella类菌种和大肠类细菌，其结果是通过点对点方法固定的多聚dT尾捕捉探针和生物素标记的扩增DNA进行杂交来检测的。张志东等检测口蹄疫病毒(FMDV)持续性感染的带毒动物，表明实时荧光定量PCR技术具有快速检测、准确、客观等优势，较优于传统的检测方法[25-26]。Metzger-Boddien et al[27]对PCR-ELISA的方法进行了评价，结果显示，样品中沙门氏菌的检出率可以达到98%。

4 展望

传统PCR技术以及衍生出来的新型PCR技术自面世以来，已被广泛应用到生命科学的各个领域。随着技术方法的不断改进与完善，荧光定量PCR技术将会逐渐完善并广泛应用。多重PCR技术在食品病原微生物、非致病微生物及环境微生物检测中具有重要作用;未来的研究主要集中在去除食品抑制因子干扰、改进样品前处理技术等方面，其次是整合应用多重PCR与其他技术，必将在未来食品微生物检测中有非常好的应用前景。

5 参考文献

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嘿嘿，首要发表的论文吧，期刊jama的 不过倒是不知道你能不能看懂了Outbreak of Salmonella Serotype TyphimuriumInfection Associated with Eating Raw GroundBeef\p=m-\Wisconsin,1994MMWR. 1995;U:905-9091 figure omittedDESPITE previously publicized out¬breaks of illness associated with and rec¬ommendations to avoid eating undercookedmeat, some persons continue toeat undercooked or raw meat. This re¬port summarizes the investigation of anoutbreak of Salmonella serotype Typhimuriumgastrointestinal illness inWisconsin associated with eating con¬taminated raw ground beef during the1994 winter holiday December 29, 1994, physicians ina group medical practice in DodgeCounty (1994 estimated population:79 360), Wisconsin, reported to the Pub¬lic Health Unit of the Dodge CountyHuman Services and Health Department(DCHSHD) that during December 27-29 they had treated 17 patients withacute gastrointestinal illness character¬ized by diarrhea and abdominal least 14 patients reported havingeaten raw ground beef that was eitherplain or seasoned with onions and anherb mix during the 72 hours beforeillness onset. Stool samples for culturewere obtained from 11 patients; Salmo¬nella serotype Typhimurium that didnot ferment tartrate was isolated fromseven specimens. Based on these reportsand findings, the DCHSHD issued a phy¬sician alert and press release that en¬couraged affected residents to reporttheir illnesses and physicians to obtainstool cultures from case-patients. In ad¬dition, DCHSHD and the Bureau of Pub¬lic Health, Wisconsin Division of Health(WDOH), initiated an investigation ofthis outbreak. A probable case of Sal¬monella infection was defined as diar¬rhea or abdominal cramps with onsetduring December 22, 1994-January 4,1995, in a resident ofor a visitor to DodgeCounty or any of the four contiguouscounties. A confirmed case was definedas a stool culture positive for tartratenegativeSalmonella and WDOH identified 107confirmed and 51 probable case-patients;ofthese, 17 (16%) were hospitalized. Pre¬dominant manifestations of illness in¬cluded diarrhea (98%), abdominal cramps(88%), chills (77%), body aches (71%),fever (65%), nausea (60%), and bloodystools (43%). The ages of ill personsranged from 2 years to 90 years; 62%were assess potential risk factors forillness, DCHSHD andWDOH conducteda case-control study including 40 casepatientswho were randomly selectedfrom the persons with a stool specimenculture positive for tartrate-negativeSalmonella Typhimurium and 40 con¬trols who were identified by random tele¬phone digit dialing. The mean ages ofcases and controls were similar (43 yearsfor cases; 47 years for controls). Of 40case-patients, 35 (88%) reported havingeaten raw ground beef during Decem¬ber 22-January 4, compared with eight(20%) of40 controls (odds ratio [OR]=28;95% confidence interval [CI]=7-117).Among the 35 who ate raw ground beef,34 (97%) had purchased the beef fromone butcher shop, compared with three(37%) of the eight controls (OR=56; 95%CI=4-1881). Knowledge of previous re¬ports of outbreaks related to eating rawor undercooked beef was less among illpersons than among controls (26 [65%]of 40 case-patients compared with 30[75%] of 40 controls [OR=; 95%CI=]). However, 22 (85%) of the26 case-patients who reported beingaware of previous outbreaks associatedwith consumption of raw ground beefcontinued this behavior compared withseven (23%) of the 30 controls with knowl¬edge of previous outbreaks (OR=;95% CI=).DCHSHD and WDOH obtained fromcase-patients six leftover samples ofrawground beef that had been purchased atthe butcher shop on five dates duringDecember 21-29 and served in differenthomes. These samples were cultured forSalmonella sp.; all grew tartrate-negativeSalmonella Typhimurium. On De¬cember 30, 1994, staffofthe Meat Safetyand Inspection Bureau (MSIB), Wiscon¬sin Department of Agriculture, Trade,and Consumer Protection (WDATCP),informed the proprietor of the butchershop of a potential problem with con¬sumption of raw ground beef from theshop and the need to properly label meatproducts. During the winter holiday sea¬son, the butcher shop sold both seasonedand unseasoned raw ground beef thathad a warning label regarding safe han¬dling of poultry. On January 2, 1995,inspectors from MSIB examined sani¬tary conditions in the butcher shop, ob¬tained invoices indicating the origin andthe quantity of the meat used to preparethe ground beef, and inspected the rawground beef production method and sell¬ing practice in the butcher from approximately 35 carcassesobtained from three different suppliershad been ground in the shop from De¬cember 21 through January 4. Leftoverproduct was reported to have been dis¬carded each day and not carried over forsale the next day. All parts of the meatgrinder except for the auger housingwere disassembled and individuallycleaned and sanitized at the end of eachday. This type of grinder allowed easydisassembly of the auger and othersmaller parts; the auger housing wasattached to the grinder with nuts andbolts and required a wrench for , the cleaning staff had not re¬ceived instructions regarding removalof the auger housing and had cleanedonly surfaces of the tunnel-like space forthe auger with a from by guest on November 23, 2009Meat remnants were present in theauger housing when the grinder wasdisassembled. Twenty environmentalswabs of the equipment and the areasrelated to the production of the groundbeefwere obtained for bacterial culture;all were negative for Salmonella specimens obtained from all fivebutchers at the shop were cultured; onewas positive for tartrate-negative Sal¬monella Typhimurium. Although thisbutcher denied illness, he had eaten rawground beef at the shop during the out¬break by: PA Frazak, MPH, Public Health Unit,Dodge County Human Svcs and Health Dept; JJKazmierczak, DVM, ME Proctor, PhD, JP Davis, MD,State Epidemiologist for Communicable Diseases, Burof Public Health, Wisconsin Div of Health; J Larson, RLoerke, Meat Safety and Inspection Bur, WisconsinDept of Agriculture, Trade, and Consumer of Bacterial and Mycotic Diseases, National Cen¬ter for Infectious Diseases; Div of Field Epidemiology,Epidemiology Program Office, Editorial Note: The investigationofthis outbreak implicated consumptionof contaminated raw ground beef as thesource of Salmonella infection. Inad¬equate cleaning and sanitization of themeat grinder probably resulted in on¬going contamination ofground beef overmany production days. The outbreak oc¬curred during the winter holiday sea¬son, and some patients reported thatconsumption of raw ground beef duringthese holidays was a practice broughtfrom Europe by their ancestors. Thedecline of cases after the holidays mayhave occurred because ground beeffromthe implicated butcher shop was nolonger consumed raw or because thegrinder was cleaned more thoroughlyafter WDATCP personnel spoke withthe proprietor of the butcher shop onDecember 30. The five persons who be¬came ill but did not report eating rawground beef may not have rememberedeating the raw ground beef, may haveeaten undercooked ground beef or foodthat was contaminated from the rawground beef, or may have become illthrough person-to-person ground beef previously has beenimplicated as a vehicle for transmissionoiSalmonella,12 and undercooked groundbeef is the most frequently recognizedvehicle for Escherichia coli 0157:H7 in¬ The prevalence of Salmonellain beef ranges from 1% for raw beef car¬casses4 to 5%-7% for ground beef ( of Agriculture, Food Safetyand Inspection Service, unpublished data,1994). Prevention measures include warn¬ing consumers of the health risks asso¬ciated with eating raw ground beef andencouraging them to thoroughly cookground beef and to adhere to safefoodhandling guidelines. Safe cooking andhandling labels on raw or partially cookedmeat and poultry are now required bythe . Department of Agriculture(USDA).However, the presence of safefoodhandling labels does not ensure ad¬herence to safe practices. For example,an investigation of risk factors for spo¬radic E. coli 0157:H7 infection indicatedthat of 43 food preparers who reportedreading the safe foodhandling label onmeat packages, 33 (77%) admitted topractices specifically discouraged on investigation in Dodge County un¬derscores that knowledge of health risksis not consistently associated with desir¬able changes in behavior. Despite publichealth warnings and publicity about re¬lated outbreaks, some consumers inDodge County and elsewhere have con¬tinued to eat raw or undercooked foods ofanimal origin. For example, a telephonesurvey of a national sample of adults con¬ducted by the Center for Food Safetyand Applied Nutrition, Food and DrugAdministration (FDA), during December1992-February 1993 indicated that 53%consumed raw eggs; 23%, undercookedhamburgers; 17%, raw clams or oysters;8%, raw sushi or ceviche; and 5%, steaktartare (raw hamburger meat).6Consumer advisories can be more ef¬fective if targeted to specific cultural orethnic groups with such high-risk di¬etary practices, and WDATCP is plan¬ning two press releases this winter holi¬day period to warn consumers of therisks associated with eating raw addition to consumer advisories,interventions to reduce the risks asso¬ciated with the consumption of groundbeef include the needs for (1) producersof ground beef to emphasize employeeeducation and training on the recom¬mended methods of cleaning and sani¬tizing meat-grinding equipment; (2)manufacturers to design meat-grindingequipment that is easily accessible forcleaning and sanitization; and (3) stateregulatory and inspection authorities toadopt and enforce FDA's Food Codemodel requirements, which offer spe¬cific recommendations for handling,cooking, and storing raw meat; cleaningand sanitizing equipment and utensils;designing and constructing equipment;and advising consumers about the risksassociated with consumption of raw orundercooked food of animal TheUSDA's Food Safety and Inspection Ser¬vice also has proposed changes in themeat and poultry inspection system toimprove assessment and control of microbialpathogens in raw meat and poul¬ Consumers can obtain more infor¬mation on safe meat handling from theUSDA's Meat and Poultry Hotline (tele¬phone [800] 535-4555).References1. Fontaine RE, Arnon S, Martin WT, et al. Raw hamburger:an interstate common source of human J Epidemiol 1978;107:. CDC. Salmonella Typhimurium\p=m-\Minnesota,Wisconsin,Michigan. MMWR 1972;21:411 . Griffin PM. Escherichia coli O157:H7 and other enterohemorrhagicEscherichia coli. In: Blaser MJ,Smith PD, Ravdin JI, Greenberg HG, Guerrant RL,eds. Infections of the gastrointestinal tract. New York:Raven Press, Ltd, . Food Safety and Inspection Service, US Departmentof Agriculture. Nationwide beef microbiological baseline data collection program: steers and heifers(October 1992-September 1993). Washington, DC: USDepartment of Agriculture, January . Mead PS, Finelli L, Spitalny K, et al. Risk factors forsporadic infection with Escherichia coli O157:H7 [Abstract].In: 44th Annual Epidemic Intelligence ServiceConference, March 27-31, 1995. Atlanta, Georgia: USDepartment of Health and Human Services, PublicHealth Service, CDC, . Klontz KC, Timbo B, Fein S, Levy A. Prevalence ofselected food consumption and preparation behaviorsassociated with increased risks of food-borne of Food Protection 1995;58:. Public Health Service. Food code, 1995. Washington,DC: US Department of Health and Human Services,Public Health Service, Food and Drug Administration,. Food Safety and Inspection Service, US Department ofAgriculture. Pathogen reduction: hazard analysis andcritical control point (HACCP) systems; proposed Register 1995;60:: Influenza Activity\p=m-\UnitedStates,1995-96 SeasonMMWR. 1996;U:937-939INFLUENZA activity in the UnitedStates increased steadily from late Oc¬tober through mid-December 1995. Thisreport summarizes influenza surveillancedata from October 1 through December16, October 1-December 16, influ¬enza viruses were isolated in 45 statesand the District of Columbia. Of the 296influenza virus isolates reported by WorldHealth Organization (WHO) collaborat¬ing laboratories in the United States, 293() were type A and three ()were type B. Of the type A isolates, 140Downloaded from by guest on November 23, 2009