单管多重PCR快速检测STD病原菌

目的建立一种同时检测解脲支原体(UU)、沙眼衣原体(CT)、淋病奈瑟菌(NG)三种病原体DNA的方法并用于临床标本检测。方法建立单管多重PCR体系,其结果与常规PCR法比较。结果可以一次性检测出解脲支原体、沙眼衣原体和淋病奈瑟菌。结论该法特异性好,单管多重PCR体系检测3种STD病原菌DNA的灵敏度最低为0.43 fg,与单独的PCR检测的灵敏度相同,将它做成体外基因诊断试剂盒能快速特异性指导单种和多重感染。     

一种多重PCR方法快速鉴定7种常见肠道病原菌

目的 建立一种能够快速检测并分型5种致泻性大肠埃希菌、志贺菌及沙门菌的多重聚合酶链反应(multiplex polymerase chain reaction,M-PCR) 方法。 方法 设计针对7种常见肠道致病菌的12对引物,通过多重PCR方法扩增后电泳观察相应条带,确定病原菌。临床标本直接划线接种于肠道选择性平皿,挑取可疑菌落直接提取核酸进行多重PCR检测。 结果 所有标准菌株均能扩增到相应目的片段,322份腹泻病标本中使用该方法共检出志贺菌24株(福氏志贺菌7株、宋内志贺菌17株)、 沙门菌5株、EPEC共12株(均为aEPEC)、ETEC共6株(elt阳性3株;est阳性3株)、EIEC共3株、VTEC共1株(stx1和stx2A均阳性)、EAEC共3株。 结论 该方法快速特异,能同时检测7种常见肠道致病菌,可用于腹泻病常见病原的快速检验。     

多重PCR快速检测3种食源性致病菌

目的建立一种能同时检测金黄色葡萄球菌、产单核李斯特菌和沙门菌3种致病菌的多重PCR检测方法。方法采用LB培养液对金黄色葡萄球菌、产单核李斯特菌和沙门菌标准菌株进行增菌。根据金黄色葡萄球菌的nuc基因、产单核李斯特氏菌的hlvA基因、沙门氏菌的invA基因设计引物,通过多重聚合酶链反应（PCR）对上述3种食源性致病菌的目的基因进行扩增,同时对反应体系进行优化。结果对平均浓度为5cfu／ml的金黄色葡萄球菌、产单核李斯特菌和沙门氏菌在LB培养液中进行8h振荡培养,可以检出阳性结果;把金黄色葡萄球菌、产单核李斯特菌、沙门菌、志贺菌、蜡样芽孢杆菌、大肠埃希菌0157、阪崎肠杆菌7种菌混合在一起提取混合基因组DNA进行PCR扩增,显示出很好的特异性结果。结论建立的多重PCR检测方法适用于金黄色葡萄球菌、产单核李斯特菌和沙门菌的快速检测,具有快速、简便、灵敏的特点,可广泛应用于食品卫生检测、食物中毒应急处理和临床检验等领域。     

用多重PCR快速检测登革病毒并分型

目的　建立一种多重聚合酶链反应 (PCR)方法 ,对不同型别登革病毒进行快速检测及分型。方法　采用 4种型别登革病毒通用的外侧引物进行逆转录PCR(RT PCR)反应 ,继用 4对 (5种 )型特异引物在单管内进行巢式PCR ,依据扩增产物的片段大小进行分型。结果　采用多重PCR ,扩增出 4 82、119、2 90及 392bp的特异片段 ,分别代表登革病毒 1～ 4型。结论　该方法快速、敏感、特异 ,有一定的推广应用价值。     

多重PCR快速鉴定19种致病真菌

摘要:目的：建立快速鉴定致病真菌的多重PCR体系。 方法：选取19种致病真菌的rDNA内部转录间隔区（ITS）序列设计引物,建立两个多重PCR体系,扩增临床与环境分离真菌菌株、人类细胞及9种常见细菌DNA,检测体系的特异性;以浓度梯度的DNA模板检测体系的灵敏度。 结果：对氟康唑天然耐药的克柔念珠菌、光滑念珠菌以及对两性霉素B耐药的土曲霉、镰刀菌、尖端赛多孢、根霉在该体系中可扩增出特异性条带,其他真菌可同时进行种属鉴定。74株被测真菌菌株的鉴定结果与常规鉴定的符合率为95.9%。两个多重PCR体系对人类基因组及9种常见细菌DNA的扩增结果均为阴性;扩增阳性的最低模板浓度均为10 fg/μL。 结论：建立的两个多重PCR体系可对致病真菌进行检测和种属鉴定。     

多重聚合酶链反应检测生殖支原体感染

目的 建立一种敏感快速的多重聚合酶链反应（ＰＣＲ）用于检测生殖支原体（Ｍｇ）。方法 性病门诊患者５１１例，正常对照５６人，分泌物（女性为宫颈，男性为尿道拭子）标作用ＮＰ－４０裂解液一步法制备模板ＤＮＡ，设计Ｍｇ种属特异性引物及功能性结蛋白基因引物，先进行标准株的特异性与敏感性试验，然后进行临床标末的ＰＣＲ扩增反应。结果两对引物良好的特异性，对Ｍｇ标准株模板敏感性可达１０＾－６μｇ，临床标本检测，广     

用多重PCR快速检测登革病毒并分型

目的 建立一种多重聚合酶链反应(PCR)方法，对不同型别登革病毒进行快速检测及分型。方法 采用4种型别登革病毒通用的外侧引物进行逆转录PCR(RT—PCR)反应，继用4对(5种)型特异引物在单管内进行巢式PCR，依据扩增产物的片段大小进行分型。结果 采用多重PCR，扩增出482、119、290及392bp的特异片段，分别代表登革病毒1～4型。结论 该方法快速、敏感、特异，有一定的推广应用价值。     

多重聚合酶链反应检测生殖支原体感染

目的建立一种敏感快速的多重聚合酶链反应（ＰＣＲ）用于检测生殖支原体（Ｍｇ）。方法性病门诊患者５１１例，正常对照５６人。分泌物（女性为宫颈、男性为尿道拭子）标本用ＮＰ－４０裂解液一步法制备模板ＤＮＡ。设计Ｍｇ种属特异性引物及功能性结蛋白基因引物，先进行标准株的特异性与敏感性试验，然后进行临床标本的ＰＣＲ扩增反应。结果两对引物有良好的特异性，对Ｍｇ标准株模板敏感性可达１０－６μｇ。临床标本检测，广东地区２６１例性病门诊患者的Ｍｇ－ＤＮＡ阳性率（５４／２６１，２０．７％）高于上海（９／８４，１０．７％）、常州（８／７０，１１．４％）及南京地区（１１／９６，１１．５％；χ２＝１６．１，Ｐ＜０．０１），后三者的感染率互相间比较，差异无显著性（χ２＝０．０５６，Ｐ＞０．０５）。性病门诊患者５１１例的Ｍｇ－ＤＮＡ总阳性率（８２／５１１，１６．１％）与５６例正常对照者（１／５６，１．８％）比较，差异有非常显著性（χ２＝７．８８２，Ｐ＜０．０１）。结论建立的ＰＣＲ技术检测Ｍｇ具有敏感、快速、特异的特点，是一种可靠的实验诊断手段。     

多重PCR同时检测病原菌及其耐药基因

目的建立一种多重PCR方法,在同一扩增体系内同时检测病原菌及其β-内酰胺类耐药基因,探讨采用多重PCR同时鉴定病原菌及其耐药基因的可行性。方法根据细菌基因序列和对β-内酰胺类抗生素耐药特点,设计一对细菌鉴定通用引物和三对β-内酰胺类耐药基因引物,并在同一扩增体系内行PCR扩增。结果 MRSA和大肠埃希菌及肺炎克雷伯菌的产酶标准菌株的bla mecA、blaTEM、blaSHV和16S-23S rRNA基因间隔区（ISR）多重扩增均为阳性,对本实验室内保存的50株多重耐药的葡萄球菌（包括40株表型筛查为MRS菌株及10株非MRS菌株）及30株ESBLs阳性的大肠埃希菌及30株肺炎克雷伯菌的多重PCR结果显示：MRS阳性菌株均同时扩增出了葡萄球菌特有的多态性及blamecA指纹图谱,而多重耐药的非MRS菌株也都扩增出了blamecA,ESBLs表型阳性的大肠埃希菌多以blaTEM为主,而肺炎克雷伯菌多以blaSHV为主。结论多重PCR与传统的培养及药敏试验相比敏感、特异、迅速,对于解决难培养或不能培养的微生物的鉴定和药敏试验,是一种很有前景的方法。     

多重PCR检测缺失型α地中海贫血

目的　针对中国人常见的缺失型α地中海贫血建立一种快速、简便、可靠的多重聚合酶链反应 (PCR)基因诊断技术。方法　设计 4对引物 ,利用单管多重PCR方法进行扩增 ,同时检测 3种缺失型和正常的α2 珠蛋白基因。结果　成功检测出正常人及 3种缺失型的杂合子、双重杂合子 ;正常α2 基因的扩增片段为 180 0bp ,东南亚型缺失基因的扩增片段为 70 8bp ,右侧缺失基因的扩增片段为 2 0 2 2bp ,左侧缺失基因的扩增片段为 16 2 8bp , α3 .7/ SEA双重杂合子的扩增片段为 2 0 2 2bp和 70 8bp , α4.2 / SEA双重杂合子的扩增片段为 16 2 8bp和 70 8bp。结论　 790例标本的基因检测结果分析表明 ,该法快速、准确 ,可用于常规临床标本的检测。     

阴道感染三种病原菌多重PCR检测方法的建立及其应用研究

目的 探讨阴道感染病原体多重PCR快速检测白念珠菌、加德纳菌和阴道滴虫的方法建立及其临床应用。方法 根据白念珠菌EO3基因、加德纳菌ITS-23srRNA基因及阴道滴虫TVK3～TVK7基因设计特异性PCR引物,采用加热煮沸裂解法制备DNA模板,进行多重PCR扩增及琼脂糖凝胶电泳检测分析,在同一体系内完成三种阴道常见病原体的快速检测。结果 经特异性试验证明所建立的方法能特异地检测出白念珠菌125 bp,加德纳菌433 bp和阴道滴虫300 bp的目的片段,而其它非目的菌株均不能扩增出相应片段; 敏感性试验证实白念珠菌为10 cfu/ml,加德纳菌为10 cfu/ml,阴道滴虫为20 cfu/ml; 对30份已知临床标本和10份模拟混合标本进行检测均可检测出相应的病原体。结论 实验证明该研究所建立的白念珠菌、加德纳菌和阴道滴虫的多重PCR体系,具有灵敏度高、特异度强,可一步同时检测阴道分泌物中三种病原体,用于阴道感染的早期快速诊断。     

多重PCR结合基因芯片技术检测11种致病菌方法的建立

目的建立一种运用多重PCR方法结合基因芯片技术快速、准确检测11种常见致病菌的方法。方法筛选志贺氏菌、肠炎沙门氏菌、伤寒沙门氏菌、大肠杆菌O157、副溶血性弧菌、普通变形杆菌、蜡样芽孢杆菌、金黄色葡萄球菌、单核细胞增生李斯特菌、产气荚膜梭菌、空肠弯曲菌的特异基因作为目的基因。设计相应的引物及探针,进行多重PCR扩增,制备寡核苷酸芯片。将多重PCR扩增产物与带有11种特异探针的基因芯片杂交。用扫描仪扫描,判定细菌种类。结果该基因芯片可特异性地检测11种致病菌,具有良好的特异性,基因组DNA检测灵敏度可达2×10-3ng。结论多重PCR结合基因芯片技术检测11种不同致病菌的方法特异性好,灵敏度高,具有较好的实用性。     

混合逆转录聚合酶链反应快速诊断6种呼吸道病毒

目的 建立对6种呼吸道病毒快速诊断的混合RT-PCR实验方法。方法通过病毒RNA的提取，同一反应管内进行逆转录及聚合酶链反应以及Southern杂交检测6种呼吸道病毒，它们是甲型及乙型流感病毒、1，2，3型副流感病毒和呼吸道合胞病毒。将临床标本分为两组，一组为已知细胞培养阳性及鉴定结果的6种病毒各10份，共60份用于方法的建立和评价；另一组为40份用作检测的临床标本，在完成混合RT-PCR，Southern杂交实验后与培养鉴定结果比较。结果对第一组60份标本均获得相应病毒的扩增条带，且特异性杂交阳性；第二组40份标本中，混合RT-PCR，Southem杂交结果显示甲、乙两型流感病毒分别为18份和9份，1，2，3型副流感病毒分别为1，3，1份，呼吸道合胞病毒8份，与培养鉴定结果完全一致。结论混合RT-PCR敏感性高，特异性强，可用于对6种呼吸道病毒的快速检测。     

多重PCR法检测苏南地区Dystrophin基因缺失

目的研究应用多重PCR法检测Dystrophin基因缺失．并了解苏南地区Dys仃ophin基因缺失的特点。方法应用多重PCR法检测29例假肥大型肌营养不良症患者基因缺失．分析苏南地区Dystrophin基因缺失的分布。结果29例患者共检出15例存在Dystrophin基因缺失．苏南地区Dystrophin基因缺失检出率为51．72％;48号为最常见缺失外显子。结论多重PCR法检测假肥大型肌营养不良症患者基因缺失可靠,可用于临床检测;苏南地区Dystrophir谌因缺失分布特点与国内其他地区差别不大。‘     

复合PCR系统检测间日疟原虫的应用研究

应用一种简易、快速的复合 PCR扩增系统检测间日疟原虫 (P.v)及混合感染。方法 :以间日疟原虫和恶性疟原虫 (P.f)小亚单位核糖体核糖核酸基因 (SSUr DNA)特定片段为靶基因 ,设计并合成引物 ,建立复合 PCR扩增系统。采用煮沸法快速制备 DNA模板研究该系统的敏感性和特异性 ,并用于临床血样的检测。结果 :从间日疟原虫和恶性疟原虫感染血样中分别扩增出分子量大小为 70 5bp和 575bp的特定扩增带。而食蟹猴疟原虫、诺氏疟原虫及正常人血样均无扩增带出现。单独检测 P.v敏感度达到 0 .1 5× 1 0 -5(约 7个原虫 /μl全血 ) ,在 P.f存在的情况下检测 P.v敏感度为 0 .1 5× 1 0 -4 。检测 1 32例疟区门诊病人冻存血标本 ,1 0 4份与镜检法结果相同。并发现 1 4份混合感染和 5份为镜检虫种鉴别失误。结论 :该系统敏感性高 ,特异性强 ,操作简单 ,并可在一次扩增中同时检出间日疟原虫和恶性疟原虫 ,具有一定的推广应用价值。     

聚合酶链反应检测粪便中的副溶血性弧菌的TDH基因

副溶血性弧菌是细菌性腹泻的主要致病因子，本研究建立一种快速标本处理和聚合酶链反应诊断方法，扩增副溶血性弧菌的热稳定直接溶血素基因。对一组５６例腹泻患者粪便便标本进行了检测，经细菌培养培养证实为副溶血性弧菌的９份标本，ＰＣＲ法均阳性；培养法阴性的３９例中，有７例检测到ＴＤＨ基因。统计学表明两种方法检测结果相当一致，但检出能力ＰＣＲ法显著高于培养法。本法简便，快速，特异，适宜临床应用。     

Efficacy of Direct Detection of Pathogens in Naturally Infected Mice by Using a High-Density PCR Array

We used a high-density array of real-time PCR assays for commonly reported rodent infectious agents (PRIA) to test naturally infected index mice and sentinel mice exposed by contact and soiled-bedding transfer. PRIA detected 14 pathogens—including viruses, bacteria, fur mites, pinworms, and enteric protozoa—in 97.2% of 28 pooled fecal samples, fur–perianal swabs, and oral swabs from 4 cages containing a total of 10 index mice. Among these pathogens, PRIA (like conventional health monitoring methods) failed to detect Mycoplasma pulmonis, Pasteurella pneumotropica, and Giardia spp. in all of the 9 contact and 9 soiled-bedding sentinels. PRIA demonstrated murine adenovirus and Cryptosporidium and Spironucleus spp. in contact but not soiled-bedding sentinels and detected Helicobacter and pinworms in fewer than half of the soiled-bedding sentinels. Of the 4 species of Helicobacter that species-specific PCR assays identified in index mice, only H. ganmani was found in soiled-bedding and contact sentinels. PRIA detected all of the pathogens in sentinels that were identified by conventional methods. Myobia musculi was detected by PCR in index and sentinel mice but missed by conventional parasitologic examinations. In summary, PRIA reproducibly detected diverse pathogens in heavily pooled specimens collected noninvasively from infected index mice antemortem. The inability of PRIA and conventional health monitoring methods (that is, parasitology, microbiology, and serology) to demonstrate transmission of some pathogens to contact sentinels and the inefficient transmission of others to soiled-bedding sentinels underscores the importance of direct PCR testing to determine the pathogen status of rodents in quarantine and during routine colony surveillance.Abbreviations: HM, health monitoring; PRIA, PCR array for rodent infectious agents; TNA, total nucleic acidA requirement for meaningful biomedical research is that the animal models used (typically mice) remain free from infection with specific pathogens, including those that rarely produce disease but still interfere with research by modulating experimental responses and contaminating biologics. To eliminate and then exclude these pathogens, rodents have been rederived or cured of infection and housed behind room- or cage-level barriers, respectively.³⁹ Because no barrier can be guaranteed 100% effective, routine health monitoring (HM) is necessary to verify the SPF status of breeding and research colonies, and imported animals in quarantine. Health monitoring of animals prior to release from quarantine has become especially important given that the genetically engineered mutant mice most frequently exchanged among investigators and institutions have been reported to harbor pathogens frequently.¹⁷Conventional rodent HM programs usually consist of monthly to quarterly serology for prevalent viruses and quarterly to yearly whole-animal assessment, including a comprehensive serologic panel, pathology, parasitology, microbiology, and—since the advent of molecular diagnostics in the 1990s—PCR testing for fastidious microorganisms such as the helicobacters and Mycoplasma pulmonis.^34,35 In addition to being determined by test sensitivity and specificity, the correspondence of HM findings to the actual health status of the population being monitored is affected by the degree to which the samples are representative of the population and specimens are suitable for the tests used. For commercial barrier rooms, HM is performed directly on colony animals of both sexes, and multiple age groups. By contrast, rodents from research colonies are rarely made available to be bled or euthanized for conventional HM; consequently, these colonies are monitored indirectly by testing sentinels. Irrespective of the diagnostic approach or test method, detection of a contamination requires that sentinels become infected with the adventitious agent.Microisolation caging systems have been widely adopted for maintaining and quarantining mice and rats because the cage-level barrier they provide has proven to be very effective at excluding and impeding the spread of adventitious agents. Microisolation cage sentinels may be cohoused with quarantined animals, but for routine surveillance, contact sentinels are impractical because they would have to be moved among colony cages, which would be labor-intensive and undermine the cage-level barrier. Instead, sentinels are kept in separate cages supplied with regular changes of soiled bedding pooled from colony cages. Typically, 1 or 2 sentinel cages are setup for a rack of cages. Reliance on soiled bedding alone to transmit infections to sentinels is problematic because infections with certain respiratory viruses, host-adapted bacteria, and parasites are transmitted inefficiently or not at all via soiled bedding.^{10,15,24,26,28,38,42} In addition, the ability of microisolation cages to control the spread of infection frequently keeps the percentage of cages with actively infected rodents low. The lower the prevalence of infection, the greater the risk that the pathogen dose in pooled bedding will be insufficient to infect sentinels. The risk may increase as sentinel mice age and become less susceptible to infection, as has been reported for murine parvovirus 1 and mouse rotavirus.^3,33In addition to increasing the risk of missing a contamination, the low prevalence of infection that can occur in microisolation-cage–maintained colonies complicates confirmation of positive findings from sentinels because samples from many cages need to be tested to have an adequate chance of detecting an infection that is limited to a few cages. PCR analysis is frequently the method of choice for this confirmatory testing because it can detect prevalent pathogens in diverse specimens, such as feces and swabs of the skin and oral cavity which can be obtained noninvasively directly from colony animals. Moreover, the high sensitivity that is characteristic of PCR assays permits specimens to be heavily pooled (for example, 10 to 1), thus facilitating broad, representative sampling of cages on a rack. Now that PCR assays have been developed for virtually all of the viruses, bacteria, fungi, and parasites included in rodent SPF exclusion lists, PCR testing of pooled specimens collected directly from colony rodents is increasingly being added to HM programs to augment sentinel testing and enhance detection of pathogens not or inefficiently transmitted in soiled bedding.One caveat regarding PCR analysis is that it can amplify genomic sequences from nonviable microorganisms . In addition, PCR testing may miss infections with viruses and other agents that are shed transiently, particularly in sentinels that are tested quarterly, although PCR has been shown to detect house hepatitis virus and mouse parvovirus in feces for weeks to months after infected mice are no longer contagious.^2,6To improve the efficiency and throughput of PCR-based HM, we developed a high-density array of PCR assays for rodent infectious agents (PRIA). For developing PRIA, we chose the OpenArray platform (Applied Biosystems, Life Technologies, Grand Island, NY) because it uses fluorogenic TaqMan PCR reactions, termed ‘real-time’ PCR analysis, because the sequence-specific signal generated by the digestion of a fluorophore-labeled internal probe is measured each amplification cycle. The number of cycles required to reach a threshold signal is inversely related to the copies of microbial DNA added to the reaction.^{9,13,20,22,46} Particularly for agents such as Helicobacter and Spironucleus spp. that are present in very high copy numbers in specimens from infected animals, estimating copy number is helpful for identifying and discounting low-copy positive results due to cross-contamination from other samples. In addition to being quantitative, the advantages of the TaqMan PCR compared with the standard qualitative gel-based PCR include better specificity due to the internal probe and the elimination of postamplification cross-contamination because reactions do not need to be opened after amplification. By separating individual PCR assays into ‘holes’ (Figure 1), the OpenArray avoids the pitfalls of homogeneous PCR multiplexing, notably competitive inhibition that can cause false-negative results, especially when there are large differences in the genomic copies of multiple agents in a sample.^21,32,44Open in a separate windowFigure 1.An OpenArray chip (18 × 0.5 × 63 mm) for PRIA. The chip contains 48 subarrays of 64 reaction holes (volume, 33 µL each), of which 56 are used to perform as many as 28 duplicate individual real-time PCR assays. The 2 chip sets, used to test for the agents listed in