Detection of Saccharopolyspora rectivirgula by quantitative real-time PCR

The thermophilic actinomycete species Saccharopolyspora rectivirgula has been associated with the exogen allergic alveolitis (EAA). EAA is caused by the inhalation of high amounts of airborne spores that can be found for example in environments of agricultural production, compost facilities, mushroom cultivation rooms, or rooms with technical air moistening. Because of the medical relevance of S. rectivirgula, a reliable detection system is needed. Therefore, a quantitative real-time polymerase chain reaction (qPCR) primer system was designed, targeting the 16S rRNA gene of the type strain S. rectivirgula DSM 43747(T) and six other S. rectivirgula reference strains. Our investigation showed that S. rectivirgula presumably own four operons of the 16S rRNA gene, which has to be considered for estimation of cell equivalents. Furthermore, the DNA recovery efficiency from these strains was tested in combination with bioaerosol or material sample as well as the influence of non-target DNA to the recovery rate. Results showed a recovery DNA efficiency of 7-55%. The recovery rate of DNA in a mixture with non-target DNA resulted in ～87%. In summary, a high amplification efficiency using real-time PCR was found, for which estimated concentrations revealed cell numbers of 2.7 × 10(5) cells m(-3) in bioaerosol and 2.8 × 10(6) cells g(-1) fw(-1) in material samples from a duck house. The specificity of the new developed quantification system was shown by generation of two clone libraries from bioarosol samples, from a duck house, and from a composting plant. Totally, the results clearly show the specificity and practicability of the established qPCR assay for detection of S. rectivirgula.     

四种食源性致病菌多重PCR检测方法的建立

目的建立一种多重PCR检测方法同时检测食品中沙门菌、志贺菌、金黄色葡萄球菌和单增李斯特菌。方法参考沙门菌侵袭蛋白A(invA)基因序列,志贺菌侵袭性质粒抗原(ipaH)基因序列,金黄色葡萄球菌引物设计参考耐热核酸酶(nuc)基因序列,单增李斯特菌引物设计参考溶血素蛋白(hly)基因序列设计引物,通过多重PCR对4种食源性致病菌的目的基因进行扩增,并对反应体系进行优化。结果对15株目标菌和17株非目标菌的检测未出现假阳性和假阴性结果,产物分子量与预期一致;4种菌的检测灵敏度分别至少达到10pg/μl;对产物的测序分析表明所得序列与目的基因序列吻合;对319份样品的检测结果显示,其中4份生鲜乳中检出金黄色葡萄球菌,1份生猪肉中检出沙门菌。结论该方法具有良好的检测特异性,可供食源性致病菌的快速检测。     

食品中沙门菌特异性二重聚合酶链反应检测方法的研究

目的建立二重聚合酶链反应(PCR)方法快速检测食品中的沙门菌(Salmonella spp.)。方法以沙门菌特异性基因invA、hilA为靶基因,选择2对引物对16株沙门菌和24株非沙门菌进行扩增,验证二重PCR的特异性。梯度稀释DNA,以不同稀释度的DNA PCR扩增。在猪肉中以不同菌量人工污染,不同增菌时间培养,提取DNA进行PCR扩增。应用该方法检测实际样品。结果以invA、hilA为靶基因的两对引物对沙门菌的检出均有很好的特异性,PCR能检出0.1332pg的沙门菌DNA,人工污染猪肉的检测限为2.5cfu/ml。本研究共检测了53份食品样品,有21份检出了沙门菌。结论建立了适用于肉类、水产品及蛋糕等食品中的沙门菌检测的快速、灵敏、特异的二重PCR方法。     

多重实时PCR检测沙门菌、志贺菌和致泻性大肠埃希菌

目的 建立多重实时荧光PCR检测沙门菌侵袭蛋白A基因（invA）、肠产毒性大肠埃希菌（ETEC）不耐热肠毒素基因（elt）、志贺菌和肠侵袭性大肠埃希菌侵袭力基因（ipaH）的方法。方法 优化多重实时荧光PCR的反应条件,检测系列10倍稀释的阳性菌DNA提取物。90份腹泻患者粪便样品经缓冲蛋白胨水（buffered peptone water,BP）增菌6h后进行多重实时荧光PCR检测,并对阳性标本进行菌株分离和鉴定。结果 多重实时PCR方法最低可以检测到10CFU／μl的福氏2aF301株、10。CFU／μl的鼠伤寒沙门菌和ETEC44815株。检测腹泻患者粪便样品,elt基因阳性率为14．4％（13／90）,ipaH基因阳性率为5．6％（5／90）;并从阳性样品中分离到3株elt基因阳性大肠埃希菌和4株ipaH基因阳性大肠埃希菌。整个检测过程可在10h内完成,其中包括BP增菌6h。结论 建立了同时检测invA、elt、ipaH毒力基因的多重实时荧光PCR方法,具有高度的特异性,可用于沙门菌、肠产毒性大肠埃希菌、志贺菌和肠侵袭性大肠埃希菌菌株的毒力基因鉴定及临床腹泻粪便标本的快速筛检。     

沙门菌基因间共有重复序列-PCR分子分型方法的优化及应用初探

目的优化沙门菌基因间共有重复序列-PCR(ERIC-PCR)分子分型方法,分析沙门菌标准菌株及流行分离株基因组DNA ERIC-PCR指纹图谱。方法提取鼠伤寒沙门菌基因组DNA作为模板,根据ERIC核心片段设计引物,运用ERIC-PCR技术扩增沙门菌基因组中的靶序列,PCR产物经琼脂糖凝胶电泳后用凝胶成像分析仪对图谱进行观察分析。反应体系中模板量、Mg2+浓度、引物浓度和退火温度的优化实验则采用对优化项目设置梯度、其它条件不变的方法进行。以优化好的ERIC-PCR方法对16株沙门菌及1株大肠杆菌进行分析。结果在总体积为25μl的反应体系中,选择DNA模板量为100ng/25μl,Mg2+浓度为2.0mmol/L,引物ERIC1R、ERIC2浓度分别为0.4μmol/L,退火温度为52℃时,扩增产物的电泳图谱条带最清晰完整。不同血清群、型和来源的沙门菌株间基因组DNA ERIC-PCR指纹图谱带型差异较大,可在250～5000bp范围内出现3～13条条带,并在250bp处出现一条特征条带。结论优化了沙门菌ERIC-PCR的重要反应条件。ERIC-PCR法能区分不同来源的沙门菌,可用于沙门菌病的流行病学调查和同源性追踪,弥补传统分型方法的不足。     

Real-time PCR quantification of Mycobacterium immunogenum in used metalworking fluids

Rapid detection and quantification of Mycobacterium immunogenum in field samples of metalworking fluids (MWFs) is important for factory fluid surveillance programs. The applicability of the developed DNA extraction and quantitative real-time PCR (qPCR) methods to detect and quantify M. immunogenum in used MWFs was evaluated. Total DNA from these samples was extracted, and M. immunogenum measured by qPCR by comparison with a standard curve derived from plasmid vectors. PCR counts were compared with bacterial culture counts. PCR counts of M. immunogenum varied from 1.42 x 10(3) to 3.68 x 10(6) cells/mL of MWFs. Recovery of M. immunogenum by bacterial culture varied from 2.5% to 70% of qPCR count in corresponding samples. Quantitative PCR could be used to measure M. immunogenum load in MWF samples with greater sensitivity and shorter processing time than the classic bacterial culture-based approach. The proposed qPCR approach could be routinely used in real-time PCR-equipped laboratories to provide early detection of M. immunogenum and to control proliferation that probably leads to hypersensitivity pneumonitis in exposed workers.     

16S rRNA基因测序分析技术在食品污染菌监测中的应用

目的 将16S rRNA基因测序分析技术应用于食品安全风险监测中非目标(相似、少见或非典型)致病菌的鉴定,为食源性疾病防控提供更多病原学证据。方法 采集18份畜类、18份禽类和9份鱼类共45份生鲜样品,目标致病菌按照GB4789标准检测,非目标菌进行16S rRNA基因测序,将全长序列在GenBank中比对确定细菌种属,通过MEGA6构建细菌16S rRNA基因进化树,判定细菌种属间的亲缘关系。结果 45份生鲜样品中检出19株目标致病菌,包括沙门氏菌、空肠弯曲菌、单增李斯特菌和副溶血弧菌,而34株非目标菌16S rRNA基因全长序列能分别比对出13种细菌,其中8种19株菌属于致病或条件致病菌,包括霍乱弧菌、溶藻弧菌、结肠弯曲菌、奇异变形杆菌、粪肠球菌和铜绿假单胞菌,以及在深圳乃至省内首次报道的布氏弓形菌和海藻希瓦氏菌。结论 深圳市售生鲜食品受到多种食源性致病菌污染,特别是非目标检出的致病菌,对此认为16S rRNA基因测序分析技术是一套有效的细菌监测适宜技术,能提高食源性疾病和食物中毒的防控水平。     

PCR产物生物发光分析技术研究

目的基于水母发光蛋白生物发光分析技术建立一种PCR产物定量检测技术。方法利用RT-PCR对CK19-mRNA进行放大,PCR引物的5’端用生物素标记,然后用交联了地高辛寡核苷酸探针与目标生物素化的DNA模板杂交。杂交复合物与包被在微孔板上的链亲合素结合,加入标记了水母发光蛋白的地高辛抗体与地高辛特异结合,再加入钙离子激发水母发光蛋白发光,发光强度正比于CK19-mRNA的量。结果探测灵敏度可达22 amol/L的RT-PCR产物。结论该定量检测技术是一种灵敏的、可靠的、非放射性的微量CK19-mRNA定量方法。     

3种食源性致病菌的多重PCR快速检测方法的建立

目的:建立快速检测食源性致病菌的多重PCR方法。方法:本研究根据肠毒性大肠埃希菌(enterotoxigenicE.coli,ETEC)的大肠杆菌不耐热毒素(Heat labileenterotoxin,LT)的B亚单位(LTB)、伤寒沙门菌(Salmonella typhi)的侵袭蛋白A(invasion protein A,invA)、福氏志贺菌(Shigella flexneri)侵袭性质粒抗原H基因(invasion plasmid antigen H,ipaH),分别设计了三对引物,预计PCR扩增的目的基因片段为194、279和435 bp。对单个基因PCR和单管多重PCR扩增的特异性、敏感性分析以及建立L16(43)正交试验对单管多重PCR扩增条件如引物浓度、dNTP浓度和Tm值等的优化,建立了快速检测肠毒性大肠埃希菌、伤寒沙门菌和福氏志贺菌的稳定的单管多重PCR方法。结果:该方法检测的灵敏度分别为:145 pg/ml肠毒性大肠埃希菌的基因组DNA、100 ng/ml伤寒沙门菌的基因组DNA、7 ng/ml福氏志贺菌的基因组DNA,与单基因PCR检测的灵敏度相同。并且模拟检测食品中的细菌,结果很稳定。结论:该方法操作简单、检验周期短、特异性和灵敏度高,能够快速地实现对食品中的多种致病菌的诊检和监控。     

Real-Time PCR探针法检测食物中毒中沙门菌方法建立及应用

目的:建立食物中毒中沙门菌Real-Time PCR检测方法并将其应用。方法:选取沙门菌侵袭蛋白A基因(in-vA)进行引物和探针设计,并对反应条件不断优化,进行灵敏度和特异度测试,建立检测沙门菌的Real-Time PCR方法。结果:DNA灵敏度检测沙门菌达到56 fg/PCR体系,菌液灵敏度检测沙门菌达到9 CFU/ml,特异度100%。用建立的方法对四起食物中毒、2000份健康从业人员体检肛拭样品、80份食品监测样品进行了检测,共检出沙门菌15株。结论:该方法具有高灵敏性和高特异性的特点,可以应用在沙门菌引起的食物中毒的快速检测中。     

致病性钩端螺旋体TaqMan Real-time PCR检测技术的建立及其应用

目的 建立致病性钩端螺旋体(钩体)TaqMan Real-time PCR检测技术.方法 以钩体16S rRNA基因的部分片段rrs基因作为靶基因,设计引物、TaqMan探针,PCR产物克隆到pMD 19-T载体,制作标准曲线,建立定量分析质控标准.利用中国15群15型致病性钩体参考菌株、16群21型非致病性钩体参考菌株、50株不同血清群致病性分离株及伯氏疏螺旋体、嗜肺军团菌、肺炎链球菌、脑膜炎奈瑟菌等27株其他常见致病菌检验引物、探针的灵敏性、特异性.将Real-timePCR、普通PCR同时应用于倍比稀释致病性钩体染色体DNA及25份现场鼠肾标本的检测.结果 建立、优化致病性钩体Real-time PCR技术,致病性钩体扩增荧光信号阳性,非致病性钩体及其他非钩体菌均无扩增.对于倍比稀释的质粒标准品,Real-time PCR和普通PCR的最低检测下限分别是10 copy/μl和104copy/μl.对于倍比稀释的钩体染色体DNA,两者的最低检测下限分别为:100 f/μl 和1 ng/μl.25份现场鼠肾标本检测显示,两种方法的检测结果一致.结论 以rrs为靶基因建立的Real-time PCR技术,具有较高的灵敏度和特异度,可用于致病性钩体的病原学检测.     

环介导等温扩增（LAMP）技术检测沙门菌属方法的建立

建立一种检测沙门菌属快速敏感的环介导等温扩增（loop-mediated isothermal amplification，LAMP）检测方法。针对沙门菌属侵袭蛋白（invA）基因序列的六个区域设计四条LAMP引物，65℃保温约60min，完成对沙门菌属的扩增，扩增产物经电泳和酶切鉴定。利用LAMP和普通PCR方法同时检测4株沙门菌和9株非沙门菌（对照组）来验证LAMP方法的特异性；将肠炎沙门菌菌液做一系列10倍稀释后用LAMP和PCR方法进行检测来比较两者敏感性。4株沙门菌扩增出LAMP特征性梯状条带，9株非沙门菌没有出现LAMP扩增，LAMP产物的特异性通过限制性内切酶得到了证实；LAMP检测沙门菌属的特异性与普通PCR相同，但其敏感性比普通PCR高10倍，LAMP检测沙门菌属的检测下限为10cfu／ml，PCR检测下限为100cfu／ml。LAMP检测速度相比PCR更快速，在60min内即可完成扩增反应。建立了一个快速、特异、敏感的沙门菌属LAMP检测方法。     

荧光实时定量PCR检测溶藻弧菌方法的建立

目的 建立荧光实时定量PCR(qPCR)定量检测溶藻弧菌的方法,检测该方法的灵敏度并与传统细菌培养比较其特异性吻合率。方法 选择溶藻弧菌的鞭毛基因fliC为目的基因设计探针引物,建立Taqman探针qPCR体系。采用双盲法设计,分别用qPCR和Biolog Microstation System对溶藻弧菌和10株相关细菌进行鉴定,以考核qPCR检测体系的特异性。同时,通过梯度稀释和绘制标准曲线,评价利用qPCR检测溶藻弧菌的灵敏度。结果 本试验建立的qPCR方法能特异、准确、快速鉴定溶藻弧菌,敏感度达102CFU/ml。结论 qPCR方法能够有效快速检测溶藻弧菌。     

Result variation and efficiency kinetics in real-time PCR

Fluorescent monitoring of DNA amplification is the basis of real-time PCR. Absolute quantification can be achieved using a standard curve method. The standard curve is constructed by amplifying known amounts of standards under identical conditions to that of the samples.The objective of the current study is to propose a mathematical model to assess the acceptability of PCR results. Four commercial standards for HCV-RNA (hepatitis C virus RNA) along with 6 patient samples were measured by real-time PCR, using two different RT-PCR reagents. The standard deviation of regression (Sy,x) was calculated for each group of standard and compared by F-Test. The efficiency kinetics was computed by logistic regression, c2 goodness of fit test was preformed to assess the appropriateness of the efficiency curves.Calculated efficiencies were not significantly different from the value predicted by logistic regression model. Reactions with more variation showed less stable efficiency curves, with wider range of amplification efficiencies.Amplification efficiency kinetics can be computed by fitting a logistic regression curve to the gathered fluorescent data of each reaction. This model can be employed to assess the acceptability of PCR results calculated by standard curve method.     

聚合酶链反应方法检测鼠疫耶尔森菌的内部对照研究

目的 避免聚合酶链反应(PCR)方法检测鼠疫菌发生假阴性。方法 通过克隆，将16srRNA引物的扩增产物与鼠疫菌F1抗原基因克隆子相连，并以其作为内部对照模板进行PCR试验。结果 得到了在包含F1抗原基因中连接有16SrRNA扩增产物的质粒，并初步确立了作为内部对照质粒的参照标准浓度。结论 在采用PCR方法检测鼠疫菌时，加入适宜浓度的内部对照质粒作为模板与待检样品同时扩增，可避免假阴性的发生。     

应用PCR技术检测致病性钩端螺旋体

目的 建立一种能特异检测我国致病性钩端螺旋体所有血清型的PCR方法。方法 从Genbank中选取钩体23SrDNA序列，设计一对种特异性引物。通过Blast验证引物的特异性和广谱性后，用PCR检测我国流行的所有18个血清群钩体代表株，观察其敏感性和特异性。探讨简便的样品处理方法，并对模拟标本进行检测。结果 18个血清群的25株钩体均出现单一482bp的特异性扩增产物，而双曲钩体及其他螺旋体、微生物、空白对照均无任何DNA扩增条带。PCR单一反应最小检出钩体数为8条。用煮沸法、试剂盒、SiO高盐吸附法及氯仿苯酚混合法处理样品对PCR结果无明显影响。对模拟标本的检测符合临床实际要求。结论 PCR方法是一种灵敏度高、特异性强、检测谱广的快速检测钩体的有效方法。     

Detection of Cryptosporidium parvum oocysts in experimentally contaminated lettuce using filtration, immunomagnetic separation, light microscopy, and PCR

Recovery of Cryptosporidium parvum oocysts in a fecal suspension that experimentally contaminated onto lettuce leaves was investigated. Material recovered from the lettuce samples by washing in detergent solutions were concentrated by filtration using the Envirochek Sampling Capsule. Oocysts were concentrated by immunomagnetic separation (IMS) and detected by microscopy following modified Ziehl-Neelsen (MZN) staining. Cryptosporidal DNA was detected using a nested-PCR assay for amplification of a fragment of the Cryptosporidium oocyst wall protein (COWP) gene, which was applied to DNA extracted from both filtrates, and material recovered from MZN stained smears on glass slides after microscopy. No Cryptosporidium were detected by microscopy or by PCR of un-inoculated lettuce leaves. After IMS, means of 0-6.5% of the total numbers of oocysts inoculated were recovered and detected by microscopy. Detection by PCR was less sensitive than microscopy. There was a strong association between successful PCR amplification, the numbers of oocysts detected by microscopy and the numbers of oocysts in the inoculum. This study confirms that C. parvum oocysts can be recovered from contaminated lettuce using filtration and IMS, and detected by microscopy and PCR. However, further developments are required to improve recovery of this parasite.     

多重PCR检测食品中的金黄色葡萄球菌、志贺菌和沙门菌

目的建立一种能同时检测食品中金黄色葡萄球菌、志贺菌和沙门菌的多重PCR检测方法。方法采用7.5%NaCl肉汤对食品样品中的金黄色葡萄球菌进行增菌,同时采用GN增菌剂对食品样品中的志贺菌和沙门菌进行增菌。根据金黄色葡萄球菌的nuc基因、志贺菌的ipaH基因、沙门菌的invA基因设计引物,通过多重聚合酶链反应(PCR)反应对食品样品中上述三种病原菌的目标基因进行扩增,同时对反应体系进行优化。结果特异性实验表明本方法的特异性良好。对污染金黄色葡萄球菌、志贺菌和沙门菌的牛奶样品进行检测,检出限为1cfu/ml。结论本实验建立的多重PCR检测方法适用于食品中金黄色葡萄球菌、志贺菌和沙门菌的快速检测,具有快速、简便、灵敏的特点。     

PCR指纹图技术鉴定临床常见酵母菌的探讨

目的探讨PCR指纹图技术用于鉴定临床常见酵母菌的方法. 方法采用PCR方法,使用随机引物M13和T3B对临床常见的7属17种酵母菌进行扩增. 结果17种酵母菌的扩增产物在数量和大小上都有其自身特征;不同种间酵母菌其PCR指纹图型存在明显的差异;对同种酵母菌内不同菌株扩增的结果表明,酵母菌的PCR指纹图分析种间变异远远大于种内变异,据此能将各种酵母菌有效地区分开. 结论控制好实验条件,PCR指纹图技术可有效地鉴定临床常见的酵母菌.     

建立Taqman探针实时荧光PCR检测食品中肠炎沙门氏菌的方法

目的利用Taqman探针实时荧光PCR检测食品中肠炎沙门氏菌。方法根据沙门氏菌属共有基因序列和肠炎沙门氏菌的特异序列分别设计2组引物及探针,运用实时荧光PCR进行特异性、灵敏性及模拟样品的检测实验。结果采用沙门氏菌属探针可检测到25种不同血清型沙门氏菌(共49株),而11株阴性对照菌株未得到扩增;利用肠炎沙门氏菌探针可从25种不同血清型的沙门氏菌(共49株)和11株阴性对照菌株中特异性的检测出全部15株肠炎沙门氏菌;以肠炎沙门氏菌系列稀释度菌液DNA为模板进行实时荧光PCR扩增,菌株的模板浓度与Ct值之间呈现良好线性关系,线性系数(R2)0.993,扩增效率87%,最低检测浓度260cfu/mL;分别接种肠炎沙门氏菌于四种样品进行模拟样品检测,实时荧光PCR检测结果与经典培养鉴定方法的检测结果相吻合。结论实时荧光PCR检测食品中肠炎沙门氏菌的方法特异性强、敏感度高,可应用于食品中肠炎沙门氏菌的检测。