土拉弗朗西斯菌胶体金免疫层析快速检测方法的建立

目的建立胶体金免疫层析技术快速定量检测土拉弗朗西斯菌。方法利用胶体金标记和双抗体夹心免疫层析技术,建立土拉弗朗西斯菌的快速检测方法,评价其特异性和敏感性,并拟合检测曲线进行定量检测。在面粉、饼干、果冻、梨汁等食品样品中添加土拉弗朗西斯菌的FopA蛋白模拟污染样品,评价该方法对固体、半固体、液体等食品样品的检测能力。结果该法可在10min内完成定性和定量检测,灵敏度为750ng／ml,线性范围750～24000ng/ml、回收率为56．7％-89．2％。结论所建立的检测土拉弗朗西斯菌的胶体金免疫层析方法,能快速、灵敏、特异、准确地检测样品中的土拉弗朗西斯菌,适用于现场快速检测。     

产气荚膜梭菌实时荧光PCR方法的建立

目的:利用荧光定量PCR技术,建立快速敏感特异的检测产气荚膜梭菌的方法。方法:以产气荚膜梭菌基因为靶序列设计引物和探针,以自产气荚膜梭菌菌株中提取的DNA为模板,优化引物和探针的浓度比,同时验证方法的特异性、敏感性。结果:建立的反应体系在上游引物浓度为0.45μmol/L、下游引物浓度为0.15μmol/L、探针浓度为0.3μmol/L时,具有良好的特异性和敏感性,与创伤弧菌等12种相关细菌均无交叉反应;对纯菌检测的灵敏度低于10 CFU/反应体系。结论:建立的实时荧光PCR方法特异、灵敏、快速,能对战时气性坏疽做出快速准确的报告,实现对这种战时高发疾病的安全、快速和定量检测。     

金标银染免疫渗滤法检测土拉弗朗西斯菌

目的:建立金标银染免疫渗滤法检测土拉弗朗西斯菌(土拉菌)的方法,评价其灵敏度、特异性、重复性及其应用。方法:以小鼠抗土拉菌脂多糖单克隆抗体作为捕获抗体包被硝酸纤维素膜、兔抗土拉菌多克隆抗体作为检测抗体标记胶体金,通过金标银染技术放大检测信号,建立金标银染免疫渗滤法检测土拉弗朗西斯菌体系;评价该方法的灵敏度、特异性和重复性;以经荧光定量PCR定量的土拉弗朗西斯菌为检测对象,比较金标银染免疫渗滤法和免疫层析法。结果:金标银染免疫渗滤法检测土拉弗朗西斯菌的最小检出量为1.0×103 CFU/mL,灵敏度高于免疫层析法;检测大肠杆菌、炭疽芽孢杆菌、布鲁菌和鼠疫耶尔森菌的结果均为阴性;密封保存的检测卡80 d内重复性良好,100 d后反应强度略有降低。结论:金标银染免疫渗滤法检测土拉弗朗西斯菌敏感性高、特异性强、重复性好,且方便快捷,不需要仪器设备,可作为快速检测土拉弗朗西斯菌的首选方法。     

一种快速定量检测益生菌制品中植物乳杆菌的方法

目的建立快速定量检测植物乳杆菌的方法,排除近缘菌的干扰。方法利用SMM系统筛选植物乳杆菌种特异序列,根据特异序列设计引物进行实时荧光定量PCR反应。结果设计的引物具有良好的种特异性,检测限为2．26×10^2CFU／mL。结论该方法快速灵敏,可以在实际生产中应用。     

PCR快速鉴定鼠疫耶尔森氏菌

建立一种简便、快速、特异的PCR检测方法,用于鼠疫耶尔森氏菌的快速鉴定。针对鼠疫耶尔森氏菌特异的一段染色体序列3a设计引物,扩增-276bp片段的鼠疫标识序列。应用该PCR反应体系,对我国17个生态型及1个待定的生态型共计275株鼠疫耶尔森氏菌及48株相关菌株的PCR扩增结果表明,实验菌株均扩增出预期的276bp片段产物带,48株相关菌株均阴性,其检测灵敏度为100pg DNA。说明该方法用于鼠疫耶尔森氏菌的检测鉴定简便、快捷,具有很高的特异性和敏感性。     

添加有扩增内标的沙门氏菌荧光定量PCR 检测体系的建立与评价

【目的】建立添加有扩增内标(IAC,Internal amplification control)的沙门氏菌EvaGreen荧光定量PCR检测体系,提高PCR检测可靠性。【方法】通过比较已有沙门氏菌属细菌的基因组序列,筛选沙门氏菌属特异检测靶点,设计特异引物;再用复合引物法构建扩增内标,优化参数,建立沙门氏菌内标PCR检测体系,利用特异性和灵敏度实验评价体系的检测性能。【结果】筛选得到的新特异靶点基因编码III型分泌系统蛋白(ssaQ)。针对该基因设计特异引物(SsaQ6),建立了添加有扩增内标的常规PCR和EvaGreen荧光定量PCR检测体系;二者对151株沙门氏菌和34株非沙门氏菌的检测符合率均达100%,对基因组DNA的检测下限达14.9拷贝/PCR和2.76拷贝/PCR;人工污染牛奶样品(初始染菌量:4-6 cfu/10 mL),増菌10 h和8 h后分别可检出沙门氏菌。【结论】本研究发掘的新靶点基因ssaQ特异性强,基于这一新靶点建立的添加有扩增内标的EvaGreen荧光定量PCR比常规内标PCR的检测限更低,重复性更好,快速方便,在12 h内即可得出检测结果,并且定量准确,有利于推进沙门氏菌PCR检测方法的标准化应用。     

粪便中肠球菌SYBR GreenI荧光定量PCR检测方法的建立

目的利用SYBR GreenI荧光定量PCR方法,建立肠球菌实时荧光PCR检测方法,并初步应用于粪便中肠球菌的检测。方法根据GenBank发表的肠球菌23S rRNA基因序列的保守区域设计合成特异性的引物;利用构建的质粒标准品绘制两种标准曲线,构建基因拷贝数、细菌数为分析指标的定量分析模型并初步应用于粪便标本的检测分析。结果所建立的SYBR GreenI荧光定量PCR方法检测灵敏度可达7个拷贝数/reaction。粪便样本根据实时荧光定量PCR方法所得的理论数值与培养菌值之间差异无显著性(P>0.05)。非炎性腹泻标本中菌数与健康成人标本中菌数差异无显著性(P>0.05)。灵敏度曲线所得的数值大于菌数标准曲线,可能由于DNA提取过程中存在部分的损失。检测粪便标本结果显示SYBR GreenI荧光定量PCR方法较平板计数法敏感、快捷、简便。结论本研究建立了一种灵敏、特异、简便易行的肠球菌定量检测方法。     

腐皮镰刀菌SYBR Green实时荧光定量PCR快速检测方法的建立

目的建立一种能够快速、灵敏、特异的鉴定腐皮镰刀菌的SYBR Green实时荧光定量PCR。方法运用SYBR Green实时荧光定量PCR反应体系检测腐皮镰刀菌,并对此方法的特异性、灵敏度和稳定性进行评价。结果通过对45例样品的检测,结果显示SYBR Green实时荧光定量PCR特异性好,其检出率高于普通PCR;灵敏度高,对重组质粒标准品的检测灵敏度为1.0×10~2copies/μL;稳定性好,对质粒为1.0×10~7copies/μL、1.0×10~5copies/μL、1.0×10~3copies/μL的标准品重复检测10次,结果显示扩增反应Ct值的变异系数为0.96%~1.68%。结论SYBR Green实时荧光定量PCR检测腐皮镰刀菌,不仅特异性好,灵敏度高,稳定性好,而且简便、快速、易操作。     

O1群霍乱弧菌实时荧光定量PCR快速检测方法的建立

目的:建立针对O1群霍乱弧菌的实时荧光定量TaqMan PCR快速检测方法,并进行模拟粪便标本的检测评价。方法:根据O1群霍乱弧菌O抗原编码基因rfb的特异性序列设计引物和TaqMan探针,建立检测O1群霍乱弧菌的实时荧光定量TaqMan PCR快速检测方法,对所建立的方法分别进行实验室内的灵敏度及特异性评价;将O1群霍乱弧菌灭活菌株悬液倍比稀释后与健康成人新鲜粪便混匀,制备成模拟带菌者粪便标本,提取DNA,进行Taq-Man PCR检测,用以评价该方法。结果:建立了快速检测O1群霍乱弧菌的实时荧光定量TaqMan PCR方法,灵敏度为每反应体系104拷贝;该方法对其他14种肠道菌DNA没有扩增;该方法对模拟粪便标本的检测灵敏度为每反应体系102 CFU。结论:建立了一种快速、高效检测O1群霍乱弧菌的荧光定量PCR检测方法,该方法可用于O1群霍乱弧菌临床粪便标本的检测。     

嗜肺军团菌实时荧光定量PCR快速检测方法的建立

目的：建立针对嗜肺军团菌Mip基因的实时荧光定量TaqMan PCR检测方法,并进行自来水和空调冷却水模拟标本的检测评价。方法：根据嗜肺军团菌Mip基因的特异性序列设计引物和TaqMan探针,建立嗜肺军团菌的实时荧光定量TaqMan PCR快速检测方法,对方法进行灵敏度及特异性评价,并对自来水和空调冷却水模拟标本中的嗜肺军团菌进行检测。结果：建立的方法对嗜肺军团菌的检测具有高度特异性,与3种非嗜肺军团菌和6种其他呼吸道病原均没有交叉反应;基因组DNA的检测灵敏度为1.6pg／μL,模拟自来水和空调冷却水标本的检测灵敏度为10CFU／mL。结论：建立的TaqMan荧光定量PCR方法特异、灵敏、快速,适于嗜肺军团菌的日常监测和暴发疫情的应急诊断。     

Real time PCR hybridization for the rapid and specific identification of Francisella tularensis

Tularemia is highly infectious and fatal zoonotic disease caused by Gram negative bacteria Francisella tularensis. The necessity to undergo medical treatment in early phase of illness in humans and possibility of making use of bacterial aerosol by terrorists in an attack create an urgent need to implement a rapid and effective method which enables to identify the agent. In our study two primers FopA F/R and hybridization probes FopA S1/S2 designed from fopA gene sequence, were tested for their potential applicability to identify F. tularensis. In this research 50 strains of F. tularensis were used and the test gave positive results. Reaction specificity was confirmed by using of non-Francisella tularensis bacterial species. The results obtained in the real-time PCR reaction with primers Tul4 F/R and hybridization probes Tul4 S1/S2, designed from tul4 gene, were comparable to the results from previous experiment with fopA - primers set. Investigation of fopA and tul4 primers and hybridization probes properties revealed characteristic Tm (melting temperature) value of the products--61 degrees C and 60 degrees C, respectively. Detection sensitivity was remarkably higher when fopA primers set was used 1 fg/microl, and for tul4 primers set, minimal detectable concentration is 10 fg/microl.     

Development of a real-time PCR assay for detection of monodon baculovirus (MBV) in penaeid shrimp

A real-time PCR method was developed to detect monodon baculovirus (MBV) in penaeid shrimp. A pair of MBV primers to amplify a 135 bp DNA fragment and a TaqMan probe were developed. The primers and TaqMan probe were specific for MBV and did not cross react with Hepatopancreatic parvovirus (HPV), White spot syndrome virus (WSSV), Infectious hypodermal and haematopoietic virus (IHHNV) and specific pathogen free (SPF) shrimp DNA. A plasmid (pMBV) containing the target MBV sequence was constructed and used for determination of the sensitivity of the real-time PCR. This real-time PCR assay had a detection limit of one plasmid MBV DNA copy. Most significantly, this real-time PCR method can detect MBV positive samples from different geographic locations in the University of Arizona collection, including Thailand and Indonesia collected over a 13-year period.     

Quantitative real-time PCR assays for detection of human adenoviruses and identification of serotypes 40 and 41

A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV41, respectively, with results obtained in less than 90 min. Using genomic equivalents, the broadly reactive TaqMan assay was able to detect 5 copies of AdV40 (which had zero mismatches with the PCR primers and probe), 8 copies of AdV41, and 350 copies of AdV3 (which had the most mismatches [seven] of any adenovirus serotype tested). For specific detection and identification of F species serotypes AdV40 and AdV41, a second real-time PCR assay was developed using fluorescence resonance energy transfer (FRET) probes that target the adenovirus fiber gene. The FRET-based assay had a detection limit of 3 to 5 copies of AdV40 and AdV41 standard DNA and was able to distinguish between AdV40 and AdV41 based on melting curve analysis. Both the TaqMan and FRET PCR assays were quantitative over a wide range of virus titers. Application of these assays for detection of adenoviruses and type-specific identification of AdV40 and AdV41 will be useful for identifying these viruses in environmental and clinical samples.     

Quantitative Real-Time PCR Assays for Detection of Human Adenoviruses and Identification of Serotypes 40 and 41

A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV41, respectively, with results obtained in less than 90 min. Using genomic equivalents, the broadly reactive TaqMan assay was able to detect 5 copies of AdV40 (which had zero mismatches with the PCR primers and probe), 8 copies of AdV41, and 350 copies of AdV3 (which had the most mismatches [seven] of any adenovirus serotype tested). For specific detection and identification of F species serotypes AdV40 and AdV41, a second real-time PCR assay was developed using fluorescence resonance energy transfer (FRET) probes that target the adenovirus fiber gene. The FRET-based assay had a detection limit of 3 to 5 copies of AdV40 and AdV41 standard DNA and was able to distinguish between AdV40 and AdV41 based on melting curve analysis. Both the TaqMan and FRET PCR assays were quantitative over a wide range of virus titers. Application of these assays for detection of adenoviruses and type-specific identification of AdV40 and AdV41 will be useful for identifying these viruses in environmental and clinical samples.     

Application of real-time PCR for quantitative detection of Clostridium botulinum type A toxin gene in food

The TaqMan real-time PCR method for the quantitative detection of C. botulinum type A was developed based on sequence-specific hybridization probes. The validity of this assay was verified by using 10 genera of 20 strains, including reference strains of C. botulinum types A, B, C, D, E and F. The detection limit of this assay was evaluated on C. botulinum type A, using a 10-fold dilution series of DNA and spores . The DNA and spores were detected up to level of 0.1 ng/ml and 10(2)spores/ml, respectively. Spore spiked food sample preparation prior to the real-time PCR was performed by two methods, heat treatment and GuSCN. The detection limits after heat treatment showed 10(2) spores/ml for spiked sausage slurry, and 10(3) spores/ml for spiked canned corn slurry, while detection limits after GuSCN precipitation showed 10(2) spores/ml in both sausage and canned corn. Therefore the real-time PCR assay after GuSCN precipitation is useful for the quantification of C. botulinum type A because it showed identical CT values in both pure spore solutions and food slurries. We suggest that quantitative analysis of C. botulinum type A by TaqMan real-time PCR can be a rapid and accurate assessment method for botulinal risk in food samples.     

Detection and quantification of Escherichia coli O157:H7 in environmental samples by real-time PCR

AIMS: To apply the real-time Polymerase chain reaction (PCR) method to detect and quantify Escherichia coli O157:H7 in soil, manure, faeces and dairy waste washwater. METHODS AND RESULTS: Soil samples were spiked with E. coli O157:H7 and subjected to a single enrichment step prior to multiplex PCR. Other environmental samples suspected of harbouring E.coli O157:H7 were also analysed. The sensitivity of the primers was confirmed with DNA from E.coli O157:H7 strain 3081 spiked into soil by multiplex PCR assay. A linear relationship was measured between the fluorescence threshold cycle (C T ) value and colony counts (CFU ml(-1)) in spiked soil and other environmental samples. The detection limit for E.coli O157:H7 in the real-time PCR assay was 3.5 x 10(3) CFU ml(-1) in pure culture and 2.6 x 10(4) CFU g(-1) in the environmental samples. Use of a 16-h enrichment step for spiked samples enabled detection of <10 CFU g(-1) soil. E. coli colony counts as determined by the real-time PCR assay, were in the range of 2.0 x 10(2) to 6.0 x 10(5) CFU PCR (-1) in manure, faeces and waste washwater. CONCLUSIONS: The real-time PCR-based assay enabled sensitive and rapid quantification of E. coli O157:H7 in soil and other environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to quantitatively determine cell counts of E.coli O157:H7 in large numbers of environmental samples, represents considerable advancement in the area of pathogen quantification for risk assessment and transport studies.     

Development of a molecular method to detect and quantify Aphanomyces euteiches in soil

A real-time PCR assay using 136F/211R primers and 161T TaqMan probe for the detection and quantification of Aphanomyces euteiches in soil is presented. The specificity of primers was tested on 105 different A. euteiches isolates, mainly from France. A calibration curve was established with a plasmid pHS1 resulting from the target region cloned into the pCR4 Topo vector (Invitrogen). The target copy number was evaluated and was constant whatever the isolate. A DNA-based method was able to discriminate between different artificial infestation levels in soil with small SDs thus validating the relevance of the extraction and amplification method in soil samples. Furthermore, a good correlation was observed between inoculum quantity in soil estimated by qPCR and root rot severity in plant evaluated by bioassays. These steps are essential when considering the feasibility of using a DNA-based method as a fast and accurate way to evaluate inoculum quantity in soil.     

荧光定量PCR检测重组新蛭素中毕赤酵母基因组DNA的残留量

目的:建立real-time PCR定量检测毕赤酵母基因组DNA残留量的方法,提高重组新蛭素的产品安全性。方法:选择拷贝数高且分布广泛的毕赤酵母5S rRNA基因为靶标基因设计扩增引物,提取酵母基因组DNA,稀释后作为扩增模板。以罗氏荧光定量PCR_LightCycler480平台为基础,建立基于SYBR GreenⅠ荧光染料的real-timePCR的检测方法,并考察用该方法检测重组新蛭素中毕赤酵母基因组残留量的灵敏度、精密度和回收率。结果:该法检测宿主DNA残留量灵敏度高,DNA浓度为0.1~1000 pg/μL范围内呈现良好的线性关系,其标准曲线的误差值小于0.2;用该法对5批注射用重组新蛭素(酵母)产品中宿主基因组DNA残留量进行了测定,结果分别为0.03、2.3、0.2、0.6、0.2 pg/mg。结论:该方法具有操作简便、灵敏度高等优点,可用于重组产品中酵母基因组残留DNA的定量测定。