多重荧光定量PCR法检测海产品中副溶血性弧菌、沙门菌和单增李斯特菌

目的建立检测海产品中副溶血性弧菌、沙门菌和单增李斯特菌的多重荧光定量PCR体系。方法针对副溶血性弧菌tlh基因,沙门菌Ompc基因和单增李斯特菌hly基因设计引物和Taq Man探针,建立多重荧光定量PCR体系,进行特异性与敏感性研究;利用该体系检测海产品中的副溶血性弧菌、沙门菌和单增李斯特菌。结果副溶血性弧菌、沙门菌和单增李斯特菌可得到特异性扩增,而共存于海产品中的其他细菌均未见扩增曲线。敏感性试验显示,该体系对副溶血性弧菌、沙门菌和单增李斯特菌的最低检测限分别为72、40、80 cfu/ml。对舟山采集的150份样品进行检测,检出32份副溶血性弧菌、11份沙门菌、5份单增李斯特菌,与国标法检测结果一致。结论本研究建立的基于Taq Man探针的多重荧光定量PCR检测方法可以特异、灵敏、简单快速地实现对海产品中副溶血性弧菌、沙门菌和单增李斯特菌的检测。     

Development of a quantitative real-time PCR method to enumerate total bacterial counts in ready-to-eat fruits and vegetables

A newly developed real-time PCR assay rapidly quantifies the total bacterial numbers in contaminated ready-to-eat vegetables and fruits compared with the standard plate count method. Primers targeting the rpoB gene, which encodes for the beta subunit of the bacterial RNA polymerase and which is common to most bacterial species, was used instead of the 16S rRNA gene, which has multiple copies and varies among bacterial species. A primer pair specific for rpoB was confirmed to amplify rpoB in a wide range of bacterial species after we assessed 49 strains isolated from five kinds of fruits and vegetables. We purchased fruits and vegetables from retail shops and enumerated the bacteria associated with them by use of real-time PCR and compared this to the number found by the culture method. We found a high correlation between the threshold PCR cycle number when compared with the plate count culture number. The real-time PCR assay developed in this study can enumerate the dominant bacterial species in ready-to-eat fruits and vegetables.     

荧光PCR检测副溶血性弧菌致病基因方法的建立

建立一种准确、快速、特异地检测副溶血性弧菌(Vibrio parahaemolyticus,VP)致病基因的荧光PCR方法。方法 用7株已知其致病基因的VP标准菌株和检出菌分别对6种VP致病基因荧光PCR检测文献方法和本文建立的方法进行验证比较,并用6种常见食源性病原菌对新建方法的特异性进行检验。结果 所建方法能够同时检测VP的3种溶血素,其检测结果与菌株的溯源结果及普通PCR结果完全一致,且与常见食源性病原菌无交叉反应。结论 本文建立的荧光PCR体系能够准确、快速、特异地检测VP的致病基因,具有较好的实用性。     

基于vvhA基因检测创伤弧菌实时荧光PCR方法的建立

为建立创伤弧菌(VV)的快速检测方法,根据vvhA基因序列设计合成引物和探针,建立实时荧光PCR方法。结果显示:所建立的方法能特异扩增出VV标准阳性菌株,而对其它14种菌株没有扩增;该方法的灵敏度为15CFU/mL;稳定性和重复性试验结果表明,同一样品重复检测4次Ct值(循环阈值)的变异系数均小于2%;利用该检测方法对采集的156份样品进行检测,共计检出2份VV阳性样品,与行标法(SN/T 1870—2007)检测结果一致。该检测方法灵敏度高、特异性强,具有良好的实用性。     

副溶血弧菌DPO-PCR检测方法的建立

以副溶血弧菌(Vibrio parahaemolyticus,VP)toxR基因为靶基因设计了一对双启动寡核苷酸(DPO)引物,建立了DPO-PCR快速检测VP的方法。结果显示,退火温度在49~69℃都能有效地扩增出目的基因,表明该检测方法对退火温度不敏感;该DPO引物仅与VP的扩增反应呈阳性,而与其他菌株无非特异性扩增反应,表明该方法特异性强;该方法的灵敏度为121 CFU/m L。利用该检测方法对采集的550份样品进行检测,共计检出19份VP阳性样品,与行标法(SN/T 1870-2007)检测结果一致,实用性良好。该DPO-PCR方法设计简单、特异性强,具有良好的实用性。     

溶藻弧菌实时荧光定量PCR快速检测方法的建立

为建立溶藻弧菌（VA）的快速检测方法,本研究以VA Collagenase基因为靶基因设计合成引物及Taq Man探针,建立了实时荧光定量PCR快速检测VA的方法。结果显示,对15株实验菌株进行荧光定量PCR检测,只有VA菌株检测为阳性,表明该检测方法特异性强;该方法的灵敏度为18 cfu/m L;稳定性和重复性实验结果表明,同一样品重复检测4次Ct值的变异系数均小于2%;利用该检测方法对采集的165份样品进行检测,共计检出2份VA阳性样品,与SN/T2564-2010行标法检测结果一致,显示了良好的实用性。该检测方法灵敏度高、特异性强,具有良好的实用性。      

食品中副溶血性弧菌toxR和tdh基因双色荧光PCR检测方法的建立

目的 建立对副溶血性弧菌（Vibrio parahaemolyticus）特异性检测toxR（跨膜转录激活蛋白）基因和tdh（热稳定性直接溶血素）毒力基因的Taqman探针双色荧光PCR检测方法。方法 根据副溶血性弧菌toxR基因和tdh基因,分别设计引物和探针,建立Taqman探针双色荧光PCR扩增体系,进行特异性、灵敏度试验;对副溶血性弧菌分离菌株实施检测,了解其tdh基因和tdh基因分布情况。结果 结果表明,副溶血性弧菌标准菌株和3株从食物中毒患者中分离获得的分离株均出现toxR基因和tdh扩增曲线,而溶藻弧菌、单增李斯特菌等31株弧菌属其他菌株和肠杆菌科的菌株未见扩增曲线。从食品中分离的37株副溶血性弧菌分离株均未携带tdh毒力基因。副溶血性弧菌检测灵敏度可达到3.6×102 cfu/mL。结论 该方法可用于同时检测食品中副溶血性弧菌的特异性和毒力基因。     

基于内参的副溶血性弧菌实时荧光定量PCR快速检测方法的建立

目的建立基于内参的副溶血性弧菌实时荧光定量PCR方法,快速检测样品中的副溶血性弧菌。方法根据Gen Bank已公布的副溶血性弧菌基因组序列,筛选特异性靶基因,设计特异性引物探针,优化反应体系,并在体系中加入内参(IAC),通过标记不同荧光基团的Taq Man探针来监测IAC,进而实时监控整个PCR反应。按照5~50 cfu/25 g的细菌量人工污染样品,以评价所建立反应的体系。结果以副溶血性弧菌基因组DNA为模板,最低检测限为1 pg/μl;以10倍梯度稀释的菌液经水煮法提取的DNA为模板,最低检测限为4×102cfu/ml;以含有gyr B的质粒为模板,最低检测极限可以达到100 copies/μl;建立gyr B和gyr B-IAC标准曲线,Ct值与模板拷贝数均呈良好线性关系(r2=0.999);人工污染初始菌量为7 cfu/25 g时,样品中副溶血性弧菌增菌6 h即可检出。结论本研究所建立的gyr B-IAC实时荧光定量PCR方法,既能有效检测食品中副溶血性弧菌,又能实时监测PCR反应过程,有效防止"假阴性"的发生,结果可靠,有利于实现海产品中副溶血性弧菌实时荧光定量PCR检测方法的标准化。     

养殖海水贝类中副溶血性弧菌的致病性及 耐药性分析

目的对从山东和辽宁沿海地区养殖海水贝类中分离到的84株副溶血性弧菌进行致病性及耐药性分析。方法通过PCR扩增及测序法检测毒力基因,通过神奈川试验测定溶血能力,采用K-B法进行药敏试验分析。结果 84株菌均含有tlh基因,均不含trh基因,有1株菌含tdh基因。tdh基因阳性菌株的神奈川试验呈阳性,其余菌株均呈阴性。菌株对氨苄西林、头孢呋辛钠和复方新诺明的耐药率分别为91.7%、6.0%和1.2%。所有菌株对阿莫西林/克拉维酸、头孢曲松、头孢吡肟、阿米卡星、庆大霉素、亚胺培南、氧氟沙星、环丙沙星、萘啶酸、氯霉素、氟苯尼考和呋喃妥因高度敏感。结论海水贝类中含有少量致病性副溶血性弧菌,菌株存在一定程度的耐药性,提示应加强对海产品中副溶血性弧菌致病性及耐药性的监控。     

PMA-qPCR定量检测VBNC副溶血弧菌方法的建立与优化

副溶血弧菌（Vibrio parahaemolyticus）在某些不利于生长的自然和食品加工条件下能够进入活的非可培养（viable but non-culturable, VBNC）状态,普通检测方式极易漏检,因此VBNC细菌检测技术的开发与优化对副溶血弧菌感染防控具有重要意义。基于叠氮溴化丙锭结合荧光定量PCR技术（propidium monoazide quantitative PCR,PMA-qPCR）能够利用活细胞的膜完整性区分活菌和死菌的原理,以副溶血弧菌ATCC 17802为对象,通过探讨PMA-qPCR方法中各项因素对活菌计数结果的影响,优化并建立了VBNC副溶血弧菌的定量检测方法。实验结果表明,当添加PMA质量浓度为15 mg/L,黑暗孵育10 min,再进行强光照射15 min后,死细胞的DNA扩增得到有效抑制,此时提取DNA模板进行qPCR扩增准确性更高,活菌数与qPCR扩增循环数呈现显著性线性相关（R²=0.99）。PMA-qPCR法测定副溶血弧菌活菌数的标准曲线显示该方法检测范围为2.90×10¹～2.90×10     

实时荧光PCR法快速检测肉类中貉源性成分

目的建立貉源性成分的实时荧光PCR快速检测手段。方法根据貉的线粒体Cytb基因序列设计引物和探针,通过优化扩增反应体系进行实时荧光PCR(RT-PCR)扩增,产物进行快速检测。结果此方法特异性良好,灵敏度可达到10~(-4)ng貉源性DNA的量。在混合或纯肉样品以及常见的肉制品中均可检测。结论本方法可以满足实际工作中肉类掺假检测的要求。     

副溶血性弧菌和溶藻弧菌的双重PCR检测技术

主要针对副溶血性弧菌(Vp BJ1997)和溶藻弧菌(Va ATCC17749)的gyrB基因的不同位点设计特异性引物,利用双重PCR技术同时检测Vp(BJ1997)和Va(ATCC17749),并对该反应体系的特异性以及灵敏度进行检测。结果显示Vp(BJ1997)灵敏度的检测下限可达2×10~2CFU/mL,Va(ATCC17749)灵敏度的检测下限可达2.03×10~2CFU/mL,双重PCR与单一PCR检测的灵敏度的数量级是相同的;与沙门氏菌(ATCC27892)、金黄色葡萄球菌(ATCC13565)、大肠杆菌(35218)、河弧菌(H265)、鳗弧菌(M936)、创伤弧菌(ATCC27562)无交叉反应;在人工污染试验中,混合菌液的检测下限2.84×10~3CFU/mL,而进一步稀释至2.84×10~2CFU/mL时,Vp(BJ1997)已检测不出,Va(ATCC17749)仍可检出。研究表明,该方法特异性强,灵敏度高,操作简单,成本低,检测速度快,为基层单位同时检测副溶血性弧菌和溶藻弧菌提供了一种快速准确的方法,对控制水产品中这两种致病性弧菌具有重大的意义。     

Density of total and pathogenic (tdh+) Vibrio parahaemolyticus in Atlantic and Gulf coast molluscan shellfish at harvest

The densities of total and pathogenic Vibrio parahaemolyticus in 671 samples of molluscan shellfish harvested in 1999 and 2000 from 14 sites in seven Gulf and Atlantic coast states were determined at 2-week intervals over a period of 12 to 16 months in each state. Changes in V. parahaemolyticus densities in shellfish between harvest and sample analysis were minimized with time and temperature controls. Densities were measured by direct plating techniques, and gene probes were used for identification. Total and pathogenic V. parahaemolyticus organisms were identified with probes for the thermolabile direct hemolysin (tlh) gene and the thermostable direct hemolysin (tdh) gene, respectively. An enrichment procedure involving 25 g of shellfish was also used for the recovery of pathogenic V. parahaemolyticus. The densities of V. parahaemolyticus in shellfish from all harvest sites were positively correlated with water temperature. Shellfish from the Gulf Coast typically had higher densities of V. parahaemolyticus than did shellfish harvested from the North Atlantic or mid-Atlantic coast. Vibrio parahaemolyticus counts exceeded 1,000 CFU/g for only 5% of all samples. Pathogenic (tdh+) V. parahaemolyticus was detected in approximately 6% of all samples by both procedures, and 61.5% of populations in the positive samples from the direct plating procedure were at the lower limit of detection (10 CFU/g). The frequency of detection of pathogenic V. parahaemolyticus was significantly related to water temperature and to the density of total V. parahaemolyticus. The failure to detect pathogenic V. parahaemolyticus in shellfish more frequently was attributed to the low numbers and uneven distribution of the organism.     

荧光PCR检测副溶血性弧菌致病性方法的建立

目的 建立一种准确、快速、特异地检测副溶血性弧菌（Vibrio parahaemolyticus,VP）致病基因的荧光PCR方法。方法 用7株已知其致病基因的VP标准菌株和检出菌分别对6种VP致病基因荧光PCR检测文献方法和本文建立的方法进行验证比较,并用6种常见食源性病原菌对新建方法的特异性进行检验。结果 所建方法能够同时检测VP的3种溶血素,其检测结果与菌株的溯源结果及普通PCR结果完全一致,且与常见食源性病原菌无交叉反应。结论 本文建立的荧光PCR体系能够准确、快速、特异地检测VP的致病基因,具有较好的实用性。     

Development of a quantitative real-time PCR assay for the detection of Aspergillus carbonarius in grapes

Aspergillus carbonarius is the main species responsible for the production of ochratoxin A (OTA) in wine grapes. To monitor and quantify A. carbonarious in grapes, a quantitative real-time PCR assay was developed as a possible tool for predicting the potential ochratoxigenic risk. DNA extraction from grape berries was performed by using conventional extraction and clean up through EZNA Hi-bond spin columns. A TaqMan probe was used to quantify A. carbonarius genomic DNA in grape berries samples. An exogenous internal positive control was used to overcome DNA recovery losses due to matrix inhibition. The quantification of fungal genomic DNA in naturally contaminated grape was performed using the TaqMan signal versus spectrophotometrically measured DNA quantities (Log10) calibration curve with a linearity range from 50 to 5 x 10(-4) ng of DNA. A positive correlation (R2=0.92) was found between A. carbonarious DNA content and OTA concentration in naturally contaminated grape samples. This is the first application of TaqMan real-time PCR for identifying and quantifying A. carbonarius genomic DNA occurring in grapes. The rapid DNA extraction method for grapes, together with the commercial availability of reagents and instrumentation, allows to perform a remarkable number of reproducible assays (96-well format) in less than 4 h.     

TaqMan实时荧光聚合酶链式反应(PCR)技术定量检测婴幼儿配方食品中的金黄色葡萄球菌

靶向于耐热核酸酶基因nuc,研究建立了Taq Man实时荧光聚合酶链式反应(PCR)(qRT-PCR)法用于婴幼儿米粉及乳粉中金黄色葡萄球菌的快速定量检测。经优化的qRT-PCR体系特异性强,仅在金黄色葡萄球菌中产生典型扩增曲线;方法灵敏度高,对标准质粒、纯菌液、模拟染菌米粉及奶粉样液的检测限分别为17.94拷贝、1.2 CFU、0.42 CFU和0.74 CFU/PCR反应体系;10倍系列梯度稀释的标准质粒、纯菌及人工染菌样液的浓度对数与qRT-PCR的Ct值线性相关度良好,拟合方程的决定系数均≥0.99;平板计数法与qRT-PCR法对模拟米粉及乳粉盲样中金黄色葡萄球菌的定量检测结果间无统计学差异。文中所建立的qRT-PCR定量法特异性强、灵敏度高,简便快捷、可靠性佳,可为婴幼儿米粉及乳粉中金黄色葡萄球菌的快速、准确定量检测提供有效手段。     

利用实时荧光定量PCR方法分析转基因水稻外源基因拷贝数

为分析转基因水稻外源基因拷贝数，利用新型、灵敏、高通量的实时荧光定量PCR方法进行转基因水稻外源基因拷贝数的分析。转基因外源基因的拷贝数通过转基因水稻外源基因(GUS和HPT基因)和水稻内标准SPS基因含量的比较计算获得。定量分析了14株T0代的转基因水稻植株，得到了外源基因插入分别为1、2、3和4的转基因植株，同时利用Southern Blot方法进行验证分析。随机选择18个已经过定量PCR检测分析的转基因水稻植株，用Southern Blot的方法分析转基因水稻植株中的HPT或GUS基因的拷贝数，Southern Blot分析结果显示有15个转基因水稻植株的分析结果与定量PCR分析的结果是一致的，3个植株定量PCR分析的转基因拷贝数稍高于Southern Blot的分析结果，主要原因是Southern Blot方法在同一个插入位点有多拷贝的T-DNA片段插入时，转基因植株的基因组在完全酶切时会产生相似的DNA片段，电泳分析时很难分辨清楚。定量PCR方法则完全避免了这种情况的发生，除非目的基因DNA片段在PCR引物处发生断裂。两种方法分析结果的比较显示定量PCR方法分析转基因拷贝数更加有效、适用。     

Evaluation of MPN method combined with PCR procedure for detection and enumeration of Vibrio parahaemolyticus in seafood

Vibrio parahaemolyticus densities in spiked and naturally contaminated seafood samples were enumerated by the MPN method combined with a PCR procedure (MPN-PCR method) targeting the species-specific thermolabile hemolysin gene (tlh), and by the MPN method using subcultivation of alkaline-peptone-water (APW) enrichment culture on thiosulfate-citrate-bile-sucrose (TCBS) agar (MPN-TCBS method). In the samples spiked with both V. parahaemolyticus and V. alginolyticus, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were similar to, or higher than the numbers of spiked cells, whereas those enumerated by the MPN-TCBS method were below the numbers of spiked cells. In naturally contaminated seafood samples, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were higher than those by the MPN-TCBS method. In the case of the MPN-TCBS method, isolation of V. parahaemolyticus from some APW cultures was difficult because of the overgrowth of many colonies other than V. parahaemolyticus (e.g., V. alginolyticus) on TCBS agar. In contrast, the PCR technique could detect tlh from APW culture without isolation of V. parahaemolyticus, so the possibility of failing to obtain a positive result in APW culture by the MPN-PCR method was considered to be lower than that by the MPN-TCBS method. Furthermore, utilization of the PCR technique reduces the time and labor required for the biochemical identification tests used in the MPN-TCBS method. For the detection and enumeration of V. parahaemolyticus in seafood, especially for samples that show many colonies other than V. parahaemolyticus on TCBS agar, the MPN-PCR method may be more convenient and reliable than the MPN-TCBS method.     

Development and validation of a real-time PCR detection method for celery in food

As from 25 November 2005 onwards, a list of ingredients with known allergenic potential has to be labeled according to Directive 2003/89/EC, including celery and products thereof. In order to provide appropriate detection methods a novel real-time polymerase chain reaction (PCR) system for the specific and sensitive detection of DNA from celery (Apium graveolens) was developed and validated. Specificity was confirmed by testing DNA derived from more than 50 food relevant organisms. Sensitivity was demonstrated on the basis of a calibration curve plotting the corresponding Ct-values against DNA amounts ranging from 1 to 1000 copies. Due to the lack of certified reference material the applicability of the method was assessed by analysis of sausages spiked with defined amounts of grounded celery seed. The limit of detection (LOD) examined exemplarily for emulsion-type sausages was 5–10 mg/kg. Analysis of celery-containing commercial products demonstrated the performance potential and limitations of the new real-time PCR system.     

A collagenase-targeted multiplex PCR assay for identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus

A multiplex PCR assay using three collagenase-targeted primer pairs for the species-specific detection of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus was developed. The results highlight the species specificity of the three primer sets designed. Because of the increasing importance of Vibrio spp. in human foodborne diseases, molecular approaches for routine microbial screening and monitoring of clinical, environmental, and food samples also have become more important. The results of this study indicate that the gene coding for collagenase should be used as an alternative molecular target to discriminate among the three Vibrio species.