Innovative Methods for Removal of PCR Inhibitors for Quantitative Detection of Plesiomonas shigelloides in Oysters by Real-Time PCR

A quantitative assay for Plesiomonas shigelloides in pure culture and oysters based on the real-time polymerase chain reaction (Rti-PCR) was developed. The methodology involved the treatment of oyster tissue homogenates with formaldehyde, differential centrifugation, and treatment of samples with activated charcoal coated with Pseudomonas fluorescens. With seeded oyster tissue homogenates, without formaldehyde or coated charcoal treatments, the lowest level of detection for P. shigelloides was 1 × 10⁷ genomic targets per gram of tissue, equivalent to 2.5 × 10⁵ genomic targets per Rti-PCR. The addition of 4% formaldehyde to tissue homogenates reduced the minimum level of detection of P. shigelloides to 1 × 10⁵ genomic targets per gram of tissue, equivalent to 2.5 × 10³ genomic targets per real-time PCR. The treatment of tissue homogenates with only activated charcoal coated with P. fluorescens reduced the minimum level of detection of P. shigelloides to 1 × 10⁴ genomic targets per gram, equivalent to 2.5 × 10² genomic targets per real-time PCR, without DNA purification or enrichment. The combination of adding 4.0% formaldehyde to oyster tissue homogenates and treating with coated charcoal reduced the level of detection of P. shigelloides to 1 × 10³ genomic targets per gram, equivalent to 25 genomic targets per real-time PCR. The linear range of detection was from 1 × 10³ to 1 × 10⁶ genomic targets per gram without enrichment.     

Quantification of Plesiomonas shigelloides in Pure Culture and Clams by Competitive PCR

A quantitative assay for Plesiomonas shigelloides in pure culture and clams based on the competitive polymerase chain reaction was developed. This is the first report for quantitative detection of P. shigelloides by competitive PCR. Forward (PS-F), reverse (PS23RV3), and hybrid primers were designed, and the specificity of the forward and reverse primer for P. shigelloides was proven. An internal standard DNA sequence was synthesized by PCR, with the hybrid primer as the forward primers and PS23RV3 as the reverse primer. A single concentration (0.588 pg/PCR) of internal standard (IS) was used for competitive PCR. The lowest level of detection of P. shigelloides was 80 CFU per PCR in pure culture, 240 CFU/g of clam tissue without enrichment, and 40 CFU/g of clam tissue after 7 hrs. nonselective enrichment at 37°C. There was a linear relationship between the log of the ratio of the relative fluorescent intensities of amplified target DNA bands to the internal standard DNA bands (IS) and the log of the CFU within a certain range in pure cultures and in clam tissue either with or without enrichment. The linear range with cells from a pure culture was the DNA derived from 8.0 × 10¹ to 8.0 × 10⁴ CFU per PCR while with clam tissue, the linear range was the DNA derived from 2.4 × 10² to 2.4 × 10⁵ CFU/g of tissue (1.2 × 10¹ to 1.2 × 10⁴ CFU per PCR) without enrichment, and 4.0 × 10¹ to 1.2 × 10⁴ CFU/g of clam tissue with enrichment, respectively.     

Factors Affecting Quantitative PCR Assay of Plesiomonas shigelloides

We have found that there are various factors that can affect the quantitative PCR assays of Plesiomonas shigelloides. Different Taq polymerase preparations, varying sets of primers, different DNA stains, and different cell lysing agents were found to significantly influence the linear relationship between the fluorescent intensities of DNA bands and the log of CFU per PCR. The primer dimers formed in the PCR can be eliminated by using different Taq polymerase preparations and different sets of primers to run the PCR.     

Factors Affecting Quantitative PCR Assay of Plesiomonas shigelloides

We have found that there are various factors that can affect the quantitative PCR assays of Plesiomonas shigelloides. Different Taq polymerase preparations, varying sets of primers, different DNA stains, and different cell lysing agents were found to significantly influence the linear relationship between the fluorescent intensities of DNA bands and the log of CFU per PCR. The primer dimers formed in the PCR can be eliminated by using different Taq polymerase preparations and different sets of primers to run the PCR.     

Quantification of Viable Plesiomonas shigelloides in a Mixture of Viable and Dead Cells Using Ethidium Bromide Monoazide and Conventional PCR

A rapid and efficient method for quantitative detection of viable Plesiomonas shigelloides in pure culture containing a mixture of viable and heat-killed cells was developed using ethidium bromide monoazide (EMA) in combination with the polymerase chain reaction (PCR). The addition of EMA (1 μg/ml) to mixtures of viable and heat-killed cells of P. shigelloides inhibited the PCR amplification of DNA derived from the dead cells, but did not inhibit the PCR amplification of DNA derived from the viable cells. EMA at 5 μg/ml or less had little or no inhibition on the PCR amplification of DNA derived from viable cells of P. shigelloides. After EMA treatment, the DNA from viable P. shigelloides cells in varying ratios of viable to dead cells could be selectively quantified by PCR. The minimum level of detection was DNA from 24 genomic targets per PCR reaction. A linear relationship was found between the relative fluorescent intensity of the DNA bands and the log of genomic targets derived from the viable cells in mixtures of viable and dead cells in the range of 2.4 × 10¹ to 2.4 × 10⁴ DNA targets from viable cells per PCR.     

Quantification of Viable Plesiomonas shigelloides in a Mixture of Viable and Dead Cells Using Ethidium Bromide Monoazide and Conventional PCR

A rapid and efficient method for quantitative detection of viable Plesiomonas shigelloides in pure culture containing a mixture of viable and heat-killed cells was developed using ethidium bromide monoazide (EMA) in combination with the polymerase chain reaction (PCR). The addition of EMA (1 μg/ml) to mixtures of viable and heat-killed cells of P. shigelloides inhibited the PCR amplification of DNA derived from the dead cells, but did not inhibit the PCR amplification of DNA derived from the viable cells. EMA at 5 μg/ml or less had little or no inhibition on the PCR amplification of DNA derived from viable cells of P. shigelloides. After EMA treatment, the DNA from viable P. shigelloides cells in varying ratios of viable to dead cells could be selectively quantified by PCR. The minimum level of detection was DNA from 24 genomic targets per PCR reaction. A linear relationship was found between the relative fluorescent intensity of the DNA bands and the log of genomic targets derived from the viable cells in mixtures of viable and dead cells in the range of 2.4 × 10¹ to 2.4 × 10⁴ DNA targets from viable cells per PCR.     

乳酸菌饮料中嗜酸乳杆菌的实时荧光定量PCR检测方法

目的：为快速准确检测乳酸菌饮料中的嗜酸乳杆菌,建立实时荧光定量聚合酶链式反应（polymerase chain reaction,PCR）检测方法。方法：根据嗜酸乳杆菌NCFM的SPIDR保守区域设计特异性引物与探针,借助建立的实时荧光定量PCR方法进行特异性、灵敏度、重复性以及抗干扰能力验证,并利用模拟样品对方法进行检验,最后对市售的实际样品进行检测。结果：该方法的特异性较好;方法的绝对灵敏度达到3pg,相对灵敏度达103 CFU/mL;重复性检测表明相对标准偏差在2.6%以下。同时进行杂菌干扰实验,在纯基因组水平和培养物水平混合大肠杆菌,扩增均无明显影响,表明建立的方法抗干扰能力较好。利用建立的实时荧光定量PCR方法对模拟样品进行检测并建立标准曲线,得出R2为0.987,线性较好,可进行实际样品的检测。对市售的11 份样品进行检测,其中6 份含有嗜酸乳杆菌菌株,5份不含嗜酸乳杆菌菌株,标记嗜酸乳杆菌的样品全部检测出嗜酸乳杆菌且含量在5.83×102～3.68×104 CFU/mL之间。结论：建立的实时荧光定量PCR方法可快速、准确地检测出乳酸菌饮料中嗜酸乳杆菌。     

Plesiomonas shigelloides - An Aquatic Food Borne Pathogen: A Review of its Characteristics,Pathogenicity, Ecology,and Molecular Detection

Plesiomonas shigelloides is a unique Gram-negative polarly flagellated pathogenic bacterium native to aquatic animals and environments. The genus Plesiomonas consists of a single homogeneous species. Its metabolism is similar to that of the genus Vibrio in that sugars are fermented with acid production but no gas. 5S rDNA sequencing has indicated the organism to be closely related to the genus Proteus. Diarrhea is the major symptom although extra intestinal infections, including septicemia, are known to occur with predisposed individuals. Oysters are the major food incriminated in outbreaks in the United States. A temperature of 42–44°C is recommended for isolation to eliminate aeromonads. The utilization of inositol with acid production is a unique characteristic of the organism that is exploited with several agar media developed for its selective and differential isolation. The organism is β-hemolytic and produces a cholera-like (CL) enterotoxin in addition to a thermostabile (TS) and a thermolabile labile (LT) enterotoxin. A large plasmid (>120 mdalton) has also been found to facilitate invasion. A variety of factors have been found to influence results obtained from application of the polymerase chain reaction (PCR) to the DNA of the organism.     

乳品检测中实时荧光定量PCR仪的研制

完成了荧光检测系统、PCR热循环扩增系统、DNA核酸定量分析诊断系统等仪器关键技术研究，针对乳品检验及临床应用要求．在国家权威部门完成了仪器性能检测、使用验证、临床试验等全过程，实现了产品化和应用示范，并达到了准确性、安全性和有效性的要求。     

实时荧光PCR定量检测肉制品中猪源性成分

为了准确可靠地对肉制品中猪源性成分进行定量检测,通过生物信息学方法筛选到猪细胞核单拷贝基因(carcinoembryonic antigen-related cell adhesion molecule 2-like,CACA).以CACA基因为扩增靶标,设计了特异性引物、TaqMan探针,建立了基于实时荧光定量PCR...     

食品中鸡源性成分实时荧光PCR检测方法的建立

目的：建立基于实时荧光聚合酶链式反应技术的食品中鸡源性成分快速检测方法。方法：以鸡线粒体细胞 色素b为目的基因,设计特异性引物和探针,通过特异性、灵敏性实验及模拟混合肉样和市售肉制品检测,对该体 系进行验证。结果：该鸡源荧光聚合酶链式反应检测体系具有很好的特异性及灵敏性,可检测3.5 pg/μL鸡源DNA 的存在;经含鸡源成分的模拟混合肉样检测,证实体系抗干扰能力强;并且通过市售食品检测表明体系可用于定性 加工食品中的鸡源成分。结论：所建立的鸡源引物探针体系具有特异性好、灵敏度高、快速高效等优点,可用于对 食品中鸡源性成分的掺假鉴别。     

实时荧光定量PCR法测定发酵乳中双歧杆菌

对实时荧光定量聚合酶链式反应(PCR)技术检测发酵乳中双歧杆菌的DNA提取方法、PCR扩增效率、标准曲线绘制进行探讨,通过比较分析碱式提取法、玻璃珠破碎法和酶解法3种提取方法对发酵乳中总DNA提取效率、4种不同参考菌株的PCR扩增效率以及单一菌株标准曲线与混合菌株标准曲线的计数结果,建立实时荧光定量PCR快速测定发酵乳制品中双歧杆菌数量方法。结果表明：酶解法提取发酵乳中总DNA效果最好,OD260/280比值基本接近1.80,且提取的质量浓度含量最高;不同菌株的PCR扩增效率不同,其中参考菌株1.2213的Ct值与其他3菌株的Ct值存在显著性差异;根据单一菌株绘制标准曲线与混合菌株绘制标准曲线计数结果无显著性差异,后者计数结果更客观、准确。采用酶解法获取样品中的菌体细胞,基于混合菌液绘制标准曲线,采用实时荧光定量PCR技术确定发酵乳中双歧杆菌种属数量,可快速、准确地测定发酵乳中双歧杆菌的数量。     

SYBR Green实时荧光定量PCR检测大豆转基因成分

采用SYBR Green实时荧光定量PCR技术,建立了食品中大豆转基因成分的定量检测方法。通过设计特异引物,扩增内源参照基因lectin和转基因靶基因CP4EPSPS,建立两种基因的拷贝数-CT标准曲线,根据标准曲线方程计算样品中的转基因含量,并且通过熔解曲线分析扩增反应特异性。结果表明,lectin和CP4EPSPS基因标准曲线线性关系好,R2值分别为0.9984和0.9953,方法的回收率为95%～110%,检测限为0.01%。本检测方法具有快速、灵敏、准确、特异、高通量等优点,可以作为食品中大豆转基因成分的定量检测方法。     

副溶血性弧菌实时荧光定量PCR标准模板的构建和检测方法的建立

目的:构建肉制品副溶血性弧菌实时荧光定量PCR的标准阳性模板和检测方法.方法:以副溶血性弧菌toxR基因上特异性片段为目标,设计并合成引物及Taqman探针,将目标片段连接到PGM-T载体上构建重组质粒,建立实时荧光定量检测体系,并考察方法的灵敏性、特异性、重复性和准确性.结果:构建出副溶血性弧菌荧光定量PCR检测方法的标准模板并建立了相应的检测方法,获得的标准曲线方程为:Y=-3.151 lgX+42.86,灵敏度40copies/反应体系,对副溶血性弧菌具有特异性,同时,该方法具有良好的重复性,变异系数小于5％.结论:使用基于Taqman探针技术的荧光定量PCR检测方法能够对食品中致病性副溶血性弧菌进行快速、简便、准确、高效的定量检测.     

实时荧光定量PCR技术在食品微生物检测和研究中的应用

实时荧光定量PCR技术是通过检测PCR产物中荧：艺讯号强度来达到定量的目的,不仅实现了对核酸信息量的分析比较,而且与常规PCR相比,它具有特异性更强、能有效解决PCR污染问题、自动化程度高等特点,近年来该技术开始作为食品微生物的研究手段。本文概述了实时荧光定量PCR技术的原理、优缺点及其在食品微生物检测中的应用与研究进展,并探讨了它的技术发展和应用前景。     

基于单拷贝核基因的荧光定量PCR技术检测驴奶的含量

本研究建立了一种基于内参基因的标准化实时聚合酶链式反应(real-time PCR)方法,能够定量检测混合掺假的灭菌乳中驴奶的含量。使用单拷贝核基因代替多拷贝线粒体基因,基于Ct值(驴特异性引物/内参引物)与驴奶含量的线性关系,对含量为5%~100% 的驴奶建立了标准曲线。该方法具有良好的线性相关(R²=0.9650)和较高的准确度,对含有20%、50%和80% 驴奶的模拟掺假样品进行定量分析,平均回收率为109.16%,平均CV值为4.68%。因此,该方法可以快速、准确地对驴奶进行定量,从而确定驴奶是否掺假以及掺假比例。

     

DNA 提取方法对实时荧光定量 PCR 检测花生过敏原 Ara h 2 基因的比较研究

目前,食物过敏是一个世界性的公共卫生问题,其中花生过敏最为严重。基于DNA的分子生物学检测方法目前已广泛应用于花生过敏原检测,样品DNA的提取质量会显著影响检测的灵敏度及准确率。本研究比较了5种DNA提取方法,包括十六烷基三甲基溴化铵（cetyl trimethyl ammonium bromide,CTAB）法、柱式法及3种基于磁珠纯化技术的DNA快速提取法,对生花生、煮花生、炸花生、烤花生、花生酥和花生酱6种不同加工方式的花生基质样品进行DNA提取,考察了不同方法获得的DNA浓度、纯度指标,并采用实时荧光定量PCR（quantitative real-time PCR, qPCR）对花生过敏原Ara h 2基因进行了检测分析。结果表明,月桂酰肌氨酸钠（Sodium Lauroyl Sarcosine）磁珠法（简称SLS磁珠法）的适用性广、提取率高,对于6种花生基质提取的DNA均能高效检出花生过敏原Ara h 2基因;对于花生含量为0.05%～1.00%的小麦粉二元混合物,SLS磁珠法的DNA提取率总体优于CTAB法,并且能有效提取出与花生共用一条生产线的燕麦片中污染的花生DNA,证实SLS磁珠法提取的实际样品DNA能够满足花生过敏原检测目的。本研究为花生及其制品DNA提取方法提供了参考,特别是磁珠类方法,高效快速,提取质量能够保障后续基于DNA的花生过敏原分子生物学方法检测结果的准确性。     

荧光PCR方法定量检测食品中香菇成分

摘要：【目的】为辨别出口商品和国内市场中谎报香菇成分、以次从好的菌菇产品,准确核定菌菇产品的价格,打击出口骗税等不法行为,特应急研究开发本方法。【方法】选取香菇基因组单拷贝核基因Hydrophobin Protein基因,设计香菇种属特异性引物,扩增107 bp的片段,用于荧光PCR定量检测。分别以3种香菇作为阳性对照和14种非香菇菌种、植物产品作为阴性对照,测试实时荧光PCR引物和探针的特异性。以香菇标准DNA进行8个浓度梯度稀释,测试本定量方法绝对定量限。制备12个梯度含量的香菇DNA样品,测试香菇相对含量的标准曲线。【结果】结果表明本研究建立的香菇荧光PCR定量检测方法对香菇物种的特异性好,与其他食物物种无交叉结果,荧光PCR扩增效率为0.90,绝对定量限LOQ为0.01 ng和含量检测LOQ为0.05％。【结论】本方法重复性和实用性好,可以满足食品和调味料中香菇含量定性定量检测要求。     

TaqMan实时荧光PCR对沙门氏菌能力验证样品的快速检测与鉴定

本研究依据GB 4789.4-2016标准对沙门氏菌ACAS-PT526能力验证样品进行常规培养法检测,同时使用TaqMan实时荧光聚合酶链反应（polymerase chain reaction,PCR）技术对预增菌培养物进行快速检测和鉴定。本研究首先以沙门氏菌特异性基因hut基因为靶基因,设计合成特异性引物和探针,提取各类食源性菌种的核酸DNA进行实时荧光PCR反应,仅沙门氏菌属出现阳性扩增,非沙门氏菌属、阴性对照和空白对照均无扩增信号,验证设计合成的引物探针具有较高的特异性。其次将能力验证样品和加标样品经预增菌、增菌、分离、纯化、生化试验和血清学鉴定,同时将预增菌培养物经实时荧光PCR测定后,18-D319和加标样品有显著的S型扩增曲线,Ct值分别为24.34和26.21,为沙门氏菌阳性,18-M906无显著荧光信号,Ct值>40.00,为沙门氏菌阴性。经API20E试剂条鉴定,18-D319为猪霍乱沙门菌亚利桑那亚种,鉴定百分率为99.90%,T值为0.97,18-M906为大肠埃希氏菌,鉴定百分率为99.80%,T值为0.94。实时荧光PCR检测结果与常规培养法检测结果一致,且更为简单快速,从预增菌到结果判定仅需12 h,结果准确度高,一批次可检测多个样品,可用于大量样品中沙门氏菌的快速筛查和对能力验证样品的检测验证。