实时荧光PCR快速筛选食品中鸭源性成分

基于鸭线粒体细胞色素Cyt b基因建立了肉制品中的鸭源性成分检测的real-time PCR方法,并对模拟样本和市售样本进行了检测分析,检出限低至1%,适用于实验室的鸭源性成分的鉴定分析。      

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。     

食品中马源性成分的实时荧光PCR检测

针对马线粒体DNA细胞色素b基因设计特异性引物和探针,建立食品中马源性成分实时荧光PCR检测方法,并经特异性和灵敏度试验验证其可行性。结果表明:该体系可扩增马DNA片段,长度为127 bp,其他常见畜、禽肉成分均无法正常扩增。该体系的检测灵敏度为1.25 pg马DNA和质量分数0.001%马肉粉。经市售食品的检测验证,表明所建立的马引物探针体系具有特异性好、灵敏度高、快速、高效等优点,可用于对食品中马源性成分的掺假鉴别检测。     

实时荧光聚合酶链式反应法检测食品中猪源性成分

针对猪线粒体细胞色素b基因序列设计特异的引物和探针,建立食品中猪源性成分实时荧光聚合酶链式反应检测方法,并经特异性和敏感性试验验证其可行性。结果表明：该体系可扩增猪肉DNA片段,长度为98bp,其他常见畜、禽肉成分均无法正常扩增。该体系灵敏度低至1pg,且阴性样品扩增后的Ct值限制在35循环以后。对于各模拟肉类样品中掺杂的猪源性成分,其检测限均达1%,经市售加工食品检测验证,表明所建立的猪引物探针体系具有特异性好、灵敏度高、快速、高效等优点,可用于对食品中猪源性成分的掺假鉴别检测。     

多重实时荧光PCR鉴别金钱白花蛇源性方法研究

目的用多重实时荧光聚合酶链式反应(多重实时荧光PCR)快速鉴别金钱白花蛇源性。方法以线粒体细胞色素C基因为目标靶基因,根据其特异性核酸序列设计引物和探针,通过优化引物及探针浓度、退火温度等PCR体系和条件,建立从活体材料和粉末制剂等中药材中检测金钱白花蛇源性的二重二色实时荧光PCR检测体系。结果通过检测市售15块金钱白花蛇药材,发现其中5块不含金钱白花蛇源性成分;10份金钱白花蛇粉末制剂中3份未检出金钱白花蛇成分;5份活体样本均为金钱白花蛇。检测结果与DNA测序结果一致。结论本研究建立的方法可用于金钱白花蛇源性的检测,能有效保障金钱白花蛇相关名贵中药及制品的安全性。     

食品中乳酸杆菌的实时荧光PCR的快速检测

本文运用实时荧光PCR(Real-time PCR)技术建立了对食品中常见的乳酸杆菌进行检测的快速方法.针对乳酸杆菌16S rDNA序列,设计了通用引物和特异性探针,进行TaqManTM实时PCR检测,以非同源性参考菌株做特异性检测;把乳酸杆菌菌株稀释成不同梯度,做灵敏度检测.实验结果表明用实时荧光PCR法检测乳酸杆菌,快速、敏感、特异性高.     

实时荧光PCR技术快速检测奶制品中掺入的大米源性成分

目的:建立基于实时荧光PCR技术的奶制品中掺入大米源性成分的快速检测方法。方法:以水稻的根部表达基因(gos9)为靶基因设计特异性引物和探针,经特异性实验和灵敏度实验验证引物探针可行性,并经模拟含大米奶粉及市售奶类制品检测验证其实际检测能力。结果:该引物探针体系只针对大米DNA进行扩增,与奶类主成分牛、羊及其它谷类和植物性食物DNA均无交叉扩增;最低能检测到0.1ng的大米DNA,对含大米粉的模拟混合奶粉样品,检出限可达0.1%(W/W)。将其应用于21份市售奶制品样品检测,对含大米源性成分的奶制品扩增阳性,检测结果与食品标签相符。结论:该实时荧光PCR检测体系具有快速、特异、灵敏的优点,可以准确鉴定出奶制品中大米成分,适用于奶类中掺加大米源性成分的检测。      

实时荧光PCR技术快速检测奶制品中掺入的大米源性成分

目的:建立基于实时荧光PCR技术的奶制品中掺入大米源性成分的快速检测方法。方法:以水稻的根部表达基因(gos9)为靶基因设计特异性引物和探针,经特异性实验和灵敏度实验验证引物探针可行性,并经模拟含大米奶粉及市售奶类制品检测验证其实际检测能力。结果:该引物探针体系只针对大米DNA进行扩增,与奶类主成分牛、羊及其它谷类和植物性食物DNA均无交叉扩增;最低能检测到0.1ng的大米DNA,对含大米粉的模拟混合奶粉样品,检出限可达0.1%(W/W)。将其应用于21份市售奶制品样品检测,对含大米源性成分的奶制品扩增阳性,检测结果与食品标签相符。结论:该实时荧光PCR检测体系具有快速、特异、灵敏的优点,可以准确鉴定出奶制品中大米成分,适用于奶类中掺加大米源性成分的检测。     

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...     

羊乳中动物源性成分的实时荧光PCR检测

建立Taqman探针实时荧光PCR检测方法。选择牛、羊线粒体16SrRNA靶序列,设计合成引物和探针,鉴别羊乳制品中的羊乳成分。结果表明,该方法最低可检测掺入2.0%牛源性成分的羊奶制品,具有灵敏、快速、高效以及高通量等优势,可作为羊奶制品羊乳成分掺假鉴别手段之一。     

菠萝成分的实时荧光PCR检测方法的建立

目的本文研究建立食品中菠萝成分的实时荧光PCR检测方法。方法根据菠萝rbcL基因设计菠萝物种特异性检测引物和荧光探针,对样品中的靶标基因片段进行检测,并进行物种特异性检测、灵敏度测试和实际应用检测。结果通过对供试的58种动植物材料进行检测,只有菠萝出现特异性扩增,其他物种材料无扩增;对不同浓度菠萝DNA样品和不同含量的菠萝粉样品进行灵敏度测试,该检测方法对菠萝成分的检测灵敏度分别为0.01 ng/μL菠萝DNA和0.1%菠萝粉;对市场销售的实际样品进行检测,能够满足于检测需求。结论该方法简单、灵敏、快速、准确,能应用于食品中菠萝成分检测。     

Analytical specificity and sensitivity of a real-time polymerase chain reaction assay for identification of bovine mastitis pathogens

Intramammary infection (IMI), also known as mastitis, is the most frequently occurring and economically the most important infectious disease in dairy cattle. This study provides a validation of the analytical specificity and sensitivity of a real-time PCR-based assay that identifies 11 major pathogen species or species groups responsible for IMI, and a gene coding for staphylococcal β-lactamase production (penicillin resistance). Altogether, 643 culture isolates originating from clinical bovine mastitis, human, and companion animal samples were analyzed using the assay. The isolates represented 83 different species, groups, or families, and originated from 6 countries in Europe and North America. The analytical specificity and sensitivity of the assay was 100% in bacterial and β-lactamase identification across all isolates originating from bovine mastitis (n = 454). When considering the entire culture collection (including also the isolates originating from human and companion animal samples), 4 Streptococcus pyogenes, 1 Streptococcus salivarius, and 1 Streptococcus sanguis strain of human origin were identified as Streptococcus uberis, and 3 Shigella spp. strains were identified as Escherichia coli, decreasing specificity to 99% in Strep. uberis and to 99.5% in E. coli. These false-positive results were confirmed by sequencing of the 16S rRNA gene. Specificity and sensitivity remained at 100% for all other bacterial targets across the entire culture collection. In conclusion, the real-time PCR assay shows excellent analytical accuracy and holds much promise for use in routine bovine IMI testing programs. This study provides the basis for evaluating the assay's diagnostic performance against the conventional bacterial culture method in clinical field trials using mastitis milk samples.     

Detection of pig derivatives in food products for halal authentication by polymerase chain reaction –restriction fragment length polymorphism

A method for detection of the presence of pig derivatives in three types of food products—sausages and casings, bread and biscuits—using polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) analysis of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene was developed. Genomic DNA of sausages and casings, bread and biscuits were extracted. The genomic DNA from the food products were found to be of good quality for the sausages and produced clear PCR products on the amplification of the mt cyt b gene of approximately 360 base pairs (bp). However, no genomic DNA was detected from the casing samples and poor quality of genomic DNA was extracted from bread and biscuits. No amplification of mt cyt b gene was produced from bread and biscuit samples. To differentiate between samples, the amplified PCR products were digested with restriction enzyme (RE) BsaJI, resulting in species‐specific RFLP. The cyt b PCR‐RFLP species identification assay gave excellent results for detection of pork adulteration in food products and is a potentially reliable technique to avoid species adulteration or fraudulent species substitution for halal authentication. Copyright © 2006 Society of Chemical Industry     

普通聚合酶链式反应鉴定牛肉及其制品中的牛源性成分

依据牛种属的线粒体细胞色素b(cytochrome b,Cytb)基因上的保守序列设计牛源特异性引物对,建立一种特异性强、灵敏性高、耗时短的鉴定食品中牛源性成分的聚合酶链式反应(polymerase chain reaction,PCR)方法。选取牦牛、水牛、黄牛品种及羊、猪、鸡、鸭、鹅、兔、马、驴、鱼、大豆、玉米等动植物源成分进行特异性试验。结果显示:所建立的方法高度特异于牛源性检测,其他非牛动植物源成分均无扩增条带。采用常见的牛品种进行灵敏性试验,结果显示所建立的方法可检测到的最低牛DNA量为0. 2 pg;将牛肉分别与猪肉、驴肉、羊肉、大豆组织混合进行PCR测定,结果表明最低检测限为鲜样品含0. 01%质量分数的牛源成分,高压121℃、0. 1MPa,20 min处理样品含0. 01%质量分数的牛源成分。对市售食品中牛源性成分检测结果与现行PCR牛源性成分检测标准方法的检测结果 100%一致。     

基于三重实时荧光聚合酶链反应构建黄牛和牦牛源性成分同步检测方法

目的 建立一种拥有内源质控的三重实时荧光聚合酶链反应(polymerase chain reaction,PCR)技术,用以快速鉴定肉和乳中黄牛和牦牛的源性成分。方法 首先对黄牛肉、牦牛肉、黄牛奶、牦牛奶等7种动物产品进行特异性检测,同时通过稀释梯度方法验证此方法的绝对检出限(limit of detection, LOD),最后通过黄牛和牦牛肉掺假模拟试验确定该方法的相对灵敏度。结果 该方法的特异性强,能特异性地检测到来源于黄牛和牦牛肉和乳的DNA,稳定扩增的内源质控有效地避免了假阴性结果,本方法针对黄牛奶的绝对LOD为2.5×10^-3～5.0×10^-3 ng,针对牦牛奶的绝对LOD为2.5×10^-3～5.0×10^-2 ng,本方法对黄牛肉和牦牛肉混合肉的相对灵敏度可达0.1%牦牛肉。结论 所建立的三重实时荧光PCR方法特异性强、灵敏度高,可实现黄牛和牦牛源性以及内源质控的同步检测,又能通过内源质控排除实验假阴性结果。