Semi-nested PCR检测饲料中牛羊源成分

加强对进口饲料中牛羊源成分的检测是防止疯牛病和痒病传播的一个重要措施。根据已发表的牛和羊特异性基因及引物序列，分别设计了1条牛和羊特异性semi-nested PCR引物，并采用semi-nested PCR技术对饲料中的牛和羊成分进行了扩增检测。结果表明，semi-nested PCR能够扩增得到247 bp的牛特异性基因条带和214 bp的羊特异性基因条带，其对饲料中牛或羊源性成分的检测灵敏度可达到0.00001%～0.0001%，比普通PCR检测灵敏度要高出103倍；对牛或羊成分DNA的检测灵敏度可以达到10-6～10-5 ng，比普通PCR检测灵敏度要高出105倍以上。该技术具有快速、灵敏和结果稳定的特点，是检测饲料中痕量牛羊源成分的一种有效方法。     

同步检测动物源性成分的五重PCR的条件优化和检出限分析

为获得肉制品中牛、羊、猪、鸡、鸭等动物源性成分的快速高效多重PCR检测方法,利用5对牛、羊、猪、鸭和鸡等动物源性成分特异性PCR引物(靶基因序列来源于线粒体基因组),对多重PCR反应条件引物浓度和退火温度进行优化,并确定该检测方法的检出限。结果表明,多重PCR方法最佳反应条件为鸭源特异性引物浓度0.12μmol·L~(-1),鸡源特异性引物浓度0.16μmol·L~(-1),猪源特异性引物浓度0.16μmol·L~(-1),羊源特异性引物浓度0.32μmol·L~(-1),牛源特异性引物浓度0.24μmol·L~(-1),退火温度58℃,d NTPs浓度0.20 mmol·L~(-1),Taq DNA聚合酶添加量5 U,循环次数40。在最佳反应条件下,所建立的牛、羊、猪、鸡和鸭等5种动物源性成分多重PCR的g DNA检出限达到20 pg。应用优化后的多重PCR方法对21份市售肉制品样本进行盲样检测,验证鉴别方法的准确性,结果与现行标准的检测结果一致,掺假率达42.86%。本试验结果为肉制品中该5种动物源性成分的快速检测提供了技术支撑。     

应用PCR技术检测饲料中的转基因成分

成功地从成品饲料中提取DNA，并采用PCR检测技术从中检出35S启动子，NOS终止子，EPSPS耐除草剂基因和CryLA(b)抗虫基因等转基因成分，同时通过扩增玉米（Zea mays)自身zein蛋白基因及大豆（Glycine max)自身lectin基因的引物和阴阳性对照，阴阳性质控，避免产生假阳性，假阴性结果。该方法已在口岸进口饲料转基因检测中得到初步应用。     

进口饲料中牛、羊源成分的PCR同时检测方法

为防止疯牛病和痒病的传入，研究提供了一种利用Chelex-100快速提取动物基因组DNA的方法，并依据牛、羊线粒体基因的同源性序列片段，设计1对通用引物，通过PCR扩增，实现了对进境饲料中牛、羊源成分的同时检测。该方法简单、快捷，在口岸检疫部门中有一定的推广价值。     

狐狸肉源性成分快速检测方法的建立

为了规范食品安全标识,防止非食用性的狐狸肉添加到肉品中导致伦理、宗教等敏感问题,迫切需要建立狐狸肉源性成分鉴定方法。本研究以狐狸、牛、羊、猪、鸭、驴和鼠肉以及模拟掺假样本为研究对象,根据狐狸线粒体基因序列设计特异性交叉引物组合,Bst DNA聚合酶反应体系经63℃恒温扩增60 min,扩增产物经电泳检测和一次性核酸试纸条检测,显示结果一致,建立了交叉引物等温扩增技术与核酸试纸条检测结合的快速检测狐狸源性成分的方法。本研究建立的狐狸肉源性成分特异性的等温扩增体系,对单一狐狸源性DNA成分的灵敏度为10 ng·μL^-1,对狐狸肉与羊肉混合物中狐狸肉成分的检出比例为1%,可为肉制品市场现场检测提供简便、经济、快速有效的技术支持。     

用复合PCR方法检测6种转基因玉米中外源DNA的特异性

利用本实验室研制的植物DNA提取试剂盒，从玉米（Zea mays）种子中提取符合PCR检测要求的DNA。根据不同转基因玉米中重组DNA的结构，分别设计引物，可对Btl1、Event176、T25、MON810、GA21和NK603等6种转基因玉米进行特异性PCR检测。在此基础上建立了针对6种转基因玉米的特异性DNA片段和玉米内参照基因（zein）的复合PCR检测方法，能同时对7对引物进行扩增，通过产物的长度差异对不同的转基因玉米进行辨别，实现了在同一个PCR反应管中同时对6种不同转基因玉米的特异性检测。     

CPA-核酸试纸条检测鸭肉源性成分方法的建立和优化

为建立简单、有效的动物源性成分定性检测方法,根据交叉引物恒温扩增技术(CPA)原理,以鸭属线粒体D-loop基因序列设计引物和探针,优化反应体系与恒温扩增条件,并配合核酸试纸条显色,建立鸭肉源性成分的快速检测方法。结果表明,建立的鸭D-loop基因CPA扩增体系中交叉引物、内引物、外引物分别为0.6、0.3、0.1μmol·L-1,Mg SO4为8 mmol·L-1。在63℃、60min恒温扩增条件下,单一鸭源性DNA成分检出限为0.1 ng·μL-1,对混合肉制品中鸭肉成分的检出比例为1%(相当于1ng·μL-1)。本研究建立的鸭源性成分检测方法为肉制品掺假鉴定提供了有效的手段。     

检测兽用疫苗中霉形体污染的PCR-ELISA技术的建立及应用

本试验建立检测兽用疫苗中霉形体污染的PCR-ELISA方法，探索其最佳试验条件及优化组合，并与PCR检测法作对比，评价其应用价值。试验依据Genbank中登录的鸡源霉形体和猪源霉形体16s rRNA基因序列，运用DNAStar软件和Primer 5.0软件设计PCR特异性引物和探针，其中引物用地高辛标记，探针用生物素标记。然后以培养的霉形体中提取的DNA为模板，依据PCR-ELISA技术原理设计试验，建立并优化PCR-ELISA反应条件。最后用建立的PCR-ELISA扩增体系和条件对从市场上随机购买的30批鸡新城疫Ⅰ系活疫苗样品、30批鸡新城疫La Sota系活疫苗样品和30批猪瘟活疫苗样品进行检测。检测结果表明：应用建立的PCR-ELISA反应体系和条件可以从疫苗样品中检测到霉形体，检测率为35.6%，比PCR法的检测率高11.2%。该方法灵敏度高，特异性强，在疫苗的霉形体污染检测中具有推广应用价值。     

反刍动物饲料中乳源性成分与牛羊肉骨粉的区分

利用奶粉的溶解性,通过水和0.5%次氯酸钠溶液洗涤混有乳源性成分和牛、羊肉骨粉的饲料样品,去除饲料中的乳粉成分后,再使用16S rDNA PCR方法进行动物源性检测。结果表明,实验所建立的方法能够完全区分饲料中的乳粉与肉骨粉。当乳粉含量分别为25%、50%和75%时,混合饲料中牛、羊肉骨粉的检出限分别为2%、0.5%和0.1%。此方法操作简单,容易掌握,可用于鉴别反刍动物饲料中非乳源性成分的牛、羊源性成分。     

牛、羊肉中水貂、猪、鼠混杂成分的多重荧光PCR鉴定方法的建立

针对现阶段肉类食品市场中出现的以劣充优的掺假问题,通过视觉、触觉、嗅觉和味觉等传统方法来鉴定肉的外观、色泽、气味和硬度,已不能满足现阶段肉类质量和源性的鉴别要求。基于现代分析仪器和生物技术的肉类掺假识别方法主要包括:基于蛋白质的检测方法、酶联免疫吸附试验(Enzyme linked immunosorbent assay,ELISA)、色谱分析方法和基于DNA序列的分子生物学检测方法。其中,基于DNA序列的分子生物学检测方法特别是荧光PCR法具有特异性强、灵敏度高,快速准确的优点。本实验运用了多重荧光PCR的技术,建立了检测牛羊肉类中掺杂水貂(Mustela lutreola)、猪(Sus scrofa)、鼠(Mus musculus)3种动物源性成分的检测方法。在3种动物源性的线粒体16S r DNA的保守区设计了一对通用引物,在可变区设计了水貂、猪、鼠3种物种特异性的分子信标探针,分别进行了探针的特异性验证、实验体系的灵敏度验证和样品的检出限实验。特异性验证中,3种探针均可有效排除其他物种DNA的干扰,特异扩增;灵敏度验证实验中,设置了7个DNA浓度稀释梯度,每个梯度6个重复,确定了该体系的灵敏度为0.01 ng/μL;样品检出限实验中,设置了3种掺杂方式,每种方式7个不同的掺杂质量比,每个质量比4个平行样,确定了本实验的样品检测灵敏度可达到1%(质量比)。本研究结果显示,本方法能特异、准确、灵敏地鉴别出牛羊肉中水貂、猪和鼠的掺杂成分,为多物种的源性鉴别提供了科学依据。     

Identification of species-specific DNA in feedstuffs

Due to the menace of transmission of spongiform encephalopathies, feed components intended for ruminant nutrition must be checked for the presence of ruminant-derived materials. A sensitive method for the identification of bovine- and ovine- and also swine- and chicken-specific mitochondrial DNA sequences based on Polymerase Chain Reaction (PCR) has been developed. The specificity of the primers for PCR has been tested using samples of DNA of other vertebrate species, which may also be present in rendering plant products intended for feed manufacture. The method allows the detection in concentrate mixtures of 0.01% of the target species derived material. The identity of a sample containing 0.1% of bovine, ovine, swine, and chicken meat-and-bone meal has further been confirmed by sequencing.     

Detection of land animal remains in fish meals by the polymerase chain reaction-restriction fragment length polymorphism technique

In the present study a technique was developed with the aim of guaranteeing the composition and security of fish meals, since it allows verification of whether these meals contain land animal remains. The method is based on polymerase chain reaction (PCR) and length polymorphism, followed by a restriction fragment length polymorphism (RFLP). Specific primers for every species were designed and calibrated, generating exclusively a PCR product with a specific size when DNA for each species was present in the sample. This technique allows the detection of land animal remains in fish meals, specifically cow, chicken, pig, horse, sheep, and goat. The identity of the PCR products can be confirmed by RFLP analysis using only one restriction enzyme. The selected restrictase generated one characteristic restriction profile for every species included in this study. The detection limit of this method was calculated by using mixtures of fish meals in different proportions and meal that exclusively contained remains of one of these land species studied. The analytical strategy herein proposed was applied to fish and meat meals, giving good results, both in the analyzed standards and in commercial samples.     

Sandwich enzyme-linked immunosorbent assay for the detection of bovine blood in animal feed

The feeding of ruminant proteins to ruminants is prohibited in most countries because the practice is thought to be responsible for the spread of bovine spongiform encephalopathy. However, currently available methods to detect ruminant blood products in rendered feedstuffs are inadequate because they lack species specificity, tissue specificity, and are not based on a thermostable analyte. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for this study that provides reliable and sensitive (0.05-0.5% v/v) detection of bovine blood materials in animal feed. The new sandwich ELISA employs two previously developed monoclonal antibodies (MAbs), Bb6G12 as the capture antibody and biotinylated MAb Bb3D6 as the detecting antibody, and is bovine-specific and blood-specific. The assay is based on the detection of a 60 kDa thermostable protein in bovine blood and provides a useful regulatory tool for monitoring fraudulent labeling or contamination of bovine blood in both heat-processed feedstuffs and unprocessed raw materials. Keywords: Bovine; blood; monoclonal antibody; sandwich ELISA.     

乳制品中金黄色葡萄球菌PCR快速检测方法的建立

为快速检测乳制品中的金黄色葡萄球菌,本研究将纳米氢氧化钛和纳米氢氧化锆固定在滤纸片上以去除PCR反应抑制因子,并以Nuc为靶基因,建立一种无需样品前增菌的PCR快速检测方法,同时确定该方法的特异性、灵敏度和实用性,并评估该方法所用试剂在冷冻保存条件下的稳定性与实用性。结果表明,该PCR快速检测方法在检测97株目标菌及83株非目标菌后未出现假阳性或假阴性结果;该方法无需增菌处理,可在4 h内完成检测,检测限为10¹ CFU·25 g^-1或10¹ CFU·25 mL^-1;该方法与传统检测方法在实际乳制品样品中金黄色葡萄球菌的检出率及活菌检测率均一致(符合率100%);此外,该检测方法所用试剂在-20℃冷冻条件下可稳定保存至少12个月。综上所述,本研究建立的PCR快速检测方法特异性强、灵敏度高,且实用性良好,能有效去除样品基质中的PCR反应抑制因子,为乳制品中金黄色葡萄球菌的快速检测提供了参考依据。     

Identification of species in animal feedstuffs by polymerase chain reaction--restriction fragment length polymorphism analysis of mitochondrial DNA

Restriction site analysis of Polymerase Chain Reaction (PCR) products of cytochrome b mitochondrial DNA was applied to identify species in meat meal and animal feedstuffs. PCR was used to amplify a variable region of cytochrome b mitochondrial DNA gene. Species differentiation was determined by digestion of the obtained 359 bp amplicon with restriction enzymes, which generated species-specific electrophoresis patterns; the sequencing of PCR products was used as confirming analysis. PCR-RFLP analysis revealed the presence of meat meal in animal feedstuffs and distinguished species of interest. The results supported the application of the method in control measures which should be adopted for meat-meal-based animal feed, as suggested by EU law. As a technical improvement, to simplify the analysis, the number of enzymes presented in this study for the detection of different species was smaller than others described in the literature; discrimination between ruminant and nonruminant species and between mammalian and poultry species was possible with few digestions.     

多重PCR检测耐除草剂转基因作物

为建立耐除草剂转基因作物的高通量检测方法,本试验以目前生产上广泛应用的5种除草剂抗性基因dmo、pat、CP4EPSPS、bar和aad1为靶标进行多重PCR(MPCR)研究。通过引物适用性测试、反应体系中的不同引物浓度和反应程序中的退火温度测试、灵敏度和特异性验证等,建立了能同时检测5种除草剂抗性基因的MPCR检测方法。结果表明,当dmo、pat、CP4EPSPS、bar、aad1基因的检测引物终浓度分别为0.2、0.2、0.3、0.4、0.2μmol·L^-1,退火温度为63℃时5种靶标扩增效果较好,且特异性条带清晰且均一。此外,MPCR检测方法具有较好的特异性,对每种靶标的检测灵敏度均可达到0.1%。适用性测试结果显示,MPCR检测方法可对含有5种除草剂抗性基因的多种转基因作物的单个品系或多个品系混合物进行筛选检测。无假阳性和假阴性结果表明,MPCR检测方法对实际样品具有很好的适用性。本试验结果为筛选高效耐除草剂转基因作物检测技术提供了一定的理论依据。     

Detection of genetically modified soybean using peptide nucleic acids (PNAs) and microarray technology

Peptide nucleic acid (PNA) microarrays for the detection of Roundup Ready soybeans in food have been prepared. PNA probes are known to be more efficient and selective in binding DNA sequences than the analogous oligonucleotides and are very suitable to be used for diagnostics in food. PNAs of different lengths were carefully designed and synthesized by solid-phase synthesis on an automatic synthesizer adopting the BOC strategy. PNAs were purified by HPLC and characterized by HPLC/MS. The probes were spotted on a functionalized surface to produce a microarray to be hybridized with PCR products. DNA extracted from reference material was amplified using Cy3- and Cy5-labeled primers, and the fluorescent PCR products obtained were hybridized on the microarray. Two protocols were adopted: the hybridization with dsDNA or with ssDNA obtained by digestion with the enzyme lambda exonuclease. The best results were obtained using a 15-mer PNA probe in combination with the ssPCR product derived from enzymatic digestion. The method was applied to the analysis of a sample of certified transgenic soybean flour.     

Event-specific qualitative and quantitative polymerase chain reaction analysis for genetically modified canola T45

Polymerase chain reaction (PCR) methods have been the main technical support for the detection of genetically modified organisms (GMOs). To date, GMO-specific PCR detection strategies have been developed basically at four different levels, such as screening-, gene-, construct-, and event-specific detection methods. Event-specific PCR detection method is the primary trend in GMO detection because of its high specificity based on the flanking sequence of exogenous integrant. GM canola, event T45, with tolerance to glufosinate ammonium is one of the commercial genetically modified (GM) canola events approved in China. In this study, the 5'-integration junction sequence between host plant DNA and the integrated gene construct of T45 canola was cloned and revealed by means of TAIL-PCR. Specific PCR primers and TaqMan probes were designed based upon the revealed sequence, and qualitative and quantitative TaqMan real-time PCR detection assays employing these primers and probe were developed. In qualitative PCR, the limit of detection (LOD) was 0.1% for T45 canola in 100 ng of genomic DNA. The quantitative PCR assay showed limits of detection and quantification (LOD and LOQ) of 5 and 50 haploid genome copies, respectively. In addition, three mixed canola samples with known GM contents were detected employing the developed real-time PCR assay, and expected results were obtained. These results indicated that the developed event-specific PCR methods can be used for identification and quantification of T45 canola and its derivates.