乳腺特异性人α-乳白蛋白真核表达载体的构建

从正常人血液中提取人基因组DNA.以其为模板,扩增人α-乳白蛋白(α-LA)2.36kb的总DNA序列,经测序鉴定后,将其连接到PSV载体SV40启动子后.构建真核表达载体LA-DNA-psv.扩增牛XS1-酪蛋白5'调控序列约1.2kb的片段.测序鉴定.切去LA-DNA-psv栽体原来的SV40启动子,连接牛αS1-酪蛋白5'调控序列约1.2kb的片段为启动子,构建真核表达载体αS1-LA-DNA-psv.为在动物乳腺中特异高效表达人α-乳白蛋白做准备研究.     

羊绒羊毛纤维线粒体DNA提取方法研究

研究和开发动物纤维的DNA检测,关键的技术难点在于DNA的提取。文章就羊绒羊毛纤维线粒体DNA的提取方法进行了实验,并通过PCR扩增的方法进行了验证。结果表明:使用Promega试剂盒,将纤维样品尽量处理成粉末状,并在样品经裂解液处理后先离心除去其中尚未完全溶解的纤维,然后进行后续操作是比较有利的,完全能够得到满足PCR扩增和DNA测序要求的线粒体DNA样品。     

基于毛细管凝胶电泳与DNA条形码技术鉴别可食用动物内脏掺假方法的研究

建立并优化了使用基于DNA条形码技术对可食用内脏制品中包括猪、牛、羊、鸡、鸭、鹅、兔7种常见动物源成分进行掺假鉴别的方法。用生理盐水清洗和真空冷冻干燥预处理后的内脏样品，经DNA提取扩增后，扩增产物经毛细管凝胶电泳分析系统进行确认，克隆测序结果提交本地数据库Viscera进行比对，同时筛选出适合7种动物源内脏DNA扩增的通用引物COI-A，优化DNA模板量和退火温度，验证考察了19个可食用内脏掺假模型的最低掺假比例。7种动物的5类内脏的PCR扩增效率均为100%，最佳的DNA模板量和退火温度为2μL和53℃，掺假成分的最低检出比例为5%。本方法灵敏度高，可靠性好，可作为常见可食用动物内脏掺假的有效检测方法。     

五重PCR检测转基因大豆

旨在建立一套准确、快速、可靠的用于转基因大豆检测及DNA提取方法;以35S启动予(Cauliflowermosaicvirus 35S, CaMV 35S)、Nos终止子(Nopalinesynthase, Nos)、NPTⅡ、CP4-EPSPS四种外源基因和大豆内源基因(Lectin)为检测的目的片段,建立五重PCR反应体系,并采用改进的方法提取DNA,进行PCR扩增,实际检测了已知转基因大豆和市售大豆;改进的DNA提取方法与经典的CTAB方法相比,提取时间至少缩短了1h,降低了提取成本.经过PCR扩增对五种外源基因进行了检测,经DNA测序证实了产物为目的扩增产物.转基因大豆的检出限为0.2%～0.5%.改进后的DNA提取方法能够快速提取用于PCR扩增的高质量模板,消除了PCR反应的抑制因子;建立的五重PCR反应体系能够高效、准确的检测转基因大豆.     

一种快速制备酵母细胞PCR扩增DNA模板的方法

报道了一种快速制备酵母细胞PCR扩增DNA模板的方法.以酿酒酵(Saccharomyces cerevisiae)为目标菌,对酵母细胞煮沸-低温冷冻预处理后,直接用于PCR扩增,获得了26S rDNAD1/D2区域序列片段,成功测序.此方法避免了复杂的DNA提取过程.     

PCR-焦磷酸测序检测单核细胞增生李斯特氏菌研究

目的 建立结合PCR-焦磷酸测序检测单核细胞增生李斯特氏菌的方法.方法 根据单核细胞增生李斯特氏菌的hly基因设计扩增引物和测序引物,特异地扩增目的片段,再制备单链模板,在测序引物引导下进行焦磷酸测序,通过测序结果与GenBank中的hly基因序列的比对进行鉴定.结果 扩增引物和测序引物表现出良好的特异性,16株单核细胞增生李斯特氏菌菌株均扩增出大小249 bp的DNA片段,焦磷酸测序结果与hly基因序列100％匹配,而阴性对照菌株均未扩增出DNA条带,焦磷酸测序结果为阴性.结论 建立的方法特异性高,是快速从DNA序列水平上检测单核细胞增生李斯特氏菌的有效手段.     

转基因水稻潮霉素标记基因PCR检测方法的建立及其在基因水平转移研究中的应用

为建立用于基因水平转移研究, 尤其是DNA经加工和消化后稳定性研究的针对转基因水稻潮霉素标记基因hpt（hygromycin phosphotransferase）的定性和实时定量PCR体系,设计针对hpt的上游通用引物多个片段定性PCR扩增体系,以植物叶绿体基因rbcl为内对照,PCR扩增产物经测序验证.将定性PCR中最小片段（236 bp）连接到质粒载体pUC18-pMD T载体上,提取质粒经验证后做外标.应用TaqMan-MGB荧光探针和引物,建立定量的外标校正曲线法,并评价方法的精密度.建立的定性PCR体系能稳定扩增出236 bp～910 bp不同大小的5个hpt片段,并经测序验证.实时定量PCR的线性范围为105～10拷贝（R^2=0.998）,最低能检出10拷贝,重复性好.本研究已成功建立了用于转基因水稻标记基因hpt基因水平转移研究的定性和定量PCR系统。     

山羊绒DNA的提取及验证

提取DNA是利用PCR技术检测纤维的关键技术难点。本文对山羊绒DNA的提取方法进行了试验,并通过PCR扩增的方法进行验证。结果表明:使用Ta Ka Ra Mini BEST Universal Genomic DNA Extraction Kit Ver.5.0试剂盒,将纤维样品尽量处理成粉末状,并在样品经裂解液处理后先离心除去其中尚未完全溶解的纤维,然后进行PCR扩增是比较有利的,完全能够得到满足PCR扩增要求的DNA样品。     

DNA条形码技术在肉品防欺诈鉴别中的应用

以DNA条形码技术鉴别进出口监督抽检的鱼肉等水产制品的种类来源,用以判别其与申报或产品标签是否相符.分别提取鱼肉等样品的基因组DNA,以目前国际上比较公认的动物线粒体细胞色素氧化酶COⅠ基因通用引物进行PCR扩增.PCR产物经测序分析后,将得到的扩增片段序列与Genbank数据库进行序列比对,同时提交Barcoding Life DNA条形码数据库(BOLD)进行鉴定分析.本批次监督抽检的16份鱼肉、鱼丸等水产制品中除1份样品未能成功获得鱼肉COⅠPCR扩增外,其余15份样品均顺利得到种类来源鉴定,鉴定结果约有31.25％的样品与产品标签标示不符.作为一种简单、快速、有效的分子鉴定技术,DNA条形码可以直接应用于鱼肉等动物源性食品的种类鉴定.     

Bacillus sp.nov SK006中纤维蛋白溶解酶的克隆及其在大肠杆菌中的表达

利用PCR技术从Bacillus sp.nov SK006中扩增得到纤维蛋白溶解酶(Fibrinolitic enzymes,FE)基因的DNA片段,将其连接到pMD-T载体上,经测序后克隆至载体PET-22b(+),转化至E.coli BL21(DE3)中,得到的重组菌能高效表达FE。诱导表达后,SDS-PAGE显示,重组FE分子量约为28 ku。     

膜芯片技术对牛、羊、牦牛、驴肉源食品的掺伪鉴别

采用膜芯片技术,通过对牛、羊、驴、牦牛、鸡、鸭、兔、貂、狐、鼠、猪11 种目标物种的检测,实现对牛、羊、驴、牦牛物种的掺伪鉴别。结果表明：该方法具有良好的特异性和适用性,检测灵敏度和掺伪灵敏度均可达到0.1%,能快速、准确地同时鉴别牛、羊、驴、牦牛、鸡、鸭、兔、貂、狐、鼠、猪11 种动物源性成分,可满足肉类食品样品中对牛、羊、牦牛、驴等掺伪鉴别的检验需求。     

Identification of chicken,duck, pigeon and pig meat by species-specific markers of mitochondrial origin

In the present study, PCR based method for meat species identification of chicken, duck, pigeon and pig was achieved by developing species-specific markers. Using mitochondrial sequences species-specific primers were designed and the sizes of them were 256 bp, 292 bp, 401 bp and 835 bp for chicken, duck, pigeon and pig, respectively. The species-specific PCR products were sequenced to confirm the specificity of the product amplified. These markers were subsequently tested for cross amplification by checking them with beef, mutton, chevon, pork, rabbit, chicken, duck, turkey and pigeon meat. DNA markers developed in this study can help identify the species of fresh, cooked and autoclaved meat of chicken, duck and pigeon and fresh and cooked meat of pig. The process of identification is simple, economical and quick as compared to other methods such as RAPD, PCR-RFLP and sequencing method of species identification.     

2013年苏州地区肉及其制品掺假情况调查

了解苏州地区肉及其制品的掺假情况,通过对肉类种源与标签明示肉源进行比对,鉴别摻假食品,为加强食品标签管理提供依据。方法 运用自建的动物源性食品种源判定Taqman实时荧光PCR检测体系对苏州地区的肉及其制品进行种源判定,与标签明示肉源进行比对,鉴别摻假食品。结果 本次调查共检验涉及32个生产单位的90份样品,总不符合率为25.6%(23/90)。检测的44份牛肉及其制品中有12份与标签不符,8份用猪肉部分替代牛肉,1份以鸭肉部分代替牛肉进行销售;此外有3份不含有牛肉成分,存在猪、鸡、鸭源性肉类之外的肉类成分。共检测羊肉及其制品16份,有2份用鸭肉代替羊肉出售,3份羊肉样品中掺入了部分猪成分,其中1份样品还存在单个样品掺杂两种外源肉类的现象(猪源性和鸭源性)。检测猪肉及其制品19份,其中2份样品含有标签未注明的鸡肉成分。在所检测的11份混合肉类样品中有4份成分与标签不符,主要是以廉价的鸡肉取代/部分取代相对高价的牛肉和猪肉。结论 肉制品掺假情况明显,用猪肉、鸭肉部分代替牛肉和羊肉仍是主要的掺假手段,牛肉掺假样品主要是熟制牛肉制品,而火锅食用羊肉卷样品则是羊肉掺假高危品,开展肉制品摻假检测对规范肉制品市场具有积极意义。此外,3份未知种源成分的牛肉样品提示在现有检测基础上还需扩大检测范围,防患于未然。     

DNA条形码在肉制品掺伪中非定向筛查技术的研究

目的对基于DNA条形码的肉制品掺伪中非定向筛选技术进行探索。方法以线粒体基因组中COI基因为靶标,设计猪、牛、羊、马、鸡、鸭、鹅、鼠8种动物源物种间的通用引物和特异性引物,建立禽畜肉的DNA条形码检测方法。同时应用该技术对50份市售的肉制品进行检测。结果本研究建立了DNA条形码筛选技术,电泳条带通过基因测序能正确识别猪、牛、羊、马、鸡、鸭、鹅、鼠8种动物源成分,结合分子克隆技术能实现对混合肉的检测。50份市售肉制品的DNA条形码结果显示,16份掺杂了除牛羊肉以外的其他禽畜肉。结论 DNA条形码技术能打破传统标准中PCR法检测目标唯一性的局限,具有良好的应用前景。     

可视化LAMP检测常见肉制品中猪肉成分

根据GenBank中公布的猪线粒体色素细胞b基因序列设计6 条特异性环介导等温扩增（loop-mediated isothermal amplification,LAMP）引物,通过简化线粒体DNA提取步骤,优化反应体系,以常见的牛、羊、鸡、鸭、马、驴肉为阴性对照验证该方法的特异性,掺假率梯度稀释以验证该LAMP法的最低检测限,建立常见肉制品中猪肉成分的可视化LAMP检测方法。结果表明：该方法提取目的基因操作简单、稳定,该可视化LAMP检测法以4-(2-吡啶偶氮)-间苯二酚钠盐为指示剂,能准确检测出常见肉类中猪肉是否掺杂,检测结果肉眼可见,灵敏度为10 pg/μL。     

Quantitative Detection of Poultry in Cooked Meat Products

ABSTRACT: There is a growing awareness of perceived harm from meat species adulteration, both intentional and accidental. The present study developed a monoclonal antibody (Mab)-based enzyme-linked immunosorbent assay (ELISA) for the quantitative detection of chicken and turkey meat adulterated in cooked (100 °C, 15 min) mammalian meat. The specificity of Mab 5D2 to different species (pork, beef, lamb, deer, horse, duck, chicken, and turkey) and tissues (serum, gizzard, heart, and liver) was studied by noncompetitive ELISA. The detection of cooked chicken in beef, and turkey in pork was accomplished by competitive and noncompetitive ELISAs. Both ELISAs were optimized to quantify cooked poultry in red meats. The new Mab-based ELISAs enabled the detection of cooked poultry in red meats at levels as low as 1% (v/v) or better. The correlation ( r > 0.994) between chicken or turkey concentrations and ELISA signals permitted the quantification of poultry adulterants in cooked non-poultry meats.     

Species identification in meat products using real-time PCR.

One of the most convenient methods for the identification of animal species in processed meat products is the examination of DNA sequences. Real-time polymerase chain reaction (qPCR) techniques are particularly suitable because even small fragments of DNA formed during heat processing of the meat can be amplified and identified. A real-time PCR method has been developed and evaluated for the identification of processed meat products. In test mixtures containing beef, pork, horse, mutton, chicken and turkey, it was possible to identify these species down to a level of 0.05%. By adjusting the number of cycles, it was possible to detect levels as low as 0.01% of these species. Cross-reactivity between these species was not found, except for pure horsemeat (250 ng DNA) in the assay for turkey meat. Cross-reactivity of deer, roe, ostrich, kangaroo, goat, domestic duck, mallard, goose, pigeon, guinea fowl, quail and pheasant was also investigated and it was found that amounts as high as 250 ng DNA of these species in the reaction vial did not result in (false) positive signals except for amounts higher than 125 ng deer DNA and higher than 50 ng pigeon DNA in the determination of chicken and beef, respectively. More than 150 meat samples were examined using DNA hybridization and real-time PCR. A comparison of the results showed a better performance of the real-time procedure compared to DNA hybridization.     

Species identification in meat products using real-time PCR

One of the most convenient methods for the identification of animal species in processed meat products is the examination of DNA sequences. Real-time polymerase chain reaction (qPCR) techniques are particularly suitable because even small fragments of DNA formed during heat processing of the meat can be amplified and identified. A real-time PCR method has been developed and evaluated for the identification of processed meat products. In test mixtures containing beef, pork, horse, mutton, chicken and turkey, it was possible to identify these species down to a level of 0.05%. By adjusting the number of cycles, it was possible to detect levels as low as 0.01% of these species. Cross-reactivity between these species was not found, except for pure horsemeat (250 ng DNA) in the assay for turkey meat. Cross-reactivity of deer, roe, ostrich, kangaroo, goat, domestic duck, mallard, goose, pigeon, guinea fowl, quail and pheasant was also investigated and it was found that amounts as high as 250 ng DNA of these species in the reaction vial did not result in (false) positive signals except for amounts higher than 125 ng deer DNA and higher than 50 ng pigeon DNA in the determination of chicken and beef, respectively. More than 150 meat samples were examined using DNA hybridization and real-time PCR. A comparison of the results showed a better performance of the real-time procedure compared to DNA hybridization.     

Species identification in meat products using real-time PCR

One of the most convenient methods for the identification of animal species in processed meat products is the examination of DNA sequences. Real-time polymerase chain reaction (qPCR) techniques are particularly suitable because even small fragments of DNA formed during heat processing of the meat can be amplified and identified. A real-time PCR method has been developed and evaluated for the identification of processed meat products. In test mixtures containing beef, pork, horse, mutton, chicken and turkey, it was possible to identify these species down to a level of 0.05%. By adjusting the number of cycles, it was possible to detect levels as low as 0.01% of these species. Cross-reactivity between these species was not found, except for pure horsemeat (250 ng DNA) in the assay for turkey meat. Cross-reactivity of deer, roe, ostrich, kangaroo, goat, domestic duck, mallard, goose, pigeon, guinea fowl, quail and pheasant was also investigated and it was found that amounts as high as 250 ng DNA of these species in the reaction vial did not result in (false) positive signals except for amounts higher than 125 ng deer DNA and higher than 50 ng pigeon DNA in the determination of chicken and beef, respectively. More than 150 meat samples were examined using DNA hybridization and real-time PCR. A comparison of the results showed a better performance of the real-time procedure compared to DNA hybridization.     

用Cytb基因序列分析鉴定肉制品掺假

目的采用Cytb基因进行肉制品真伪鉴定的尝试性探索。方法随机采集21份肉制品样品,首先采用CTAB法方法提取样品DNA,基于Cytb基因的通用引物对21份样品DNA进行PCR扩增,把扩增产物进行测序,然后采用DNAMAN软件进行序列的比对分析。结果 CTAB方法适合肉制品的DNA提取。10份牛肉样品中9份与标注吻合,1份检测出是猪肉;2份羊肉样品中1份与标注吻合,1份检测是猪肉;2份猪肉样品中1份与标注吻合,1份检测是鸡肉;1份鸡肉样品与标注吻合;6份牦牛肉样品中2份与标注吻合,4份检测是水牛肉。结论用Cytb基因序列分析比对方法适合肉制品的真伪鉴定,市场上肉制品存在掺假问题。