Analysis of pork adulteration in commercial meatballs targeting porcine-specific mitochondrial cytochrome b gene by TaqMan probe real-time polymerase chain reaction

A test for assessing pork adulteration in meatballs, using TaqMan probe real-time polymerase chain reaction, was developed. The assay combined porcine-specific primers and TaqMan probe for the detection of a 109 bp fragment of porcine cytochrome b gene. Specificity test with 10 ng DNA of eleven different species yielded a threshold cycle (Ct) of 15.5 ± 0.20 for the pork and negative results for the others. Analysis of beef meatballs with spiked pork showed the assay can determine 100-0.01% contaminated pork with 102% PCR efficiency, high linear regression (r(2) = 0.994) and ≤ 6% relative errors. Residuals analysis revealed a high precision in all determinations. Random analysis of commercial meatballs from pork, beef, chicken, mutton and goat, yielded a Ct between 15.89 ± 0.16 and 16.37 ± 0.22 from pork meatballs and negative results from the others, showing the suitability of the assay to determine pork in commercial meatballs with a high accuracy and precision.     

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...     

可视化跨越式滚环等温扩增技术在猪肉掺假检测中的应用

目的:建立了一种跨越式等温扩增技术(SRCA)结合荧光染料SYBR Green I检测肉制品中猪肉掺假的方法。方法:根据NCBI中猪的线粒体序列,通过DNAMAN等软件设计SRCA的特异性引物,分别采用普通SRCA和荧光可视化SRCA的方法建立肉制品中猪肉成分的检测方法,对9个不同物种的新鲜肌肉组织样本进行实验从而验证SRCA方法的特异性,并对66份未标识猪肉成分商业肉制品进行猪肉成分的检测。结果:SRCA荧光可视法检测猪肉DNA的灵敏度为6.5 fg/μL,与传统PCR方法相比,SRCA荧光可视法的灵敏度提高了1000倍。在人工添加猪肉的混合样品中,SRCA方法检测猪肉含量为0.01%(w/w)。与NY/T 3309-2018行业标准相比,SRCA方法检测肉制品中猪肉的敏感性、特异性和符合率分别为100%、96.66%、98.48%。结论:SRCA是一种检测肉制品中猪肉掺假的灵敏、直观的方法,具有很大的应用潜力。     

基于COI序列的DNA微条形码技术鉴别熟肉制品中11种肉掺假的研究

建立并优化了基于COI序列的DNA微条形码技术（mini-barcoding）检测熟肉制品中11种常见肉类掺假的方法。样品经超声与真空冷冻干燥处理,提取DNA模板和PCR扩增后,目标扩增物经切胶纯化后进行克隆测序,并将测序结果提交GenBank数据库Blast比对。筛选出适合猪、牛、羊、鸡、鸭、鸽子、马、驴、鹅、兔、鼠11种肉类的扩增的通用引物COI-A,对PCR扩增条件进行优化,并建立18个掺假模式,对低经济价值肉类（猪、鸡、鸭、马、驴、鼠）的掺入的最低比例进行考察。结果表明:11种熟肉的DNA经通用引物COI-A扩增后,其扩增效率均为100%,18个掺假模式中,牛肉和羊肉中掺入6种低经济价值肉类的最低检出比例为5%,而掺入鸡肉的最低检出比例为10%,而鹅肉、鸽子肉和兔肉的掺假模式中最低检出比例为10%;利用本方法检测30批次市售样品,发现有18批次的熟肉制品存在掺假情况。该方法前处理简单,灵敏度合适,重现性好,可作为高经济价值熟肉制品中掺假低经济价值肉类的有效检测方法。     

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。     

Polymerase chain reaction assay targeting cytochrome b gene for the detection of dog meat adulteration in meatball formulation

A polymerase chain reaction (PCR) assay for the assessment of dog meat adulteration in meatballs was developed. The assay selectively amplified a 100-bp region of canine mitochondrial cytochrome b gene from pure, raw, processed and mixed backgrounds. The specificity of the assay was tested against 11 animals and 3 plants species, commonly available for meatball formulation. The stability of the assay was proven under extensively autoclaving conditions that breakdown target DNA. A blind test from ready to eat chicken and beef meatballs showed that the assay can repeatedly detect 0.2% canine meat tissues under complex matrices using 0.04 ng of dog DNA extracted from differentially treated meatballs. The simplicity, stability and sensitivity of the assay suggested that it could be used in halal food industry for the authentication of canine derivatives in processed foods.     

微滴式数字PCR对肉制品中羊肉和猪肉定量分析

为了实现肉制品中羊肉和猪肉的量化研究,本文应用微滴式数字PCR对肉制品进行定量检测。通过在看家基因上设计单拷贝特异性引物,能更好的鉴别是人为因素的掺假还是在加工过程中无意的带入。根据dd PCR的结果显示,在一定范围肉质量和DNA的浓度,DNA浓度和DNA拷贝数(C)成现一定的线性关系。通过人为掺假及市售样品检测以验证此方法。以DNA浓度作为中间换算值,得到肉质量和DNA拷贝数之间的换算关系M_羊=0.12C-10.6 M_猪=0.07C+4。通过对已知掺假比例的肉样及市售样品进行检测,能够较好的得到掺假肉的质量。目前市场上存在一定的掺假现象且该检测方法具有一定市场应用前景,本文建立的微滴式数字PCR在羊肉和猪肉及其制品中的掺假检测具有较强的应用潜力,对目前混乱的肉制品掺假的市场监管提供了科学的依据。     

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。     

Identification of pork in beef meatballs using Fourier transform infrared spectrophotometry and real-time polymerase chain reaction

A study on development of Fourier-transform infrared spectrophotometric method combined with principle component analysis as well as real-time polymerase chain reaction for determination of pork–beef mixture in meatballs has been performed. A lipid component extracted from pork and beef in meatballs is analyzed using Fourier-transform infrared spectroscopy, while DNA extracted from meatball was analyzed using real-time polymerase chain reaction. The correlation between actual and predicted concentration of lard using Fourier-transform infrared spectroscopy was performed by aid of partial least squares, while grouping of lard and beef fat components in meatball was carried out by Fourier-transform infrared spectra coupled with principle component analysis. The results showed that Fourier-transform infrared spectra at wavenumbers of 1000–1200 cm^?1 coupled with partial least square and principle component analysis are successfully used for quantification and classification of pork in beef meatballs. The relationship between actual value and predicted value of lard (lipid fraction obtained from meatballs containing pork) with Fourier-transform infrared spectrophotometric method revealed good correlation, with coefficient determination (R²) value of 0.997 and standard error of calibration of 0.04%. Principle component analysis is able to classify samples containing pork and beef meatballs. Fourier-transform infrared spectroscopy using normal spectra is fast technique for identification and quantification of lard extracted from pork in meatball. In addition, real-time polymerase chain reaction using Leptin Primer–AJ 865080 can be used for amplification of pork DNA specifically in meatballs containing pork.     

DNA条形码COI序列在常见肉类鉴别中的应用研究

为了对常见的4种肉类及相关肉制品进行掺假鉴定,判别与产品标签是否相符,本研究以COI基因为靶基因,建立了4种动物源性食品DNA条形码鉴别技术。分别提取牛、羊、猪、鸭四大物种的基因组DNA为模板,以其COI基因的保守序列区设计6对通用引物,结合文献报道及数据库提供的7对通用引物进行PCR扩增,并将测序结果提交Gen Bank数据库Blast比对,评价不同DNA条形码的检测鉴别能力。筛选出COI-A为最优序列,在4个物种中扩增效率100%。对抽检的20个批次的肉加工品样品进行检测,鉴定结果约有90%的样品与产品标签标示的成分相符。其中1个批次的牛丸制品因肉类成分含量低未扩增成功,1个批次的牛丸制品检出鸭源成分,判定掺假。DNA条形码技术快速有效,本研究筛选的COI-A序列可直接用于牛、羊、猪、鸭及其肉制品的鉴定,并为其它常见动物源性食品的种类鉴定提供一定参考依据。     

基于便携式荧光定量PCR仪定量检测驴肉中掺假鸭肉

为建立快速方便的驴肉制品分子鉴定方法,本文以驴肉和常见的掺假肉类(鸭肉)为研究对象,筛选特异性引物和TaqMan探针,利用便携式Mini8 Plus实时荧光定量PCR仪进行灵敏度和特异性实验,通过绘制扩增标准曲线及确定驴肉和鸭肉的质量与DNA比值常数,对不同掺入比例(加入定量的鸭肉制成含量分别为20%、40%、60%、80%)的模拟样品和实际驴肉样品进行检测。结果显示,该方法对驴、鸭肉均具有良好的特异性,可以与马、猪、山羊、梅花鹿、牛、鸡、狗肉明显区分;对驴源性DNA成分的检出限为0.01 ng/μL,鸭源性DNA成分的检出限为0.1 ng/μL,对驴肉与鸭肉混合物中鸭肉成分的灵敏度为0.1%(w/w);所建立的标准曲线线性关系良好,驴肉DNA扩增标准曲线:y=-3.584x+27.003,R²=0.9982;鸭肉DNA扩增标准曲线:y=-3.538x+30.907,R²=0.9991;采用已建立的方法对35份驴肉样本进行市场试点调查,发现6份(17.1%)驴肉样本中含有鸭肉成分。以上研究结果说明,该实时荧光定量PCR方法可用于驴肉产品中其他...     

DETECTION OF SPECIES ADULTERATION IN PORK PRODUCTS USING AGAR-GEL IMMUNODIFFUSION AND ENZYME-LINKED IMMUNOSORBENT ASSAY

Mixing undeclared species in meat products is illegal under food labeling regulations. This study compared the conventional agar-gel immunodiffusion (AGID) with the Enzyme-Linked Immunosorbent Assay (ELISA) for detecting species adulteration and assessed the species adulteration problem in raw ground pork products in Alabama retail markets. Forty-two ground pork and 87 fresh pork sausage samples collected throughout Alabama were examined by AGID and ELISA for four species: pork, beef, poultry and sheep. Using ELISA, 91 % of the ground pork samples were found to contain other meats while 71 % were found to be contaminated using AGID. Using ELISA, 54% of the sausage samples were found to contain undeclared species while none were found to be contaminated using AGID. The major adulterating species in the pork products was beef followed by poultry and sheep. Reliable analytical methods, such as ELISA, must be used as a regulatory tool to discourage the meat species adulteration problem in retail markets.     

Effect of Modified Wheat Gluten on Boiling Resistance Capacity of Pork Meatballs

The effect of the modified wheat gluten (MWG) extender, prepared by alcalase‐based hydrolysis and transglutaminase cross‐linking, on meatballs was analyzed in this study. Here, we studied the effect of MWG addition on the boiling resistance capacity of pork meatballs (MB‐MWG) at high temperature (100 °C) and increasing cooking time; meatballs with added soy protein isolates (MB‐SPI) and raw wheat gluten (MB‐WG) were used as references. The cooking loss, water‐holding capacity (WHC), and textural properties of meatballs were investigated. The results revealed that MB‐MWG showed lower cooking loss, which decreased by 49.16% compared to meatballs without added extenders when treated for 30 min. The WHC of MB‐MWG significantly increased from 80.68% to 95.42%. The hardness, springiness, and chewiness (textural properties) of MB‐MWG were also significantly increased by 97.05%, 6.68%, and 121.96%, respectively. The addition of MWG increased the cross‐linking in meatballs during the cooking process, as indicated by the higher G′. SDS‐PAGE indicated an obvious decrease in myosin heavy chain in MB‐MWG cooked for 30 min at 100 °C, which was attributed to the interaction of myofibrillar proteins in pork meat with MWG. The nuclear magnetic resonance T₂ relaxation time patterns indicated that MWG addition caused an increase in the bound water content, and decrease in the free water content, of meatballs. An analysis of the microstructures revealed that the MB‐MWG formed the most regular and compact network. Therefore, MWG could be used as an ingredient to facilitate the processing of meat products.     

一种基于荧光PCR法检测虹鳟鱼源性成分方法的建立

目的:建立一种基于荧光聚合酶链式反应技术鉴别虹鳟鱼的检测方法.方法:以细胞色素C氧化酶I为目的基因,设计特异性引物和探针,通过测试引物特异性和模拟检测虹鳟鱼与三文鱼鱼肉混合物验证该体系的检测效果.结果:该虹鳟鱼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,可特异性地检测出含1％(w/w)虹鳟鱼的DNA存在.说明该...     

肉制品中猪肉源成分的Proofman-梯型熔解温度等温扩增检测

建立一种基于Proofman探针（proofreading enzyme-mediated probe cleavage）的梯型熔解温度等温扩增（ladder-shape melting temperature isothermal amplification,LMTIA）方法检测肉制品中的猪肉成分。选取猪细胞核中的特异性基因PRLR为靶基因,设计LMTIA引物和Proofman探针。通过优化反应体系,对所建立的方法进行特异性、灵敏度和最低检测限结果评价。结果表明：所建立的Proofman-LMTIA方法可在30 min内完成检测;相对于从鸡肉、鸭肉、牛肉、羊肉、猫肉、狗肉、玉米淀粉、红薯淀粉、木薯淀粉、绿豆淀粉、小麦粉中提取的基因组DNA,可特异性检测猪基因组DNA;检测灵敏度为1 ng/μL,对人工模拟的混合肉样中猪肉的最低检测限为0.1%。所建立的Proofman-LMTIA方法对猪肉成分有较好的特异性,能够快速、准确检测出肉制品中的猪肉成分,可对猪肉掺假进行快速检测。     

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。     

Real-time PCR based on single-copy housekeeping genes for quantitative detection of goat meat adulteration with pork

Adulteration of goat meat with cheaper meat such as pork has been frequently found. Conventional PCR methods to distinguish goat meat from adulteration are qualitative, which easily produce false-positive results due to contamination but not adulteration. To address this problem, real-time PCR based on single-copy housekeeping genes encoding replication protein A1 was developed for goat meat and pork. By calculating the Ct ratio of goat meat/pork, the goat meat content in a suspected adulteration could be deduced by a good linear correlation (R² = 0.9868) in a range from 5% to 80%. Analysis of the simulated samples of goat meat showed high accuracy with recoveries of 104.91% and 105.00% for the goat meat contents of 40% and 60%, respectively, and coefficient of variations were as low as 9.24% and 9.09%. Thus, the developed assay supplied a useful tool for food regulation authorities to achieve quantitative authentication of goat meat.     

肉制品中羊源性成分的定性和定量检测

通过检测肉制品中DNA的来源进行动物源性检测是打击鲜肉及加工肉制品制假掺假的重要技术手段。依据GB/T 25165—2010《明胶中牛、羊、猪源性成分定性检测方法 实时荧光PCR法》合成用于TaqMan 实时荧光聚合酶链式反应的引物和探针,利用鲜肉及加工肉制品进行羊源性成分定性和定量检测方法研究。首先利用12 种不同动物鲜肉组织的DNA检测方法的特异性,然后以羊源DNA梯度稀释液为模板进行灵敏度实验,最后在加工肉制品中检测方法的适用性和定量检测能力。结果表明：此方法具有羊源性成分检测特异性,对其他动物来源的鲜肉DNA均无扩增信号,可以检出羊肉和猪肉混合样品中0.1%的羊肉;方法灵敏度高,可以检出10 pg的羊源DNA;方法适用性广,可以对加工肉制品（羊肉干）进行羊源性成分的定性和定量检测。     

响应面法优化低脂猪肉丸加工工艺研究

以低脂猪肉丸为研究对象,通过响应面法研究了大豆分离蛋白、卡拉胶及脂肪对低脂猪肉丸感官评分值的影响。建立了二次多项模型,且模型高度显著、拟合度良好。研究结果表明,优化后生产低脂猪肉丸的工艺参数为:大豆分离蛋白添加量3.23%,卡拉胶添加量0.11%,脂肪添加量10.44%。     

Identification of pork genome in commercial meat extracts for Halal authentication by SYBR green I real‐time PCR

The identification of pork DNA in meat extracts is very important for Halal authentication and Muslim consumers demand protection from falsely labelled meat products. A pig‐specific SYBR green I real‐time PCR assay has been developed to address this issue. Using specific primers for pig mitochondrial DNA, successful amplification has been obtained by DNA extracted from control meat samples. With SYBR green I real‐time PCR, the specificity of the amplification was showed by Tm value. Detection limit of the real‐time PCR was down to 0.1 ng of porcine DNA. An appropriate linearity was obtained by construction of a standard curve based on Ct value and different concentrations of porcine DNA. By conventional PCR, no amplification was shown by porcine DNA less than 0.1 ng. The established method was conducted on commercially available meat extracts for detection and quantification of porcine DNA. The results showed the SYBR green I real‐time PCR could be considered a robust method for Halal authentication of meat extracts.