Identification of tuna species by a real-time polymerase chain reaction technique

Tuna are highly priced fishes that are often used in processed products. For effective fishery management and protection of consumers’ rights, it is important to develop a molecular method to identify the species of the tuna products. In this study we have developed a molecular method based on real-time polymerase chain reaction (real-time PCR) technology for the rapid identification of four tuna species. Four species-specific TaqMan probes were designed to identify bigeye tuna (Thunnus obesus), Pacific bluefin tuna (Thunnus orientalis), southern bluefin tuna (Thunnus maccoyii), and yellowfin tuna (Thunnus albacares). A SYBR green system was also designed to enhance the authentication of T. obesus. Both systems can distinguish target species from others in an efficient and high-throughput manner and can be applied to species identification of tuna products.     

Detection of pathogenic and spoilage micro-organisms in food with the polymerase chain reaction

Traditional biochemical and immunochemical methods for the detection of micro-organisms in food have been supplemented by a number of DNA-based methods during the last decade. Besides the development of direct hybridization techniques, emphasis has been laid onin vitroamplification methods.In vitromethods such as the self-sustained sequence replication (3SR) or the nucleic acid sequence based amplification (NASBA), the Q-beta replicase amplification and the ligase chain reaction (LCR) have had, until now, only limited practical relevance for food monitoring and control. The most developedin vitroamplification method is the polymerase chain reaction (PCR) that allows rapid and selective identification of micro-organisms. Because PCR is increasingly raising interest as a detection method in food hygiene and control, an overview of PCR-systems for the detection of bacteria and viruses in food is provided with this paper. Although the PCR method has advantages (particular specificity, sensitivity) it also has limitations, one of which is its inability to allow differentiation between viable and non-viable micro-organisms. Furthermore, the inhibition of PCR by food-derived inhibitors can result in false negative results. Possible reasons for PCR inhibition and ways to differentiate between viable and dead micro-organisms are discussed in this review.     

食品中腐败酵母的实时荧光PCR鉴定

为研究食品中腐败酵母实时荧光PCR快速鉴定方法,设计和筛选出了可用于腐败酵母鉴定的多条探针和引物,并建立了针对酿酒酵母、鲁氏接合酵母、斯巴达克毕赤酵母和布鲁塞尔德克酵母等4种腐败酵母菌的实时荧光PCR鉴定方法。用文中建立的方法对从糕点、蜂蜜、饮料等市售食品中分离出的60余株酵母菌进行了鉴定,发现其中4株为酿酒酵母,21株为鲁氏酵母,其他为与上述4种不同的酵母。以实时荧光PCR方法鉴定酵母,全过程仅需约3 h,与常用的生化鉴定方法相比,简化了鉴定步骤,提高了鉴定准确性,缩短了鉴定时间。     

潲水油聚合酶链式反应鉴定技术研究

目的建立特异的潲水油聚合酶链式反应(PCR)鉴定方法。方法以猪肉、牛肉、羊肉、鸡肉、哺乳动物等动物源性基因及大米物种特异性基因作为靶基因,采用PCR及荧光PCR方法鉴定潲水油。结果 6个自制的粗制潲水油样品全部被鉴定为潲水油,7个疑似潲水油样品中的5个被鉴定为潲水油,而4个正常食用植物油全部被鉴定为非潲水油。结论以潲水油中可能混杂的动物源性基因和大米基因作为靶标,采用普通PCR或荧光PCR方法可有效鉴定潲水油。     

DNA fingerprinting of thermophilic lactic acid bacteria using repetitive sequence-based polymerase chain reaction

DNA fingerprints of lactic acid bacteria were generated by polymerase chain reaction using a primer based on the repetitive elements found in the genome of Streptococcus pneumoniae (BOX-PCR). The method made it possible to identify 37 isolates from raw milk. industrial starters and yogurt. Differentiation at species, subspecies and strain level was possible for Lactobacillus delbrueckii subsp. lactis, Lb. delbrueckii subsp bulgaricus and Str. thermophilus. BOX-PCR was also applied to studying the strain composition of a starter culture and the direct detection of strains in commercial fermented milk.     

Identification of yeast strains using the polymerase chain reaction

Commonly used techniques for the identification of industrial yeast strains are usually time-consuming and cumbersome. Moreover, some of these methods may give ambiguous results. A novel strategy has been developed for identifying yeast strain employing polymerase chain reaction technology. Using customised oligonucleotides, some regions of the yeast genome between δ elements are amplified to give an ‘amplified’ sequence polymorphisml (Skolnick and Wallace 1988) characteristic of the strains. With this technique it is possible to identify individual strains of Saccharomyces cerevisiae.     

Identification of cow's milk in "buffalo" cheese by duplex polymerase chain reaction

A duplex polymerase chain reaction (PCR) was developed to identify the milk of bovine and buffalo species in cheese products, particularly in mozzarella cheese, a typical Italian cheese made from buffalo's milk. Two sets of primers were designed on the basis of the alignment of the sequence codifying mitochondrial cyt b available in the GenBank database. The primers proved to be species-specific, giving rise to 279-bp (bovine) and 192-bp (buffalo) amplified fragments. Since the amplification conditions for bovine and buffalo primers were identical, a duplex PCR was successfully applied to identify the two species in a single reaction step. This technique, when used to test cheese products from the retail trade, allowed the detection of partial or even total substitution of cow's milk for buffalo's milk, in some cases in samples of cheese misleadingly labeled "pure buffalo" mozzarella.     

PCR方法快速检测变形杆菌的研究

建立了一种快速、准确检测变形杆菌属的PCR方法.采用传统分离培养法、全自动微生物分析系统检测法和常规PCR方法对66株变形杆菌样本分离株进行鉴定.三种检测方法结果一致,但PCR方法检测时间只需5～6h,比传统分离方法节省40h左右.通过对13株非变形杆菌属菌株的特异性实验和标准菌株的灵敏度实验,进一步表明,PCR方法具有操作简便、准确可靠以及灵敏特异的特点,优于传统分离培养法和全自动微生物分析系统检测法.     

Detection of Penicillium expansum by polymerase chain reaction

Penicillium expansum is a major causative agent of postharvest decay in a variety of fruits, including apples, peaches, nectarines, and cherries. It causes significant economic losses to the fruit industry and is also of potential public health significance, since it produces patulin, a mycotoxin known to cause harmful effects in animals. Rapid and specific detection of P. expansum is important for ensuring microbiological quality and safety of fruits and fruit juices. The traditional methods for identification of P. expansum are time-consuming and labor-intensive. In this study, we report a polymerase chain reaction utilizing primers based on the polygalacturonase gene of P. expansum. The PCR amplified a 404-bp DNA product from all the P. expansum isolates tested, but not in other common foodborne Penicillium species and Escherichia coli. Experiments to determine the sensitivity of the PCR indicated that it can detect the DNA equivalent from as low as 25 spores of P. expansum. The PCR could potentially be used as a rapid tool for screening fruits for the presence of P. expansum.     

实时荧光PCR鉴定婴幼儿配方食品中克罗诺杆菌的研究

目的 建立一种能够快速准确鉴定克罗诺杆菌的实时荧光聚合酶链式反应（PCR）方法,并对食品样品和人工污染样品进行克罗诺杆菌检验。方法 根据克罗诺杆菌DNA旋转酶B亚基（gyrB）基因保守区序列设计引物和探针。通过特异性试验、绝对灵敏度、相对灵敏性试验、抗干扰试验对所建立方法进行方法学验证。采用人工污染样品增菌液进行方法灵敏度试验。结果 本研究建立的方法能够特异性扩增7种克罗诺杆菌,但对与其亲缘关系较近的其他肠杆菌及食品中较为常见的其他致病菌均无扩增,表明本研究建立的方法具有很好的特异性和抗干扰能力。采用阪崎克罗诺杆菌验证绝对灵敏度达1～10 pg,相对灵敏度可以达到103 CFU/mL。在基因组水平和培养物水平均具有很好的抗干扰能力。人工污染样品在36℃增菌24 h后检测灵敏度可以达到100 CFU/mL。结论本研究所建立的实时荧光PCR方法对婴幼儿配方食品样品中的克罗诺杆菌的检测具有快速、特异、灵敏和稳定的特点,可以为传统婴幼儿配方食品中的克罗诺杆菌的检验提供技术参考。     

Identification of the bacterial biodiversity in koumiss by denaturing gradient gel electrophoresis and species-specific polymerase chain reaction

Bacterial biodiversity in traditional koumiss fermented milk was studied by denaturing gradient gel electrophoresis (DGGE). Target DNA bands were identified according to the reference species ladder, constructed in this study. Comigrating bands present in the DGGE profiles were resolved by species-specific PCR. The results revealed a novel bacterial profile and extensive bacterial biodiversity in koumiss. The dominant lactic acid bacteria included Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus fermentum, and Lactobacillus kefiranofaciens. Frequently encountered bacterial species were Enterococcus faecalis, Lactococcus lactis, Lactobacillus paracasei, Lactobacillus kitasatonis, and Lactobacillus kefiri. Leuconostoc mesenteroides, Streptococcus thermophilus, Lactobacillus buchneri, and Lactobacillus jensenii were occasionally found in this product. In addition, L. buchneri, L. jensenii, and L. kitasatonis, which were never previously isolated by culture-dependent methods, were identified for the first time in the Xinjiang koumiss. Furthermore, conventional cultivation was performed by plating samples on M17, de Man-Rogosa-Sharpe, Halligan-Pearce, and Kenner fecal media. The results revealed that lactobacilli were the dominant species in the koumiss ecosystem, which was consistent with the results obtained by the DGGE analysis. This is the first systematic study of the microbial composition in koumiss, and our findings will be helpful in selecting appropriate strains for the manufacture of this product at the industrial level.     

PCR技术检测空肠弯曲菌的研究进展

空肠弯曲菌(Campylobacter jejuni)是一种人畜共患病病原菌,可以使人和动物引发多种疾病。目前,检测C.jejuni采用的国标方法是传统的培养法,但C.jejuni培养条件苛刻,且培养法存在操作繁琐、特异性不强、费时等缺点。聚合酶链式反应(polymerase chain reaction,PCR)以其快速、准确、灵敏度高、特异性强的特点,现已广泛应用于C.jejuni的检测,并成为目前快速检测临床与食品中C.jejuni最常用的的方法。本文综述了近年来利用RCR技术,包括常规PCR、多重PCR、巢式PCR、实时荧光定量PCR、PCR-酶联免疫吸附法、最大几率数-PCR、PCR-限制性片段长度多态性、PCR-变性高效液相色谱、PCR-变性梯度凝胶电泳和磁捕获-荧光PCR方法检测C.jejuni的研究进展,并针对这些PCR技术的原理、检测效果、优点和缺点等方面进行了分析比较,为有效控制和预防该菌引起的疾病提供重要信息。     

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。     

利用PCR方法检测水稻及其加工产品中转基因水稻

市场上的转基因产品以及深加工产品越来越多,其安全性引起全世界的极大争论,很多国家的检验机构相继开展了转基因产品的检测工作。以转基因水稻为材料,以聚合酶链式反应(PCR)方法为基础,选择适用于转基因食品安全性检验的核酸检测技术,针对转入苏云金杆菌(Bacillus thuringiensis,简写为Bt)基因水稻的Cry1Ac片段进行PCR检测,建立适合转基因水稻的检测方法。该方法简便快速、检测结果与标准及其他文献资料相符。     

聚合酶链反应在融合子鉴定中的应用

以粟酒裂殖酵母和酿酒酵母为出发菌株,进行了原生质体融合试验,并对检出的融合子采用聚合酶链反应(PCR)扩增亲本的特定基因序列,以此达到鉴定融合子的目的.在检出的3株融合子中,均扩增出亲本特定基因,证明了该法在融合子鉴定中的可行性.     

实时荧光聚合酶链式反应法检测食品中狗源性成分

根据狗线粒体NADH氧化还原酶亚基2基因(ND2)中的序列设计了狗特异性引物和Taqman探针,建立了食品中狗源性成分的实时荧光聚合酶链式(Real-time PCR)检测方法。实验表明,该方法可以很好地区分狗与其他常见的14个物种,引物、Taqman探针特异性良好,检测灵敏度可达100fg,适用于食品中狗源性成分的快速鉴定。     

Analysis of the inhibition of food spoilage yeasts by vanillin

The antimicrobial potential of vanillin, the major component of vanilla flavour, was examined against the growth of three yeasts associated with food spoilage, Saccharomyces cerevisiae, Zygosaccharomyces bailii and Zygosaccharomyces rouxii. Minimum inhibitory concentration (MIC) values of 21, 20 and 13 mM vanillin were determined for the three yeast strains, respectively. The observed inhibition was found to be biostatic. During fermentation, the bioconversion of sub-MIC levels of vanillin in the culture medium was demonstrated. The major bioconversion product was identified as vanillyl alcohol, however low levels of vanillic acid were also detected. Neither the vanillyl alcohol nor the vanillic acid was found to be antagonistic to yeast cell growth. The results indicate the importance of the aldehyde moiety in the vanillin structure regarding its antimicrobial activity and that the bioconversion of vanillin could be advantageous for the yeasts, but only at levels below MIC. These bioconversion activities, presumably catalysed by non-specific dehydrogenases, were shown to be expressed constitutively. It was observed that increased vanillin concentrations inhibited its own bioconversion suggesting that the activity required intact cells with metabolic capacity.     

微滴式数字聚合酶链式反应定量检测食品中金黄色葡萄球菌方法的研究

目的 建立微滴数字聚合酶链式反应(ddPCR)快速定量检测食品中金黄色葡萄球菌的方法.方法 以金黄色葡萄球菌nu基因为靶序列,筛选出同时适用于实时荧光定量PCR(qPCR)和微滴数字PCR(ddPCR)的引物探针,建立食品中金黄色葡萄球菌ddPCR快速定量检测方法,并对该方法进行特异性、灵敏度、准确性和重复性实验.结果...     

牛奶中志贺氏菌PCR检测方法的建立

根据Genbank志贺氏菌侵袭性质粒抗原H (ipaH)基因序列, 自行设计引物, 扩增特异的326 bp核酸片段, 经过优化PCR扩增条件, 建立了志贺氏菌特异、敏感、快速的PCR检测方法, 并对牛奶中的志贺氏菌进行了检测.特异性试验结果表明, 志贺氏菌参考菌株均能扩增出特异的核酸片段, 大肠杆菌、巴氏杆菌、金黄色葡萄球菌、沙门氏菌、蜡样芽孢杆菌、变形杆菌、绿脓杆菌的扩增结果均为阴性.敏感性试验结果表明, 采用试剂盒提取基因组, 该方法的敏感性可达到1.75×102 cfu/mL.人工污染牛奶的模拟检测结果表明, PCR方法的检测限为1.75×103 cfu/mL.     

Detection of Salmonella spp. in tropical seafood by polymerase chain reaction

The incidence of Salmonella spp. in tropical seafood was studied using standard microbiological techniques and polymerase chain reaction (PCR). Six of 20 finfish (30%), 4 of 20 clams (20%) and 1 of 20 shrimp (5%) were positive by culture techniques and by PCR. In a comparative study of different selective enrichment broths and selective plating media, more than one enrichment broth and selective agar were found to be necessary for efficient detection of Salmonella from seafood. Selenite cystine broth (SCB) was found to be more efficient compared to tetrathionate broth (TTB) while both bismuth sulfite agar (BSA) and hektoen enteric agar (HEA) were equally effective as selective plating media for fish. In the case of clams, HEA was found to be more effective. The presence of Salmonella spp. could be detected by PCR amplification of DNA extracted directly from the enrichment broths. In two cases, enrichment broths that were positive by PCR did not yield Salmonella by conventional methods.