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.     

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.     

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.     

Establishment and application of a polymerase chain reaction for the identification of beef

A polymerase chain reaction (PCR) based method for the identification of beef by amplification of bovine 1.709 satellite DNA was established. The method not only was able to amplify raw beef DNA, but also cooked or autoclaved meat DNA. The sequence selected for amplification consisted of a 218 bp DNA fragment lying in the 1.709 satellite DNA of bovine. A pair of synthetic oligonucleotides flanking this sequence were used as printers, and genomic DNA extracted from beef samples employed as templates. Each batch of reaction mix contained Taq DNA polymerase, a buffer component, deoxynucleotide triphosphates, genomic DNA template and a pair of bovine oligodeoxynucleotide primers in a final volume of 50 μl. The amplification of bovine DNA was performed by using 33 cycles of denaturation at 94°C (40 s), annealing at 53.5°C (50 s) and extension at 72°C (60 s), with a 7 min extension at 72°C in the last cycles. The amplified products were subjected to rapid electrophoresis in 3% agarose gel and visualized under ultraviolet illumination after ethidium bromide staining. A Hae Ш restriction endonuclease test was done to verify the specificity of the PCR amplification, and the expected DNA fragments were produced. The specificity test demonstrated that this method was positive for bovine, buffalo and yak meat DNA, but negative for equine, sheep, goat, camel, swine, deer and mouse meat DNA, etc. At least 33.6 fg of DNA from raw beef samples and 0.32 pg of DNA from cooked or autoclaved beef samples were detected, respectively, by PCR. We tested 103 beef samples by PCR and obtained 100% correct identification. The method needed only 6 h for detection of meat products of all kinds. The results showed that the PCR method was sensitive, specific, convenient and rapid, so it may be suitable for rapid identification of beef.     

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.     

Development of a polymerase chain reaction assay for species identification of goose and mule duck in foie gras products

Polymerase chain reaction amplification of a conserved region of the -actin gene has been used for the specific identification of goose (Anser anser) and mule duck (Anas platyrhynchos×Cairina moschata) foie gras. Universal primers were used for the amplification of a DNA fragment containing three introns and four exons of the -actin gene in goose and mule duck. Sequence analysis of the amplified fragments was necessary for the design of forward species-specific primers in the goose and mule duck -actin genes. The use of species-specific forward primers, together with a reverse universal primer, produced amplicons of different length, allowing clear identification of goose and mule duck foie gras samples. Analysis of experimental mixtures demonstrated that 1% of duck can be easily detected in goose foie gras using the PCR method developed here. This genetic marker can be very useful for the accurate identification of these two species in foie gras products.     

基于rpoB靶基因的PCR法快速检测弓形菌

建立了一种弓形菌(Arcobacter)快速、敏感、特异的PCR检测方法。采用PCR技术扩增弓形菌编码RNA聚合酶β亚基的rpoB基因,并对该方法进行特异性和敏感性测试。结果只有弓形菌在205bp处出现目的扩增带,其他菌株扩增结果均为阴性。该法对弓形菌的检测限可达8.20×102cfu/mL。建立的PCR方法具有操作简便、准确可靠以及灵敏特异的特点,为食品中弓形菌的快速检测提供了新的手段。      

Mitochondrial markers for the detection of four duck species and the specific identification of Muscovy duck in meat mixtures using the polymerase chain reaction

A polymerase chain reaction (PCR) assay for the qualitative detection of four duck species in meat mixtures, and a second PCR assay for the specific identification of Muscovy duck, have been developed based on oligonucleotide primers targeting the 12S rRNA mitochondrial gene. The specificity of both assays was tested against a wide range of animal species. The technique was applied to raw and sterilized muscular binary mixtures, with a detection limit that ranged from 0.1% to 1.0% (w/w). The short length (less than 100 bp) of the DNA fragments amplified with these primer pairs was found to be essential for the successful amplification in samples with highly degraded DNA, and consequently, it could be very useful in inspection programmes to enforce labelling regulation of heat and pressure-processed products, for which other methods cannot be applied.     

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。     

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。     

Universal primers for species authentication of animal foodstuff in a single polymerase chain reaction

BACKGROUND: There are many DNA‐based systems for detecting animal species present in food and food products, applicable for food quality control and authentication. However, most (if not all) methods require more than one pair of primers and cannot be applied over a wide taxonomic range, e.g. identifying vertebrates and invertebrates with the same primers and protocols. RESULTS: A pair of primers is described here that allows in a single polymerase chain reaction the identification of animal species in food and processed (precooked, canned or smoked) food products over a wide taxonomic range. CONCLUSION: These primers permit the identification of most animal taxa employed in human nutrition, from invertebrates such as molluscs to higher vertebrates, distinguishing between species of the same genus. The short fragment amplified within the 16S rDNA exhibits phylogenetic value and could be considered universal based on the wide taxonomic range assayed. The primers are easy to use and accessible for laboratories with a modest budget, as well as being valuable for consumer information and to reveal food fraud. Copyright © 2012 Society of Chemical Industry     

Detection of genetically modified soybean and its product tou-kan by polymerase chain reaction with dual pairs of DNA primers

Since genetically modified (GM) crops and foods began to appear in market, the detection of these GM foods has become an important issue. Efficient and reliable methods are urgently needed to analyze the GM content of a large quantity of foods. However, most foods are processed through heating or cooking, and their proteins are denatured. Therefore, DNA rather than protein is the major target for detecting GM foods. In this report, polymerase chain reaction (PCR) was performed with dual sets of DNA primers, which co-amplified the soybean-specific lectin gene and the transgenic petunia transit peptide sequence (CTP) in the 5′ end of the 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS) gene within the same reaction. PCR with these two sets of primers reacted specifically with lectin gene and CTP, and can detect soybean with GM content less than 1%. PCR detecting both lectin gene and CTP was also applied to a soybean-derived product tou-kan, a traditional oriental food. Results indicated that although DNA was partially degraded in tou-kan, this PCR method was still able to detect the presence of EPSPS gene. However, when the DNA within tou-kan was destroyed badly, neither lectin nor EPSPS genes were detected by the PCR suggested here. Finally, an examination procedure for the Roundup Ready soybean was suggested according to the results of PCR with dual pairs of DNA primers. The proposed PCR method has proved to be a reliable and efficient method for detecting the GM content of the processed foods from Roundup Ready soybean. An erratum to this article can be found at     

食品中单核细胞增生李斯特菌株的分离及聚合酶链式反应鉴定

目的 建立单核细胞增生李斯特菌的聚合酶链式反应(PCR)检测方法,了解市售食品中单核细胞增生李斯特菌的污染情况.方法 采集成都市市售生畜肉、生禽肉、熟肉制品、水产品、生食蔬菜以及其他熟食等食品样品共135份,采用李氏增菌肉汤(LB1,LB2)进行初增菌,应用选择性分离培养基PALCAM和在TSA-YE平板上进行分离,利用单增李斯特显色平板进行鉴定;根据李斯特菌的特异性基因iap基因设计引物,采用PCR方法检测所有分离的李斯特菌株;根据单增李斯特菌的特异性基因hly基因和prfA基因设计引物检测单核细胞增生李斯特菌株.结果 135份样品中共检出李斯特菌17株,检出率为12.6％;其中单核细胞增生李斯特菌4株,检出率为3.0％.结论 本研究建立的PCR方法具有特异性,本市市售食品不同程度受到李斯特菌的污染.     

数字聚合酶链式反应法检测贝类和浆果中甲肝病毒

目的建立一种数字聚合酶链式反应(PCR)检测贝类和浆果中甲肝病毒的方法。方法样品经蛋白酶K消化-聚乙二醇法进行甲肝病毒富集后,使用高纯度病毒核酸试剂盒进行RNA提取,之后对甲肝病毒进行数字PCR检测。结果本方法对甲肝病毒有典型扩增,重复性和稳定性良好,对于草莓样品中甲肝病毒的检测灵敏度为25.30 CCID_(50)/20 g,树莓样品中甲肝病毒的检测灵敏度为6.32 CCID_(50)/20 g,贝类样品中甲肝病毒的检测灵敏度为12.54 CCID_(50)/2 g,表明其灵敏度高。结论该方法快速、准确、灵敏,适合测定贝类和浆果食品中甲肝病毒。     

Identification of spoilage yeasts in a food-production chain by microsatellite polymerase chain reaction fingerprinting

A survey of yeast strains present in the production chain of mayonnaise and salad dressings was carried out over a period of 14 months. Attempts were made to identify the isolated yeasts with the API system, but identification of all species involved was not possible. In the investigation the performance of the microsatellite polymerase chain reaction (PCR) fingerprinting analysis with the microsatellite oligonucleotide primers (GAC)₅and (GTG)₅appeared to be superior. Several yeast species were encountered in the production lines but only the speciesZygosaccharomyces bailiiandZygosaccharomyces bisporuswere present in the final products. Only microsatellite PCR fingerprinting analysis allowed discrimination between species of theZygosaccharomycesgenus. In addition, the PCR-based technique allowed the discrimination of different types within theZ. bailiispecies.Z. bailiistrains isolated from spoiled products displayed PCR-fingerprinting types that appeared to be identical to some of those generated by strains isolated from one specific production line. This suggested that microsatellite PCR fingerprinting is useful in tracing back the origin of spoilage outbreaks, and that it can be applied in microbiological quality assurance monitoring systems in industrial environments.     

A rapid polymerase chain reaction (PCR)-based assay for the identification of Listeria monocytogenes in food samples.

A rapid and simple assay has been developed which allows specific detection of Listeria monocytogenes within 3.5 h in cultures prepared from suspect food samples and propagated 48 h in selective medium. The assay is based on PCR technology, and uses a specific primer set derived from sequences located down-stream of the hlyA gene. The specificity of the primer set was confirmed by testing 115 L. monocytogenes, 14 L. innocua, 5 L. seeligeri and 4 L. ivanovii isolates. The assay was compared to standard microbiological tests and gave identical results for 83 food samples, including 32 positives. These field trials indicate that the assay developed provides an alternative detection system for L. monocytogenes in foods, which can be used by the food industry.     

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.     

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。     

Evaluation of the polymerase chain reaction method for identification of Vibrio vulnificus isolated from marine environments.

The polymerase chain reaction (PCR) method for identification of Vibrio vulnificus in the marine environment was evaluated by comparing it to both the conventional and DNA-DNA hybridization methods. Of 13,325 isolates obtained from seawater and sediment samples, and oyster and goby specimens collected from the coastal waters of Tokyo Bay, Japan, only 61 isolates were identified as V. vulnificus on the basis of phenotypic characteristics and the amplification of the cytotoxin-hemolysin gene by the PCR method. All 61 isolates were further confirmed to be V. vulnificus by a DNA-DNA hybridization method and the API 20E system although they were divided into 13 groups on the basis of their API 20E profiles. These results strongly suggest that the PCR method is useful for identification of this organism.