Genetic Identification of Lophius budegassa and L. piscatorius by PCR-RFLP Analysis of a Mitochondrial tRNAGlu/Cytochrome bSegment

Identification of Lophius budegassa(black‐bellied angler) and L. piscatorius(angler) (Lophiiformes) was carried out on the amplification of a 486 bp tRNA^Glu/cytochrome b segment using the polymerase chain reaction (PCR). Direct DNA sequencing of 6 PCR products was carried out. Six restriction endonucleases (AluI, CfoI, HaeIII, HinfI, Mae, and ScrFI) with different species‐specific restriction fragment length polymorphism (RFLP) were selected. Digestions of PCR products from 30 individuals showed no intraspecific polymorphism. Double digestions (CfoI and HinfI, and HaeIII and ScrFI) were simpler and more rapid than single digestions. This technique is suitable for distinguishing tails of both Lophius species.     

Bt新菌株GGD-4的特性研究与杀虫晶体蛋白基因鉴定

从连云港地区的土壤中分离得到一株苏云金芽孢杆菌新菌株GGD-4,对此菌株的个体形态、生理生化特点进行了研究,并通过PCR-RFLP鉴定体系分析了此菌株中cry基因类型,为进一步利用该菌株奠定了基础.     

肉制品中牛源性成分的PCR-RFLP检测

根据牛线粒体DNA特异性片段设计出引物,应用PCR-RFLP技术检测出7种生肉和4种加工肉制品中的牛源性成分,并根据线粒体中Cytb保守序列设立内源参照基因检测DNA提取效率。方法简便、快速、适合实验室常规物种检测。     

保健食品建泽泻及其混淆品的PCR-RFLP分子鉴别

目的:寻找简易可重复的分子标记方法对保健食品建泽泻及其混淆品进行鉴定。方法:对建泽泻及其混淆品的rDNA ITS区进行PCR扩增、测序,运用ClustalX、Mega3.0、DNAMAN 4.0等软件对ITS区进行序列分析和PCR限制性酶切图谱分析(PCR-RFLP)。结果:依据建泽泻及其混品完整的rDNA ITS区片段长度,将慈菇和甘薯两种混淆品首先区分开;再通过PCR-RFLP分析,可将建泽泻从川泽泻、窄叶泽泻、小泽泻和芋等混淆品中成功鉴别出来。结论:rDNA ITS片段长度不足以用于鉴别保健食品建泽泻原料的真伪;PCR-RFLP可对保健食品建泽泻及其混淆品进行简便而快捷的分子鉴别。     

Novel multiplex PCR-RFLP assay discriminates bovine,porcine and fish gelatin substitution in Asian pharmaceuticals capsule shells

Gelatin is widely used in pharmaceuticals as a protective coating, such as soft and hard capsule shells. However, the animal source of gelatin is a sensitive issue because certain gelatins such as porcine and bovine gelatins are not welcome in Halal, Kosher and Hindus’ consumer goods. Recently, we have documented DNA barcoding and multiplex PCR platforms for discriminating porcine, bovine and fish gelatins in various fish and confectionary products; but those assays were not self-authenticating and also not tested in highly refined pharmaceutical products. To address this knowledge gap, here we report a self-authenticating multiplex PCR-restriction fragment length polymorphism (RFLP) assay to identify animal sources of various gelatin in pharmaceutical capsules. Three different restriction enzymes, BsaAI, Hpy188I and BcoDI were used to yield distinctive RFLP patterns for gelatin-based bovine (26, 94 bp), fish (97, 198 bp) and porcine (17, 70 bp) DNA in control experiments. The specificity was cross-tested against 16 non-target species and the optimised assay was used to screen gelatin sources in 30 halal-branded pharmaceuticals capsule shells. Bovine and porcine DNA was found in 27 and 3 of the 30 different capsules products. The assay was suitable for detecting 0.1 to 0.01 ng total DNA extracted from pure and mixed gelatins. The study might be useful to authenticate and monitor halal, kosher, vegetarian and Hindu compliant pharmaceuticals, foods and cosmetics.     

PCR-RFLP Analysis of the Internal Transcribed Spacer (ITS) Region for Identification of 3 Clam Species

ABSTRACT Restriction site analysis of the internal transcribed spacer (ITS) region amplified by the polymerase chain reaction (PCR) was used for the specific identification of 3 clam species: Ruditapes decussatus (grooved carpet shell), Venerupis pullastra (pullet carpet shell), and Ruditapes philippinarum (Japanese carpet shell). PCR amplification using primers based on nucleotide sequences of Mytilus ITS regions produced a fragment of 1195 bp in R. decussatus, 1074 bp in V. pullastra, and 1188 bp inR. philippinarum. Digestion of the PCR products with endonucleases HinfI and Rsa I, followed by agarose gel electrophoresis of the digested products, yielded specific restriction profiles that enabled direct visual identification of the species analyzed.     

Molecular Identification of Commercial Spicy Pollack Roe Products by PCR-RFLP Analysis

ABSTRACT: Spicy pollack roe products are a popular seafood item made from fish eggs that should be made with salt-cured mature roes of walleye pollack Theragra chalcogramma . Because of high demand and poor catch of walleye pollack, however, spicy pollack roe products are often susceptible to substitution with roes of closely related codfish. In this study, a simple method identifying the ingredients of commercial spicy pollack roe products was developed to differentiate walleye pollack from codfish substitutes such as gray cod Gadus macrocephalus using PCR-RFLP (Restriction Fragment Length Polymorphism) analysis. PCR amplification of the mitochondrial cytochrome b gene yielded single fragments commonly from pollack and cod. Direct digestion of the PCR products with Mph 11031 restriction enzyme showed an unique restriction fingerprint only in pollack. This PCR-RFLP analysis enabled the reliable identification of commercial spicy pollack roe products made by only pollack roes from products padded with cod roes. It thus can be useful to expose substitution of pollack roes with lower valued codfish roes in commercial spicy pollack roe products.     

Development of a molecular method for the rapid discrimination of red seaweeds used for agar production

In Tunisia, agar is produced from the species Gracilaria verrucosa. Other species, such as Gracilariopsis sp. which have a very similar morphology to Gracilaria, can be harvested in a mixture with Gracilaria with the result that the quantity and quality of agar extracted is different than initially expected. In this study, we tested the use of ITS sequences from 5.8s rDNA for species discrimination between G. verrucosa and Gracilariopsis harvested from two production sites (Bizerte and Tunis Lagoons). The amplification of genomic ITS sequences and sequencing shows that the samples collected from Tunis can be identified by PCR fragment of 1124 bp whereas the samples from Bizerte are characterized by the presence of two fragments of 1124 and 983 bp, respectively. A BLAST investigation in Genbank shows that the sequence similarities between the fragment of 1124 pb and G. verrucosa was 85% and between the fragment of 983 pb and Gracilariopsis sp. Plymoutn was 94%. Gracilariopsis sp. possesses a PCR fragment with a length of 983 bp, which discriminates it from G. verrucosa when these two species are collected in a mixture as in the lagoon of Bizerte. The RFLP method after EcoRI application provides a characteristic pattern for G. verrucosa, which is composed of two main fragments with respective sizes of 823 and 301 bp whereas amplified DNA of Gracilariopsis was uncut under these conditions.     

Presence of non-Saccharomyces yeasts in cellar equipment and grape juice during harvest time

The purpose of this study was to analyze the presence of different yeasts in the facilities of four wineries from the D.O.Ca. Rioja region in Spain. The study was conducted through the identification of the yeasts via the PCR-RFLP technique of the ITS region of rDNA. The diversity of non-Saccharomyces yeasts found in wineries has previously only been studied to a limited extent, despite the fact that these yeasts take part both in the start of spontaneous fermentation and in the changes which occur in the wines during their subsequent conservation. Most earlier studies carried out on cellar ecosystems have focussed on the clonal diversity of Saccharomyces cerevisiae. The results obtained in this study indicated that the presence of non-Saccharomyces yeasts in facilities is higher than that of the S. cerevisiae, with percentages of over 60% in all the wineries analyzed. Yeasts belonging to 10 genera and 18 species were isolated, but the only genera present in all four wineries were Cryptococcus, Pichia, and Saccharomyces. The Zygosaccharomyces bailii yeast responsible for taint was detected in one cleaned winery, in both the winemaking equipment and the fermenting must. It was also noted that the quantity and type of yeasts present in the facilities are related to the product used for cleaning them. It is also necessary to point out that the cleaning of the cellars prior to the reception of the grapes does not completely eliminate the yeasts present, so that these can subsequently become part of the vinification process.     

Survey of the authenticity of prawn and shrimp species in commercial food products by PCR-RFLP analysis of a 16S rRNA/tRNA mitochondrial region

A novel PCR-RFLP method was evaluated as a tool to assess the incidence of incorrect labelling of prawns and shrimps in commercial food products. The whole method can be performed in less than 8 h in only one day of work. PCR amplification with primers 16Scru4/16Scru3, targeted to the amplification of a ca. 530 bp region of 16S rRNA and tRNA^Val mitochondrial genes, was coupled to restriction analysis with AluI, TaqI or HinfI. Forty-one commercial food products were considered. The molecular method considered allowed the identification of up to 17 different prawn and shrimp species in all the processed products considered. Seven (28%) of the 25 food products declaring one or more species in their labels were incorrectly labelled. Authentication was successfully assessed in commercial peeled products subjected to industrial processing, in which none of the products displayed labelling at species level. Overall, incorrect labelling was detected in 10 (24.4%) of the 41 commercial products tested, while another 16 samples (39%) exhibited incomplete labelling. The molecular method evaluated in this study proved to be a rapid and easy-to-perform two-step analytical approach to achieve species identification of commercial whole specimens of frozen prawns and shrimps and in peeled processed products where such raw materials are included as added-value ingredients.