Detection and Identification of Probiotic Lactobacillus plantarum Strains by Multiplex PCR Using RAPD-Derived Primers

Lactobacillus plantarum 2035 and Lactobacillus plantarum ACA-DC 2640 are two lactic acid bacteria (LAB) strains that have been isolated from Feta cheese. Both display significant potential for the production of novel probiotic food products. The aim of the present study was the development of an accurate and efficient method for the molecular detection and identification of the above strains in a single reaction. A multiplex PCR assay was designed for each strain, based on specific primers derived from Random Amplified Polymorphic DNA (RAPD) Sequenced Characterized Amplified Region (SCAR) analysis. The specificity of the assay was tested with a total of 23 different LAB strains, for L. plantarum 2035 and L. plantarum ACA-DC 2640. The multiplex PCR assay was also successfully applied for the detection of the above cultures in yogurt samples prepared in our lab. The proposed methodology may be applied for monitoring the presence of these strains in food products, thus evaluating their probiotic character. Moreover, our strategy may be adapted for other novel LAB strains with probiotic potential, thus providing a powerful tool for molecular discrimination that could be invaluable to the food industry.     

植物种子DNA快速提取新方法

本研究提出了一种提取植物种子DNA的快速、简便、有效的新方法。基本过程包括选取饱满植物种子3-5粒并研成细粉．用提取缓冲液提取、离心分离、异丙醇沉淀、TE缓冲液保存得到的DNA。结果表明：所获得的DNA浓度和质量均较高．经扩增与琼脂糖凝胶电泳后得到了非常清晰的DNA谱带，特别适合序列特征扩增区段（Sequence-Characterize Amplified Region，SCAR）检测。     

双孢蘑菇SCAR标记的建立及在菌株群鉴定中的应用

根据15条具有菌株群特异性的SRAP、ISSR、RAPD扩增产物DNA条带测序结果,设计64对SCAR引物,其中6对引物能成功扩增出目的条带,即有6条特异片段(A、C、E、G、H、J)被成功转化为SCAR标记.通过这6个SCAR标记的不同组合,将40个双孢蘑菇菌株分为9个类群和6个类群,与基于SRAP、ISSR、RAPD资料的聚类分析结果的组间距离值取13和16时的分类结果完全吻合.对A片段的SCAR-PCR检测结果是:可从40个双孢蘑菇菌株中鉴别出双孢蘑菇333号菌株(编号01).     

一株酿酒酵母的特征区域序列扩增标记

以23株不同来源的酿酒酵母为研究对象,分别提取基因组DNA,试用50条随机引物对其进行随机扩增多态性DNA分析,筛选到两条具有菌株鉴别能力的随机引物。P09可以从2.412,ST—01,SK—26,ZD—01四株酿酒酵母基因组DNA中扩增出长度为433bp的Sc—433片断Sc—433;P46可以从2.1882,ST—01,NJ—02三株酿酒酵母基因组DNA中扩增出长度为665bp的Sc—665片断,其中仅有目的菌株ST—01能稳定的扩增出这2个标记。把这2个片断分别克隆到pUCmT质粒载体中,经过酶切鉴定后测序,根据序列设计特异性引物,把RAPD标记转化为特征区域序列扩增标记,为酿酒酵母菌株的分子鉴别提供了新的借鉴。     

Development and Application of SCAR Marker for the Detection of Papaya Seed Adulteration in Traded Black Pepper Powder

The present study reports the development of a specific, sensitive, and reproducible Sequence Characterized Amplified Region (SCAR) marker to detect papaya seed powder adulteration in traded black pepper powder. A putative RAPD marker (449 bp) specific to papaya seed was identified, cloned, and sequenced to design the SCAR primers. This specific SCAR marker could detect the presence of papaya seed in all the analyzed simulated standards and in one of five branded market samples of black pepper powder tested. The analytical strategy being very simple could be used for large scale screening of powdered black pepper market samples intended for export and domestic uses.     

紫菜自由丝状体SCAR标记的获得

应用RAPD技术对27个紫菜自由丝状体材料进行遗传多样性分析，获得7个紫菜材料的7个特异标记。对这7个特异片段进行了回收、克隆，对其中5个片段已完成了序列测定，根据这5个序列的两端顺序合成了5对SCAR—PCR引物，经过优化PCR反应条件成功地将紫菜材料Porphyra tenuipedalis的RAPD扩增片段OPZ-19140和P．yezoensis qd-8的RAPD扩增片段OPJ-18488转换成两个SCAR标记。因为这两个标记的序列已全部测通，故又称为STS标记。这两个SCAR标记(STS标记)可以作为紫菜P．tenuipedalis和P.yezoensis qd-8自由丝状体材料种质鉴定、优良品系选育和产权保护的分子标记。     

Genetic Dominant Variants in STUB1, Segregating in Families with SCA48, Display In Vitro Functional Impairments Indistinctive from Recessive Variants Associated with SCAR16

Variants in STUB1 cause both autosomal recessive (SCAR16) and dominant (SCA48) spinocerebellar ataxia. Reports from 18 STUB1 variants causing SCA48 show that the clinical picture includes later-onset ataxia with a cerebellar cognitive affective syndrome and varying clinical overlap with SCAR16. However, little is known about the molecular properties of dominant STUB1 variants. Here, we describe three SCA48 families with novel, dominantly inherited STUB1 variants (p.Arg51_Ile53delinsProAla, p.Lys143_Trp147del, and p.Gly249Val). All the patients developed symptoms from 30 years of age or later, all had cerebellar atrophy, and 4 had cognitive/psychiatric phenotypes. Investigation of the structural and functional consequences of the recombinant C-terminus of HSC70-interacting protein (CHIP) variants was performed in vitro using ubiquitin ligase activity assay, circular dichroism assay and native polyacrylamide gel electrophoresis. These studies revealed that dominantly and recessively inherited STUB1 variants showed similar biochemical defects, including impaired ubiquitin ligase activity and altered oligomerization properties of the CHIP. Our findings expand the molecular understanding of SCA48 but also mean that assumptions concerning unaffected carriers of recessive STUB1 variants in SCAR16 families must be re-evaluated. More investigations are needed to verify the disease status of SCAR16 heterozygotes and elucidate the molecular relationship between SCA48 and SCAR16 diseases.     

松杨栅锈菌小种特异性DNA片段的SCAR标记转换

用SCAR标记技术对松杨栅锈菌生理小种特异性DNA片段进行了转化和检测研究.通过对RAPD随机引物的扩增筛选,发现随机引物BAO118扩增产生的约700bp的DNA片段与松杨栅锈菌小种特异性相关联.将该片段进行回收、TA克隆、双向测序和序列整合后,序列全长685bp,3'端有BAO118随机引物序列.根据整合后序列,用Primerselect软件设计了一对包含随机引物序列的简并引物对.用该引物对对供试菌样进行PCR扩增,成功获得了松杨栅锈菌小种685bp的SCAR标记.用该标记进行田间混合孢子菌样、单孢子接种菌样DNA检测,均得到了685bp的DNA片段,不受寄主DNA和孢子菌系类型的影响,具有较好的稳定性和特异性.     

Developing an SCAR and ITS reliable multiplex PCR-based assay for safflower adulterant detection in saffron samples

Saffron (Crocus sativus L.), one of the most important and expensive medicinal spice products traded internationally, is subject to adulteration by design or default with safflower stamens and corn stigmas, leading to poor quality of saffron samples. The present study aims at the development of specific, sensitive and reproducible PCR-based markers to detect these adulterants in traded saffron. Six putative RAPD markers generated by random primers, OPA-14, MG-11, MG-12 and AJ-05, were identified as saffron specific by comparative RAPD analysis of genuine saffron, safflower and corn. These specific RAPD markers were cloned, sequenced and six pairs of SCAR primers were designed. Specific designed primers were able to amplify reproducible saffron DNA with expected sizes and no amplification in corn and safflower DNA. In this study, a primer pair was also designed based on ITS sequences for specific amplification of safflower DNA. PCR reactions were also specifically amplified 613 bp of ITS region in safflower genome. The multiplex PCR assays were further established for the joint use of some SCAR and ITS markers efficiently. The special feature of this new molecular method was technically rapid and convenient practically and suitable for analyzing large numbers of samples. Thus, the simple rapid PCR-based molecular method could be used as a helpful assistant tool for the identification of adulterant saffron samples. This study described the development of a new SCAR and ITS maker-based multiplex PCR assay for the rapid molecular detection of substitutes in saffron.