A polymerase chain reaction-restriction fragment length polymorphism method based on the analysis of a 16S rRNA/tRNA(Val) mitochondrial region for species identification of commercial penaeid shrimps (Crustacea: Decapoda: Penaeoidea) of food interest

A simple PCR-RFLP method has been developed for the identification of 19 penaeid shrimp species of food interest belonging to the superfamily Penaeoidea. Preliminary amplification, sequencing and alignment of a 960 bp fragment of the 16S rRNA/tRNA(Val)/12S rRNA mitochondrial region allowed the design of 16Scru4/16Scru4 primers, constructed on well-conserved mitochondrial sequences of the penaeid shrimp species considered. Such primers afforded the amplification of an internal 515-535 bp region of the 16S rRNA/tRNA(Val) genes that, when subjected to cleavage with AluI, TaqI and HinfI, provided species-specific restriction patterns. Moreover, the proposed method also allowed the definition of different intraspecific restriction types between different populations of Litopenaeus vannamei, Farfantepenaeus notialis, Fenneropenaeus merguiensis, Metapenaeus sp., Melicertus latisulcatus and Pleoticus muelleri of different origins. The method described here was also successfully applied for the identification of penaeid shrimps in complex processed precooked foods, where this type of shellfish is used as an added-value food ingredient. Sequencing analysis provided new information about the genetic relationships among shrimps not only at the levels of species and genus, but also among different populations at intraspecific level. The 16S rRNA/tRNA(Val) fragment considered in this study seems to be accurate for shrimp species identification in raw and processed foodstuffs and for phylogenetic analysis among penaeid shrimp species.     

A study of comparability in amplified fragment length polymorphism profiling using a simple model system

A simple amplified fragment length polymorphism (AFLP) model, using the bacteriophage lambda genome, was developed to test the reproducibility of this technique in an international comparative study. Using either non-selective or selective primers, nine fragments or subsets of two or three fragments, respectively, were predicted using in silico software. Under optimized conditions, all predicted fragments were experimentally generated. The reproducibility of the AFLP model was tested by submitting both "unknown" DNA template that had been restricted and ligated with AFLP linkers (R/L mixture) and corresponding primer pairs to nine laboratories participating in the study. Participants completed the final PCR step and then used either slab gel electrophoresis or CE to detect the AFLP fragments. The predicted fragments were identified by the majority of participants with size estimates consistently up to 3 base pair (bp) larger for slab gel electrophoresis than for CE. Shadow fragments, 3 bp larger than the predicted fragments, were often observed by study participants and organizers. The nine AFLP fragments exhibited relative intensities ranging from less than 3% to 22% and, apart from the two weakest fragments, with a % CV of 16 to 25. Fragments containing the highest guanine-cytosine (GC) content of 50-56% showed the greatest stability in the AFLP profiles.     

Capillary electropherograms for restriction fragment length polymorphism of Helicobacter pylori

Rapid identification of Helicobacter pylori strains is of importance for diagnosis and then treatment of duodenal and gastric ulcers. We developed a CE approach for the analysis of RFLP of the PCR products of urease (UreAB) gene and flagellin A (FlaA) gene fragments. Prior to CE analysis, the 2.4-kbp UreAB and 1.5-kbp FlaA PCR products were digested with the restriction enzymes HaeIII and HhaI, respectively. The DNA fragments were then separated by CE in conjunction with laser-induced fluorescence detection using poly(ethylene oxide) in the presence of electroosmotic flow. The DNA fragments range in sizes 259-1831 bp and 12-827 bp for UreAB and FlaA restriction fragments, respectively. Of 27 samples, the CE approach provided five and ten different RFLP patterns of the HaeIII and HhaI digests. The RFLP of PCR products of the two genes allow great sensitivity of identification of H. pylori strains. When compared with slab gel electrophoresis, the present CE approach provides advantages of rapidity (within 6 min per run), simplicity, and automation. The preliminary results have shown great practicality of the CE approach for screening H. pylori strains.     

Rapid identification of pathogenic bacteria by capillary electrophoretic analysis of rRNA genes

Molecular diagnosis is playing an increasingly important role in the rapid detection and identification of pathogenic organisms in clinical samples. The genetic variation of ribosomal genes in bacteria offers an alternative to culturing for the detection and identification of these organisms. Here 16S rRNA and 16S-23S rRNA spacer region genes were chosen as the amplified targets for single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) capillary electrophoresis analysis and bacterial identification. The multiple fluorescence based SSCP method for the 16S rRNA gene and the RFLP method for the 16S-23S rRNA spacer region gene were developed and applied to the identification of pathogenic bacteria in clinical samples, in which home-made short-chained linear polyacrylamide (LPA) was used as a sieving matrix; a higher sieving capability and shorter analysis time were achieved than with a commercial sieving matrix because of the simplified template preparation procedure. A set of 270 pathogenic bacteria representing 34 species in 14 genera were analyzed, and a total of 34 unique SSCP patterns representing 34 different pathogenic bacterial species were determined. Based on the use of machine code to represent peak patterns developed in this paper, the identification of bacterial species becomes much easier.     

聚合酶链式反应-限制性片段长度多态性和芯片生物分析技术用于台湾海峡常见石斑鱼和鲷鱼的品种鉴定

应用聚合酶链式反应-限制性片段长度多态性分析(PCR-RFLP)和芯片生物分析系统建立了台湾海峡常见石斑鱼和鲷鱼的分子生物学品种鉴定新方法。首先提取鱼的基因组脱氧核糖核酸(DNA)进行细胞色素b基因特定片段的PCR扩增,然后用DdeI、HaeIII和NlaIII 3种限制性内切酶进行酶切,在Agilent DNA 1000芯片上对酶切片段进行分离。该方法成功鉴定了台湾海峡常见的8种石斑鱼品种和5种鲷鱼品种,是一种快速、简便、有效的鱼类品种鉴定分析手段。     

Polymerase chain reaction-restriction fragment length polymorphism analysis of mitochondrial cytb gene from species of dairy interest

Species identification plays an important role in food allergy prevention and food substitution detection that can reduce the commercial value of a product. For these reasons, many molecular methods have been developed to determine species origin; among them, polymerase chain reaction (PCR)-based methods were successfully applied to processed or unprocessed foodstuffs. An updated PCR-RFLP (restriction fragment length polymorphism) method of the cytb gene was developed for the identification of the 4 species of main interest in the dairy industry (Bos, Ovis, Capra, Bubalus). The comparative analysis of the 92 cytb sequences available in the database belonging to the 4 species allowed identification of 2 highly conserved regions, which were used to design 2 oligonucleotides for the PCR amplification of a 275 base-pair (bp) cytb fragment. The in silico analysis allowed identification of a set of species-specific restriction endonucleases (HaeIII, TaqI, and MwoI), which generated easily analyzable species-specific restriction profiles of the 275 bp cytb DNA fragment. The system was developed for both purified DNA and DNA extracted from meat or dairy products and finally tested on mixed samples, indicating its applicability to foodstuffs.     

Integration of combined heteroduplex/restriction fragment length polymorphism analysis on an electrophoresis microchip for the detection of hereditary haemochromatosis

This work describes an integrated method of enzymatic digestion, heteroduplex analysis (HA) and electrophoretic sizing on a microfluidic chip. HA techniques based on microchip electrophoresis are capable of the high sensitivity detection of subtle mutations such as single nucleotide polymorphisms (SNPs) but are not readily able to detect homozygous mutant genotypes. Such homozygous conditions are commonly encountered with the gene implicated in hereditary haemochromatosis, HFE. We employed the restriction fragment length polymorphism (RFLP) method of mutation detection to complement the HA method in a rapid novel on-chip procedure that separated digested PCR fragments to reliably determine the presence or absence of the most important mutations associated with haemochromatosis. This method was able to distinguish the homozygous mutant, heterozygous and homozygous wildtype genotypes. The mutations investigated here (C282Y, H63D and S65C) are often the mutation targets used in the genetic testing for haemochromatosis. This method provides the extremely specific digestion methods needed for the analysis of the known and relatively common mutations that have a significant probability of occurring in a homozygous form. However, the high sensitivity of the HA method is useful in detecting other mutations of lesser likelihood which, by virtue of their rarity, are likely to be present only in a heterozygous form. Although the conventional methods of analysing these mutations require as much as a day to perform, this microchip method, even without robotics or multiplexed operation, can be performed in about 10 min per sample.     

Determination of locust bean gum and guar gum by polymerase chain reaction and restriction fragment length polymorphism analysis

A polymerase chain reaction (PCR) was developed to differentiate the thickening agents locust bean gum (LBG) and the cheaper guar gum in finished food products. Universal primers for amplification of the intergenic spacer region between trnL 3' (UAA) exon and trnF (GAA) gene in the chloroplast (cp) genome and subsequent restriction analysis were applied to differentiate guar gum and LBG. The presence of <5% (w/w) guar gum powder added to LBG powder was detectable. Based on data obtained from sequencing this intergenic spacer region, a second PCR method for the specific detection of guar gum DNA was also developed. This assay detected guar gum powder in LBG in amounts as low as 1% (w/w). Both methods successfully detected guar gum and/or LBG in ice cream stabilizers and in foodstuffs, such as dairy products, ice cream, dry seasoning mixes, a finished roasting sauce, and a fruit jelly product, but not in products with highly degraded DNA, such as tomato ketchup and sterilized chocolate cream. Both methods detected guar gum and LBG in ice cream and fresh cheese at levels <0.1%.     

Capillary electrophoresis with dual laser detection in separation of amplified fragment length polymorphism fragments

We are presenting the application of CE technique with dual‐channel LIF detection for the simultaneous separation of DNA fragments labeled with two different fluorescence dyes. The optimal conditions of the analysis were determined for the separation of amplified fragment length polymorphism (AFLP) fragments labeled with 5′‐6‐carboxyfluorescein (6‐FAM) and the DNA size standard labeled with sulfoindocyanine succinimidyl ester (Cy‐5). CE equipped with both argon ion and diode lasers is a good alternative for sequencers and might be applied in analyses of PCR products generated by various fingerprinting methods.     

Notl-Msell methylation-sensitive amplied fragment length polymorhism for DNA methylation analysis of human cancers

We have applied a methylation-sensitive restriction endonuclease, NotI, to the existing amplified fragment length polymorphism (AFLP) method and developed NotI-MseI methylation-sensitive-AFLP (MS-AFLP). NotI-MseI MS-AFLP allows the analysis of DNA methylation alterations at the NotI sites scattered over the genome. Hypermethylation and hypomethylation are visualized by the decrease and increase in the band intensity of DNA fingerprints. Identification of consistent changes can be facilitated through parallel electrophoresis of multiple samples. DNA fragments exhibiting alterations can be cloned from fingerprint bands by amplification of gel-eluted DNA with the same pair of primers used for radioactive fingerprint presentation. Fluorescent NotI-MseI MS-AFLP offers a safer method of studying the alterations in DNA methylation, and may be applied to the hybridization of DNA microarrays in the future. Using NotI-MseI MS-AFLP, we observed frequent hypomethylation of a satellite DNA repeat sequence in a majority of breast tumors.     

Application of silver staining to the rapid typing of the polymorphism of HLA-DQ alleles by enzymatic amplification and allele-specific restriction fragment length polymorphism.

A rapid and highly sensitive silver staining method, originally developed for the detection of proteins, was slightly modified to detect nucleic acids in polyacrylamide gels. The second exons of the histocompatibility antigen HLA-DQA 1 and DQB 1 genes were selectively amplified from genomic DNA by the polymerase chain reaction (PCR). Digestion of the PCR products by endonucleases, followed by their size-separation on polyacrylamide gels and visualization by silver staining, allowed us to define the HLA-DQ alleles of the genomic DNA. The intensity of staining of digested PCR-amplified DNA is linear from at least 8 to 18 ng for fragments of lengths ranging from approximately 40 to 200 bp. Thus, silver staining in combination with PCR and allele-specific restriction fragment length polymorphism provides a simple, safe, and rapid method for accurate definition of HLA-DQ alleles at the nucleotide level in the clinical typing laboratory.     

Identification of hallucinogenic fungi from the genera Psilocybe and Panaeolus by amplified fragment length polymorphism

Unambiguous identification of the hallucinogenic fungi of the genera Psilocybe and Panaeolus is required by national and international drug control legislation. We report on a DNA-based test using the technique of amplified fragment length polymorphism (AFLP). AFLP can differentiate species of the two genera Psilocybe and Panaeolus by using different primer sets. The identification of hallucinogenic fungi using a DNA-based test, which can be used in conjunction with morphological features, will assist in forensic investigations.     

Identification and classification of seafood‐borne pathogenic and spoilage bacteria: 16S rRNA sequencing versus MALDI‐TOF MS fingerprinting

The present study aims to compare two molecular technologies, 16S rRNA sequencing and MALDI‐TOF MS, for bacterial species identification in seafood. With this aim, 70 reference strains from culture collections, including important seafood‐borne pathogenic and spoilage bacterial species, and 50 strains isolated from commercial seafood products, were analysed by both techniques. Genomic analysis only identified the species of 50% of the isolated strains, proving to be particularly poor at identifying members of the Pseudomonas and Bacillus genera. In contrast, MALDI‐TOF MS fingerprinting identified 76% of the strains at the species level. The mass spectral data were submitted to the SpectraBank database ( http://www.spectrabank.org ), making this information available to other researchers. Furthermore, cluster analysis of the peak mass lists was carried out with the web application SPECLUST and the calculated groupings were consistent with results determined by a phylogenetic approach that is based on the 16S rRNA sequences. However, the MALDI‐TOF MS analysis demonstrated more discriminating potential that allowed for better classification, especially for the Pseudomonas and Bacillus genera. This is of importance with respect to the varying pathogenic and spoilage character at the intragenus and intraspecies level. In this sense, MALDI‐TOF MS demonstrated to be a competent bacterial typing tool that extends phenotypic and genotypic approaches, allowing a more ample classification of bacterial strains.     

16S rRNA gene-based metagenomic analysis reveals differences in bacteria-derived extracellular vesicles in the urine of pregnant and non-pregnant women

Recent evidence has indicated that bacteria-derived extracellular vesicles (EVs) are important for host–microbe communication. The aims of the present study were to evaluate whether bacteria-derived EVs are excreted via the urinary tract and to compare the composition of bacteria-derived EVs in the urine of pregnant and non-pregnant women. Seventy-three non-pregnant and seventy-four pregnant women were enrolled from Dankook University and Ewha Womans University hospitals. DNA was extracted from urine EVs after EV isolation using the differential centrifugation method. 16S ribosomal RNA (16S rRNA) gene sequencing was performed using high-throughput 454 pyrosequencing after amplification of the V1–V3 region of the 16S rDNA. The composition of 13 taxa differed significantly between the pregnant and non-pregnant women. At the genus level, Bacillus spp. EVs were more significantly enriched in the urine of the pregnant women than in that of the non-pregnant women (45.61% vs 0.12%, respectively). However, Pseudomonas spp. EVs were more dominant in non-pregnant women than in pregnant women (13.2% vs 4.09%, respectively). Regarding the compositional difference between pregnant women with normal and preterm delivery, EVs derived from Ureaplasma spp. and the family Veillonellaceae (including Megasphaera spp.) were more abundant in the urine of preterm-delivered women than in that of women with normal deliveries. Taken together, these data showed that Bacillus spp. EVs predominate in the urine of pregnant women, whereas Pseudomonas spp. EVs predominate in the urine of non-pregnant women; this suggests that Bacillus spp. EVs might have an important role in the maintenance of pregnancy.     

Glycosyl linkage characteristics and classifications of exo-polysaccharides of some regionally different strains of Lentinula edodes by amplified fragment length polymorphism assay and cluster analysis

We report here the first combined amplified fragment length polymorphism (AFLP) analysis of genomic DNA fingerprinting data and cluster analysis of the exo-polysaccharide glycosyl linkage data of 10 regionally different strains of Lentinula edodes to compare their genetic and structural similarities and differences. In addition, the monosaccharide compositions, molecular weights, glycosyl structural linkages were investigated for the exo-polysaccharides extracted from these different phylogenetic groups of regionally different L. edodes. All exo-polysaccharides had similar molecular weight distribution between 1 × 10⁴ and 3 × 10⁶ Da and the monosaccharide composition analysis revealed the presence of heterogeneous materials containing glucose, mannose, xylose, galactose, fucose, rhamnose and arabinose in different ratios. Among these monosaccharides, the glucose contents are the highest for all but one strain, indicating that glucose probably is the building block of the backbones of these exo-polysaccharides. The AFLP assay data helped to classify the 10 L. edodes strains into three distinct genetic groups. Gas chromatographic and mass spectrometric (GC-MS) data revealed five different glycosyl linkage types for these exo-polysaccharides. Most of the exo-polysaccharide backbone structures contain (1 → 4)-linked-d-glucopyranosyl and (1 → 6)-linked-d-glucopyranosyl moieties. Arabinose 1 → 4 linkages and mannose 1 → 2 linkages also exist in all strains. The only differences among these linkages are their monosaccharide compositions leading to different degree of backbone and branch formations. Cluster analyses of the GC-MS data of the exo-polysaccharides of the 10 strains resulted in 10 dendrograms. However, four of the 10 dendrograms were identical and were obtained using the average, Ward and weighted linkage type method of Manhattan distance and using the Ward method of Euclidean distance. The results of cluster analyses were not very much different from that of the AFLP assay and allowed the comparison of genetic and structural similarities and differences.     

Molecular docking and 3D-QSAR study of pyranmycin derivatives against 16S rRNA A site

To understand pharmacophore properties of pyranmycin derivatives and to design novel inhibitors of 16S rRNA A site, comparative molecular field analysis (CoMFA) approach was applied to analyze three-dimensional quantitative structure–activity relationship (3D-QSAR) of 17 compounds. AutoDock 3.0.5 program was employed to locate the orientations and conformations of the inhibitors interacting with 16S rRNA A site. The interaction mode was demonstrated in the aspects of inhibitor conformation, hydrogen bonding and electrostatic interaction. Similar binding conformations of these inhibitors and good correlations between the calculated binding free energies and experimental biological activities suggest that the binding conformations of these inhibitors derived from docking procedure were reasonable. Robust and predictive 3D-QSAR model was obtained by CoMFA with q² values of 0.723 and 0.993 for cross-validated and non-cross-validated, respectively. The 3D-QSAR model built here will provide clear guidelines for novel inhibitors design based on the Pyranmycin derivatives against 16S rRNA A site.     

Tobramycin与16S rRNA A位点复合物的分子动力学模拟

采用分子动力学方法(Molecular dynamics, MD)对托普霉素(Tobramycin)与16S rRNA的A位点复合物的特异性识别机制进行了理论模拟研究, 模拟时间为3.6 ns. 结果表明, A位点中波动最大的部位是两个环外碱基A1492和A1493; tobramycin的环Ⅰ和环Ⅱ是其最保守的结构单元, 可能参与了Tobramycin与16S rRNA的A位点之间的特异性识别. 另外, 发现一个残存时间为3.6 ns的“结构化”水分子, 它桥接了Tobramycin环Ⅱ的N3与环Ⅰ的N6'之间的氢键, 稳定了Tobramycin的结构; Tobramycin周围水合密度较高的位点出现在环Ⅰ和环Ⅱ附近, 这也正是晶体结构中形成较多水媒介氢键及动力学模拟中结构化水分子出现的位置. 动力学模拟证实Tobramycin与16S rRNA间的结合是大量氢键及水分子相互作用的结果, 这有助于设计和开发以Tobramycin为基础, 具有高亲和力及特异性的16S rRNA抑制剂.