Species identification in salted products of red snappers by semi-nested PCR-RFLP based on the mitochondria 12S rRNA gene sequence

A molecular approach was developed to distinguish species of red snappers among commercial salted fish products. The specific fragments of the mitocliondrial 12S rRNA gene, which were about 450 bp, were obtained using the semi-nested polymerase chain reaction (semi-nested PCR). Subsequently, PCR amplicons were sequenced, aiming to select restriction endonucleases that generated species-specific restriction fragment length polymorphism (RFLP) profiles. Discrimination of red snappers Lutjanus sanguineus, L. erythopterus from L. argentintaculatus, L. malabarius and other morphologically similar fishes such as Lethrinus leutjanus and Pinjalo pinjalo was feasible by one restriction digestion reaction with three endonucleases Hae III, Sca I and SnaB I, however, for differentiation of L. sattguineus and L. erythopterus, another restriction digestion reaction with single restriction endonuclease Mae II was needed. The seminested PCR-RFLP was demonstrated to be reliable in species identification of salted fish products in this study. (c) 2005 Published by Elsevier Ltd.

Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli

Allophycocyanin (APC), a cyanobacterial photosynthetic phycobiliprotein, functions in energy transfer as a light-harvesting protein. One of the prominent spectroscopic characteristics of APC is a strong red-shift in the absorption and emission maxima when monomers are assembled into a trimer. Previously, holo-APC alpha and beta subunits (holo-ApcA and ApcB) were successfully synthesized in Escherichia coli. In this study, both holo-subunits from Synechocystis sp. PCC 6803 were co-expressed in E. coli, and found to self-assemble into trimers. The recombinant APC trimer was purified by metal affinity and size-exclusion chromatography, and had a native structure identical to native APC, as determined by characteristic spectroscopic measurements, fluorescence quantum yield, tryptic digestion analysis, and molecular weight measurements. Combined with results from a study in which only the monomer was formed, our results indicate that bilin synthesis and the subsequent attachment to apo-subunits are important for the successful assembly of APC trimers. This is the first study to report on the assembly of recombinant ApcA and ApcB into a trimer with native structure. Our study provides a promising method for producing better fluorescent tags, as well as a method to facilitate the genetic analysis of APC trimer assembly and biological function.

cDNA cloning and mRNA expression of a selenium-dependent glutathione peroxidase from Zhikong scallop Chlamys farreri

The glutathione peroxidases are essential enzymes of the cellular antioxidant defence system. In the present study, the full-length cDNA sequence encoding an extracellular glutathione peroxidase (designated CfGPx3) was isolated from Zhikong scallop Chlamys farreri. The complete cDNA was of 1194 bp, containing a 5' untranslated region (UTR) of 50 bp, a 3' UTR of 490 bp and an open reading frame (ORF) of 654 bp encoding a polypeptide of 217 amino acids. CfGPx3 possessed all the conserved features critical for the fundamental structure and function of glutathione peroxidase, such as the selenocysteine encoded by stop codon UGA, the GPx signature motif ((96)LGVPCNQFI(103)) and the active site motif ((WNFEKF184)-W-179). The high similarity of CfGPx3 with GPx from other organisms indicated that CfGPx3 should be a new member of the glutathione peroxidase family. By fluorescent quantitative real-time PCR, the CfGPx3 mRNA was universally detected in the tissues of haemocytes, gill, gonad, muscle and hepatopancreas with the highest expression in hepatopancreas. After scallops were challenged by Listonella anguillarum, the expression level of CfGPx3 transcript in haemocytes was significantly up-regulated (P<0.05) at 8 h post challenge. These results suggested that CfGPx3 was potentially involved in the immune response of scallops and perhaps contributed to the protective effects against oxidative stress. (C) 2010 Elsevier Inc. All rights reserved.

AiC1qDC-1, a novel gC1q-domain-containing protein from bay scallop Argopecten irradians with fungi agglutinating activity

The globular C1q-domain-containing (C1qDC) proteins are a family of versatile pattern recognition receptors via their globular C1q (gC1q) domain to bind various ligands including several PAMPs on pathogens. In this study, a new gC1q-domain-containing protein (AiC1qDC-1) gene was cloned from Argopecten irradians by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNA of AiC1qDC-1 was composed of 733 bp, encoding a signal peptide of 19 residues and a typical gC1q domain of 137 residues containing all eight invariant amino acids in human C1qDC proteins and seven aromatic residues essential for effective packing of the hydrophobic core of AiC1qDC-1. The gC1q domain of AiC1qDC-1, which possessed the typical 10-stranded beta-sandwich fold with a jelly-roll topology common to all C1q family members, showed high homology not only to those of Cl qDC proteins in mollusk but also to those of C1qDC proteins in human. The AiC1qDC-1 transcripts were mainly detected in the tissue of hepatopancreas and also marginally detectable in adductor, heart, mantle, gill and hemocytes by fluorescent quantitative real-time PCR. In the microbial challenge experiment, there was a significant up-regulation in the relative expression level of AiC1qDC-1 in hepatopancreas and hemocytes of the scallops challenged by fungi Pichia pastoris GS115, Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Listonella anguillarum. The recombinant AiC1qDC-1 (rAiC1qDC-1) protein displayed no obvious agglutination against M. luteus and L. anguillarum, but it aggregated P. pastoris remarkably. This agglutination could be inhibited by D-mannose and PGN but not by LPS, glucan or D-galactose. These results indicated that AiC1qDC-1 functioned as a pattern recognition receptor in the immune defense of scallops against pathogens and provided clues for illuminating the evolution of the complement classical pathway. (C) 2010 Elsevier Ltd. All rights reserved.

Species identification in salted products of red snappers by semi-nested PCR-RFLP based on the mitochondrial 12S rRNA gene sequence

A molecular approach was developed to distinguish species of red snappers among commercial salted fish products. The specific fragments of the mitochondrial 12S rRNA gene, which were about 450bp, were obtained using the semi-nested polymerase chain reaction (semi-nested PCR). Subsequently, PCR arnplicons were sequenced, aiming to select restriction endonucleases that generated species-specific restriction fragment length polymorphism (RFLP) profiles. Discrimination of red snappers Lutjanus sanguineus, Lutjanus erythopterus from Lutjanus argentimaculatus, Lutjanus malabarius and other morphologically similar fishes such as Lethrinus leutjanus and Pinjalo pinjalo was feasible by one restriction digestion reaction with three endonucleases Hae III, Sca I and SnaB I, however, for discrimination of L. sanguineus and L. erythopterus, another restriction digestion reaction with single restriction endonuclease Mae II was needed. The semi-nested PCR-RFLP was demonstrated to be reliable in species identification of salted fish products in this study. (c) 2005 Elsevier Ltd. All rights reserved.

Evaluation of a combined wavelet and a combined principal component analysis classification system for BCG diagnostic problem

Heart disease is one of the main factor causing death in the developed countries. Over several decades, variety of electronic and computer technology have been developed to assist clinical practices for cardiac performance monitoring and heart disease diagnosis. Among these methods, Ballistocardiography (BCG) has an interesting feature that no electrodes are needed to be attached to the body during the measurement. Thus, it is provides a potential application to asses the patients heart condition in the home. In this paper, a comparison is made for two neural networks based BCG signal classification models. One system uses a principal component analysis (PCA) method, and the other a discrete wavelet transform, to reduce the input dimensionality. It is indicated that the combined wavelet transform and neural network has a more reliable performance than the combined PCA and neural network system. Moreover, the wavelet transform requires no prior knowledge of the statistical distribution of data samples and the computation complexity and training time are reduced.

应用PCR-DGGE技术分析长江口低氧区和黄海冷水团的细菌群落组成

利用PCR-DGGE技术对长江口外低氧区海域和黄海冷水团海域的细菌群落组成进行了分析。 长江口外低氧区海域的细菌群落组成分析结果为：对获得的25条DGGE条带进行了克隆、测序，所得到的序列进行了系统进化分析（细菌16S rRNA基因V3区序列），分别归属于4个细菌类群：变形菌门(Proteobacteria)、拟杆菌门(Bacteroides)、厚壁菌门(Firmicutes)和蓝细菌门(Cyanobacteria)。其中有16条分别与变形细菌亚群的γ和δ-Proteobacteria相似。通过时空分析发现，低氧水体的细菌群落组成与非低氧水体的组成是不同的。低氧水体的优势菌群是拟杆菌门(Bacteroides)中的Flavobacteria。 黄海冷水团海域的细菌群落组成和优势菌群分析结果为：细菌16S rDNA V3区特征片段经DGGE分离、条带切割，共得到24条DGGE条带，克隆、测序后，将所得序列进行系统进化分析，分别归属于2个细菌类群：变形细菌门(Proteobacteria)和拟杆菌门(Bacteroides)。在24条序列中有16条分别与变形细菌亚群的γ和δ-Proteobacteria相似，有5条与拟杆菌门相似。通过时空分析发现，10月份（冷水团存在期），冷水团内部水体的细菌群落组成包括γ-Proteobacteria、δ-Proteobacteria和Bacteroides，而冷水团外部的水体的细菌群落组成包括γ-Proteobacteria和Bacteroides。冷水团内部水层的优势菌群为γ-Proteobacteria。4月份虽然冷水团没有形成，但是所调查的海域海水温度都不高，在7℃－12℃范围内，所以4月份所有站位，不管是底层的还是总的的细菌群落组成都与10月份冷水团内部（海水温度低于10℃）水体的相同，与10月份冷水团外部（海水温度大于19℃）的不同。

应用单细胞PCR方法研究胶州湾一株鳍藻的遗传特征

近年来，分子生物学技术与方法被越来越多地应用于有害赤潮研究。其中，单细胞PCR方法是对难以室内培养的有害赤潮藻种进行遗传特征研究的一项重要技术。本实验尝试建立微藻的单细胞PCR方法，并将其应用于鳍藻研究。 应用室内培养的亚历山大藻，研究了微藻的单细胞PCR方法，并对藻细胞的固定和保存方法进行了比较。结果表明，浮游植物研究中常用的甲醛固定方法只能用于短期保存样品（少于5天），而乙醇固定、鲁格氏液固定、或者在-20℃下冷冻保存的样品，在较长的时间后（60天）仍可以得到比较理想的PCR扩增结果。 应用单细胞PCR方法，对青岛近海采集的鳍藻进行了研究，扩增并测定了包括核糖体大亚基（LSU）rDNA的5’端D1-D2区序列，以及5.8S rDNA和ITS区的部分序列信息。通过分析软件对所得到的鳍藻序列信息与基因库中已知的鳍藻序列信息进行了分析与对比。根据ITS和LSU序列信息构建的系统进化树都显示，本文采集的鳍藻藻种与国外报道的圆形鳍藻聚为一支，初步确定采集的鳍藻应为圆形鳍藻，对该藻种形态学特征的观察也支持这一结果。该藻种部分LSU rDNA序列与欧洲同种鳍藻相似度达到99%,与其它等鳍藻遗传距离在18%-20%之间。测定的ITS区序列与同种圆形鳍藻有62个碱基的差异，与LSU rDNA序列相比，ITS序列变异更大。应用LC-MS方法对该鳍藻的进行了DSP毒素分析，结果未检测到OA或DTX1毒素。 这是我国首次应用单细胞PCR方法对鳍藻开展的研究工作，首次报道了我国鳍藻的核糖体部分序列信息。圆形鳍藻在青岛近海海域是初次报道，显示了单细胞PCR方法在鳍藻研究中的重要意义。

PCR-DGGE analysis of intestinal bacteria and effect of Bacillus spp. on intestinal microbial diversity in kuruma shrimp (Marsupenaeus japonicus)

In this study, the intestinal microbiota of kuruma shrimp (Marsupenaeus japonicus) was examined by molecular analysis of the 16S rDNA to identify the dominant intestinal bacteria and to investigate the effects of Bacillus spp. on intestinal microbial diversity. Samples of the intestines of kuruma shrimp fed normal feed and Bacillus spp. amended feed. PCR and denaturing gradient gel electrophoresis (DGGE) analyses were then performed on DNA extracted directly from the guts. Population fingerprints of the predominant organisms were generated by DGGE analysis of the universal V3 16S rDNA amplicons, and distinct bands in the gels were sequenced. The results suggested that the gut of kuruma shrimp was dominated by Vibrio sp. and uncultured gamma proteobacterium. Overall, the results of this study suggest that PCR-DGGE is a possible method of studying the intestinal microbial diversity of shrimp.

Identification of Crassostrea ariakensis and related oysters by multiplex species-specific PCR

Genetic markers are needed for rapid and reliable identification of oysters. In this study, we developed multiplex genus- and species-specific PCR markers for the identification of oysters from China. We used the mitochondrial cytochrome oxidase I (COI) and nuclear 28S ribosomal RNA genes for marker development. DNA sequences from different species were obtained from GenBank or by direct sequencing. Sequences were aligned, and genus- and species-specific nucleotides were identified. Primers were designed for genus/species-specific amplification to generate fragments of different sizes. A multiplex set of genus- and species-specific primers from the 28S gene was able to separate C. ariakensis and C. hongkongensis from other species and assign oysters to four genera. A set of species-specific COI primers provided positive identification of all five Crassostrea species from China, C. ariakensis, C. hongkongensis, C. angulata, C. gigas, and C. sikamea in a single PCR. The multiplex PCR assays do not require fluorescence-labeling or post-PCR enzyme digestion, providing a simple, fast and reliable method for the identification of oysters from China.

Molecular authentication of the traditional Tibetan medicinal plant Swertia mussotii

Swertia mussotii is an important species in Tibetan folk medicine. However, it is quite expensive and frequently adulterated, so reliable methods for authentication of putative specimens and preparations of the species are needed to protect consumers and to support conservation measures. We show here that the chloroplast (cp) DNA rpl16 intron has limited utility for differentiating S. mussotii from closely related species, since the cpDNA rpl16 sequences are identical in S. mussotii and two other species of Swertia. However, the rDNA internal transcribed spacer (ITS) sequences differ significantly between S. mussotii and all of 13 tested potential adulterants. Thus, the ITS region provides a robust molecular marker for differentiating the medicinal S. mussotii from related adulterants. Therefore, a pair of allele-specific diagnostic primers based on the divergent ITS region was designed to distinguish S. mussotii from the other species. Authentication by allele-specific diagnostic PCR using these primers is convenient, effective and both simpler and less time-consuming than sequencing the ITS region.

Development of a sensitive fluorescent derivatization reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) and its application for determination of amino acids from seeds and bryophyte plants using high-performance liquid chromatography with fluorescence detection and identification with electrospray ionization mass spectrometry

A pre-column derivatization method for the sensitive determination of amino acids and peptides using the tagging reagent 1,2-benzo-3,4dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent was replaced by 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEOC. BCEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z (M + H)(+) under electrospray ionization (ESI) positive-ion mode with an exception being Tyr detected at negative mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 246.2 corresponding to the cleavage of C-O bond of BCEOC molecule. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% are observed with a 3-4-fold molar reagent excess. Derivatives exhibit strong fluorescence and extracted detzvatization solution with n-hexane/ethyl acetate (10:1, v/v) allows for the direct injection with no significant interference from the major fluorescent reagent degradation by-products, such as 1,2-benzo-3,4-dihydrocarbazole-9-ethanol (BDC-OH) (a major by-product), mono- 1,2-benzo-3,4-dihydrocarbazole-9-ethyl carbonate (BCEOC-OH) and bis-(1,2-benzo-3,4-dihydrocarbazole-9-ethyl) carbonate (BCEOC)(2). In addition, the detection responses for BCEOC derivatives are compared to those obtained with previously synthesized 2-(9-carbazole)-ethyl chloroformate (CEOC) in our laboratory. The ratios AC(BCEOC)/AC(CEOC) = 2.05-6.51 for fluorescence responses are observed (here, AC is relative fluorescence response). Separation of the derivatized peptides and amino acids had been optimized on Hypersil BDS C-18 column. Detection limits were calculated from 1.0 pmol injection at a signal-to-noise ratio of 3, and were 6.3 (Lys)-177.6 (His) fmol. The mean interday accuracy ranged from 92 to 106% for fluorescence detection with mean %CV < 7.5. The mean interday precision for all standards was < 10% of the expected concentration. Excellent linear responses were observed with coefficients of > 0.9999. Good compositional data could be obtained from the analysis of derivatized protein hydrolysates containing as little as 50.5 ng of sample. Therefore, the facile BCEOC derivatization coupled with mass spectrometry allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids and peptides from biological and natural environmental samples. (c) 2005 Elsevier B.V. All rights reserved.

基因芯片PCR温度模糊自整定PID控制算法的研究

在核酸扩增反应仪中,基因芯片核酸扩增反应过程要求实现温度高精度快速跟踪控制,常规温控方案和算法难以实现。将模糊推理系统与常规PID控制方式相结合,采用模糊自整定PID控制算法实现了温度快速跟踪控制。实验结果表明:模糊自整定PID控制算法比常规PID算法具有更强的鲁棒性,能够克服控制对象热惯性参数时变性的影响,降低了输出温度最大超调量,提高了稳态精度。

硫酸盐还原菌类群分离培养和PCR分析鉴定

探讨分离培养和聚合酶链反应（PCR）在硫酸盐还原菌类群分析鉴定中的应用。［方法］采用分离培养和PCR 对贵州阿哈湖沉积物中硫酸盐还原菌类群进行分析，并对分离纯化的一株硫酸盐还原菌进行鉴定。 ［结果］ 贵州阿哈湖沉积物硫酸盐还原菌类群为脱硫肠菌属、脱硫叶菌属和脱硫球菌- 脱硫线菌- 脱硫八叠菌属；纯化菌株经PCR 扩增初步鉴定为脱硫叶菌属,菌株形态与鉴定结果相符。 ［结论］ 采用分离培养结合PCR，可以对硫酸盐还原菌类群进行分析和鉴定。

Análise da variabilidade intra-espécie dos genes pld e rpoB de Corynebacterium pseudotuberculosis por meio de marcadores PCR-RFLP.

Corynebacterium pseudotuberculosis é o agente etiológico da linfadenite caseosa em ovinos e caprinos, uma doença infectocontagiosa crônica caracterizada por abscessos em linfonodos superficiais ou internos. Os animais acometidos podem apresentar lesões internas ou externas, o que representa perdas econômicas graves na caprinovinocultura, como o comprometimento da produção de lã em ovinos, deficiência reprodutiva, condenação da carne ou morte do animal. Uma vez estabelecida a infecção no rebanho, torna-se difícil sua erradicação. Com o objetivo de avaliar a variabilidade genética intra-espécie em C. pseudotuberculosis, 31 isolados provenientes de seis municípios do Estado de Pernambuco, sendo 23 de caprinos e oito de ovinos, tiveram os seus DNAs genômicos extraídos e analisados por reação em cadeia da polimerase e análise de polimorfismos por tamanho de fragmentos de restrição (PCR-RFLP). Os locis escolhidos foram os genes pld e rpoB, cujas seqüências dos oligonucleotídeos amplificaram fragmentos de 924 e 447 pares de bases (pb), respectivamente. Os produtos da amplificação foram digeridos com as enzimas Pst I e Msp I (gene pld) e HpyCh4 IV e Msp I (gene rpoB). Os fragmentos de restrição foram corridos em gel de poliacrilamida a 15%, os quais foram corados com brometo de etídeo. Para o gene pld, digerido com a enzima Pst I, foram obtidos fragmentos de 566, 195, 91 e 72 pb; e com a enzima Msp I, fragmentos de 395, 369, 108 e 52 pb. O gene rpoB digerido com a enzima HpyCh4 IV apresentou fragmentos de 235, 126 e 86 pb; e com a enzima Msp I apresentou fragmentos de 222, 93, 78 e 54 pb. O padrão de bandas obtido para os 31 isolados de C. pseudotuberculosis, analisados neste estudo, foi monomórfico, não havendo, portanto, polimorfismo entre os diferentes isolados, independentes da espécie hospedeira ou da área geográfica estudada, o que corrobora com o padrão homogêneo da infecção para a região.