Application of molecular techniques on heterotrophic hydrogen production research

This paper reviews the application of molecular techniques in heterotrophic hydrogen production studies. Commonly used molecular techniques are introduced briefly first, including cloning-sequencing after polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), terminal-restriction fragment length polymorphism (T-RFLP), fluorescence in situ hybridization (FISH) and quantitative real-time PCR. Application of the molecular techniques in heterotrophic hydrogen production studies are discussed in details, focusing on identification of new isolates for hydrogen production, characterization of microbial compositions in bioreactors, monitoring microbial diversity variation, visualization of microbial distribution in hydrogen-producing granular sludge, and quantification of various microbial populations. Some significant findings in recent hydrogen production studies with the application of molecular techniques are discussed, followed by a research outlook of the heterotrophic biohydrogen field.     

AFLP Fingerprinting for Assessing Intraspecific Variation and Genome Mapping in Mites

Molecular genetic techniques have come a long way in the last decade. With the advent of PCR, genetic markers are now accessible for all organisms, including mites. However, there is usually a trade-off between the accuracy of the molecular technique or genetic marker and expediency. In mites, many molecular techniques are not applicable due to their small size. Here we describe a relatively new molecular fingerprinting technique, amplified fragment length polymorphism (AFLP), which is currently used widely in plant genomic research. We outline the AFLP procedure adapted for mites, show results using this technique from our own research and discuss the benefits and limitations of AFLPs for assessing genetic variation and for genome mapping. It is our intention to highlight the possible use of AFLPs as genetic markers with a broad application in acarological research.     

随机引物在分子生物学研究中的应用

随机引物指非特异序列的寡聚核苷酸作为DNA合成过程中的引物, 是相对于特异引物的概念.90年代它与PCR技术结合衍生了几项新技术:采用不同长度随机引物进行DNA指纹分析而衍生出的RAPD、AP-PCR及DAF方法; 进行mRNA多态分析的“差异显示”; 以及rPCR, T-PCR等技术. 以RAPD为例介绍了随机引物PCR的技术特点及其在分子生物学研究中的应用.     

Molecular approaches for the detection and identification of bifidobacteria and lactobacilli in the human gastrointestinal tract

In this review an overview of various molecular techniques and their application for the detection and identification of bifidobacteria and lactobacilli in the gastrointestinal (GI) tract is presented. The techniques include molecular typing techniques such as amplified ribosomal DNA restriction analysis (ARDRA), randomly amplified polymorphic DNA (RAPD), pulsed field gel electrophoresis (PFGE), ribotyping and community profiling techniques such as PCR coupled to temperature and denaturing gradient gel electrophoresis (PCR-TGGE and PCR-DGGE, respectively). Special attention is given to oligonucleotide probes and primers that target the ribosomal RNA (rRNA) sequences and their use in PCR and different hybridisation techniques such as DNA microarrays and fluorescent in situ hybridisation (FISH). In addition, recent findings based on the molecular studies of bifidobacteria and lactobacilli in the GI-tract are reviewed.     

157 Examining Phytoplankton Communities with Environmental PCR

Recent advances in biotechnology have taken much of the guesswork out of many molecular biological procedures, and promote a high rate of success even for relative beginners. In designing the laboratory component of my Phycology course, I have taken advantage of these improved methods to develop a semester-long, molecular-based class project, using "environmental PCR" to examine phytoplankton diversity. Students learn a number of common molecular techniques within the context of dynamic and unrehearsed phycological research. This provides a greater appreciation of the general utility of molecular technology, and its application to algal systems in particular. A series of easy to follow protocols are linked together, allowing students to PCR amplify sequences from mixed environmental samples, clone the resulting fragments to separate individual sequences, screen the clones by restriction enzyme digests and sequence clones with clearly different restriction patterns. The resulting sequences then are used in BLAST searches of GenBank to determine the nearest available match among sequences annotated previously. The techniques allow both qualitative and quantitative estimates of the organisms present, as well as comparative analyses of the diversity observed through direct visual observations versus those recovered by PCR. Each step of the procedure can be accomplished in the duration of a typical 3 hour lab period, and most involve intervals of incubation that permit adequate time for other lab exercises, such as a survey algal diversity, on which traditional phycological laboratories are based.     

Basics of PCR and related techniques applied in veterinary parasitology

We attempte through the following overall review pertaining to the basics of PCR techniques (Polymerase Chain Reaction), to introduce the main applications used in veterinary parasitology. A major problem restricting the application possibilities of molecular biology techniques is of quantitative nature. Amplification techniques represent a real revolution, for it makes possible the production of tens, even hundreds of nanogrammes of sequences when starting from very small quantities. The PCR technique has dramatically transformed the strategies used so far in molecular biology and subsequently research and medical diagnosis.     

Detection of Toxoplasma gondii DNA by polymerase chain reaction in experimentally desiccated tissues

Despite toxoplasmosis being a common infection among human and other warm-blooded animals worldwide, there are no findings about Toxoplasma gondii evolutionary forms in ancient populations. The molecular techniques used for amplification of genetic material have allowed recovery of ancient DNA (aDNA) from parasites contained in mummified tissues. The application of polymerase chain reaction (PCR) to paleoparasitological toxoplasmosis research becomes a promising option, since it might allow diagnosis, acquisition of paleoepidemiological data, access to toxoplasmosis information related origin, evolution, and distribution among the ancient populations. Furthermore, it makes possible the analysis of parasite aDNA aiming at phylogenetic studies. To standardize and evaluate PCR applicability to toxoplasmosis paleodiagnostic, an experimental mummification protocol was tested using desiccated tissues from mice infected with the ME49 strain cysts, the chronic infection group (CIG), or infected with tachyzoites (RH strain), the acute infection group (AIG). Tissues were subjected to DNA extraction followed by PCR amplification of T. gondii B1 gene. PCR recovered T. gondii DNA in thigh muscle, encephalon, heart, and lung samples. AIG presented PCR positivity in encephalon, lungs, hearts, and livers. Based on this results, we propose this molecular approach for toxoplasmosis research in past populations.     

Detection and enumeration of coliforms in drinking water: current methods and emerging approaches.

The coliform group has been used extensively as an indicator of water quality and has historically led to the public health protection concept. The aim of this review is to examine methods currently in use or which can be proposed for the monitoring of coliforms in drinking water. Actually, the need for more rapid, sensitive and specific tests is essential in the water industry. Routine and widely accepted techniques are discussed, as are methods which have emerged from recent research developments.Approved traditional methods for coliform detection include the multiple-tube fermentation (MTF) technique and the membrane filter (MF) technique using different specific media and incubation conditions. These methods have limitations, however, such as duration of incubation, antagonistic organism interference, lack of specificity and poor detection of slow-growing or viable but non-culturable (VBNC) microorganisms. Nowadays, the simple and inexpensive membrane filter technique is the most widely used method for routine enumeration of coliforms in drinking water.The detection of coliforms based on specific enzymatic activity has improved the sensitivity of these methods. The enzymes beta-D galactosidase and beta-D glucuronidase are widely used for the detection and enumeration of total coliforms and Escherichia coli, respectively. Many chromogenic and fluorogenic substrates exist for the specific detection of these enzymatic activities, and various commercial tests based on these substrates are available. Numerous comparisons have shown these tests may be a suitable alternative to the classical techniques. They are, however, more expensive, and the incubation time, even though reduced, remains too long for same-day results. More sophisticated analytical tools such as solid phase cytometry can be employed to decrease the time needed for the detection of bacterial enzymatic activities, with a low detection threshold.Detection of coliforms by molecular methods is also proposed, as these methods allow for very specific and rapid detection without the need for a cultivation step. Three molecular-based methods are evaluated here: the immunological, polymerase chain reaction (PCR) and in-situ hybridization (ISH) techniques. In the immunological approach, various antibodies against coliform bacteria have been produced, but the application of this technique often showed low antibody specificity. PCR can be used to detect coliform bacteria by means of signal amplification: DNA sequence coding for the lacZ gene (beta-galactosidase gene) and the uidA gene (beta-D glucuronidase gene) has been used to detect total coliforms and E. coli, respectively. However, quantification with PCR is still lacking in precision and necessitates extensive laboratory work. The FISH technique involves the use of oligonucleotide probes to detect complementary sequences inside specific cells. Oligonucleotide probes designed specifically for regions of the 16S RNA molecules of Enterobacteriaceae can be used for microbiological quality control of drinking water samples. FISH should be an interesting viable alternative to the conventional culture methods for the detection of coliforms in drinking water, as it provides quantitative data in a fairly short period of time (6 to 8 h), but still requires research effort.This review shows that even though many innovative bacterial detection methods have been developed, few have the potential for becoming a standardized method for the detection of coliforms in drinking water samples.     

Techniques and statistical data analysis in molecular population genetics

Following the development of PCR methods, molecular techniques have become widely used for detecting genetic variation in natural populations. Most nucleotide changes can be detected by these techniques. Many of these changes probably reflect silent substitutions that are likely to be selectively neutral, making them particularly suitable to population genetic studies. In this paper, we review the published literature on molecular population genetics, with respect to the genome assayed (nuclear, mitochondrial or chloroplast), the organisms studied, the molecular techniques used, and the biological problems addressed. Several molecular techniques are then compared using experimental results obtained from a population genetic study of the Mytilus complex in the North Atlantic and Mediterranean. Finally, the most appropriate theoretical tools to analyse molecular population genetic data are discussed.     

Molecular Genotyping of Macrophomina phaseolina Isolates: Comparison of Microsatellite Primed PCR and Repetitive Element Sequence-based PCR

Seventy isolates of Macrophomina phaseolina recovered from different host plants were assessed for DNA polymorphism using two molecular techniques: microsatellite primed polymerase chain reaction (MSP‐PCR) under both touchdown (T) and non‐touchdown (NT) PCR conditions and primers corresponding to disperse repetitive sequence‐based polymerase chain reaction (rep‐PCR). Fingerprints obtained by rep‐PCR were compared with those of MSP‐PCR. Even though these methods yielded intraspecific polymorphisms, yet different levels of discrimination could be obtained. A partial correlation was apparent between the molecular techniques used. Some of the genetic groups/genotypes were supported by both the molecular markers employed in the study, thus confirming their relationship. Thirty nine MSP (T), 55 MSP (NT) and 53 rep‐PCR genotypes were identified with discrimination indices of 0.962, 0.993 and 0.99, respectively. Our results have shown that rep‐PCR is a rapid, inexpensive technique that is highly reproducible and almost as discriminatory as MSP‐PCR for genotyping M. phaseolina isolates and is highly suitable for understanding disease epidemiology at molecular level. Suggesting, thereby, that it is a robust technique employed for genotypical and phylogenetic studies for determining taxonomical diversity and phylogenetic structure of the economically important fungal pathogen of cluster bean. The data presented here will help researchers to design effective strategies for deployment of resistant germplasm in cluster bean (Cymopsis tetragonoloba) growing regions in the country and worldwide.     

现代生物技术在食品检测中的应用

介绍了DNA探针、PCR技术、免疫检测技术在食品微生物及转基因成分检测中的应用。着重阐述了PCR技术的工作原理、应用及其发展前景。同时简要介绍了生物芯片及其在食品检测中的应用前景     

微生物分子生态学技术及其在环境污染研究中的应用

较为系统地概述了核酸探针检测技术、利用引物的PCR技术、DNA序列分析技术和电泳分离及显示技术在国内外的研究进展,并探讨了这些技术在环境污染研究中的应用及其方向。结果表明,这些被认为是重要的微生物分子生态学技术,在探索微生物与污染环境之间的相互关系中发挥了重要作用。促进了污染环境的微生物遗传适应进化机制的研究,污染物的微生物降解有关基因的定位及微生物工程菌的构建等方面的工作,从而推进了污染环境微生物修复的分子生态学的发展。     

Parasitoids, predators and PCR: the use of diagnostic molecular markers in biological control of Arthropods

Abstract:  The polymerase chain reaction (PCR) revolutionized the field of diagnostics, and today it has routine applications in medical, veterinary, forensic and botanical sciences. The fields of biological control and insect pest management have generally been slow to adopt PCR-based diagnostics in comparison with other fields of science. However, there has been increasing interest in the use of molecular diagnostic tools in arthropod biological control. In applied entomology, molecular techniques have generally been used for insect identification and systematics; however, PCR-based techniques are increasingly becoming recognized as valuable tools in ecological studies. Here, we review research that has used PCR-based techniques for parasitoid and predator/prey identification and detection, and place these studies in the context of their contributions to biological control of arthropods. The status and future directions of diagnostic molecular markers in applied entomology and insect pest management are also discussed.     

PCR-based methodology for molecular microchimerism detection and quantification

Peripheral blood microchimerism after pregnancy or solid organ transplantation has been widely studied, but a consensus on its detection has not yet been adopted. The objective of this study was to establish a panel of reproducible molecular polymerase chain reaction (PCR)-based methods for detection and quantification of foreign cells in an individual. We analyzed length polymorphisms generated by short tandem repeat (STR) and variable number tandem repeat (VNTR) markers. Human leukocyte antigen (HLA)-A and -B polymorphisms were detected by reference strand conformation analysis (RSCA). Class II polymorphisms on HLA-DRB1 locus were analyzed both by classical PCR-sequence-specific primers (SSP) and by quantitative PCR (Q-PCR). Also, sex-determining region-y gene (SRY) gene allowed specific male donor discrimination and quantification by Q-PCR in female recipients. Binomial statistical distribution analysis was used for each molecular technique to determine the number of PCR replicates of each sample. This analysis allowed the detection of the lowest detectable microchimerism level, when present. We could detect microchimerism in more than 96% and more than 86% of cases at levels as low as 1:10(5) and 1:10(6) donor per recipient cells (DPRC), respectively, using Q-PCR for SRY or for nonshared HLA-DRB1 alleles. These techniques allowed as low as 1 genome-equivalent cell detection. Lower levels (nanochimerism) could be detected but not quantified because of technique limitations. However, classical PCR methods allowed detection down to 1:10(4) DPRC for HLA-DRB1 PCR-SSP. The clinical application of these techniques in solid organ transplanted recipients showed microchimerism levels ranging from 1:10(4) to 1:10(6) DPRC after kidney or heart transplantation, and 1 log higher (1:10(3) to 1:10(6) DPRC) after liver transplantation. In conclusion, the standardization of molecular microchimerism detection techniques will allow for comparable interpretation of results in microchimerism detection for diagnostic or research studies.     

淡水浮游植物功能类群分类法的提出、发展及应用

浮游植物分类方法是揭示浮游植物群落演替规律、开展淡水生态研究的工作基础和重要工具.林氏分类法和分子鉴定法在生态学应用上存在的不足促进了浮游植物功能类群分类法的发展.功能类群分类法是一种以浮游植物个体生态学为依据的生态分类法.本文概述了浮游植物功能类群(functional group,FG)、生态功能类群(morpho-functional group,MFG)和形态功能类群(morphology-based functional group,MBFG)等浮游植物分类方法的理论基础和分类依据,分析比较了这3种分类方法的优势与不足,认为FG是目前应用于水生生态学研究和水环境生物评价中相对适用的浮游植物分类方法.介绍了功能类群分类法在国内外淡水浮游植物生态学研究中的应用现状,简述了以FG为基础提出的Q指数法和Q-R指数法两种水质评价法的评价标准及存在的不足.
     

高通量测序技术在野生动物食性分析中的应用

食性研究是动物生态学颇受关注的一个重要内容,而食性分析方法由于受到技术和适用范围的限制,也在不断改进和更新。随着高通量测序技术的发展,该技术逐渐扩展到野生动物的食性分析,使食性分析的效率得到极大提升,并拓宽了食性分析的应用范围。尽管高通量测序应用于食性分析在数据量、灵敏度和分辨率方面的优势较为明显,但由于涉及到的步骤较多,受到的影响因素较为复杂,目前高通量测序应用于食性分析还属于研究比较薄弱的领域。概述了高通量测序技术应用于食性分析的基本流程,总结了该技术在食物组成分析、种内和种间食性关系、食物与栖息地、行为关系方面的研究动态,分析了PCR、污染和定量分析对该技术应用性的影响,提出了相应的解决对策和建议,并对其应用前景进行了展望。     

REACTIVE OXYGEN METABOLISM IN THE TROPICAL BROWN ALGA DICTYOTA DICHOTOMA

There is a growing understanding that phagotrophic ciliates are often important members of aquatic communities in terms of their trophic role and mobilization of small cell production to higher consumers. As formidable consumers of small phytoplankton species they are likely to be also important in determining the community composition of the pico- and nanophytoplankton assemblages. Dilution method experiments were conducted during the winter and summer in the South Slough, an arm of the Coos Bay on the southern Oregon coast, to assess the impact of ciliate grazing on two size fractions of chlorophyll (0.2 to 5 mm and> 5 mm) and on the growth and abundance of specific phytoplankton groups, particularly cryptophytes and Synechococcus sp. The premise of the dilution technique is that grazers are diluted with their food and the observed rate of change in chlorophyll or phytoplankton abundance is linearly related to the dilution factor. Results from previous studies using the dilution technique have been given in terms of the grazing impact of microzooplankton on total chlorophyll. The findings of the research presented using a more rigorous application of the dilution method suggest that ciliates are differential in their grazing of phytoplankton, targeting small phytoplankton biomass and preying selectively on components of the assemblage that constitute this biomass.     

现代分子生物学技术在瘤胃微生态系统研究中的应用

瘤胃中栖息着大量的微生物,由于这些微生物组成复杂且有些细菌在体外无法培养,目前对这些微生物的了解仍然很少。现代分子生物学技术的发展为研究瘤胃微生物提供了有效的方法,利用核酸探针、基因序列分析、遗传指纹技术、全细胞杂交和实时定量PCR等技术可以对瘤胃微生物的分类及进化关系、区系结构图、重要酶的表达以及目的微生物的准确定量进行更为深入和透彻的研究。发展和利用这些技术不仅可以研究微生物之间的关系以及微生物与饲料颗粒之间时间与空间的关系,还能直接在细菌自然生长的环境中对其各种特征进行研究。     

Methods in molecular cardiology: the polymerase chain reaction

Several polymerase chain reaction (PCR) techniques are described in this review to give insight into the potential applications for cardiovascular research. Although PCR can be performed in several ways, all applications are based on the same general principle, the amplification of DNA or RNA by the enzyme polymerase. This amplification provides the opportunity to detect, identify and multiply a single copy of DNA or RNA, in or outside the cell. This powerful technique can be used in several directions of DNA and RNA research resulting in the ability to specifically detect the presence and activity of genes. The use of these techniques in cardiovascular research is discussed here.     

The use of multiplex PCR to detect and differentiate food- and beverage-associated microorganisms: a review

Regarding food safety, rapid detection of microbial species is crucial to develop effective preventive and/or adjustment measures. Classical methods for determining the presence of certain species are time-consuming and labor-intensive, hence, molecular methods, which offer speed, sensitivity and specificity, have been developed to address this problem. Multiplex PCR (MPCR) is widely applied in the various fields of microbiology for the rapid differentiation of microbial species without compromising accuracy. This paper describes the method and reports on the state-of-the-art application of this technique to the identification of microorganisms vehiculated with foods and beverages. The identification of both pathogens and probiotics and the species important for food fermentation or deterioration will be discussed. Applications of MPCR in combination with other techniques are also reviewed. Potentials, pitfalls, limitations and future prospects are summarised.