昆虫核酸分子系统学研究进展

本文从研究对象、方法、类群、内容等方面综述了近十年来昆虫核酸分子系统学研究进展概况。文中首先介绍了ＲＦＬＰＡ、探针杂交及ＤＮＡ指纹、ＰＣＲ与ＲＡＰＤ－ＰＣＲ、顺序分析方法及应用情况,列举了在双翅目、膜翅目、同翅目、直翅目等目昆虫中的研究进展,并从居群遗传结构、分类学研究、系统发育和分子进化４个方面总结了昆虫核酸分子系统学的研究内容和主要成果,最后指出ＲＡＰＤ－ＰＣＲ与ＲＦＬＰ联合用于测序是近期昆虫分子系统学上最有应用价值的方法。     

未培养微生物的研究与微生物分子生态学的发展*

近年来现代分子技术和基因组学逐渐渗透到有关生命科学的整个领域,也为微生物生态学提供了新的研究方法和机遇。16S rRNA基因序列分析、DNA-DNA杂交、核酸指纹图谱以及宏基因组学等分子技术检查自然环境中的微生物,可以克服传统纯培养技术的不足,是一条探知未培养微生物、寻找新基因及其产物的新途径,开启了我们认识微生物多样性和获得新资源的大门。     

Molecular methods used in clinical laboratory: prospects and pitfalls

The role of molecular detection, identification and typing or fingerprinting of microorganisms has shifted gradually from the academic world to the routine diagnostic laboratory. Molecular methods have been used increasingly over the past decade to improve the sensitivity, specificity and turn-around time in the clinical laboratory. Molecular methods have also been used to identify new and nonculturable agents. Many high-throughput molecular tests are now available commercially, which impacts on the infrastructure in many of the diagnostic laboratories. In this paper, we take an overall look at the use of molecular methods (prospects vs. pitfalls) based on our clinical and public health experience, particularly as they related to Borrelia burgdorferi, a vector-borne pathogen, Treponema pallidum, a re-emerging sexually transmitted global pathogen, and West Nile virus, a newly recognized virus in North America.     

New molecular screening tools for analysis of free-living diazotrophs in soil

Free-living nitrogen-fixing prokaryotes (diazotrophs) are ubiquitous in soil and are phylogenetically and physiologically highly diverse. Molecular methods based on universal PCR detection of the nifH marker gene have been successfully applied to describe diazotroph populations in the environment. However, the use of highly degenerate primers and low-stringency amplification conditions render these methods prone to amplification bias, while less degenerate primer sets will not amplify all nifH genes. We have developed a fixed-primer-site approach with six PCR protocols using less degenerate to nondegenerate primer sets that all amplify the same nifH fragment as a previously published PCR protocol for universal amplification. These protocols target different groups of diazotrophs and allowed for direct comparison of the PCR products by use of restriction fragment length polymorphism fingerprinting. The new protocols were optimized on DNA from 14 reference strains and were subsequently tested with bulk DNA extracts from six soils. These analyses revealed that the new PCR primer sets amplified nifH sequences that were not detected by the universal primer set. Furthermore, they were better suited to distinguish between diazotroph populations in the different soils. Because the novel primer sets were not specific for monophyletic groups of diazotrophs, they do not serve as an identification tool; however, they proved powerful as fingerprinting tools for subsets of soil diazotroph communities.     

New Molecular Screening Tools for Analysis of Free-Living Diazotrophs in Soil

Free-living nitrogen-fixing prokaryotes (diazotrophs) are ubiquitous in soil and are phylogenetically and physiologically highly diverse. Molecular methods based on universal PCR detection of the nifH marker gene have been successfully applied to describe diazotroph populations in the environment. However, the use of highly degenerate primers and low-stringency amplification conditions render these methods prone to amplification bias, while less degenerate primer sets will not amplify all nifH genes. We have developed a fixed-primer-site approach with six PCR protocols using less degenerate to nondegenerate primer sets that all amplify the same nifH fragment as a previously published PCR protocol for universal amplification. These protocols target different groups of diazotrophs and allowed for direct comparison of the PCR products by use of restriction fragment length polymorphism fingerprinting. The new protocols were optimized on DNA from 14 reference strains and were subsequently tested with bulk DNA extracts from six soils. These analyses revealed that the new PCR primer sets amplified nifH sequences that were not detected by the universal primer set. Furthermore, they were better suited to distinguish between diazotroph populations in the different soils. Because the novel primer sets were not specific for monophyletic groups of diazotrophs, they do not serve as an identification tool; however, they proved powerful as fingerprinting tools for subsets of soil diazotroph communities.     

Comparative PCR-based fingerprinting of Vibrio cholerae isolated in Malaysia

Vibrio cholerae, the causative agent of cholera, is endemic in many parts of the world, especially in countries poor in resources. Molecular subtyping of V. cholerae is useful to trace the regional spread of a clone or multidrug-resistant strains during outbreaks of cholera. Current available PCR-based fingerprinting methods such as Random Amplified Polymorphic DNA (RAPD)-PCR, Enterobacterial Repetitive Intergenic Consensus Sequence (ERIC)-PCR, and Repetitive Extragenic Palindromic (REP)-PCR were used to subtype V. cholerae. However, there are problems for inter-laboratory comparison as these PCR methods have their own limitations especially when different PCR methods have been used for molecular typing. In this study, a Vibrio cholerae Repeats-PCR (VCR-PCR) approach which targets the genetic polymorphism of the integron island of Vibrios was used and compared with other PCR-based fingerprinting methods in subtyping. Forty-three V. cholerae of different serogroups from various sources were tested. The PCR-fingerprinting approaches were evaluated on typeability, reproducibility, stability and discriminatory power. Overall, Malaysian non-O1/non-O139 V. cholerae were more diverse than O1 strains. Four non-O1/non-O139 strains were closely related with O1 strains. The O139 strain in this study shared similarity with strains of both O1 and non-O1/non-O139 serogroups. ERIC-PCR was the most discriminative approach (D value = 0.996). VCR-PCR was useful in discriminating non-O1/non-O139 strains. RAPD-PCR and REP-PCR were less suitable for efficient subtyping purposes as they were not reproducible and lacked stability. The combination of the ERIC-PCR and VCR-PCR may overcome the inadequacy of any one approach and hence provide more informative data.     

Computational Structure-Based De Novo Design of Hypothetical Inhibitors against the Anti- Inflammatory Target COX-2

Cyclooxygenase-2 (COX-2) produces prostaglandins in inflamed tissues and hence has been considered as an important target for the development of anti-inflammatory drugs since long. Administration of traditional non-steroidal anti-inflammatory drugs (NSAIDs) and other COX-2 selective inhibitors (COXIBS) for the treat of inflammation has been found to be associated with side effects, which mainly includes gastro-intestinal (GI) toxicity. The present study involves developing a virtual library of novel molecules with high druglikeliness using structure-based de novo drug designing and 2D fingerprinting approach. A library of 2657 drug like molecules was generated. 2D fingerprinting based screening of the designed library gave a unique set of compounds. Molecular docking approach was then used to identify two compounds highly specific for COX-2 isoform. Molecular dynamics simulations of protein-ligand complexes revealed that the candidate ligands were dynamically stable within the cyclooxygenase binding site of COX-2. The ligands were further analyzed for their druglikeliness, ADMET properties and synthetic accessibility using knowledge based set of rules. The results revealed that the molecules are predicted to selectively bind to COX-2 enzyme thereby potentially overcoming the limitations posed by the drugs in clinical use.     

The use of molecular techniques based on ribosomal RNA and DNA for rumen microbial ecosystem studies: a review

This paper analyses the research progress in the use of molecular techniques based on ribosomal RNA and DNA (rRNA/rDNA) for rumen microbial ecosystem since first literature by Stahl et al. (1988). Because rumen microbial populations could be under-estimated by adopting the traditional techniques such as roll-tube technique or most-probable-number estimates, modern molecular techniques based on 16S/18S rRNA/rDNA can be used to more accurately provide molecular characterization, microbe populations and classification scheme than traditional methods. Phylogenetic-group-specific probes can be used to hybridize samples for detecting and quantifying of rumen microbes. But, competitive-PCR and real-time PCR can more sensitively quantify rumen microbes than hybridization. Molecular fingerprinting techniques including both denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and restriction fragment length polymorphisms (RFLP) can used to explore diversity of bacteria, protozoa and fungi in the rumen ecosystem. By constructing clone libraries of 16S/18S rRNA/rDNA of rumen microbes, more new microbes can be discovered and identified. For fungi, internal transcribed spacers (ITS) of fungi are better than 18S rRNA/rDNA for discriminating operational taxonomic units. In conclusion, 16S/18S rRNA/rDNA procedures have been used with success in rumen microbes and are quickly gaining acceptance for studying rumen microbial ecosystem, and will become useful methods for rumen ecology research. However, molecular techniques based on 16S/18S rRNA/rDNA don't preclude classical and traditional microbiological techniques. It should used together to acquire accurate and satisfactory results.     

Comparison of molecular fingerprinting methods for analysis of soil microbial community structure

Denaturing gradient gel electrophoresis (DGGE), terminal-restriction fragment length polymorphism (T-RFLP) analysis, and automated ribosomal intergenic spacer analysis (ARISA) have been widely used as molecular fingerprinting methods for analysis of microbial communities. To find suitable methods, we compared the three fingerprinting methods by analyzing soil fungal communities in four differing land-use types: bare ground, crop fields, grasslands, and forests. We also examined optimal primer pairs for DGGE analysis by comparing single and mixed DNA samples of cultured fungal populations. Principal coordinate analysis (PCO), nonmetric multidimensional scaling method (NMDS), and analysis of similarities (ANOSIM), which are major multivariate statistical analyses for quantifying fingerprint patterns, were compared. All three fingerprinting methods yielded clear discrimination of soil fungal communities among the four land-use types, irrespective of statistical methods. The advantages and disadvantages of the three fingerprinting methods were discussed.     

Assessment of discriminatory power of three different fingerprinting methods based on killer toxin sensitivity for the differentiation of Saccharomyces cerevisiae strains

AIMS: A panel composed of 44 taxonomically certified strains of Saccharomyces cerevisiae of different origin was used to evaluate the discriminatory power of three different fingerprinting methods based on sensitivity towards 24 killer toxins. METHODS AND RESULTS: Binary data matrix (BDM), triplet data matrix (TDM) and numerical data matrix (NDM) were used as fingerprinting methods. NDM possessed the highest discriminatory power, assessed through the Simpson's, and Hunter and Gaston's indices for the measurement of diversity. The upper limits of fingerprinting ability expressed by the three above methods have been also discussed. CONCLUSIONS: NDM determined a significant increase of discriminatory power than the use of BDM or TDM, in terms of an effective amplification of their fingerprinting efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: The NDM fingerprinting method could find application in control laboratories for the discrimination of yeast strains of industrial importance or covered by patent.     

Genome physical mapping with large-insert bacterial clones by fingerprint analysis: methodologies, source clone genome coverage, and contig map quality

Genome physical mapping with large-insert clones by fingerprint analysis is becoming an active area of genomics research. Here, we report two new capillary electrophoresis-based fingerprinting methods for genome physical mapping and the effects of different fingerprinting methods and source clone genome coverage on quality physical map construction revealed by computer simulations and laboratory experiments. It was shown that the manual sequencing gel-based two-enzyme fingerprinting method consistently generated larger and more accurate contigs, followed by the new capillary electrophoresis-based three-enzyme method, the new capillary electrophoresis-based five-enzyme (SNaPshot) method, the agarose gel-based one-enzyme method, and the automatic sequencing gel-based four-enzyme method, in descending order, when 1% or fewer questionable clones were allowed. Analysis of clones equivalent to 5x, 8x, 10x, and 15x genomes using the fingerprinting methods revealed that as the number of clones increased from 5x to 10x, the contig length rapidly increased for all methods. However, when the number of clones was increased from 10x to 15x coverage, the contig length at best increased at a lower rate or even decreased. The results will provide useful knowledge and strategies for effective construction of quality genome physical maps for advanced genomics research.     

Challenges in Laboratory Detection of Fungal Pathogens in the Airways of Cystic Fibrosis Patients

Study of the clinical significance of fungal colonization/infection in the airways of cystic fibrosis (CF) patients, especially by filamentous fungi, is challenged by the absence of standardized methodology for the detection and identification of an ever-broadening range of fungal pathogens. Culture-based methods remain the cornerstone diagnostic approaches, but current methods used in many clinical laboratories are insensitive and unstandardized, rendering comparative studies unfeasible. Guidelines for standardized processing of respiratory specimens and for their culture are urgently needed and should include recommendations for specific processing procedures, inoculum density, culture media, incubation temperature and duration of culture. Molecular techniques to detect fungi directly from clinical specimens include panfungal PCR assays, multiplex or pathogen-directed assays, real-time PCR, isothermal methods and probe-based assays. In general, these are used to complement culture. Fungal identification by DNA sequencing methods is often required to identify cultured isolates, but matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is increasingly used as an alternative to DNA sequencing. Genotyping of isolates is undertaken to investigate relatedness between isolates, to pinpoint the infection source and to study the population structure. Methods range from PCR fingerprinting and amplified fragment length polymorphism analysis, to short tandem repeat typing, multilocus sequencing typing (MLST) and whole genome sequencing (WGS). MLST is the current preferred method, whilst WGS offers best case resolution but currently is understudied.     

BOX-PCR is an Adequate Tool for Typing <Emphasis Type="Italic">Aeromonas</Emphasis> spp.

PCR-based methods of fingerprinting take advantage of the presence of repetitive sequences that are interspersed throughout the genome of diverse bacterial species. They include the repetitive extragenic palindromic (REP) sequence, the enterobacterial repetitive intergenic consensus sequence (ERIC) and the 154-bp BOX element. The combination of the three methods is used for fine discrimination of strains and is designated as rep-polymerase chain reaction (PCR). REP-PCR and ERIC-PCR have been shown to be useful for typing Aeromonas strains. To our knowledge, rep-PCR fingerprinting method using the BOXA1R primer has never been tested on aeromonads. In this study, the BOX-PCR fingerprinting technique was evaluated for the discrimination of strains of some Aeromonas species. All strains were typeable and the majority showed unique banding patterns. Four strains from culture collections were used to investigate the reproducibility of the method. According to our results, BOX-PCR fingerprinting is applicable for typing of Aeromonas strains and can be considered as a useful complementary tool for epidemiological studies of members of this genus.     

Molecular characterization of phenol-degrading bacteria isolated from different Egyptian ecosystems

Twelve selected phenol-degrading bacterial isolates were obtained on phenol agar plates using culture enrichment technique. Molecular identification of the isolates was performed using eubacterial 16S rRNA PCR specific primers. Based on 16S rDNA sequence analysis, the results revealed that the majority of the isolates (8 out of 12) are affiliated to the g-subdivision of Proteobacteria. Four out of the eight isolates are closely related to the genus Acinetobacter. Molecular heterogeneity among the phenol-degrading isolates was further investigated by using rep-PCR chromosomal fingerprinting and correlated with plasmid and antibiotic profile analysis. Rep-PCR results strongly confirmed that the bacterial isolates from different environmental sites produced different fingerprinting patterns. The mineralization of phenol by all isolates was evaluated using 14C-labeled phenol assay. Phenol mineralization ranged from 55% (W-17) to 0.4% (Sea-9). This was further confirmed by the detection of several monoaromatic and polyaromatic degrading genes, e.g., pheA, MopR, XylE, and NahA. In addition, catalytic enzymes such as catalase and dioxygenase were also monitored.     

Advances in the systematics and evolution of western Mediterranean representative species of the Pentanema conyzae clade through genetic fingerprinting

Forty-five populations of Pentanema corresponding to seven species included in the Pentanema conyzae clade have been studied using AFLP fingerprinting. The results show that allopolyploidization could have been involved in the diversification of this group, specifically in species P. langeanum and P. maletii. Molecular data confirm the presence of P. britannicum in the Iberian Peninsula and key steps are provided to identify the species that are morphologically the most challenging.     

Characterization of genetic markers for in vitro cell line identification of the marine sponge Axinella corrugata

The marine sponge Axinella corrugata is being developed as a model organism for in vitro marine invertebrate research. Molecular genetics methods such as DNA fingerprinting [amplified fragment length polymorphism (AFLP) and single-stranded conformation polymorphism (SSCP)] and single-locus DNA sequence analyses were applied to this model to meet the primary objective of identifying positive A. corrugata-specific molecular markers that will aid in verifying cell identity in vitro and distinguish sponge cells from potential microbial contaminants. The extent of intra- and interspecific variation in these markers from geographically distinct samples of A. corrugata and closely related sponge taxa was also assessed. Two novel nuclear loci along with intervening transcribed spacer (ITS) regions of nuclear rRNA were characterized, although the latter appeared to better meet primary marker criteria, such as taxonomic specificity and high frequency of detection (via polymerase chain reaction [PCR]) from different individuals (n > 40) and cell cultures. Phylogenetic and phylogeographic analyses of ITS DNA sequences helped clarify taxonomies and also suggested species boundaries between and among western Atlantic and eastern Atlantic/Indian Ocean A. corrugata and Axinellidae samples. Patterns of genetic variation have important implications for the systematics, evolution, and chemical ecology of A. corrugata and related axinellids and are discussed.     

DNA fingerprinting: parentage studies in natural populations and the importance of linkage analysis.

It has been suggested that a full linkage analysis is a prerequisite for confident paternity testing, by using DNA fingerprinting, in natural populations. These fears are based on a confusion between linkage and linkage disequilibrium and a misplaced assumption that linkage between bands will necessarily reduce the effective number of paternal-specific bands. Several methods for detecting linkage without resorting to the analysis of large sibships are considered, for example, by analysing half-sibships, by band-association, and by altering the experimental conditions used. Even if linkage is present, the magnitude of its effects are unlikely to undermine the accuracy of the technique, given the average levels of variability being detected. We conclude that the effects of linkage are only likely to present a problem when sample sizes are very small or when closely related individuals are being tested together.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.     

Fingerprinting closely related xanthomonas pathovars with random nonamer oligonucleotide microarrays

Current bacterial DNA-typing methods are typically based on gel-based fingerprinting methods. As such, they access a limited complement of genetic information and many independent restriction enzymes or probes are required to achieve statistical rigor and confidence in the resulting pattern of DNA fragments. Furthermore, statistical comparison of gel-based fingerprints is complex and nonstandardized. To overcome these limitations of gel-based microbial DNA fingerprinting, we developed a prototype, 47-probe microarray consisting of randomly selected nonamer oligonucleotides. Custom image analysis algorithms and statistical tools were developed to automatically extract fingerprint profiles from microarray images. The prototype array and new image analysis algorithms were used to analyze 14 closely related Xanthomonas pathovars. Of the 47 probes on the prototype array, 10 had diagnostic value (based on a chi-squared test) and were used to construct statistically robust microarray fingerprints. Analysis of the microarray fingerprints showed clear differences between the 14 test organisms, including the separation of X. oryzae strains 43836 and 49072, which could not be resolved by traditional gel electrophoresis of REP-PCR amplification products. The proof-of-application study described here represents an important first step to high-resolution bacterial DNA fingerprinting with microarrays. The universal nature of the nonamer fingerprinting microarray and data analysis methods developed here also forms a basis for method standardization and application to the forensic identification of other closely related bacteria.