Capillary Electrophoresis Single-strand Conformation Polymorphism Research Articles

Past, Present, and Future of DNA Typing for Analyzing Human and Non-Human Forensic Samples

Forensic DNA analysis has vastly evolved since the first forensic samples were evaluated by restriction fragment length polymorphism (RFLP). Methodologies advanced from gel electrophoresis techniques to capillary electrophoresis and now to next generation sequencing (NGS). Capillary electrophoresis was and still is the standard method used in forensic analysis. However, dependent upon the information needed, there are several different techniques that can be used to type a DNA fragment. Short tandem repeat (STR) fragment analysis, Sanger sequencing, SNapShot, and capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) are a few of the techniques that have been used for the genetic analysis of DNA samples. NGS is the newest and most revolutionary technology and has the potential to be the next standard for genetic analysis. This review briefly encompasses many of the techniques and applications that have been utilized for the analysis of human and nonhuman DNA samples.

Quantitative Monitoring of Microbial Species during Bioleaching of a Copper Concentrate.

Monitoring of the microbial community in bioleaching processes is essential in order to control process parameters and enhance the leaching efficiency. Suitable methods are, however, limited as they are usually not adapted to bioleaching samples and often no taxon-specific assays are available in the literature for these types of consortia. Therefore, our study focused on the development of novel quantitative real-time PCR (qPCR) assays for the quantification of Acidithiobacillus caldus, Leptospirillum ferriphilum, Sulfobacillus thermosulfidooxidans, and Sulfobacillus benefaciens and comparison of the results with data from other common molecular monitoring methods in order to evaluate their accuracy and specificity. Stirred tank bioreactors for the leaching of copper concentrate, housing a consortium of acidophilic, moderately thermophilic bacteria, relevant in several bioleaching operations, served as a model system. The microbial community analysis via qPCR allowed a precise monitoring of the evolution of total biomass as well as abundance of specific species. Data achieved by the standard fingerprinting methods, terminal restriction fragment length polymorphism (T-RFLP) and capillary electrophoresis single strand conformation polymorphism (CE-SSCP) on the same samples followed the same trend as qPCR data. The main added value of qPCR was, however, to provide quantitative data for each species whereas only relative abundance could be deduced from T-RFLP and CE-SSCP profiles. Additional value was obtained by applying two further quantitative methods which do not require nucleic acid extraction, total cell counting after SYBR Green staining and metal sulfide oxidation activity measurements via microcalorimetry. Overall, these complementary methods allow for an efficient quantitative microbial community monitoring in various bioleaching operations.

Bacterial Community Structure and Functional arrA Gene Diversity Associated with Arsenic Reduction and Release in an Industrially Contaminated Soil

ABSTRACTThis study aimed at evaluating potential arsenic (As) mobility in an industrially contaminated soil (64 mg/kg of As) of the Meuse River basin, and at identifying key bacterial groups that drive soil As dynamics. Both speciation and release of As from this soil was followed under anaerobic conditions using a laboratory batch experiment. In the presence of exogenous carbon sources, AsV initially present in the soil matrix and/or adsorbed on synthetic hydrous ferric oxides were solubilized and mainly reduced to AsIII by indigenous soil microflora. After a 1-month incubation period in these biotic conditions, AsIII accounted for 80–85% of the total dissolved As and more than 60% of the solid-phase As. Bacterial community structure (i.e., 16S rDNA-based capillary electrophoresis single-strand conformation polymorphism profiles) changed with incubation time and As amendment. The detection of distantly related arsenate respiratory reductase genes (arrA), as functional markers of AsV respirers, indicates that novel dissimilatory AsV-reducing bacteria may be involved in As biotransformation and mobility in anoxic soils. Since As and iron were concomitantly released, a crucial role of indirect As-mobilizing bacteria on As behavior was also revealed. Our results show that the majority of As within the soil matrix was bioavailable and bioaccessible for heterotrophic AsV reduction to AsIII, which may increase As toxicity and mobility in the contaminated soils.

DEVELOPMENT AND EVALUATION OF A NOVEL PROBE MICROARRAY FOR GENOTYPING CITRUS TRISTEZA VIRUS USING AN INTEGRATED LAB-ON-CHIP DEVICE

An In-Check system was designed for Citrus tristeza virus (CTV) isolates genotyping. The system includes a set of 44 probes and enables a RT-PCR and microarray hybridization to be handled in a miniaturized silicon lab-on-chip (LoC) device. The 44 probes were designed based on 5'UTR, ORF1a, RdRp, p20, p23 and p33 genes. A probe on the p27 gene was used as a positive control. Capture probes were designed on variable regions of five phylogenetic groups corresponding to T36, T30, VT, NZ-RB, T68 CTV strains and the emerging HA16-5 and NZM16 isolates. Twenty-two isolates collected in Italy, inducing a range of symptoms spanning from vein clearing and slight to strong stem pitting on alemow, to seedling yellows of sour orange, were tested by the LoC device leading to the identification of T30, VT and T30+VT (mixed infections) groups. Eleven out of 23 probes specific for these genotypes reacted producing different patterns among and inside the groups. Results using the LoC device were comparable with capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis and multiple molecular marker (MMM) assays. The preliminary results demonstrate that the device is highly robust and fast.

Identification of poultry species using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) methods

In the last decades, animal species identification became more important to prevent food adulteration. Here, we demonstrate the identification of seven poultry species, chicken, guinea fowl, pheasant, turkey, goose, duck and muscovy duck, through the use of the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) methods. DNA were isolated from poultry meat and meat products and were amplified with universal primers, designed for the mitochondrial 12S rRNA. Species-specific patterns and the reliable detection limit were identified as 0.5% for PCR and CE applications. Analyses of commercially available poultry products revealed fraud, as 6 of 36 contained undeclared species. The above-mentioned techniques are sensitive, reproducible and reliable for poultry species identification from foodstuffs.

SNaPshot and CE-SSCP: Two Simple and Cost-Effective Methods to Reveal Genetic Variability Within a Virus Species.

The multiplex SNaPshot and the capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) procedures are here used for rapid and high-throughput description of the molecular variability of viral populations. Both approaches are based on (1) standard amplification of genomic sequence(s), (2) labeled primers or labeled single-stranded DNA, and (3) migration of fluorescent-labeled molecules in capillary electrophoresis system. The SNaPshot technology was used to describe the diversity of 20 targeted single nucleotide polymorphisms (SNPs) selected from alignment of viral genomic sequences retrieved from public database. The CE-SSCP procedure was applied to identify the polymorphisms of two small (<500 bases in length) genomic regions of viral genomes. The different steps of SNaPshot and CE-SSCP setup procedures are presented using Potato virus Y (PVY, Potyvirus) and Plum pox virus (PPV, Potyvirus) RNA viruses as molecular targets, respectively.

CAPILLARY ELECTROPHORESIS-SINGLE STRAND CONFORMATION POLYMORPHISM ANALYSIS AND MULTIPLE MOLECULAR MARKER GENOTYPING ALLOW A RAPID DIFFERENTIATION OF CITRUS TRISTEZA VIRUS ISOLATES

According to recent studies, characterization of Citrus tristeza virus (CTV) isolates by molecular technologies is a prerequisite for developing a rational control strategy based on eradication or preimmunization (cross-protection). In this work we used the recently developed capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) method and the multiple molecular markers (MMM) analysis to evaluate a number of CTV isolates collected in different locations of Sicily. The screening, performed after a preliminary testing on indicator plants, led to the differentiation of three main CTV sub-groups or clusters: (i) severe isolates widespread in the area, (ii) mild isolates, and (iii) mixed isolates including both severe and mild CTV variants.

Spatial distribution of microbial communities in the shallow submarine alkaline hydrothermal field of the Prony Bay, New Caledonia.

The shallow submarine hydrothermal field of the Prony Bay (New Caledonia) discharges hydrogen- and methane-rich fluids with low salinity, temperature (< 40°C) and high pH (11) produced by the serpentinization reactions of the ultramafic basement into the lagoon seawater. They are responsible for the formation of carbonate chimneys at the lagoon seafloor. Capillary electrophoresis single-strand conformation polymorphism fingerprinting, quantitative polymerase chain reaction and sequence analysis of 16S rRNA genes revealed changes in microbial community structure, abundance and diversity depending on the location, water depth, and structure of the carbonate chimneys. The low archaeal diversity was dominated by few uncultured Methanosarcinales similar to those found in other serpentinization-driven submarine and subterrestrial ecosystems (e.g. Lost City, The Cedars). The most abundant and diverse bacterial communities were mainly composed of Chloroflexi, Deinococcus-Thermus, Firmicutes and Proteobacteria. Functional gene analysis revealed similar abundance and diversity of both Methanosarcinales methanoarchaea, and Desulfovibrionales and Desulfobacterales sulfate-reducers in the studied sites. Molecular studies suggest that redox reactions involving hydrogen, methane and sulfur compounds (e.g. sulfate) are the energy driving forces of the microbial communities inhabiting the Prony hydrothermal system.

Biofilm development during the start‐up period of anaerobic biofilm reactors: the biofilm Archaea community is highly dependent on the support material

To evaluate the impact of the nature of the support material on its colonization by a methanogenic consortium, four substrata made of different materials: polyvinyl chloride, 2 polyethylene and polypropylene were tested during the start-up of lab-scale fixed-film reactors. The reactor performances were evaluated and compared together with the analysis of the biofilms. Biofilm growth was quantified and the structure of bacterial and archaeal communities were characterized by molecular fingerprinting profiles (capillary electrophoresis-single strand conformation polymorphism). The composition of the inoculum was shown to have a major impact on the bacterial composition of the biofilm, whatever the nature of the support material or the organic loading rate applied to the reactors during the start-up period. In contrast, the biofilm archaeal populations were independent of the inoculum used but highly dependent on the support material. Supports favouring Archaea colonization, the limiting factor in the overall process, should be preferred.

Application of capillary electrophoresis single-stranded conformation polymorphism (CE-SSCP) analysis for identification of fungal communities in cheese

As major contributors of the ripening process, yeasts and filamentous fungi play a fundamental role in cheese-making. Still, there is no rapid and affordable identification method available for both yeasts and filamentous fungi encountered in cheeses. In the present study, we developed a method based on CE-SSCP analysis of nuclear ribosomal DNA ITS amplicons, along with a species pattern database comprising 37 fungal species. By combining analyses of the ITS1 and ITS2 conformers, 25 out of 37 species were discriminated using CE-SSCP analysis. This reproducible and sensitive method was applied to determine the fungal community composition of 36 cheeses including blue-veined, pressed-cooked, pressed-uncooked, red-smear and surface-mould ripened cheeses. Overall, each cheese contained between 1 and 6 fungal species and 23 different species of fungi were detected including 8 yeast species, 9 filamentous species and 6 unidentified species. Comparison of the fungal diversity obtained after cloning and sequencing (rDNA ITS) versus CE-SSCP for 8 cheeses showed that CE-SSCP was at least as exhaustive as cloning and sequencing of thirty clones per cheese. In conclusion, this CE-SSCP method was an effective tool to identify the fungi present in various cheese varieties and may be of interest for the cheese industry to rapidly describe the composition of cheese fungal communities.

Effect of temperature gradients on single-strand conformation polymorphism analysis in a capillary electrophoresis system using Pluronic polymer matrix

Capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) analysis is a prominent bioseparation method based on the mobility diversity caused by sequence-induced conformational differences of single-stranded DNA. The use of Pluronic polymer matrix has opened up new opportunities for CE-SSCP, because it improved the resolution for various genetic analyses. However, there still exists a challenge in optimizing Pluronic-based CE-SSCP, because the physical properties of Pluronic solutions are sensitive to temperature, particularly near the gelation temperature, where the viscoelasticity of Pluronic F108 solutions sharply changes from that of a Newtonian fluid to a hydrogel upon heating. We have focused on a set of experiments to control the ambient temperature of the CE system with the aim of enhancing the reliability of the CE-SSCP analysis by using the Applied Biosystems ABI 3130xl genetic analyzer with Pluronic F108 solution matrix. The ambient temperature control allowed us to vary the inlet and outlet portion of the capillary column, while the temperature of the column was kept at 35°C. The resolution to separate 2 single-base-pair-differing DNA fragments was significantly enhanced by changing the temperature from 19 to 30°C. The viscoelastic properties of the F108 solution matrix upon heating were also investigated by ex situ rheological experiments with an effort to reveal how the development of gels in Pluronic solutions affects the resolution of CE-SSCP. We found that the column inlet and outlet temperatures of the capillary column have to be controlled to optimize the resolution in CE-SSCP by using the Pluronic matrix.

Spatial variability of particle-attached and free-living bacterial diversity in surface waters from the Mackenzie River to the Beaufort Sea (Canadian Arctic)

Abstract. We explored the patterns of total and active bacterial community structure in a gradient covering surface waters from the Mackenzie River to the coastal Beaufort Sea in the Canadian Arctic Ocean, with a particular focus on free-living (FL) vs. particle-attached (PA) communities. Capillary electrophoresis–single-strand conformation polymorphism (CE-SSCP) showed significant differences when comparing river, coast and open sea bacterial community structures. In contrast to the river and coastal waters, total (16S rDNA-based) and active (16S rRNA-based) communities in the open sea samples were not significantly different, suggesting that most present bacterial groups were equally active in this area. Additionally, we observed significant differences between PA and FL bacterial community structure in the open sea, but similar structure in the two fractions for coastal and river samples. Direct multivariate statistical analyses showed that total community structure was mainly driven by salinity (a proxy of dissolved organic carbon and chromophoric dissolved organic matter), suspended particles, amino acids and chlorophyll a. Pyrosequencing of 16S rRNA genes from selected samples confirmed significant differences between river, coastal and sea samples. The PA fraction was only different (15.7% similarity) from the FL one in the open sea sample. Furthermore, PA samples generally showed higher diversity (Shannon, Simpson and Chao indices) than FL samples. At the class level, Opitutae was most abundant in the PA fraction of the sea sample, followed by Flavobacteria and Gammaproteobacteria, while the FL sea sample was dominated by Alphaproteobacteria. Finally, for the coast and river samples and both PA and FL fractions, Betaproteobacteria, Alphaproteobacteria and Actinobacteria were dominant. These results highlight the coexistence of particle specialists and generalists and the role of particle quality in structuring bacterial communities in the area. These results may also serve as a basis to predict further changes in bacterial communities should climate change lead to further increases in river discharge and related particle loads.

Microbes on the Cliff: Alpine Cushion Plants Structure Bacterial and Fungal Communities

Plants affect the spatial distribution of soil microorganisms, but the influence of the local abiotic context is poorly documented. We investigated the effect of a single plant species, the cushion plant Silene acaulis, on habitat conditions, and microbial community. We collected soil from inside (In) and outside (Out) of the cushions on calcareous and siliceous cliffs in the French Alps along an elevation gradient (2,000–3,000 masl). The composition of the microbial communities was assessed by Capillary-Electrophoresis Single Strand Conformation Polymorphism (CE-SSCP). Univariate and multivariate analyses were conducted to characterize the response of the microbial beta-diversity to soil parameters (total C, total N, soil water content, , and pH). Cushions affected the microbial communities, modifying soil properties. The fungal and bacterial communities did not respond to the same abiotic factors. Outside the cushions, the bacterial communities were strongly influenced by bedrock. Inside the cushions, the bacterial communities from both types of bedrock were highly similar, due to the smaller pH differences than in open areas. By contrast, the fungal communities were equally variable inside and outside of the cushions. Outside the cushions, the fungal communities responded weakly to soil pH. Inside the cushions, the fungal communities varied strongly with bedrock and elevation as well as increases in soil nutrients and water content. Furthermore, the dissimilarities in the microbial communities between the In and Out habitats increased with increasing habitat modification and environmental stress. Our results indicate that cushions act as a selective force that counteracts the influence of the bedrock and the resource limitations on the bacterial and fungal communities by buffering soil pH and enhancing soil nutrients. Cushion plants structure microbial communities, and this effect increases in stressful, acidic and nutrient-limited environments.

Effects of temperature and fertilization on the structure of total versus active bacterial communities from sub-Antarctic seawater exposed to crude oil and diesel fuel

Polar environments are exposed to the risk of oil pollution. However, there is limited knowledge regarding how the variation of physicochemical factors influencing biodegradation may affect bacterial community structure. The effects of temperature (4, 10 and 20°C) and organic fertilization (Inipol EAP 22) on community structure and diversity of bacteria inhabiting Kerguelen sub-Antarctic waters were studied in crude- and diesel-amended microcosms. Dynamics of total (i.e., 16S rDNA-based) and metabolically active (i.e., 16S rRNA-based) bacterial community structure and diversity were monitored using capillary-electrophoresis single-strand conformation polymorphism. Results showed that total and active community structures were differently influenced by temperature and fertilization in the presence of hydrocarbons. Both fertilization and temperature induced changes in total community structure in the presence of crude oil and diesel. However, temperature showed a limited influence on active community structure, and fertilization induced changes in the presence of crude oil only. Simpson’s index decreased for total bacterial communities at all temperatures in the presence of crude oil and diesel, whereas a lower reduction was observed for active bacterial populations. In the presence of fertilizer, the diversity of the whole community approached control values after seven incubation weeks; this was not observed for the active bacterial community. This study evidenced qualitative differences in total and active bacterial community structures of Kerguelen seawaters in the presence of hydrocarbons and different responses relative to variation in temperature and fertilization. These factors and hydrocarbons composition have to be taken into account to understand bacterial community dynamics after an oil spill. Keywords : Oil hydrocarbons; Inipol EAP 22; temperature; 16S rDNA/rRNA; sub-Antarctic seawater Citation: Polar Research 2013, 32 , 18521, http://dx.doi.org/10.3402/polar.v32i0.18521

Modification of activities of the ruminal ecosystem and its bacterial and protozoan composition during repeated dietary changes in cows1

Dietary change alters the ruminal ecosystem and can be regarded as a disturbance. Studying the response to a disturbance can help us understand the behavior of the ecosystem. Our work is concerned with the response of the ruminal ecosystem (composition and activities) during the application of repeated dietary disturbances to 6 dry Holstein cows. For 2 mo, the cows received a hay-based diet [experimental period (EP) 0], followed by 3 EP of successive changes (EP 1, 2, and 3) comprised of 2 parts: the first (10 d) with a corn silage-based diet and the second (25 d) with a hay-based diet. The measurements and samplings were done on the last days of EP 0 and of each part of EP 1 through 3, with the results of EP 0 used as covariables in the statistical models. The physicochemical measurements (pH and redox potential) and the fermentation variables (VFA, ammonia) were determined hourly between the morning and evening meals (n = 8 measurements/d). Samples of ruminal contents were taken 3 h after the morning meal to determine enzymatic activity [amylase, carboxymethyl cellulase (CMCase), and xylanase], to count the main protozoan genera and to quantify the bacteria by quantitative PCR, and to determine its structure by the capillary electrophoresis single-strand conformation polymorphism process. The pH fell for the corn silage-based diet with the EP (P < 0.05) but not for the hay-based diet. The VFA concentration decreased for both diets with the EP (P < 0.001), with the primary changes in acetate and propionate. The ammonia concentration increased for the corn silage-based diet with the EP (P < 0.05), whereas for the hay-based diet the highest value was observed for EP 2 (P < 0.05). The total quantity of bacteria decreased between EP 1 and 3 (P < 0.05) for both diets. The structure of the bacterial community was not affected by the disturbances for the corn silage-based diet, whereas for the hay-based diet large differences were evident between EP 1 and 3 (P < 0.05) and 2 and 3 (P < 0.01). The number of protozoa increased over the EP, with a more marked effect for the corn silage-based diet (diet × EP interaction, P < 0.05). The specific amylase, CMCase, and xylanase activities decreased over the EP for both diets (P < 0.05). The dietary changes applied in our experiment involved strong modifications of the ruminal ecosystem and alterations of ruminal fermentation and enzymatic activities. These alterations were reinforced with the repetition of the dietary changes.

Capillary electrophoresis single-strand conformation polymorphism for the monitoring of gastrointestinal microbiota of chicken flocks

The objective of the present study was to evaluate the capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) to characterize poultry gut microbiota and the ability of this molecular method to detect modifications related to rearing conditions to be used as an epidemiological tool. The V3 region of the 16S rRNA gene was selected as the PCR target. Our results showed that this method provides reproducible data. The microbiota analysis of individuals showed that variability between individual fingerprints was higher for ileum and cloaca than for ceca. However, pooling the samples decreased this variability. To estimate the variability within and between farms, we compared molecular gut patterns of animals from the same hatchery reared under similar conditions and fed the same diet in 2 separate farms. Total aerobic bacteria, coliforms, and lactic acid bacteria were enumerated using conventional bacteriological methods. A significant difference was observed for coliforms present in the ceca and the cloaca depending on the farm. Ileal contents fingerprints were more closely related to those of cloacal contents than to those of ceca contents. When comparing samples from the 2 farms, a specific microbiota was highlighted for each farm. For each gut compartment, the microbiota fingerprints were joined in clusters according to the farm. Thus, this rapid and potentially high-throughput method to obtain gut flora fingerprints is sensitive enough to detect a "farm effect" on the balance of poultry gut microbiota despite the birds being fed the same regimens and reared under similar conditions.

PCR-CE-SSCP used to authenticate edible oils

A useful procedure for the qualitative determination of edible vegetable oils adulteration has been developed. A CE-SSCP (Capillary Electrophoresis-Single Strand Conformation Polymorphism) method based on PCR (Polymerase-Chain-Reaction) technology was established to distinguish olive, soybean, sunflower, peanut, sesame, maize and corresponding edible oils, which are the most common edible oils in China. Two rbcL (ribulose 1,5-bisphosphate carboxylase large subunit) gene fragments were amplified from these 6 oil plants by 2 pairs of universal primers (uni4 and rbcl1) respectively and the amplicons were identified by CE-SSCP on ABI 3100 automatic sequence analyzer. Six oil plants were successfully differentiated by combined pattern of uni4 and rbcl1. Sensitivity of the method detecting DNA and oil mixture at different ratio was at level as low as 10% (V/V). The method developed was very suitable for the determination of modeled adulterants but it may also reveal an adulteration even if it does not derive from the adulterants employed in this study.

Effects of storage conditions on soil molecular biology for forensic science purposes

The object of this project is to identify changes in soil microbial community DNA and Fatty Acid profiles caused by storage, using capillary electrophoresis single‐strand conformation polymorphism (CE‐SSCP) and fatty acid methyl ester (FAME) analysis.There were six different storage treatments tested (−80°C, −20°C, 4°C, freeze dried, air dried, oven dried) on soil samples collected from 4 different locations in Nebraska. Samples were taken at a depth of 0.0 cm to 5.0 cm from three biological replicates at each collection site. After collection, soils were sieved and subsamples were placed into each storage treatment for five weeks then analyzed, with the exception of one subsample (fresh) which had DNA and fatty acids extracted within 36 hours of collection. Additional fresh samples were collected and processed two weeks after initial collection and seasonally.The CE‐SSCP and FAME analysis will illustrate the microbial community and its diversity for an individual soil sample through the number of peaks, molecular size of peaks and relative peak heights. Using statistical analysis it can be determined which storage treatments altered the soil microbial community profile when compared to the fresh subsample. Results will be presented to show the effectiveness of these two methods to detect small variations in the soil microbial community. Funded by National Institute of Justice (25‐6228‐0159‐001).

Precise H1N1 swine influenza detection using stuffer-free multiplex ligation-dependent probe amplification in conformation-sensitive capillary electrophoresis

The H1N1 influenza virus has spread worldwide to become pandemic. Here, we developed a new method to discriminate various types of influenza A, including H1N1, using stuffer-free multiplex ligation-dependent probe amplification based on a conformation-sensitive separation method, namely capillary electrophoresis–single-strand conformation polymorphism. Unlike conventional methods, our approach precisely detects five relevant gene markers permitting confirmation of infection.

Characterization of isolates of Citrus tristeza virus by sequential analyses of enzyme immunoassays and capillary electrophoresis-single-strand conformation polymorphisms

Citrus tristeza virus (CTV) is the causal agent of tristeza disease, which is one of the most devastating diseases of citrus crops worldwide. This paper describes a method for the rapid detection and genotyping of naturally spreading CTV isolates. This method uses ELISA or dot-blot immunological tests to detect trees infected with CTV. The reaction wells or membrane spots for which there is a positive reaction are sequentially treated by (i) washing and elution of viral RNA from the trapped samples, (ii) one-step synthesis of cDNA and PCR and (iii) automated fluorescence-based capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis of amplification products. Comparative CE-SSCP results are presented for CTV RNA extracted directly from infected leaves and ELISA plates or from membranes. In the analyses of all of these RNA samples, the p18, p27 and p23 CTV genes were targeted for amplification. Specific profiles of forward and reverse strands were obtained from a group of eight CTV isolates collected in Sicily, each with distinct biological characteristics, which were analyzed using the conventional two-step procedure (immunological detection followed by CE-SSCP molecular characterization after RNA isolation) or in a continuous process of ELISA/CE-SSCP or dot-blot/CE-SSCP starting from infected plant material. The combined method is simple, highly sensitive and reproducible, thus allowing the processing of numerous field samples for a variety of epidemiological needs. The sequential processing of an ELISA or dot-blot/ELISA followed by CE-SSCP is expected to allow the rapid detection of recent CTV infections along with the simultaneous characterization of the genetic diversity and structure of the population of newly invading CTV.