RESUMO
BACKGROUND: The 5' region of cytochrome oxidase I (COI) is the standard marker for DNA barcoding. However, COI has proved to be of limited use in identifying some species, and for some taxa, the coding sequence is not efficiently amplified by PCR. These deficiencies lead to uncertainty as to whether COI is the most suitable barcoding fragment for species identification of ticks. METHODS: In this study, we directly compared the relative effectiveness of COI, 16S ribosomal DNA (rDNA), nuclear ribosomal internal transcribed spacer 2 (ITS2) and 12S rDNA for tick species identification. A total of 307 sequences from 84 specimens representing eight tick species were acquired by PCR. Besides the 1,834 published sequences of 189 tick species from GenBank and the Barcode of Life Database, 430 unpublished sequences representing 59 tick species were also successfully screened by Bayesian analyses. Thereafter, the performance of the four DNA markers to identify tick species was evaluated by identification success rates given by these markers using nearest neighbour (NN), BLASTn, liberal tree-based or liberal tree-based (+threshold) methods. RESULTS: Genetic divergence analyses showed that the intra-specific divergence of each marker was much lower than the inter-specific divergence. Our results indicated that the rates of correct sequence identification for all four markers (COI, 16S rDNA, ITS2, 12S rDNA) were very high (> 96%) when using the NN methodology. We also found that COI was not significantly better than the other markers in terms of its rate of correct sequence identification. Overall, BLASTn and NN methods produced higher rates of correct species identification than that produced by the liberal tree-based methods (+threshold or otherwise). CONCLUSIONS: As the standard DNA barcode, COI should be the first choice for tick species identification, while 16S rDNA, ITS2, and 12S rDNA could be used when COI does not produce reliable results. Besides, NN and BLASTn are efficient methods for species identification of ticks.
Assuntos
Código de Barras de DNA Taxonômico , Complexo IV da Cadeia de Transporte de Elétrons/genética , Carrapatos/classificação , Animais , Sequência de Bases , Primers do DNA/genética , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Marcadores Genéticos/genética , Variação Genética , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da Espécie , Carrapatos/genéticaRESUMO
To control the spread of tick-borne diseases, there is an urgent need to develop a reliable technique that can distinguish different species of ticks. DNA barcoding has been proved to be a powerful tool to identify species of arthropods, but this technique has not yet been developed for identifying ticks. Here, we screened and analyzed 1082 sequences of ticks from BOLD system and GenBank, consisting of 647 16S, 325 COI, and 110 18S. These sequences are reported in previous studies and considered to be correctly identified at the species level. Through the analyses of genetic divergences and neighbor-joining (NJ) phylogenetic relationships between the species of ticks, our results show that COI and 16S are reliable in discriminating species of ticks and the 18S could discriminate ticks at the genera level. New universal primers for 16S, 18S, and COI of ticks were designed and a DNA barcoding system for the Ixodida was developed. To assess the performance of this system, 57 specimens of ticks were collected within China. Our results show that DNA barcoding system could correctly identify the species of specimens in adult and subadult stages. This system would assist non-taxonomists to conveniently identify the species of Ixodida based on DNA sequences rather than morphological traits. However, there are still serious deficiencies in the information of 16S and COI of some species of ticks, and additional research is needed to resolve this problem.
Assuntos
Código de Barras de DNA Taxonômico , Carrapatos/genética , Animais , Complexo IV da Cadeia de Transporte de Elétrons/genética , Variação Genética , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , RNA Ribossômico 18S/genética , Especificidade da EspécieRESUMO
An integration event-specific fluorescent liquid bead array was developed for the simultaneous identification of 10 genetically modified (GM) maize, including Bt176, Bt11, MON810, NK603, GA21, MON88017, MON89034, MIR604, T25 and MIR162, as well as one non-GM maize. The system comprised 11 specific oligonucleotide probes labeled with an amino group and coupled to fluorescence-encoded microspheres. To enable fluorescence detection, 11 pairs of primers labeled with biotin at the 5' ends were used. The hybridization signal of biotinylated PCR product to the probe-coupled microspheres was then detected. The limit of detection of this assay was 0.1% for GM maize, which is lower than the current labeling threshold levels enforced in the EU (0.9%). The results of the positive and negative controls were consistent with their expected situation, which showed that the method was highly specific. We detected GM maize in 20 of the 1370 commercial food samples tested, which were labeled as containing maize. The overall sensitivity, specificity, rapidity and high throughput capacity of this liquid chip system suggest that it could provide a significant improvement over current methods, and potentially offer an improved platform for further research into the detection of other GM plants.
Assuntos
DNA de Plantas/análise , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Plantas Geneticamente Modificadas/genética , Zea mays/genética , Fluorescência , Análise de Alimentos/métodos , Reação em Cadeia da Polimerase/métodos , Sensibilidade e EspecificidadeRESUMO
Rapid diagnosis of novel emerging subtypes of influenza viruses is vital for effective global influenza surveillance. To this end, a novel microarray based surveillance was developed for subtyping all influenza A viruses on one chip. Using reference strains of different influenza subtypes and samples from different areas, the results show that all the subtypes of the influenza A virus could be identified simultaneously on this microchip with high sensitivity. There was no cross-hybridization reaction with other viruses, indicating that the microarray is specific for influenza A viruses. Such a diagnostic microarray will undoubtedly be useful for identifying novel influenza A virus subtypes.
Assuntos
Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Aves , DNA Viral/genética , Genoma Viral , Humanos , Influenza Aviária/virologia , Influenza Humana/virologia , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , RNA Viral/genética , Sensibilidade e EspecificidadeRESUMO
As a natural reservoir of manifold zoonotic viruses, fruit bats have been involved in at least three emerging zoonoses in recent years. This paper aims to introduce the epidemiological characteristics of these diseases emerged in the Australasian region between 1994 and 1999, transmission pathways of the newly discovered viruses and the relationship between the changed entironment of fruit bats and occurrences of these emerging diseases and provide a clue for the epidemiological investigations of SARS.
