Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 25
Filtrar
Más filtros

Banco de datos
Tipo del documento
País de afiliación
Intervalo de año de publicación
1.
J Med Virol ; 95(1): e28116, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36056469

RESUMEN

Increasing evidence supports inter-species transmission of SARS-CoV-2 variants from humans to domestic or wild animals during the ongoing COVID-19 pandemic, which is posing great challenges to epidemic control. Clarifying the host range of emerging SARS-CoV-2 variants will provide instructive information for the containment of viral spillover. The spike protein (S) of SARS-CoV-2 is the key determinant of receptor utilization, and therefore amino acid mutations on S will probably alter viral host range. Here, to evaluate the impact of S mutations, we tested 27 pseudoviruses of SARS-CoV-2 carrying different spike mutants by infecting Hela cells expressing different angiotensin-converting enzyme 2 (ACE2) orthologs from 20 animals. Of these 27 pseudoviruses, 20 bear single mutation and the other 7 were cloned from emerging SARS-CoV-2 variants, including D614G, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (B.1.429), and Mu (B.1.621). Using pseudoviral reporter assay, we identified that the substitutions of T478I and N501Y enabled the pseudovirus to utilize chicken ACE2, indicating potential infectivity to avian species. Furthermore, the S mutants of real SARS-CoV-2 variants comprising N501Y showed significantly acquired abilities to infect cells expressing mouse ACE2, indicating a critical role of N501Y in expanding SARS-CoV-2 host range. In addition, A262S and T478I significantly enhanced the utilization of various mammal ACE2. In summary, our results indicated that T478I and N501Y substitutions were two S mutations important for receptor adaption of SARS-CoV-2, potentially contributing to the spillover of the virus to many other animal hosts. Therefore, more attention should be paid to SARS-CoV-2 variants with these two mutations.


Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Animales , Ratones , SARS-CoV-2/genética , Enzima Convertidora de Angiotensina 2/genética , Células HeLa , Especificidad del Huésped , Pandemias , Glicoproteína de la Espiga del Coronavirus/genética , Mutación , Unión Proteica , Mamíferos
2.
BMC Vet Res ; 17(1): 48, 2021 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-33485335

RESUMEN

BACKGROUND: Brucellosis is a serious zoonosis disease that frequently causes significant economic loss in animal husbandry and threatens human health. Therefore, we established a rapid, accurate, simple and sensitive fluorescent immunochromatographic strip test (ICST) based on quantum dots (QDs) for detection the antibodies of Brucella infection animals serum. RESULTS: The test strips were successfully prepared by quantum dot fluorescent microspheres (QDFM) as tracers, which were covalently coupled to an outer membrane protein of Brucella OMP22. The outer membrane protein OMP28 and monoclonal antibodies of OMP22 were separately dispensed onto a nitrocellulose membrane as test and quality control lines, respectively. The critical threshold for determining negative or positive through the ratio of the fluorescent signal of the test line and the control line (HT / HC) is 0.0492. The repeatability was excellent with an overall average CV of 8.78%. Under optimum conditions, the limit of detection was 1.05 ng/mL (1:512 dilution). With regard to the detection of brucellosis in 150 clinical samples, the total coincidence rate of ICST and Rose Bengal plate test (RBPT) was 97.3%, the coincidence rate of positive samples was 98.8%, the coincidence rate of negative samples was 95.3%, the sensitivity of RBPT is 1:32, and no cross reaction with the sera of other related diseases was observed. CONCLUSION: In our present study, the QDFM has promising application for on-site screening of brucellosis owing to its high detection speed, high sensitivity, high specificity and low cost.


Asunto(s)
Brucella/inmunología , Brucelosis/veterinaria , Puntos Cuánticos/química , Animales , Anticuerpos Antibacterianos/química , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Brucelosis/diagnóstico , Brucelosis/inmunología , Bovinos , Cromatografía de Afinidad/métodos , Cromatografía de Afinidad/veterinaria , Cabras , Microesferas , Tiras Reactivas , Sensibilidad y Especificidad , Ovinos
3.
Korean J Parasitol ; 58(1): 37-46, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-32145725

RESUMEN

Livestock husbandry is vital to economy of the Tarim Basin, Xinjiang Autonomous Region, China. However, there have been few surveys of the distribution of ixodid ticks (Acari: Ixodidae) and tick-borne pathogens affecting domestic animals at these locations. In this study, 3,916 adult ixodid ticks infesting domestic animals were collected from 23 sampling sites during 2012-2016. Ticks were identified to species based on morphology, and the identification was confirmed based on mitochondrial 16S and 12S rRNA sequences. Ten tick species belonging to 4 genera were identified, including Rhipicephalus turanicus, Hyalomma anatolicum, Rh. bursa, H. asiaticum asiaticum, and Rh. sanguineus. DNA sequences of Rickettsia spp. (spotted fever group) and Anaplasma spp. were detected in these ticks. Phylogenetic analyses revealed possible existence of undescribed Babesia spp. and Borrelia spp. This study illustrates potential threat to domestic animals and humans from tick-borne pathogens.


Asunto(s)
Ácaros y Garrapatas/genética , Ácaros y Garrapatas/patogenicidad , Animales Domésticos/parasitología , Análisis de Secuencia de ADN , Enfermedades por Picaduras de Garrapatas/parasitología , Animales , China , ARN Ribosómico , ARN Ribosómico 16S
4.
Exp Appl Acarol ; 73(3-4): 493-499, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-29181674

RESUMEN

Rhipicephalus turanicus is an important tick species potentially carrying tick-borne pathogens. Several tick species have obvious subspecies divergence. However few studies aimed to examine the existence of divergence within R. turanicus. Therefore, a detailed morphological and molecular analysis was conducted for comparing R. turanicus from the Mediterranean Basin (represented by Albania) and Central Asia (Northwestern China). Altogether 315 adult ticks of R. turanicus (103 from Albania and 212 from China) were morphologically and molecularly analysed. DNA samples were used for mitochondrial 16S rRNA and cox1 gene sequences analysis. In addition, as potentially genetic markers, three fragments including partial nad1-16S rRNA, nad2-cox1, cox1-tRNA-Lys, were designed and then phylogenetically analyzed. Based on detailed morphological observations, only basis capituli length:width ratio (females), the length, the width and the length:width ratio of the scutum (males) had differences between R. turanicus from China and Albania. Gene divergences of 16S rRNA, cox1, partial nad1-16S rRNA, nad2-cox1 and cox1-tRNA-Lys from China and Albania ticks were 3.53-4.84, 3.57-4.92, 3.57-4.07, 3.57-4.39 and 3.18-4.69%, respectively. The evaluated five genetic markers revealed two phylogenetic branches in R. turanicus. Obvious differences exist within R. turanicus based on morphological and genetic analysis. Three newly designed genetic markers (partial nad1-16S rRNA, nad2-cox1 and cox1-tRNA-Lys) in this study may be suitable genetic tools for identification and analysis in R. turanicus. Subspecies analysis of R. turanicus from other regions of the world should be initiated in the future.


Asunto(s)
Proteínas de Artrópodos/genética , Rhipicephalus/anatomía & histología , Rhipicephalus/genética , Albania , Animales , China , Complejo IV de Transporte de Electrones/genética , Femenino , Marcadores Genéticos/genética , Masculino , Filogenia , ARN Ribosómico 16S/genética , Rhipicephalus/clasificación , Rhipicephalus/enzimología , Análisis de Secuencia de ADN
5.
Parasitol Res ; 115(3): 987-96, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26584827

RESUMEN

Gastrointestinal nematodes within the subfamily Ostertagiinae (Teladorsagia, Ostertagia, and Marshallagia et al.) are among the most common infections of domesticated livestock. These parasites are of particular interest, as many of the species within this group are of economic importance worldwide. Traditionally, nematode species designations have been based on morphological criteria. However, this group possesses poorly defined species. There is an urgent need to develop a reliable technique that can distinguish species of Ostertagiinae. DNA barcoding has been proved to be a powerful tool to identify species of birds, mammals, and arthropods, but this technique has not yet been examined for identifying species of Ostertagiinae. In this study, a total of 138 mitochondrial DNA (mtDNA) cytochrome c oxidase subunit I (COI) sequences from individuals representing 11 species of Ostertagiinae were acquired by PCR for the first time. The specimens were collected from pastoral area of northern China. Genetic divergence analyses showed that mean interspecific Kimura two-parameter distances of COI (13.61 %) were about four times higher than the mean value of the intraspecific divergence (3.69 %). Then, the performance of the COI to identify species of Ostertagiinae was evaluated by identification success rates using nearest neighbor (NN) and BLASTn. The results indicated that the rates of correct sequence identification for COI were high (>80 %) when using the NN and BLASTn methods. Besides, the deep lineage divergences are detected in Teladorsagia circumcincta. Meanwhile, the analyses also detected no genetic differentiation between some species such as Ostertagia hahurica and Ostertagia buriatica. These results indicate that the traditional status of species within Ostertagiinae should be closely examined based on the molecular data.


Asunto(s)
Enfermedades de los Bovinos/parasitología , Enfermedades de las Ovejas/parasitología , Trichostrongyloidea/clasificación , Trichostrongyloidea/aislamiento & purificación , Tricostrongiloidiasis/veterinaria , Animales , Bovinos , China , Código de Barras del ADN Taxonómico/métodos , ADN de Helmintos/genética , ADN Mitocondrial/genética , Ovinos , Trichostrongyloidea/genética , Tricostrongiloidiasis/parasitología
6.
Mol Cell Probes ; 29(4): 244-53, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26013296

RESUMEN

Schmallenberg virus (SBV) is a newly emerged orthobunyavirus that predominantly infects livestock such as cattle, sheep, and goats. Its nucleocapsid (N) protein is an ideal target antigen for SBV diagnosis. In this study, a stable BHK-21 cell line, BHK-21-EGFP-SBV-N, constitutively expressing the SBV N protein was obtained using a lentivector-mediated gene transfer system combined with puromycin selection. To facilitate the purification of recombinant SBV N protein, the coding sequence for a hexa-histidine tag was introduced into the C-terminus of the SBV N gene during construction of the recombinant lentivirus vector pLV-EGFP-SBV-N. The BHK-21-EGFP-SBV-N cell line was demonstrated to spontaneously emit strong enhanced green fluorescent protein (EGFP) signals that exhibited a discrete punctate distribution throughout the cytoplasm. SBV N mRNA and protein expression in this cell line were detected by real-time RT-PCR and western blot, respectively. The expressed recombinant SBV N protein carried an N-terminal EGFP tag, and was successfully purified using Ni-NTA agarose by means of its C-terminal His tag. The purified SBV N protein could be recognized by SBV antisera and an anti-SBV monoclonal antibody (mAb) 2C8 in an indirect enzyme-linked immunosorbent assay and western blot analyses. Indirect immunofluorescence assays further demonstrated that the stable cell line reacts with SBV antisera and mAb 2C8. These results suggest that the generated cell line has the potential to be used in the serological diagnosis of SBV.


Asunto(s)
Proteínas de la Nucleocápside/metabolismo , Orthobunyavirus/metabolismo , Línea Celular , Vectores Genéticos , Lentivirus/genética , Proteínas de la Nucleocápside/aislamiento & purificación
7.
Protein Expr Purif ; 92(1): 1-8, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23988909

RESUMEN

Schmallenberg virus (SBV) is a novel orthobunyavirus that primarily infects ruminants such as cattle, sheep and goats. The nucleocapsid (N) protein of SBV has been shown to be an ideal target antigen for serological detection. To prepare a monoclonal antibody (mAb) against the N protein, the full-length coding sequence of the SBV N gene was cloned into pET-28a-c(+) and pMAL-c5X vectors to generate two recombinant plasmids, which were expressed in Escherichia coli BL21 as histidine (His)-tagged (His-SBV-N) and maltose-binding protein (MBP)-tagged (MBP-SBV-N) fusion proteins, respectively. After affinity purification of His-SBV-N with Ni-NTA agarose and MBP-SBV-N with amylose resin, His-SBV-N was used to immunize BALB/c mice, while MBP-SBV-N was utilized to screen for mAb-secreting hybridomas. Six hybridoma cell lines stably secreting mAbs against N were obtained. Clone 2C8 was selected for further study because of its rapid growth characteristics in vitro and good reactivity with recombinant SBV N proteins in enzyme-linked immunosorbent assays. The epitope recognized by 2C8 is located at amino acids 51-76 of the SBV N protein. Western blot analyses showed that 2C8 reacts with both recombinant SBV N proteins and SBV isolates. It is also cross-reactive with the N proteins of genetically related Shamonda, Douglas and Akabane viruses, but not with the Rift Valley fever virus N protein. The successful preparation of recombinant N proteins and mAbs provides valuable materials that can be used in the serological diagnosis of SBV.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Clonación Molecular/métodos , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/inmunología , Orthobunyavirus/genética , Orthobunyavirus/inmunología , Animales , Especificidad de Anticuerpos , Infecciones por Bunyaviridae/veterinaria , Infecciones por Bunyaviridae/virología , Bovinos/virología , Línea Celular , Femenino , Cabras/virología , Ratones , Ratones Endogámicos BALB C , Proteínas de la Nucleocápside/química , Orthobunyavirus/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Ovinos/virología
8.
Dis Aquat Organ ; 106(1): 85-91, 2013 Sep 24.
Artículo en Inglés | MEDLINE | ID: mdl-24062556

RESUMEN

The protozoan parasite Bonamia ostreae is a destructive pathogen of flat oysters and has been reported to be widespread in Europe and North America. The biological characteristics of this unicellular parasite are still not fully understood. In this study, 104 Ostrea edulis imported from the USA to the Guangdong province of China for consumption were examined for Bonamia infection. PCR assay, combined with restriction fragment length polymorphism, sequencing and BLAST analysis, showed that B. ostreae DNA could be detected in 1 of the 104 oyster samples. Light microscopy revealed Bonamia-like organisms in the oyster. PCR assay and fluorescent in situ hybridization showed that B. ostreae organisms were present and retained their integrity after 4 wk in culture. Acridine orange-ethidium bromide staining indicated that the B. ostreae were still alive. In conclusion, B. ostreae was present in oysters imported to China. More importantly, the parasite was able to survive for at least 4 wk of in vitro culture at 4°C, which further implied a long-term transmission risk of B. ostreae. Considering the wide culture beds of Crassostrea ariakensis and C. gigas in China, and that C. ariakensis and C. gigas are susceptible hosts or reservoirs of B. ostreae, our study highlights the potential risk of introducing B. ostreae by importing O. edulis from a Bonamia endemic area.


Asunto(s)
Haplosporidios/fisiología , Interacciones Huésped-Parásitos , Ostrea/parasitología , Animales , China , Comercio
9.
Dis Aquat Organ ; 104(2): 141-8, 2013 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-23709467

RESUMEN

Perkinsus is a genus of unicellular protozoan parasite responsible for mass mortality of several commercially valuable mollusks. Surveillance and inspection of its epidemiology in the field calls for convenient and rapid detection methods. Here, a loop-mediated isothermal amplification (LAMP) assay was developed to detect the presence of Perkinsus spp. in mollusks. Specific LAMP primers were designed targeting the conserved internal transcribed spacer 2 (ITS-2) region of the rRNA gene of Perkinsus spp. Using ITS-2 recombinant plasmid as a template, we optimized the LAMP reaction system and conditions and then evaluated the analytical sensitivity and specificity of the assay. The LAMP assay was validated using clam samples collected from coastal areas in eastern China and oysters imported to China and compared with the traditional Ray's fluid thioglycollate culture method (RFTM). Our results showed that the LAMP detection method for Perkinsus was successful. The detection limit was 10 copies of plasmid DNA. Compared to the RFTM assay, the LAMP detection method was more sensitive (56 versus 52 positive out of 60 samples). P. olseni and P. marinus from infected hosts were successfully detected by this method. The LAMP method is rapid, sensitive, and specific for Perkinsus spp. detection, and could be used to screen for perkinsosis both on farms and at ports.


Asunto(s)
Alveolados/aislamiento & purificación , Bivalvos/parasitología , Técnicas de Amplificación de Ácido Nucleico/métodos , Alveolados/genética , Animales , Interacciones Huésped-Parásitos , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad
10.
Ecol Evol ; 12(3): e8663, 2022 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-35261750

RESUMEN

The phylogenetic structure of the genus Niviventer has been studied based on several individual mitochondrial and nuclear genes, but the results seem to be inconsistent. In order to clarify the phylogeny of Niviventer, we sequenced the complete mitochondrial genome of white-bellied rat (Niviventer andersoni of the family Muridae) by next-generation sequencing. The 16,291 bp mitochondrial genome consists of 22 transfer RNA genes, 13 protein-coding genes (PCGs), two ribosomal RNA genes, and one noncoding control region (D-Loop). Phylogenetic analyses of the nucleotide sequences of all 13 PCGs, PCGs minus ND6, and the entire mitogenome sequence except for the D-loop revealed well-resolved topologies supporting that N. andersoni was clustered with N. excelsior forming a sister division with N. confucianus, which statistically rejected the hypothesis based on the tree of cytochrome b (cytb) gene that N. confucianus is sister to N. fulvescens. Our research provides the first annotated complete mitochondrial genome of N. andersoni, extending the understanding about taxonomy and mitogenomic evolution of the genus Niviventer.

11.
J AOAC Int ; 105(5): 1437-1446, 2022 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-35377440

RESUMEN

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread over the world since its emergence. Although the dominant route of SARS-CoV-2 infection is respiratory, a number of studies revealed infection risk from contaminated surfaces and products, including porcine-derived food and other products. The SARS-CoV-2 outbreak has been severely threatening public health, and disrupting porcine products trade and the pig industry. Swine acute diarrhea syndrome coronavirus (SADS-CoV), which was responsible for large-scale, fatal disease in piglets, emerged in 2017 and has caused enormous economic losses in the pig industry. Currently, reverse transcription real-time PCR (RT-rPCR) is the gold standard method for SARS-CoV-2 diagnosis and is most commonly used for SADS-CoV detection. However, inaccurate detection of the SARS-CoV-2 infection obtained by RT-rPCR is increasingly reported, especially in specimens with low viral load. OBJECTIVE: This study aimed to develop an accurate reverse transcription droplet digital PCR (RT-ddPCR) assay for the detection of SARS-CoV-2 and SADS-CoV simultaneously. METHODS: Two pairs of primers and one double-quenched probe targeting the RNA-dependent RNA polymerase (RDRP) region of the open reading frame 1ab (ORF1ab) gene of SARS-CoV-2 and the corresponding ORF1ab region of SADS-CoV were designed to develop the RT-ddPCR assay. The sensitivity, specificity, repeatability, and reproducibility were tested using complementary RNAs (cRNAs) and clinical specimens. RESULTS: The detection limits of RT-ddPCR were 1.48 ± 0.18 and 1.38 ± 0.17 copies in a 20 µL reaction for SARS-CoV-2 and SADS-CoV cRNAs, respectively (n = 8), showing approximately 4- and 10-fold greater sensitivity than the RT-rPCR assay. This assay also exhibited good specificity, repeatability, and reproducibility. CONCLUSION: The established RT-ddPCR assay was shown to be a highly effective, accurate, and reliable method for the sensitive detection of SARS-CoV-2 and SADS-CoV. HIGHLIGHTS: This RT-ddPCR assay could be used to detect both SARS-CoV-2 and SADS-CoV in a sample with one double-quenched probe, and is also the first reported RT-ddPCR assay for SADS-CoV detection.


Asunto(s)
COVID-19 , SARS-CoV-2 , Alphacoronavirus , Animales , COVID-19/diagnóstico , Prueba de COVID-19 , Humanos , ARN Viral/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reproducibilidad de los Resultados , Transcripción Reversa , SARS-CoV-2/genética , Sensibilidad y Especificidad , Porcinos
12.
J AOAC Int ; 104(5): 1389-1393, 2021 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-33769495

RESUMEN

BACKGROUND: The diseases caused by the Capripoxvirus species have very similar symptoms and are difficult to distinguish clinically. According to a recent report, Capripoxvirus are not strictly host specific. OBJECTIVE: This study aimed to identify the viruses from ovine (include sheep and goat) or bovine, which will assist in selecting the appropriate vaccine and correct measures to control diseases. METHOD: Universal primers for all Capripoxvirus and specific probes for lumpy skin disease virus, sheeppox virus, and goatpox virus were designed and analyzed to identify the viruses from ovine (including sheep and goats) or bovine species. The parameters of the system, such as the annealing temperatures and the quantities of primers and probes used, were optimized. The sensitivity, specificity, and reproducibility were tested. RESULTS: Each probe showed a specific fluorescent signal, with no cross reaction with other pathogens that cause symptoms similar to those of the poxviruses. The LOD was 102 copies of the target genome DNA. The 557 local clinical samples and samples from Ethiopia were successfully detected and the results were consistent with a restriction fragment length polymorphism PCR analysis of the P32 and RPO30 genes and gene sequencing. CONCLUSIONS: This optimized real-time PCR detection system has good diagnostic sensitivity and specificity and can be used for the rapid and effective differential diagnosis of these diseases in goats, sheep, and cattle. HIGHLIGHTS: It is a rapid detection method to distinguish the viruses from ovine (include sheep and goat) or bovine.


Asunto(s)
Capripoxvirus , Enfermedades de las Cabras , Virus de la Dermatosis Nodular Contagiosa , Infecciones por Poxviridae , Enfermedades de las Ovejas , Animales , Capripoxvirus/genética , Bovinos , Enfermedades de las Cabras/diagnóstico , Cabras , Virus de la Dermatosis Nodular Contagiosa/genética , Infecciones por Poxviridae/diagnóstico , Infecciones por Poxviridae/veterinaria , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Ovinos , Enfermedades de las Ovejas/diagnóstico
13.
Mitochondrial DNA B Resour ; 5(1): 443-444, 2020 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-33366593

RESUMEN

Uruguayan beef is one of the most popular products in the export market. In this study, we report the complete mitochondrial genome sequence of Uruguayan native cattle for the first time. The total mitochondrial genome sequence is 16,339 bp in length with the base composition of 33.4% for A, 27.2% for T, 26.0% for C, and 13.4% for G. The description of all genes is similar to the typical mitochondrial genomes of cattle. The annotated mitochondrial genome of Uruguayan native cattle would serve as an important genetic data set for further study.

14.
Biomed Res Int ; 2019: 5046958, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31016191

RESUMEN

Sorghum comprises 31 species that exhibit considerable morphological and ecological diversity. The phylogenetic relationships among Sorghum species still remain unresolved due to lower information on the traditional DNA markers, which provides a limited resolution for identifying Sorghum species. In this study, we sequenced the complete chloroplast genomes of Sorghum sudanense and S. propinquum and analyzed the published chloroplast genomes of S. bicolor and S. timorense to retrieve valuable chloroplast molecular resources for Sorghum. The chloroplast genomes ranged in length from 140,629 to 140,755 bp, and their gene contents, gene orders, and GC contents were similar to those for other Poaceae species but were slightly different in the number of SSRs. Comparative analyses among the four chloroplast genomes revealed 651 variable sites, 137 indels, and nine small inversions. Four highly divergent DNA regions (rps16-trnQ, trnG-trnM, rbcL-psaI, and rps15-ndhF), which were suitable for phylogenetic and species identification, were detected in the Sorghum chloroplast genomes. A phylogenetic analysis strongly supported that Sorghum is a monophyletic group in the tribe Andropogoneae. Overall, the genomic resources in this study could provide potential molecular markers for phylogeny and species identification in Sorghum.


Asunto(s)
Cloroplastos/genética , Genoma del Cloroplasto/genética , Sorghum/genética , Composición de Base/genética , ADN de Cloroplastos/genética , Orden Génico/genética , Marcadores Genéticos/genética , Genómica/métodos , Filogenia , Poaceae/genética , Análisis de Secuencia de ADN/métodos
15.
J Vet Diagn Invest ; 31(3): 415-425, 2019 May.
Artículo en Inglés | MEDLINE | ID: mdl-30947641

RESUMEN

Advances in molecular testing and microfluidic technologies have opened new avenues for rapid detection of animal viruses. We used a centrifugal microfluidic disk (CMFD) to detect 6 important swine viruses, including foot-and-mouth disease virus, classical swine fever virus, porcine reproductive and respiratory swine virus-North American genotype, porcine circovirus 2, pseudorabies virus, and porcine parvovirus. Through integrating the loop-mediated isothermal amplification (LAMP) method and microfluidic chip technology, the CMFD could be successfully performed at 62℃ in 60 min. The detection limit of the CMFD was 3.2 × 102 copies per reaction, close to the sensitivity of tube-type LAMP turbidity methods (1 × 102 copies per reaction). In addition, the CMFD was highly specific in detecting the targeted viruses with no cross-reaction with other viruses, including porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine rotavirus. The coincidence rate of CMFD and conventional PCR was ~94%; the CMFD was more sensitive than conventional PCR for detecting mixed viral infections. The positive detection rate of 6 viruses in clinical samples by CMFD was 44.0% (102 of 232), whereas PCR was 40.1% (93 of 232). Thirty-six clinical samples were determined to be coinfected with 2 or more viruses. CMFD can be used for rapid, sensitive, and accurate detection of 6 swine viruses, offering a reliable assay for monitoring these pathogens, especially for detecting viruses in widespread mixed-infection clinical samples.


Asunto(s)
Técnicas de Amplificación de Ácido Nucleico/veterinaria , Porcinos/virología , Virus/clasificación , Virus/aislamiento & purificación , Animales , Límite de Detección , Técnicas de Amplificación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Virus/genética
16.
Parasit Vectors ; 11(1): 442, 2018 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-30064470

RESUMEN

BACKGROUND: Insect-borne diseases could induce severe symptoms in human and clinical signs in animals, such as febrility, erythra, arthralgia and hemorrhagic fever, and cause significant economic losses and pose public health threat all over the world. The significant advantages of Luminex xMAP technology are high-throughput, high parallel and automation. This study aimed to establish a liquid bead array based on Luminex xMAP technology that was able to simultaneously detect multiple insect-borne pathogens. METHODS: Specific probes and primers to detect the nucleic acid of 10 insect-borne pathogens were designed. Probes were coupled with fluorescent carboxylated microspheres. The parameters of the system were optimized, including ratio of forward/reverse primers (1:2), hybridization temperature (50 °C) and duration (30 min) and quantity of PCR product (2 µl). The sensitivity and specificity of the system were also evaluated. Moreover mixed nucleic acid of 10 insect-borne pathogens, including Bluetongue virus, Epizootic hemorrhagic disease virus of deer, Coxiella burnetii, African swine fever virus, West Nile fever virus, Borrelia burgdorferi, vesicular stomatitis virus, Rift Valley fever virus, Ebola virus and Schmalenberg's disease virus, and 3000 clinical samples were tested for practicability. RESULTS: The optimized detection system showed high sensitivity, specificity and reproducibility. Each probe showed specific fluorescence signal intensity without any cross-hybridization for the other insect-borne pathogens tested, which included dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus, Xinjiang hemorrhagic fever virus, spotted fever group rickettsiae, ehrlichiae and chikungunya virus. The limit of detection was 10 copies of target gene. Insect-borne pathogens were successfully detected among the 3000 clinical samples, and the results were consistent with those obtained using gold-standard assays or commercial nucleic acid detection kits. CONCLUSIONS: This optimized liquid array detection system was high-throughput and highly specific and sensitive in screening of the insect-borne pathogens. It was promising in detection of these pathogens for molecular epidemiological studies.


Asunto(s)
Borrelia/aislamiento & purificación , Coxiella burnetii/aislamiento & purificación , Insectos/microbiología , Virus/aislamiento & purificación , Animales , ADN/aislamiento & purificación , Ácidos Nucleicos , ARN/aislamiento & purificación , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Garrapatas/microbiología
17.
Sci Rep ; 8(1): 1865, 2018 01 30.
Artículo en Inglés | MEDLINE | ID: mdl-29382871

RESUMEN

Ticks host a wide range of zoonotic pathogens and are a significant source of diseases that affect humans and livestock. However, little is known about the pathogens associated with bat ticks. We have collected ectoparasites from bat carcasses over a seven year period. Nucleic acids (DNA and RNA) were extracted from 296 ticks removed from bats and the species designation was confirmed in all ticks as Argas (Carios) vespertilionis. A subset of these samples (n = 120) were tested for the presence of zoonotic pathogens by molecular methods. Babesia species, Rickettsia spp., within the spotted fever group (SFG), and Ehrlichia spp. were detected in ticks removed from 26 bats submitted from 14 counties across England. The prevalence of Rickettsia spp. was found to be highest in Pipistrellus pipistrellus from southern England. This study suggests that the tick species that host B. venatorum may include the genus Argas in addition to the genus Ixodes. As A. vespertilionis has been reported to feed on humans, detection of B. venatorum and SFG Rickettsia spp. could present a risk of disease transmission in England. No evidence for the presence of flaviviruses or Issyk-Kul virus (nairovirus) was found in these tick samples.


Asunto(s)
Argas/genética , Babesia/genética , Quirópteros/microbiología , Quirópteros/parasitología , Rickettsia/genética , Infestaciones por Garrapatas/veterinaria , Zoonosis/epidemiología , Animales , Argas/clasificación , Babesia/clasificación , Inglaterra , Genes Bacterianos , Interacciones Huésped-Patógeno , ARN Ribosómico 16S/genética , Rickettsia/clasificación , Infestaciones por Garrapatas/epidemiología , Zoonosis/microbiología
18.
PLoS One ; 13(12): e0208615, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30586406

RESUMEN

Argasid ticks (Acari: Argasidae) carry and transmit a variety of pathogens of animals and humans, including viruses, bacteria and parasites. There are several studies reporting ixodid ticks (Acari: Ixodidae) and associated tick-borne pathogens in Xinjiang, China. However, little is known about the argasid ticks and argasid tick-associated pathogens in this area. In this study, a total of 3829 adult argasid ticks infesting livestock were collected at 12 sampling sites of 10 counties in the Peripheral Oases, which carry 90% of the livestock and humans population, around the Tarim Basin (southern Xinjiang) from 2013 to 2016. Tick specimens were identified to two species from different genera by morphology and sequences of mitochondrial 16S rRNA and 12S rRNA were derived to confirm the species designation. The results showed that the dominant argasid ticks infesting livestock in southern Xinjiang were Ornithodoros lahorensis (87.86%, 3364/3829). Ornithodoros lahorensis was distributed widely and were collected from 10 counties of southern Xinjiang. Argas japonicus was collected from Xinjiang for the first time. In addition, we screened these ticks for tick-associated pathogens and showed the presence of DNA sequences of Rickettsia spp. of Spotted fever group and Anaplasma spp. in the argasid ticks. This finding suggests the potential role for Argas japonicus as a vector of pathogens to livestock and humans.


Asunto(s)
Anaplasma/aislamiento & purificación , Argas/microbiología , Ornithodoros/microbiología , Rickettsia/aislamiento & purificación , Anaplasma/clasificación , Anaplasma/genética , Anaplasma/patogenicidad , Animales , Argas/clasificación , Argas/genética , Bovinos , China , Vectores de Enfermedades , Mitocondrias/genética , Ornithodoros/clasificación , Ornithodoros/genética , Filogenia , ARN Ribosómico/clasificación , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN Ribosómico 16S/clasificación , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Rickettsia/clasificación , Rickettsia/genética , Rickettsia/patogenicidad , Análisis de Secuencia de ADN , Ovinos , Infestaciones por Garrapatas/parasitología , Infestaciones por Garrapatas/patología , Infestaciones por Garrapatas/veterinaria
19.
Cytotechnology ; 69(1): 145-156, 2017 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-28083834

RESUMEN

Schmallenberg virus (SBV) is a Culicoides-transmitted orthobunyavirus that poses a threat to susceptible livestock species such as cattle, sheep and goats. The nucleocapsid (N) protein of SBV is an ideal diagnostic antigen for the detection of viral infection. In this study, a stable Vero cell line, Vero-EGFP-SBV-N, constitutively expressing the SBV-N protein was established using a lentivirus system combined with puromycin selection. This cell line spontaneously emitted green fluorescent signals distributed throughout the cytoplasm, in which the expression of SBV-N fusion protein was confirmed by western blot analysis. The expression of SBV-N protein in Vero-EGFP-SBV-N cells was stable for more than fifty passages without puromycin pressure. The SBV-N fusion protein contained both an N-terminal enhanced green fluorescent protein (EGFP) tag and a C-terminal hexa-histidine (6 × His) tag, by which the N protein was successfully purified using Ni-NTA affinity chromatography. The cell line was further demonstrated to be reactive with SBV antisera and an anti-SBV monoclonal antibody in indirect immunofluorescence assays. Taken together, our results demonstrate that the Vero-EGFP-SBV-N cell line has potential for application in the serological diagnosis of SBV infection.

20.
Front Plant Sci ; 8: 1854, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29118779

RESUMEN

Rice is the most important crop in the world as the staple food for over half of the population. The wild species of Oryza represent an enormous gene pool for genetic improvement of rice cultivars. Accurate and rapid identification of these species is critical for effective utilization of the wild rice germplasm. In this study, we developed valuable chloroplast molecular markers by comparing the chloroplast genomes for species identification. Four chloroplast genomes of Oryza were newly sequenced on the Illumina HiSeq platform and other 14 Oryza species chloroplast genomes from Genbank were simultaneously taken into consideration for comparative analyses. Among 18 Oryza chloroplast genomes, five variable regions (rps16-trnQ, trnTEYD, psbE-petL, rpoC2 and rbcL-accD) were detected for DNA barcodes, in addition to differences in simple sequence repeats (SSR) and repeat sequences. The highest species resolution (72.22%) was provided by rpoC2 and rbcL-accD with distance-based methods. Three-marker combinations (rps16-trnQ + trnTEYD + rbcL-accD, rps16-trnQ + trnTEYD + rpoC2 and rpoC2 + trnTEYD + psbE-petL) showed the best species resolution (100%). Phylogenetic analysis based on the chloroplast genome provided the best resolution of Oryza. In the comparison of chloroplast genomes in this study, identification of the most variable regions and assessment of the focal regions of divergence were efficient in developing species-specific DNA barcodes. Based on evaluation of the chloroplast genomic resources, we conclude that chloroplast genome sequences are a reliable and valuable molecular marker for exploring the wild rice genetic resource in rice improvement.

SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA