A real time Taqman RT-PCR for the detection of rabbit hemorrhagic disease virus 2 (RHDV2)

As key prey, the wild rabbit downsize constitutes a major drawback on the endangered Iberian lynx (Lynx pardinus) re-introduction in the Iberia. Several captive breeding units mostly located in Alentejo, endeavour the wild rabbit repopulation of depleted areas assigned for the lynx re-introduction. Here we report an RHDV2 outbreak that occurred in early 2016 in a wild rabbit captive breeding unit located in Barrancos municipality. The estimated mortality rate between March and April 2016 was approximately 8.67%. Anatomopathologic examination was carried out for 13 victimized rabbits. Molecular characterization was based on the complete vp60 capsid gene. The 13 rabbit carcasses investigated showed typical macroscopic RHD lesions testing positive to RHDV2- RNA. Comparison of the vp60 nucleotide sequences obtained from two specimens with others publically available disclosed similarities below 98.22% with RHDV2 strains originated in the Iberia and Azores and revealed that the two identical strains from Barrancos-2016 contain six unique single synonymous nucleotide polymorphisms. In the phylogenetic analysis performed, the Barrancos-2016 strains clustered apart from other known strains, meaning they may represent new evolutionary RHDV2 lineages. No clear epidemiological link could be traced for this outbreak where the mortalities were lower compared with previous years. Yet, network analysis suggested a possible connection between the missing intermediates from which the strains from Barrancos 2013, 2014 and 2016 have derived. It is therefore possible that RHDV2 has circulated endemically in the region since 2012, with periodic epizootic occurrences. Still, six years after its emergence in wild rabbits, RHDV2 continues to pose difficulties to the establishment of natural wild rabbit populations that are crucial for the self-sustainability of the local ecosystems.

Otimização de marcadores desenvolvidos para PCR convencional para uso em qPCR visando a diagnose de Xanthomonas campestris pv viticola em videira.

No Brasil, Xanthomonas campestris pv. viticola (Xcv), causadora do cancro bacteriano em videira, é uma praga quarentenária A2, com ocorrência no Semiárido Nordestino. A bactéria pode ser disseminada de plantas assintomáticas pela distribuição de material propagativo e ocorrências restritas da doença em outras regiões foram identificadas. Para diagnose confiável por PCR convencional, o DNA deve ser extraído de culturas de bactérias isoladas de tecido com sintomas suspeitos. Com a técnica, é possível detectar até 0,25 pg de DNA bacteriano total. Atualmente, métodos que empregam tecidos assintomáticos não estão disponíveis. O objetivo deste trabalho foi desenvolver um protocolo sensível à detecção de Xcv por qPCR, empregando iniciadores disponíveis da técnica convencional.

Multiplex PCR para a identificação de cochonilhas farinhentas (Hemiptera Pseudococcidae) presentes na cultura da videira no Brasil.

Uma alta diversidade de espécies de cochonilhas farinhentas (Hemiptera:Pseudococcidae) tem sido observada na cultura da videira. Algumas espécies são inclusive consideradas pragas quarentenárias para outros países. A identificação de cochonilhas farinhentas é um dos fatores limitantes para o manejo destes insetos no campo, em decorrência da grande similaridade morfológica observada entre espécies próximas e da variação morfológica que ocorre em alguns grupos em decorrência do hospedeiro e da temperatura de desenvolvimento. Além disto, a identificação destes insetos baseia-se em características morfológicas presentes apenas em fêmeas adultas, realizada por poucos especialistas. O objetivo deste trabalho foi desenvolver um ?kit? de identificação molecular das principais espécies de cochonilhas farinhentas presentes na cultura da videira no Brasil [(Dysmicoccus brevipes (Cockerell), Phenacoccus solenopsis (Tinsley), Planococcus citri (Risso), Planococcus ficus (Signoret) e Pseudococcus viburni (Signoret)]. Cochonilhas farinhentas foram coletadas na Serra Gaúcha, RS, Vale do São Francisco (Polos Juazeiro- BA e Petrolina-PE) e em cidades produtoras de uvas do Paraná.

Processamento de liquido cefalo-raquidiano para extração de RNA genômico do vírus da artrite-encefalite caprina e diagnóstico molecular por RT-nested PCR.

Este comunicado descreve os procedimentos de coleta, processamento do líquido céfalo-raquidiano, para a extração de RNA genômico viral, e de detecção do vírus através da técnica de RT-nested PCR, potencial método de diagnóstico molecular da CAE.

Processamento de sangue e líquido sinovial para extração de RNA genomico do vírus da artrite-encefalite caprina e diagnóstico molecular por RT-nested PCR.

Este comunicado descreve os procedimentos de coleta e processamento de líquido sinovial e sangue seguido pela extração de RNA genômico e, finalmente, o diagnóstico molecular do vírus pela técnica de RT-nested PCR.

Processamento de sêmen para extração de RNA genômico do vírus da artrite-encefalite caprina e diagnóstico molecular por RT-nested PCR.

O presente comunicado descreve os procedimentos necessários para a coleta e processamento de amostras de sêmen de caprinos infectados pelo CAEV para posterior extração do RNA viral por meio de um método baseado em centrifugação em coluna de sílica. A avaliação da presença de RNA no sêmen será feita, diretamente, por meio da reação de RT-nested PCR, portencial método de diagnóstico molecular da CAE.

Occurrence of apple stem grooving capillovirus in Santa Catarina, Brazil, detected by RT-PCR.

1999

Diagnóstico molecular por PCR-RFLP e gene LYS2 para identificação de Fusarium decemcellulare.

Fusarium decemcellulare é encontrado como agente causal de doenças com diferentes sintomas em diversas espécies de plantas em regiões tropicais e subtropicais. Em guaranazeiro, espécie nativa da Amazônia de importância econômica e social, a doença denominada de complexo superbrotamento é atualmente um dos principais problemas da cultura. Técnicas moleculares são cada vez mais requeridas para identificação rápida e segura de patógenos como complemento as técnicas convencionais. O objetivo do trabalho foi desenvolver métodos moleculares por PCR e PCR-RFLP para rápida identificação de F. decemcellulare.

Caracterização de Fusarium decemcellulare isolado de guaranazeiro (Paullinia cupana var. sorbilis) por meio de ERIC-PCR.

A presente pesquisa visa analisar a diversidade genética De F. decemcellulare isolado de mudas e plantas adultas de guaranazeiro com sintomas de superbrotamento, hipertrofia floral ou galhas por meio do marcador molecular ERIC-PCR.

RAPD em pau-rosa (Aniba rosaeodora Ducke): adaptação do método para coleta de amostras in situ, ajuste das condições de PCR e apresentação de um processo para selecionar bandas reprodutíveis.

Os objetivos deste trabalho foram: (1) validar um método para a coleta de material vegetal de pau-rosa; (2) selecionar um método para a extração de DNA de folhas de pau-rosa, em quantidade e com qualidade adequadas para a obtenção de padrões RAPD e (3) desenvolver e validar um critério, baseado no grau de reprodutibilidade para selecionar bandas RAPD para as análises genéticas.

Detecção de oito vírus em videiras por meio de RT-PCR em tempo real.

2011

Mucopolysaccharidosis Type Ii: Identification Of 30 Novel Mutations Among Latin American Patients.

In this study, 103 unrelated South-American patients with mucopolysaccharidosis type II (MPS II) were investigated aiming at the identification of iduronate-2-sulfatase (IDS) disease causing mutations and the possibility of some insights on the genotype-phenotype correlation The strategy used for genotyping involved the identification of the previously reported inversion/disruption of the IDS gene by PCR and screening for other mutations by PCR/SSCP. The exons with altered mobility on SSCP were sequenced, as well as all the exons of patients with no SSCP alteration. By using this strategy, we were able to find the pathogenic mutation in all patients. Alterations such as inversion/disruption and partial/total deletions of the IDS gene were found in 20/103 (19%) patients. Small insertions/deletions/indels (<22 bp) and point mutations were identified in 83/103 (88%) patients, including 30 novel mutations; except for a higher frequency of small duplications in relation to small deletions, the frequencies of major and minor alterations found in our sample are in accordance with those described in the literature.

Nat2, Xrcc1 And Hogg1 Polymorphisms, Cigarette Smoking, Alcohol Consumption And Risk Of Upper Aerodigestive Tract Cancer.

To evaluate associations between polymorphisms of the N-acetyltransferase 2 (NAT2), human 8-oxoguanine glycosylase 1 (hOGG1) and X-ray repair cross-complementing protein 1 (XRCC1) genes and risk of upper aerodigestive tract (UADT) cancer. A case-control study involving 117 cases and 224 controls was undertaken. The NAT2 gene polymorphisms were genotyped by automated sequencing and XRCC1 Arg399Gln and hOGG1 Ser326Cys polymorphisms were determined by Polymerase Chain Reaction followed by Restriction Fragment Length Polymorphism (PCR-RFLP) methods. Slow metabolization phenotype was significantly associated as a risk factor for the development of UADT cancer (p=0.038). Furthermore, haplotype of slow metabolization was also associated with UADT cancer (p=0.014). The hOGG1 Ser326Cys polymorphism (CG or GG vs. CC genotypes) was shown as a protective factor against UADT cancer in moderate smokers (p=0.031). The XRCC1 Arg399Gln polymorphism (GA or AA vs. GG genotypes), in turn, was a protective factor against UADT cancer only among never-drinkers (p=0.048). Interactions involving NAT2, XRCC1 Arg399Gln and hOGG1 Ser326Cys polymorphisms may modulate the risk of UADT cancer in this population.

Albumin Is Synthesized In Epididymis And Aggregates In A High Molecular Mass Glycoprotein Complex Involved In Sperm-egg Fertilization.

The epididymis has an important role in the maturation of sperm for fertilization, but little is known about the epididymal molecules involved in sperm modifications during this process. We have previously described the expression pattern for an antigen in epididymal epithelial cells that reacts with the monoclonal antibody (mAb) TRA 54. Immunohistochemical and immunoblotting analyses suggest that the epitope of the epididymal antigen probably involves a sugar moiety that is released into the epididymal lumen in an androgen-dependent manner and subsequently binds to luminal sperm. Using column chromatography, SDS-PAGE with in situ digestion and mass spectrometry, we have identified the protein recognized by mAb TRA 54 in mouse epididymal epithelial cells. The ∼65 kDa protein is part of a high molecular mass complex (∼260 kDa) that is also present in the sperm acrosomal vesicle and is completely released after the acrosomal reaction. The amino acid sequence of the protein corresponded to that of albumin. Immunoprecipitates with anti-albumin antibody contained the antigen recognized by mAb TRA 54, indicating that the epididymal molecule recognized by mAb TRA 54 is albumin. RT-PCR detected albumin mRNA in the epididymis and fertilization assays in vitro showed that the glycoprotein complex containing albumin was involved in the ability of sperm to recognize and penetrate the egg zona pellucida. Together, these results indicate that epididymal-derived albumin participates in the formation of a high molecular mass glycoprotein complex that has an important role in egg fertilization.

Overlapped Sequence Types (sts) And Serogroups Of Avian Pathogenic (apec) And Human Extra-intestinal Pathogenic (expec) Escherichia Coli Isolated In Brazil.

Avian pathogenic Escherichia coli (APEC) strains belong to a category that is associated with colibacillosis, a serious illness in the poultry industry worldwide. Additionally, some APEC groups have recently been described as potential zoonotic agents. In this work, we compared APEC strains with extraintestinal pathogenic E. coli (ExPEC) strains isolated from clinical cases of humans with extra-intestinal diseases such as urinary tract infections (UTI) and bacteremia. PCR results showed that genes usually found in the ColV plasmid (tsh, iucA, iss, and hlyF) were associated with APEC strains while fyuA, irp-2, fepC sitDchrom, fimH, crl, csgA, afa, iha, sat, hlyA, hra, cnf1, kpsMTII, clpVSakai and malX were associated with human ExPEC. Both categories shared nine serogroups (O2, O6, O7, O8, O11, O19, O25, O73 and O153) and seven sequence types (ST10, ST88, ST93, ST117, ST131, ST155, ST359, ST648 and ST1011). Interestingly, ST95, which is associated with the zoonotic potential of APEC and is spread in avian E. coli of North America and Europe, was not detected among 76 APEC strains. When the strains were clustered based on the presence of virulence genes, most ExPEC strains (71.7%) were contained in one cluster while most APEC strains (63.2%) segregated to another. In general, the strains showed distinct genetic and fingerprint patterns, but avian and human strains of ST359, or ST23 clonal complex (CC), presented more than 70% of similarity by PFGE. The results demonstrate that some zoonotic-related STs (ST117, ST131, ST10CC, ST23CC) are present in Brazil. Also, the presence of moderate fingerprint similarities between ST359 E. coli of avian and human origin indicates that strains of this ST are candidates for having zoonotic potential.