PCR em tempo real para caracterização de fontes alimentares de flebotomíneos (Diptera: Psychodidae)

Os flebotomíneos são insetos hematófagos de grande importância médica e veterinária atuando como vetores de parasitas como Leishmania. O estudo do padrão alimentar desses vetores pode ajudar a compreender a sua interação com potenciais reservatórios de Leishmania. Neste estudo, desenvolvemos ensaios de PCR em tempo real para identificação de sangue em flebotomíneos. Seis pares de primers foram desenhados com base no gene citocromo b de sequencias disponíveis no GenBank dos seguintes hospedeiros potenciais: cão, gato, cavalo, galinha, rato e humano. Primeiramente, os ensaios de PCR em tempo real utilizando SYBR Green foram conduzidos usando uma curva padrão com oito concentrações diferentes (i.e., 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg e 1 fg por 2 µl) de amostras do DNA extraído do sangue com EDTA a partir de cada espécie de animal. Em seguida, o DNA foi extraído de 100 fêmeas de flebotomíneos ingurgitadas de campo pertencentes a três espécies (i.e., Lutzomyia longipalpis, L. migonei e L. lenti) foram testadas pelos protocolos aqui padronizados. Fêmeas de flebotomíneos foram experimentalmente alimentadas em um rato (Rattus rattus) e utilizadas para avaliar a detecção do ensaio. Os protocolos funcionaram de forma eficiente com limites de detecção de 10 pg a 100 fg. Fêmeas de flebotomíneos ingurgitadas coletadas no campo estavam alimentadas de humanos (73 por cento), galinhas (23 por cento), cães (22 por cento), cavalos (15 por cento), ratos (11 por cento) e gatos (2 por cento). Curiosamente, 76,1 por cento das fêmeas de L. longipalpis foram positivas para o sangue humano. No total, 48 por cento das fêmeas testadas estavam alimentadas em uma única fonte, 31 por cento em duas e 12 por cento em três. A análise do curso de tempo mostrou que a técnica de PCR em tempo real visando o DNA de roedor foi capaz de detectar pequenas quantidades de DNA do hospedeiro até 5 dias após o repasto sanguíneo. Esses protocolos representam ferramentas promissoras para a identificação da fonte alimentar de flebotomíneos de campo

Otimização da RT-PCR Multiplex para detecção de vírus dengue em amostras de Aedes aegypti infectados artificialmente

A dengue é a mais importante doença viral transmitida por mosquitos, no que diz respeito à morbidade e mortalidade, que afeta os seres humanos. Este vírus é transmitido pelos vetores Aedes albopictus e Aedes aegypti, este último é o principal vetor nas Américas. O controle da doença se baseia na vigilância laboratorial e vigilância entomológica. A vigilância laboratorial visa aprimorar a capacidade do diagnóstico, detectando precocemente a circulação viral e monitorando os sorotipos circulantes. Dentro deste tipo de vigilância, a RT-PCR é um método bastante usado no diagnóstico da doença em humanos e mosquitos, porém, a má conservação do material pode comprometer a integridade do RNA e trazer resultados falso-negativos. O desenvolvimento de melhores métodos de vigilância do vírus dengue (DENV) em mosquitos é de grande valor para os programas de controle. Desta maneira, o presente projeto visou otimizar a técnica de RT-PCR Multiplex para detecção de DENV em amostras de Ae. aegypti infectadas artificialmente pelo vírus. Primers que amplificam uma região de 80 pb do gene rpL8 de mosquito foram desenhados no site Primer3 e avaliados na ferramenta online Multiple Primer Analyzer, junto com primers que amplificam os sorotipos DENV. Não houve competição de primers e foi observado bandas distintas no gel de agarose. Foi avaliado o efeito de diferentes formas de preservação do material genético das amostras (RNAlater®, freezer -80°C e nitrogênio líquido) por 7 dias, onde não houve diferenças significativas em relação à integridade do RNA. O efeito de diferentes formas de extração de RNA (Kit da QIAGEN® , TRIzol® e Chomczymski-Sacchi) também foi avaliado e o método ChomczymskiSacchi obteve o melhor desempenho. A otimização desta técnica permitirá uma maior confiabilidade nos resultados, já que além da detecção dos sorotipos, haverá uma confirmação da qualidade do RNA, aprimorando a capacidade do diagnóstico e auxiliando a prevenção e controle da transmissão da dengue.

Pseudomonas syringae pv. coryli, the causal agent of bacterial twig dieback of Corylus avellana

Thirty-eight bacterial strains isolated from hazelnut (Corylus avellana) cv. Tonda Gentile delle Langhe showing a twig dieback in Piedmont and Sardinia, Italy, were studied by a polyphasic approach. All strains were assessed by fatty acids analysis and repetitive sequence-based polymerase chain reaction (PCR) fingerprinting using BOX and ERIC primer sets. Representative strains also were assessed by sequencing the 16S rDNA and hrpL genes, determining the presence of the syrB gene, testing their biochemical and nutritional characteristics, and determining their pathogenicity to hazelnut and other plants species or plant organs. Moreover, they were compared with reference strains of other phytopathogenic pseudomonads. The strains from hazelnut belong to Pseudomonas syringae (sensu latu), LOPAT group Ia. Both fatty acids and repetitive-sequence-based PCR clearly discriminate such strains from other Pseudomonas spp., including P. avellanae and other P. syringae pathovars as well as P. syringae pv. syringae strains from hazelnut. Also, the sequencing of 16S rDNA and hrpL genes differentiated them from P. avellanae and from P. syringae pv. syringae. They did not possess the syrB gene. Some nutritional tests also differentiated them from related P. syringae pathovars. Upon artificial inoculation, these strains incited severe twig diebacks only on hazelnut. Our results justify the creation of a new pathovar because the strains from hazelnut constitute a homogeneous group and a discrete phenon. The name of P. syringae pv. coryli is proposed and criteria for routine identification are presented.

The development of a real-time PCR to detect pathogenic Leptospira species in kidney tissue

A LightCycler(R) real-time PCR hybridization probe-based assay that detects a conserved region of the 16S rRNA gene of pathogenic but not saprophytic Leptospira species was developed for the rapid detection of pathogenic leptospires directly from processed tissue samples. In addition, a differential PCR specific for saprophytic leptospires and a control PCR targeting the porcine beta-actin gene were developed. To assess the suitability of these PCR methods for diagnosis, a trial was performed on kidneys taken from adult pigs with evidence of leptospiral infection, primarily a history of reproductive disease and serological evidence of exposure to pathogenic leptospires (n = 180) and aborted pig foetuses (n = 24). Leptospire DNA was detected by the 'pathogenic' specific PCR in 25 tissues (14%) and the control beta-actin PCR was positive in all 204 samples confirming DNA was extracted from all samples. No leptospires were isolated from these samples by culture and no positives were detected with the 'saprophytic' PCR. In a subsidiary experiment, the 'pathogenic' PCR was used to analyse kidney samples from rodents (n = 7) collected as part of vermin control in a zoo, with show animals with high microagglutination titres to Leptospira species, and five were positive. Fifteen PCR amplicons from 1 mouse, 2 rat and 14 pig kidney samples, were selected at random from positive PCRs (n = 30) and sequenced. Sequence data indicated L. interrogans DNA in the pig and rat samples and L. inadai DNA, which is considered of intermediate pathogenicity, in the mouse sample. The only successful culture was from this mouse kidney and the isolate was confirmed to be L. inadai by classical serology. These data suggest this suite of PCRs is suitable for testing for the presence of pathogenic leptospires in pig herds where abortions and infertility occur and potentially in other animals such as rodents. Crown Copyright (C) 2007 Published by Elsevier Ltd. All rights reserved.

Evaluation and comparison of SYBR Green I Real-Time PCR and TaqMan Real-Time PCR methods for quantitative assay of Listeria monocytogenes in nutrient broth and milk

Specific traditional plate count method and real-time PCR systems based on SYBR Green I and TaqMan technologies using a specific primer pair and probe for amplification of iap-gene were used for quantitative assay of Listeria monocytogenes in seven decimal serial dilution series of nutrient broth and milk samples containing 1.58 to 1.58×107 cfu /ml and the real-time PCR methods were compared with the plate count method with respect to accuracy and sensitivity. In this study, the plate count method was performed using surface-plating of 0.1 ml of each sample on Palcam Agar. The lowest detectable level for this method was 1.58×10 cfu/ml for both nutrient broth and milk samples. Using purified DNA as a template for generation of standard curves, as few as four copies of the iap-gene could be detected per reaction with both real-time PCR assays, indicating that they were highly sensitive. When these real-time PCR assays were applied to quantification of L. monocytogenes in decimal serial dilution series of nutrient broth and milk samples, 3.16×10 to 3.16×105 copies per reaction (equals to 1.58×103 to 1.58×107 cfu/ml L. monocytogenes) were detectable. As logarithmic cycles, for Plate Count and both molecular assays, the quantitative results of the detectable steps were similar to the inoculation levels.

The development of a ligase mediated PCR with potential for the differentiation of serovars within Leptospira interrogans

A ligase mediated polymerase chain reaction (LMPCR) was developed to amplify between the repetitive element, IS1533, of Leptospira and adjacent chromosomally located BglII restriction endonuclease enzyme sites. To do this, complimentary oligonucleotide linkers designed to anneal together with an overhanging BglII end were ligated to BglII digested DNA from 35 leptospiral reference strains and field isolates, This ligated DNA was used as template for PCR with oligonucleotide primers specific for the linker and for the repetitive element IS1533. The resultant amplicon profile hybridised a 102 hp region derived from the terminus of IS1533 thus confirming that amplicons generated by LMPCR contained part of IS1533. The number of fragments generated containing IS1533 was significantly fewer than that generated by RFLP but the LMPCR method has the potential to use far less template DNA and be quicker than standard RFLP. Obvious and reproducible interserovar differences were demonstrated by LMPCR whereas for 20 of 21 L. hardjo-bovis isolates tested no intraserovar differences were observed. Of those serovars known to possess IS1533 homologues and tested here by LMPCR, each produced a unique amplicon profile which hybridised the IS1533 terminus probe. The limited heterogeneity amongst hardjo-bovis isolates is discussed as is the potential contribution of this method to diagnosis, differentiation and the phylogenetics of the Leptospires.

PCR-based markers diagnostic for spring and winter seasonal growth habit in barley

Toward the ultimate goal of replacing field-based evaluation of seasonal growth habit, we describe the design and validation of a multiplex polymerase chain reaction assay diagnostic for allelic status at the barley (Hordeum vulgare ssp. vulgare L.) vernalization locus, VRN-H1 By assaying for the presence of all known insertion–deletion polymorphisms thought to be responsible for the difference between spring and winter alleles, this assay directly tests for the presence of functional polymorphism at VRN-H1 Four of the nine previously recognized VRN-H1 haplotypes (including both winter alleles) give unique profiles using this assay. The remaining five spring haplotypes share a single profile, indicative of function-altering deletions spanning, or adjacent to, the putative “vernalization critical” region of intron 1. When used in conjunction with a previously published PCR-based assay diagnostic for alleles at VRN-H2, it was possible to predict growth habit in all the 100 contemporary UK spring and winter lines analyzed in this study. This assay is likely to find application in instances when seasonal growth habit needs to be determined without the time and cost of phenotypic assessment and during marker-assisted selection using conventional and multicross population analysis.

Characterization of the complex locus of bean encoding polygalacturonase-inhibiting proteins reveals subfunctionalization for defense against fungi and insects.

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant inhibitors of fungal endopolygalacturonases (PGs) that belong to the superfamily of Leu-rich repeat proteins. We have characterized the full complement of pgip genes in the bean (Phaseolus vulgaris) genotype BAT93. This comprises four clustered members that span a 50-kb region and, based on their similarity, form two pairs (Pvpgip1/Pvpgip2 and Pvpgip3/Pvpgip4). Characterization of the encoded products revealed both partial redundancy and subfunctionalization against fungal-derived PGs. Notably, the pair PvPGIP3/PvPGIP4 also inhibited PGs of two mirid bugs (Lygus rugulipennis and Adelphocoris lineolatus). Characterization of Pvpgip genes of Pinto bean showed variations limited to single synonymous substitutions or small deletions. A three-amino acid deletion encompassing a residue previously identified as crucial for recognition of PG of Fusarium moniliforme was responsible for the inability of BAT93 PvPGIP2 to inhibit this enzyme. Consistent with the large variations observed in the promoter sequences, reverse transcription-PCR expression analysis revealed that the different family members differentially respond to elicitors, wounding, and salicylic acid. We conclude that both biochemical and regulatory redundancy and subfunctionalization of pgip genes are important for the adaptation of plants to pathogenic fungi and phytophagous insects.

Detection of tobacco mosaic tobamovirus in cigarettes through RT-PCR

TMV was tried to recover from a variety of branded cigarettes and cigars. Tobacco from six different brands of cigarettes and cigar were processed and reverse transcriptase polymerase chain reaction was employed for the detection of TMV. RTPCR confirmed the presence of TMV in tobacco from one brand of cigarette and one brand of cigar. Bean plants (Phaseolus vulgaris) were inoculated manually with tobacco sap of cigarettes resulting in the production of localized disease lesions. Together, these results showed that tobacco used to make cigarettes and cigars can function as an effective disease vector, potentially aiding the movement of infectious TMV between countries. This is an important finding prompting a need to test smoking tobacco for other virus particles that infect tobacco plants and survive processing as well as considering biosecurity measures to limit virus transmission