Molecular diagnosis of Anaplasma marginale in cattle: quantitative evaluation of a real-time PCR (Polymerase Chain Reaction) based on msp5 gene

The rickettsia Anaplasma marginale is considered the main agent of bovine anaplasmosis. Due the nonspecific clinical signs of the anaplasmosis, the diagnosis of infection depends of laboratory confirmation. In recent years, molecular diagnostic methods have been used to detect A. marginale in cattle. However, the existence of a large number of assays of different sensitivity and cost makes the choice of an appropriate test difficult. In the present study, a real-time Polymerase Chain Reaction (PCR) based on the msp5 target gene was quantitatively assessed and compared to an end point PCR. Both reactions were subjected to sensitivity and specificity evaluation using plasmid DNA and samples from cattle experimentally infected with A. marginale. A comparative field trial of the tests was carried out using samples of cattle from a stable enzootic area for A. marginale. The real-time PCR showed a higher sensitivity than the end point PCR. This reaction (i.e. real-time PCR) was able to detect one copy of the msp5 gene in 100 ηg of plasmidial DNA, and more than 80% of its results were positive among experimentally infected animals seven days after infection. In addition, based on in silico analysis, the real-time PCR evaluated in the present study appears to be useful for the detection of A. ovis.

Genetic purity certificate in seeds of hybrid maize using molecular markers

One of the main features that confer high quality to the seed is its genetic purity, in which one of the major causes of contamination is the self-pollination of the female parent. Up to date, there is no accurate and fast methods for detecting such contamination. Thus, this work was carried out to certify the genetic purity in seeds of hybrid maize using different biochemical and DNA-based markers. Two single-cross hybrids and their parental lines derived from the maize breeding program at UFLA were evaluated by isoenzymatic pattern of alcohol dehydrogenase (ADH), esterase (EST), acid phosphatase (ACP), glutamate-oxaloacetate transaminase (GOT), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), phosphoglucomutase (PGM), 6-phosphoglucomate dehydrogenase (PGDH), catalase (CAT) and ß-glucosidade (ßGLU) and by microsatellites markers. The enzymatic systems that were able to distinguish the hybrids from their parental line were the catalase, the isocitrate dehydrogenase and the esterase. The esterase showed a Mendelian segregation pattern for UFLA 8/3 hybrid, that enables a safer genetic purity certificate. Microsatellites were able to differentiate the hybrid lines and the respective parental lines. Moreover, this technique was fast, precise and without environment effects. For microsatellites, the amplification pattern was identical when young leaves or seeds were used as DNA source. The possibility of using seeds as DNA source would accelerate and facilitate the role process of the genetic purity analysis.