PCR associated with agar gel immunodiffusion assay improve caprine arthritis-encephalitis (CAEV) control

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

PCR detection of staphylococcal enterotoxin genes in Staphylococcus aureus strains isolated from raw and pasteurized milk

Milk is considered a nutritious food because it contains several important nutrients including proteins and vitamins. Conversely, it can be a vehicle for several pathogenic bacteria such as Staphylococcus aureus. This study aimed to analyze the frequency of genes encoding the staphylococcal enterotoxins (SEs) SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI and SEJ in S. aureus strains isolated from raw or pasteurized bovine milk. S. aureus was found in 38 (70.4%) out of 54 raw milk samples at concentrations of up to 8.9 X 10(5) CFU/ml. This microorganism was present in eight samples of pasteurized milk before the expiration date and in 11 samples analyzed on the expiration date. of the 57 strains studied, 68.4% were positive for one or more genes encoding the enterotoxins, and 12 different genotypes were identified. The gene coding for enterotoxin A, sea, was the most frequent ( 16 strains, 41%), followed by sec (8 strains, 20.5%), sed (5 strains, 12.8%). seb (3 strains. 7.7%) and see (2 strains, 5.1%). Among the genes encoding the other enterotoxins, seg was the most frequently observed (11 strains. 28.2%), followed by sei (10 strains) and seh and sej (3 strains each). With the recent identification of new SEs, the perceived frequency of enterotoxigenic strains has increased. suggesting that the pathogenic potential of staphylococci may be higher than previously thoughts however, further studies are required to assess the expression of these new SEs by S. aureus. and their impact in foodborne disease. The quality of Brazilian milk is still low. and efforts from the government and the entire productive chain are required to attain consumer safety. (C) 2008 Elsevier B.V. All rights reserved.

PCR primed with minisatellite core sequences yields species-specific patterns and assessment of population variability in fishes of the genus Brycon

Minisatellite core sequences were used as single primers in polymerase chain reaction (PCR) to amplify genomic DNA in a way similar to the random amplified polymorphic DNA methodology. This technique, known as Directed Amplification of Minisatellite-region DNA, was applied in order to differentiate three neotropical fish species (Brycon orbignyanus, B. microlepis and B. lundii ) and to detect possible genetic variations among samples of the threatened species, B. lundii , collected in two regions with distinct environmental conditions in the area of influence of a hydroelectric dam. Most primers generated species-specific banding patterns and high levels of intraspecific polymorphism. The genetic variation observed between the two sampling regions of B. lundii was also high enough to suggest the presence of distinct stocks of this species along the same river basin. The results demonstrated that minisatellite core sequences are potentially useful as single primers in PCR to assist in species and population identification. The observed genetic stock differentiation in B. lundii associated with ecological and demographic data constitute a crucial task to develop efficient conservation strategies in order to preserve the genetic diversity of this endangered fish species.

Discrimination of Shark species by simple PCR of 5S rDNA repeats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genetic identification of lamniform and carcharhiniform sharks using multiplex-PCR

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identification of hybrids between Neotropical fish Leporinus macrocephalus and Leporinus elongatus by PCR-RFLP and multiplex-PCR: Tools for genetic monitoring in aquaculture

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genetic identification of the sharks Rhizoprionodon porosus and R. lalandii by PCR-RFLP and nucleotide sequence analyses of 5S rDNA

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identification of the shark species Rhizoprionodon lalandii and R-porosus (Elasmobranchii, Carcharhinidae) by multiplex PCR and PCR-RFLP techniques

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Forensic identification of the guitarfish species Rhinobatos horkelli, R-percellens and Zapteryx brevirostris using multiplex-PCR

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Faecal examination and PCR to detect Strongyloides venezuelensis in experimentally infected Lewis rats

More sensitive methodologies are necessary to improve strongyloidiasis diagnosis. This study compared the sensitivities of the McMaster modified technique and polymerase chain reaction (PCR) assays, both performed in faecal samples. Lewis rats were subcutaneously infected with 4,000, 400 or 40 infective third-stage larvae, considered as high, moderate or low infection, respectively. Seven days later, they were euthanized to count adult nematodes recovered from the small intestine. Stool samples were used to count the number of eggs per gram (EPG) of faeces and to detect parasite DNA by PCR performed with a species and a genus primer pair. The sensitivity of these assays depended upon parasite burden and the primer specificity. All assays presented 100% sensitivity at the highest parasite load. In the moderate infection, EPG and PCR with the genus primer maintained 100% specificity, whereas PCR sensitivity with the species primer decreased to 77.7%. In low infection, the sensitivity was 60% for EPG, 0% for PCR with the species primer and 90% for PCR done with the genus primer. Together, these results suggest that PCR with a genus primer can be a very sensitive methodology to detect Strongyloides venezuelensisin faeces of Lewis rats infected with very low parasite burden.

Quantification of bovine cytokine gene expression using real-time RT-PCR methodology

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Diagnosis of Giardia infections by PCR-based methods in children of an endemic area

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Advantages of using nested collision induced dissociation/post-source decay with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: sequencing of novel peptides from wasp venom

We have examined the applicability of the 'nested' collision induced dissociation/post-source decay (CID/PSD) method to the sequencing of novel peptides from solitary wasps which have neurotoxic venom for paralyzing other insects. The CID/PSD spectrum of a ladder peptide derived from an exopeptidase digest was compared with that of the intact peptide. The mass peaks observed only in the CID/PSD spectrum of a ladder peptide were extracted as C-terminal fragment ions. Assignment of C-terminal fragment ions enabled calculation of N-terminal fragment masses, leading to differentiation between N-terminal fragment ions and internal fragment ions. This methodology allowed rapid and sensitive identification by removing ambiguity in the assignment of the fragment ions, and proved useful for sequencing unknown peptides, in particular those available as natural products with a limited supply. Copyright (C) 2000 John Wiley & Sons, Ltd.

A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements

We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2'deoxycytidine (DAC), where we found a 1-16% decrease in Alu element and 18-60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation.