Polymerase chain reaction and real-time PCR for diagnosing of Leishmania infantum chagasi in dogs

A importância do cão como reservatório de L. infantum chagasi no meio urbano tem estimulado a realização de inúmeros trabalhos de avaliação de técnicas de diagnóstico, uma vez que este procedimento, quando realizado corretamente, torna-se um importante passo na prevenção da doença em humanos. Dentre os métodos de diagnóstico, as técnicas moleculares têm adquirido destaque. Objetivou-se neste trabalho verificar o desempenho da Reação em Cadeia da Polimerase (PCR) e da PCR em tempo real (qPCR) para diagnóstico da Leishmaniose Visceral Canina (LVC) utilizando diferentes amostras biológicas. Para tanto foram utilizados 35 cães provenientes de uma área endêmica para LVC, onde foram utilizados para o diagnóstico molecular, aspirado de medula óssea, fragmentos de linfonodo e baço. Neste estudo a qPCR foi capaz de detectar um maior número de animais positivos quando comparada com a PCR. Já entre as diferentes amostras biológicas utilizadas não foi observada diferença significativa na detecção de DNA de L. infantumchagasi por meio da PCR e qPCR. Mesmo assim, considerando a facilidade de obtenção, o linfonodo pode ser considerada como a melhor amostra para diagnóstico molecular da infecção por L. infantum chagasi.

Identification of reference genes for expression analysis by real-time quantitative PCR in drought-stressed soybean

O objetivo deste trabalho foi validar, pela técnica de PCR quantitativo em tempo real (RT-qPCR) genes para serem utilizados como referência em estudos de expressão gênica em soja, em ensaios de estresse hídrico. Foram avaliados quatro genes comumente utilizados em soja: Gmβ-actin, GmGAPDH, GmLectin e GmRNAr18S. O RNA total foi extraído de seis amostras: três amostras de raízes em sistema de hidroponia com diferentes intensidades de déficit hídrico (0, 25, 50, 75 e 100 minutos de estresse hídrico), e três amostras de folhas de plantas cultivadas em areia com diferentes umidades do solo (15, 5 e 2,5% de umidade gravimétrica). Os dados brutos do intervalo cycle threshold (Ct) foram analisados, e a eficiência de cada iniciador foi calculada para uma analise da Ct entre as diferentes amostras. A aplicação do programa GeNorm foi utilizada para a avaliação dos melhores genes de referência, de acordo com a estabilidade. O GmGAPDH foi o gene menos estável, com o maior valor médio de estabilidade de expressão (M), e os genes mais estáveis, com menor valor de M, foram o Gmβ-actin e GmRNAr18S, tanto nas amostras de raízes como nas de folhas. Estes genes podem ser usados em RT-qPCR como gens de referência para análises de expressão gênica.

Genetic grouping of avian infectious bronchitis virus isolated in Brazil based on RT-PCR/RFLP analysis of the S1 gene

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

Quantitative real-time PCR identifies a critical region of deletion on 22q13 related to prognosis in oral cancer

Quantitative real time PCR was performed on genomic DNA from 40 primary oral carcinomas and the normal adjacent tissues. The target genes ECGFB, DIA1, BIK, and PDGFB and the microsatellite markers D22S274 and D22S277, mapped on 22q13, were selected according to our previous loss of heterozygosity findings in head and neck tumors. Quantitative PCR relies on the comparison of the amount of product generated from a target gene and that generated from a disomic reference gene (GAPDH-housekeeping gene). Reactions have been performed with normal control in triplicates, using the 7700 Sequence Detection System (PE Applied Biosystems). Losses in the sequences D22S274 (22q13.31) and in the DIA1 (22q13.2-13.31) gene were detected in 10 out of 40 cases (25%) each. Statistically significant correlations were observed for patients with relative copy number loss of the marker D22S274 and stages T3-T4 of disease (P=0.025), family history of cancer (P = 0.001), and death (P = 0.021). Relative copy number loss involving the DIA1 gene was correlated to family history of cancer (P<0.001), death (P=0.002), and consumption of alcohol (P=0.026). Log-rank test revealed a significant decrease in survival (P=0.0018) for patients with DIA1 gene loss. Relative copy number losses detected in these sequences may be related to disease progression and a worse prognosis in patients with oral cancer.

The incidence of Shiga toxin-producing Escherichia coli in cattle with mastitis in Brazil

Aims: To determine the prevalence and molecular characteristics of Shiga toxin-producing Escherichia coli (STEC) isolates from bovine mastitic milk in Brazil.Methods and Results: A total of 2144 milk samples from dairy cattle showing mastitis were screened for the presence of E. coli. A total of 182 E. coli isolates were selected and examined. All were subjected to dot blot analysis using the CVD419 probe for the detection of the enterohaemolysin (hly) gene, and to a multiplex PCR for the detection of stx1, stx2 and eaeA genes. STEC were isolated from 22 (12.08%) milk samples. All the STEC isolates were tested for sensibility to 10 antimicrobials; the resistances most commonly observed were to cephalothin (86.3%), tetracycline (63.6%) and doxycycline (63.6%).Conclusion: STEC isolates were found in bovine mastitic milk in Brazil.Significance and Impact of the Study: STEC isolates from mastitic milk were potentially pathogenic for human in that they belonged to serogroups associated with diarrhoea and haemolytic-uraemic syndrome, some of them were stx2, eaeA and hly positive.

Diagnosis of canine brucellosis: Comparison between serological and microbiological tests and a PCR based on primers to 16S-23S rDNA interspacer

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol-chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.

A SIMPLE TECHNIQUE FOR ISOLATION OF DNA SUITABLE FOR PCR AMPLIFICATION FROM CYTOGENETIC PREPARATIONS

In order to rescue molecular information from chromosome preparations, we describe a rapid procedure to obtain DNA from cytogenetic preparations in microscope slides, stored for one to live years at room temperature. This technique was modified from previously described procedures and the DNA obtained was shown to be suitable for PCR amplification.

A novel PCR-RFLP assay for the detection of the single nucleotide polymorphism at position+1440 in the human CXCR2 gene

We designed a novel PCR-RFLP assay to genotype for the CXCR2 +1440 (G/A) single nucleotide polymorphism, which provides a simple, low-cost, practical, and reproducible method. Allele frequencies in healthy Brazilian individuals were found to be 0.65% for allele A and 0.35% for allele G.

Use of PCR-RFLP (Polymerase Chain Reaction-Restricted Fragment Length Polymorphism) in the gene of the enzyme Stearoyl-CoA-Desaturase in Bubalus bubalis

The milk is an important food because it contents Conjugated Linoleic Acids (CIA). These fatty acids are synthesized in mammary gland under action of the enzyme Stearoyl CoA-Desaturase (SCD) and have showed some positive effects in human disease prevention and treatments. A variation of CLA in milk fat exists and can be partially explained by the different levels of expression of SCD. The aim was to study part of the encoding regions of SCD's gene using PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). Genomic DNA was extracted from lactating Murrah females. After this, PCR reactions were made by using primers Z (sic) (sic) D1 that encloses exon I, II and intron I. The fragments amplified are composed by 938 pb. Then, RFLP techniques were applied in the fragments using the restriction enzymes Pst I and Sma I. The enzyme Pst I has generated fragments of 788pb and 150bp and the Sma I has generated fragments of 693pb and 245pb. All the animals showed the same migration standard for both enzymes, characterizing a genetic monomorphism for this region of SCD gene. The analysis determined that there aren't genetic differences between these animals in the studied regions by using Pst I and Sma I enzymes.

Epstein-Barr Virus (EBV) detection and typing by PCR: a contribution to diagnostic screening of EBV-positive Burkitt's lymphoma

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Real-time PCR assay targeting the actin gene for the detection of Cryptosporidium parvum in calf fecal samples

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

Parentage testing in alpacas (Vicugna pacos) using semi-automated fluorescent multiplex PCRs with 10 microsatellite markers

The aim of this study was to assess and apply a microsatellite multiplex system for parentage determination in alpacas. An approach for parentage testing based on 10 microsatellites was evaluated in a population of 329 unrelated alpacas from different geographical zones in Peru. All microsatellite markers, which amplified in two multiplex reactions, were highly polymorphic with a mean of 14.5 alleles per locus (six to 28 alleles per locus) and an average expected heterozygosity (H-E) of 0.8185 (range of 0.698-0.946). The total parentage exclusion probability was 0.999456 for excluding a candidate parent from parentage of an arbitrary offspring, given only the genotype of the offspring, and 0.999991 for excluding a candidate parent from parentage of an arbitrary offspring, given the genotype of the offspring and the other parent. In a case test of parentage assignment, the microsatellite panel assigned 38 (from 45 cases) offspring parentage to 10 sires with LOD scores ranging from 2.19 x 10(+13) to 1.34 x 10(+15) and Delta values ranging from 2.80 x 10(+12) to 1.34 x 10(+15) with an estimated pedigree error rate of 15.5%. The performance of this multiplex panel of markers suggests that it will be useful in parentage testing of alpacas.