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1.
Article in English | MEDLINE | ID: mdl-24808911

ABSTRACT

BACKGROUND: Molecular biological methods have become increasingly relevant to the diagnosis and control of infectious diseases, such as leishmaniasis. Since various factors may affect the sensitivity of PCR assays, including DNA yield and purity, an optimal extraction method is pivotal. Losses of a parasite's DNA during extraction may significantly impair its detection by PCR and lead to false-negative results. This study proposes a triplex PCR assay targeting the parasite's DNA, an external control (pUC18) and an internal control (G3PD) for accurate diagnosis of leishmaniasis. RESULTS: Two primer pairs were designed to detect the plasmid pUC18 and a triplex PCR assay targeting the Leishmania braziliensis kinetoplast DNA, the external control and the internal control was standardized. The triplex PCR assay was assessed for its ability to detect the three target DNA fragments simultaneously. PCR products from pUC18 DNA resulted in bands of 368 (P1) and 316 (P2) base pairs (bp). The triplex PCR optimized with the chosen external control system (P1) allowed the simultaneous detection of the internal control (G3PD - 567 bp) as well as of small quantities (10 pg) of the target parasite's DNA, detected by amplification of a 138 bp product. CONCLUSIONS: The new tool standardized herein enables a more reliable interpretation of PCR results, mainly by contributing to quality assurance of leishmaniasis diagnosis. Furthermore, after simple standardization steps, this protocol could be applied to the diagnosis of other infectious diseases in reference laboratories. This triplex PCR enables the assessment of small losses during the DNA extraction process, problems concerning DNA degradation (sample quality) and the detection of L. braziliensis kDNA.

2.
Article in English | LILACS | ID: lil-724683

ABSTRACT

Molecular biological methods have become increasingly relevant to the diagnosis and control of infectious diseases, such as leishmaniasis. Since various factors may affect the sensitivity of PCR assays, including DNA yield and purity, an optimal extraction method is pivotal. Losses of a parasite’s DNA during extraction may significantly impair its detection by PCR and lead to false-negative results. This study proposes a triplex PCR assay targeting the parasite’s DNA, an external control (pUC18) and an internal control (G3PD) for accurate diagnosis of leishmaniasis.


Subject(s)
Animals , Diagnosis , False Negative Reactions , Leishmaniasis/pathology , Polymerase Chain Reaction
3.
J. venom. anim. toxins incl. trop. dis ; 20: 1-6, 04/02/2014. tab, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1484572

ABSTRACT

Molecular biological methods have become increasingly relevant to the diagnosis and control of infectious diseases, such as leishmaniasis. Since various factors may affect the sensitivity of PCR assays, including DNA yield and purity, an optimal extraction method is pivotal. Losses of a parasite’s DNA during extraction may significantly impair its detection by PCR and lead to false-negative results. This study proposes a triplex PCR assay targeting the parasite’s DNA, an external control (pUC18) and an internal control (G3PD) for accurate diagnosis of leishmaniasis.


Subject(s)
Animals , Diagnosis , Leishmaniasis/pathology , Polymerase Chain Reaction , False Negative Reactions
4.
DNA Repair (Amst) ; 7(11): 1882-92, 2008 Nov 01.
Article in English | MEDLINE | ID: mdl-18761429

ABSTRACT

Mammalian DNA polymerase beta is a nuclear enzyme involved in the base excision and single-stranded DNA break repair pathways. In trypanosomatids, this protein does not have a defined cellular localization, and its function is poorly understood. We characterized two Trypanosoma cruzi proteins homologous to mammalian DNA polymerasebeta, TcPolbeta and TcPolbetaPAK, and showed that both enzymes localize to the parasite kinetoplast. In vitro assays with purified proteins showed that they have DNA polymerization and deoxyribose phosphate lyase activities. Optimal conditions for polymerization were different for each protein with respect to dNTP concentration and temperature, and TcPolbetaPAK, in comparison to TcPolbeta, conducted DNA synthesis over a much broader pH range. TcPolbeta was unable to carry out mismatch extension or DNA synthesis across 8-oxodG lesions, and was able to discriminate between dNTP and ddNTP. These specific abilities of TcPolbeta were not observed for TcPolbetaPAK or other X family members, and are not due to a phenylalanine residue at position 395 in the C-terminal region of TcPolbeta, as assessed by a site-directed mutagenesis experiment reversing this residue to a well conserved tyrosine. Our data suggest that both polymerases from T. cruzi could cooperate to maintain mitochondrial DNA integrity through their multiple roles in base excision repair, gap filling and translesion synthesis.


Subject(s)
DNA Polymerase beta/metabolism , DNA, Mitochondrial/metabolism , Trypanosoma cruzi/enzymology , p21-Activated Kinases/metabolism , Amino Acid Sequence , Animals , Biochemistry/methods , Cloning, Molecular , DNA Primers/chemistry , Microscopy, Confocal , Models, Biological , Molecular Sequence Data , Mutagenesis, Site-Directed , Recombinant Proteins/chemistry , Sequence Homology, Amino Acid
5.
Exp Parasitol ; 116(1): 25-34, 2007 May.
Article in English | MEDLINE | ID: mdl-17189633

ABSTRACT

Using a functional complementation strategy, we have isolated a Schistosoma mansoni cDNA that complemented Escherichia coli mutant strains which are defective in the DNA base excision repair pathway. This cDNA partially complemented the MMS-sensitive phenotype of these strains. The sequence of the isolated cDNA was homologous to genes involved in the RNA metabolism pathway, especially ScIMP4 of Saccharomyces cerevisiae. To establish whether the S. mansoni cDNA clone could complement yeast ScIMP4-defective mutants, we constructed a yeast haploid strain that coded for a truncated Imp4p protein. This mutant strain was treated with different DNA damaging agents, but showed only MMS sensitivity. The functional homology between the ScIMP4 gene and the cDNA from S. mansoni was verified by partial complementation of the mutant yeast with the worm's gene. This gene appears to be involved in DNA repair and RNA metabolism in both S. mansoni and S. cerevisiae.


Subject(s)
Alkylating Agents/pharmacology , DNA Repair/genetics , Methyl Methanesulfonate/pharmacology , Ribosomal Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Schistosoma mansoni/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary/chemistry , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/radiation effects , Gene Library , Genetic Complementation Test , Hydroxyurea/pharmacology , Molecular Sequence Data , Mutation , Nucleic Acid Synthesis Inhibitors/pharmacology , Phenotype , Polymerase Chain Reaction , Ribosomal Proteins/chemistry , Ribosomal Proteins/physiology , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/radiation effects , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/physiology , Schistosoma mansoni/drug effects , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid
6.
Mol Biochem Parasitol ; 149(2): 191-200, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16828179

ABSTRACT

The Rad51 gene encodes a highly conserved enzyme involved in DNA double-strand break (DSB) repair and recombination processes. We cloned and characterized the Rad51 gene from Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. This gene is expressed in all three forms of the parasite life cycle, with mRNA levels that are two-fold more abundant in the intracellular amastigote form. The recombinase activity of the TcRad51 gene product was verified by an increase in recombination events observed in transfected mammalian cells expressing TcRad51 and containing two inactive copies of the neomycin-resistant gene. As a component of the DSB repair machinery, we investigated the role of TcRad51 in the resistance to ionizing radiation and zeocin treatment presented by T. cruzi. When exposed to gamma irradiation, different strains of the parasite survive to dosages as high as 1 kGy. A role for TcRad51 in this process was evidenced by the increased expression of its mRNA after irradiation. Furthermore, transfected parasites over-expressing TcRad51 have a faster kinetics of recovery of the normal pattern of chromosomal bands after irradiation as well as a higher resistance to zeocin treatment than do wild-type cultures.


Subject(s)
Genes, Protozoan , Protozoan Proteins/genetics , Rad51 Recombinase/genetics , Trypanosoma cruzi/genetics , Trypanosoma cruzi/radiation effects , Amino Acid Sequence , Animals , Base Sequence , CHO Cells , Cricetinae , DNA, Protozoan/genetics , Gamma Rays , Humans , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Protozoan/genetics , RNA, Protozoan/metabolism , Radiation Tolerance/genetics , Recombination, Genetic , Sequence Homology, Amino Acid , Trypanosoma cruzi/enzymology , Trypanosoma cruzi/pathogenicity
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