Your browser doesn't support javascript.

Portal de Pesquisa da BVS Veterinária

Informação e Conhecimento para a Saúde

Home > Pesquisa > ()
Imprimir Exportar

Formato de exportação:



Adicionar mais destinatários

Enviar resultado
| |

Molecular identification of Clostridium chauvoei from common filter paper

Farias, Luana D'Avila; Botton, Sônia de Avila; Costa, Mateus Matiuzzi da; Castagna de Vargas, Agueda Palmira.
Acta sci. vet. (Impr.); 40(4): Pub. 1075, 2012. ilus
Artigo em Inglês | VETINDEX | ID: biblio-1377731


Background: Blackleg is an acute and often fatal infection in bovine caused by the bacterium Clostridium chauvoei. The absence of conclusive diagnosis of blackleg usually occurs due to absence of practical and economical methods to send samples to microbiology laboratory. The goal of this work was to verify the possibility of using ordinary filter paper as a practical and economically feasible method for collecting, storing and shipping material to the laboratory to be used in a rapid and direct PCR approach to detect Clostridium chauvoei DNA. Materials, Methods & Results: The PCR technique for the diagnosis of blackleg from common filter paper was tested for specificity, sensitivity and feasibility. To test the specificity, the papers were impregnated with a suspension of the following microorganisms: C. chauvoei, C. perfringens, C. septicum, Bacillus anthracis, Staphylococcus aureus, and Escherichia coli. To test the sensitivity different concentration of C. chauvoei (ATCC 10092) were pipetted on common filter paper. To both test, DNA extraction of impregnated ordinary filter paper and their respective controls followed the method previously described and tested under different storage times (0 h, 24 h, 72 h and a week later). To test the feasibility, 12 bovine livers were collected and tissues samples were impregnate on common filter paper with suspension of C. chauvoei. The filter paper was stored for 48 h, 72 h and one week. Subsequently, a rapid and direct PCR approach to detect C. chauvoei was performed. All procedures were performed in triplicate and was performed by PCR using the same primers employed to amplify the flic gene encoding flagellin (FliC). There was no cross reaction with any tested microorganism, confirming the specificity of the flic gene previously studied. It was possible to visualize the amplification until the corresponding to 100 CFU. Specific PCR amplification products were visualized in 100% of the trials at 48 h, 70% at 72 h, and 90% within one week of storage at room temperature using direct PCR. Discussion: This report describes a rapid, highly sensitive method for the detection of C. chauvoei DNA from liver tissue bovine samples stored on filter papers. It was observed a high sensitivity and a specificity of 100%. The selection of hepatic tissue was based on previous studies that identified C. chauvoei in this tissue by PCR assays. Besides, blackleg in visceral form can be detected in hepatic tissue but does not in muscle. According to others researchers, the direct PCR procedure exhibits several advantages, such as costs and time reduction through omission of DNA extraction as well as avoid any cross contamination with other agents. However, current substances in the blood and tissues may inhibit the PCR amplification. For this reason, a methanol fixation and preheating the samples before the direct PCR assay was performed, mainly because the amplicon is relatively large (535 bp). Some authors consider the use of direct PCR from filter paper simple and inexpensive which offer a handy tool for epidemiologic studies and to clinicians, particularly in many tropical countries where collection and storage of clinical specimens for this purpose are logistically complicated. Furthermore, this procedure can simplify the material shipment for laboratory diagnosis, since it can also be transported in standard envelops by regular mail. The current results propose the use of the direct PCR from common filter paper as practical and economical alternative to diagnosis of blackleg.
Biblioteca responsável: BR68.1