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| Author(s): |
Marcela da Silva Rubio
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Jaboticabal. 2018-01-24. |
| Institution: | Universidade Estadual Paulista (Unesp). Faculdade de Ciências Agrárias e Veterinárias. Jaboticabal |
| Defense date: | 2017-12-11 |
| Advisor: | Angelo Berchieri Junior; Rafael Antonio Casarin Penha Filho |
| Abstract | |
Poultry p roducts are often associated with food outbreaks from Salmonella spp. and c urrently there are 2659 Salmonella serovars. Existing programs for disease prevention and control are complex due to the various sources of contamination within the poultry industry . Obtaining a rapid diagnostic test for identification of bacteria is cruci al in order to rapidly implement control measures to contain bacterial spread, to reduce losses in animal production and to avoid risks from food - borne infections to human health. The objective of the present study was to perform a standardization and application of the real - time PCR for indirect diagnosis and quantification of bacterial load in organs and feces of commercial birds infected by Salmonella enterica subesp. enterica so rovares Enteritidis, Typhimurium and Gallinarum (biovares Gallinarum and Pullorum), a t different post - infection moments. The present work describes the development of two multiplex qPCR using a low - cost DNA dye (SYBr Green) , one for quantification and diff erential diagnosis between S . Gallinarum and S . Pullorum , and another to quantify and differentiate S . Enteritidis and S . Typhimurium . After qPCR standardization , f our experimental groups were inoculated with each strains , orally at the 13th week of life of the birds. At four and seven days post challenge, t h ree birds from each group were euthanasia for liver and cecal content sample s collection. After the sampling , was carried out b acterial identification and quantification by real - time PCR and conventional microbiology . According to the multiplex qPCR standardization, the melting temperatures ( Tm ) was evaluated to obtain the differential diagnosis . For the first reaction, t he pSGP amplicon (97 bp) showed Tm of 78°C for both biovars. The pSG amplicon (273 bp) showed a Tm of 86.2°C for S . Gallinarum and pSP amplicon (260 bp) dissociated at 84.8°C for S . Pullorum identification. For the second reaction, a Tm of 85°C ( 206bp ) amplified product for S . Enteritidis and Tm of 79°C ( 62bp ) amplified product for S . Typhimurium . The standard curve showed the high sensitivity of the proposed test, since it was possible to obtain eight quantification points, vii starting at 10 8 CFU/mL and ending at 10 1 CFU/mL. When the reaction s were applied in samples from birds infected with each respective bacteria, it was possible to evidence the high sens itivity and specificity of qPCR , especially when the samples came from feces, because obtained positive results even from samples with a large number of contaminants and low bacterial load. As a result of the present study, multiplex qPCR reaction s with high sensitivity, specificity and based on the fluorescence of SYBr Green was standardi zed. In addition, this methodology aligns low cost to the faster diagnostic result, making it attractive for application in routine laboratory analyzes. (AU) | |
| FAPESP's process: | 14/05496-7 - Use of quantitative Real-Time PCR for differential diagnosis of Salmonella enterica subsp. enterica serovar Enteritidis, Typhimurium and Gallinarum (biovars Gallinarum and Pullorum) in domestic poultry (Gallus Gallus) |
| Grantee: | Marcela da Silva Rubio |
| Support Opportunities: | Scholarships in Brazil - Doctorate |