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Establishment and Application of TaqMan Probe Real-time PCR for DRV

Author: YanJin
Tutor: WuBaoCheng；LiangQuanShun；GuanYuFang
School: Fujian Agriculture and Forestry University
Course: Clinical Veterinary Medicine
Keywords: Muscovy duck reovirus TaqMan probe Real-time fluorescent RT-PCR Detect
CLC: S858.32
Type: Master's thesis
Year: 2009
Downloads: 89
Quote: 0
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Abstract


Muscovy duck reovirus infection in recent years in the China Fujian, Zhejiang, Jiangsu, Guangdong, China Southern Muscovy duck feeding area a new Muscovy duck blight, also known as the Muscovy duck liver white spot disease, commonly known as \white spot disease \Higher morbidity and mortality, causing serious economic losses to the Muscovy duck farming. Therefore, the establishment of a practical and rapid method for the diagnosis of waterfowl breeding industry at home and abroad more and more concern. Ducks on the subject to the laboratory isolated and identified save the solitary virus YB isolate reovirus research material, using a TaqMan probe method against Muscovy duck reovirus real-time fluorescence RT-PCR diagnostic methods and conducted a preliminary Application. Of this study include: 1. Optimization of reaction conditions on the real-time fluorescent RT-PCR diagnostic method with muscovy embryo amplification virus extraction of viral nucleic acid to optimize the reaction conditions with the nucleic acid. Through the test showed that the annealing / extension temperature of 57 ℃, the end of the upstream primer and the downstream primer concentration of 0.7 um, and the TaqMan probe of a final concentration of 0.5 uM, can be obtained Preferably the CT value and the Delta Rn value. 2 Preparation and repeatability of the standard test the target fragment obtained by RT-PCR Gel Extraction, the recovered fragments were ligated PMD18-T Vector and then ligated product was transformed into DH5α competent cells cultured recombinant bacteria, and then extract recombinant plasmid. Standard positive control, the recombinant plasmid can do for the diagnosis and recombinant bacteria easy to store, the recombinant plasmid easy access, more convenient way than to save the virus. Measure the concentration of its copy number of the recombinant plasmid, was diluted to 1.0 × 1010, save as a standard. After the standard 10-fold dilution, selected three concentrations of standard, each concentration four times repeated tests to verify the stability of the method, the test results show that the smaller the coefficient of variation, and good stability. 3 Construction of the standard curve in the standard 10-fold dilution selected five concentration gradient, each gradient do four replications to make the standard curve. The slope of the standard curve (Slope) is -3.1, and the correlation coefficient (R2) was 0.994, and the standard curve, a good correlation. 4 a higher sensitivity of the diagnostic method is applied, can be detected at least 100 copies / UL standard goods and 100 times higher than the ordinary PCR sensitivity. By the specificity of the test showed that the diagnostic methods are not subject to interference from other common duck virus. Muscovy Duck artificial attack drug, its detection of liver tissue, the detection rate of 100%. The application of the diagnosis method can also be of clinical samples collected for testing.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Poultry > Duck
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