Related Experiment Videos
Dot-blot hybridization using digoxigenin-labeled cDNA probe complementary to the S1 gene of avian infectious
H Nagano1, H Hashimoto, Y Tanaka
1Research Center for Veterinary Science, Kitasato Institute, Chiba, Japan.
Abstract:
Digoxigenin-dUTP-labeled DNA probe was prepared from a cDNA clone complementary to the gene encoding S1 region of the spike protein of infectious bronchitis coronavirus (IBV) strain M41. The probe exclusively reacted with four strains at 56 degrees C which were grouped to the same serotype as the strain used for the probe. In contrast, at 68 degrees C, the probe reacted only with the homologous strain and did not react even with the strains belonging to the same serotype. The dot-blot hybridization thus appeared serotype-specific at 56 degrees C and strain-specific at 68 degrees C. In addition, it was revealed that the S1 gene has some nucleotide sequence variation even among strains in the same serotype. This technique should be applied to determining serotypes of the virus isolates and to differentiating field isolates from the vaccine strain.
Insights
A digoxigenin-dUTP-labeled DNA probe targeting the infectious bronchitis coronavirus (IBV) S1 gene can differentiate viral serotypes at 56°C and specific strains at 68°C. This method aids in identifying IBV serotypes and distinguishing field from vaccine strains.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Infectious bronchitis virus (IBV) is a significant pathogen in poultry.
- Accurate differentiation of IBV strains is crucial for effective disease control and vaccine development.
- The spike protein's S1 region is a key determinant of viral serotype and host interaction.
Purpose of the Study:
- To develop a molecular method for differentiating IBV strains.
- To assess the serotype- and strain-specificity of a DNA probe targeting the IBV S1 gene.
- To evaluate the utility of this method for identifying field isolates and vaccine strains.
Main Methods:
- Preparation of a digoxigenin-dUTP-labeled DNA probe from a cDNA clone of the IBV M41 strain S1 gene.
- Dot-blot hybridization assays performed at two different temperatures (56°C and 68°C).
- Testing the probe's reactivity against multiple IBV strains from the same and different serotypes.
Main Results:
- The DNA probe demonstrated serotype-specific reactivity at 56°C, hybridizing with IBV strains of the same serotype.
- At 68°C, the probe exhibited strain-specific reactivity, hybridizing only with the homologous IBV M41 strain.
- Nucleotide sequence variation within the S1 gene was observed even among strains of the same serotype.
Conclusions:
- Dot-blot hybridization using an S1 gene probe is a valuable tool for serotype and strain differentiation of IBV.
- The temperature-dependent specificity allows for flexible application in diagnostics.
- This technique can aid in monitoring IBV epidemiology and distinguishing field isolates from vaccine strains.