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Rapid Detection of Infectious Laryngotracheitis Virus by Standardization of Polymerase Chain Reaction Targeting a Relatively Conserved Region of the Thymidine Kinase Gene

S. Chakma
Department of Pathology, Bangladesh Agricultural University, Mymensingh

S. Sarker
Department of Animal Husbandry and Veterinary Science, University of Rajshahi, Rajshahi

S. Talukder
Department of Animal Science and Nutrition, Chittagong Veterinary and Animal Sciences University, Khulshi, Chittagong

M.H. Haque
Department of Animal Husbandry and Veterinary Science, University of Rajshahi, Rajshahi

E.H. Chowdhury
Department of Pathology, Bangladesh Agricultural University, Mymensingh

and A.S.M. Bari
Department of Pathology, Bangladesh Agricultural University, Mymensingh

Abstract/ Executive Summary:

Infectious laryngotracheitis is an acute upper respiratory tract infection of chickens caused by infectious laryngotracheitis virus. The study was conducted to standardize the polymerase chain reaction targeting a relatively conserved region of the thymidine kinase gene for the rapid detection of infectious laryngotracheitis virus. The vaccine samples were collected from two renowned company of Bangladesh. DNA was extracted from diluted vaccine samples by using Wizard® Genomic DNA purification kit and thymidine kinase gene was amplified by using PCR system 9600 Thermocycler. Two vaccine samples were positively amplified by polymerase chain reaction. A procedure was developed for rapid detection of infectious laryngotracheitis virus by polymerase chain reaction of the conserved region of viral thymidine kinase gene containing DNA fragments. The results obtained in this study suggested that the polymerase chain reaction procedure could serve as a fast and sensitive method for the detection of vaccine strains of infectious laryngotracheitis viruses.

Keywords: Infectious laryngotracheitis virus, viral thymidine kinase (TK) gene, polymerase chain

Location:

Start Date:

End Date:

Program Area: Pest Management

Commodity:

Objectives:

The present study was carried out to standardize the polymerase chain reaction (PCR) targeting a relatively conserved region of the thymidine kinase (TK) gene for the rapid detection of ILT virus.

Methodology:

The Nobilis ILT and Gallivac LT freeze dried vaccine samples were collected respectively from the Intervet, Bengal Overseas Limited and Advance Animal Science Company Limited, Dhaka, Bangladesh, and carried in an ice box to the Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural PCR amplification was performed in a final volume of 10μl containing 2μl of extracted DNA template, Taq DNA polymerase buffer (10x) 1μl, dNTPs 1μl, primer-F (50n mol) 0.5μl, primer-R (50n mol) 0.5μl, Taq DNA polymerase 0.2μl and 4.8μl nuclease free water (Science Park Rd.#01-23, The Geni S’pore). 2μl water was added instead of DNA to the water control tube. Amplification was carried out in Gene amplification PCR system 9600 Thermocycler (eppendorf, Germany), using condition modified from Abbas et al., (1996). The pre-mix was then mixed well through spinning. Initial denaturation was at 94°C for 1 min, again denaturation was at 95°C for 1 minute, annealing at 50°C for 1 minute and extension at 72°C for 1.5 minutes, with a final extension at 72°C for 5 minutes for total 35 cycles and held for 4°C in refrigerator until electrophoresis.University, Mymensingh. The vaccine was diluted with the given sterile buffered and colored solvent and stored at 2-80C.DNA was extracted from diluted vaccine samples by using Wizard Genomic DNA purification kit (Promegra Corporation. 2800 Woods Hollow Road. Madison, USA). The extracted DNA was quantified using a spectrophotometer’s (SpectronicGeneticsTM New York, USA) and expressed in ng/μl.An appropriate primer sequence for PCR was selected to amplify thymidine kinase (TK) gene (Griffin & Boursnell, 1990) of ILT virus. The primer sequences were synthesized commercially by Sangamo Biosciences, Inc., Singapore.The amplified PCR products were separated by electrophoresis on 1.5% agarose gel containing 5μl ml-1 ethidium bromide with a 100 bp ladder (Promega, Madison, WI, USA) as molecular weight marker (Oliveira et al., 2003). After electrophoresis, the gel was taken out carefully from the gel chamber and placed on the UV Transilluminator in the dark chamber of the Image Documentation System (Labortechnik, Germany).

Source of Information: Univ. j. zool. Rajshahi. Univ. Vol. 29, 2010 pp. 61-64 ISSN 1023-6104

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In spite of the use of vaccines for the control of ILTV, the disease continues to be a problem in commercial poultry. The use of live and modified live vaccines has been demonstrated to lead to spread of the virus, particularly within 7-10 days of vaccination. Carriers may result, and it is possible that the infection become indigenous and thus the vaccine strains of the virus may be involved in ILTV outbreaks. These findings provide support for the requirement of continuous monitoring of the vaccine strains and the development of molecular techniques that may allow simple and reliable identification of different vaccine strains.

Knowledge Management: Journal