P Antony, G Nair, V Jayaprakasan, M Mini, T Aravindakshan
P Antony, G Nair, V Jayaprakasan, M Mini, T Aravindakshan. Nucleic Acid Based Differentiation Of Pasteurella Multocida Serotypes. The Internet Journal of Veterinary Medicine. 2006 Volume 2 Number 2.
Knowledge of the serotype of Pasteurella multocida (P. multocida) involved in an outbreak is essential in order to constitute effective control measures. Antigenic characterization of P. multocida is accomplished by capsular serogrouping and somatic serotyping. These typing techniques are carried out by reference laboratories only. This often leads to delay in knowing the serotype of the isolate. DNA based techniques for differentiation of serotypes can provide an alternative to conventional. A PCR-REA technique was standardized to distinguish P. multocida serotypes A:1, A:3 and B:2
To treat a particular disease and to adopt effective preventive measures against the same, the causative agent has to be found out as quickly as possible. Conventional methods for diagnosis of pasteurellosis rely on the detection of the organism by microscopy and its isolation and identification. However as far as pasteurellosis is concerned it is not just sufficient to know the identity of the organism. To constitute effective control measures it is important to know the serotype of the organism.
Recently a multiplex Polymerase Chain Reaction was introduced as a rapid alternative to capsular serotyping system. (Townsend
The PCR-Restriction fragment length polymorphism (PCR-RFLP) is a technique wherein DNA sequence variation is identified by amplification of the region using PCR, followed by digestion of amplified product with a restriction endonuclease. The restriction fragments vary in size and can be revealed as different sized bands on agarose/acrylamide gels.
This technique has been used by several workers to detect polymorphism within a gene segment and such information has been useful for serotyping of isolates.
Till date no PCR-RFLP based techniques have been used for determination of both the capsular and somatic serotyping of
Materials And Methods
Pasteurella multocida strains
Two oligonucleotides based on the sequence of
OmpH 1 5'-GCG TTT CAT TCA AAG CAT CTC-3' - 21 mer and
OmpH 2 5'-ATG ACC GCG TAA CGA CTT TC -3'- 20 mer
Preparation of template DNA from cultures
Polymerase Chain Reaction was conducted using bacterial culture lysates as template DNA. A pure colony of
Amplification of OmpH gene
A 50 µl reaction mixture was prepared in 0.2 ml thin walled PCR tube. Five microlitres of template DNA was added to a reaction mixture containing 40 pmoles each of primer OmpH 1 and OmpH 2, 200 µM of each dNTP, 1 X
Restriction Enzyme Analysis Of OmpH-PCR Product
The amplified PCR products were subjected to restriction enzyme digestion using restriction enzymes
The primer pairs OmpH 1 and OmpH 2, designed to amplify the
Specificity of the Primers
Primer pairs OmpH 1 and OmpH 2 did not amplify the DNA prepared from unrelated bacterial species such as
Restriction enzyme analysis
Restriction analysis of the amplified products of serotypes A:1, A:3 and B:2 were carried out using the same restriction endonucleases
The custom designed primers could successfully amplify the
REA of amplified products of OmpH-PCR with
However, further studies have to be carried out with all the different serotypes to know whether profiles unique to each serotype are obtained, before the technique can be put for routine use. To the best of our knowledge this is the first report of the use of PCR-RFLP for differentiation of
The authors are grateful to the Indian Council of Agricultural Research, New Delhi, India for providing financial support under the “All India Network Programme on Haemorrhagic Septicaemia”, and the Dean, College of Veterinary and Animal Sciences, Mannuthy, Kerala, India for providing facilities to conduct this study.
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