VPB 321: Animal Biotechnology (2+1)

DETECTION OF INFECTIOUS BRONCHITIS VIRUS BY RT-PCR

Aim

• To demonstrate RT-PCR for diagnosis of infectious bronchitis of chicken

Materials required

• For IB diagnostic purposes, two primer pairs (N784 to N1145 and N791 to N1129, respectively) are used to detect IBV in the birds.

Primer sequences

• N784 FP : AATTTTGGTGATGACAAGATGA
• N1145RP : CATTGTTCCTCTCCTCATCTG
• N791NFP : GTGATGACAAGATGAATGAGGA
• N1129NRP : CAGCTGAGGTCAATGCTTTATC

Procedure

• The amplification reaction is carried out in 25 m l mixtures containing the following :
  • 10 X PCR buffer (10 mM Tris.HCl (pH 9.0); 50 mM KCl; 2.5 mM MgCl) : 2.5 m l
  • dNTP (0.2 mM each) : 1.0 u l
  • Primer N784 (10 pmol) : 1.0 u l
  • Primer N1145 (10 pmol) : 1.0 u l
  • Taq DNA polymerase (2.5 U / m l) : 0.5 u l
  • cDNA : 5.0 u l
  • Nuclease free water : 14.0 u l
• Amplification is performed with a thermal profile of 94 ^o C for 45 sec ; 60^o C for 1 min ; 72^o C for 2 min. This cycle profile is repeated 35 times with a final extension at 72^o C for 7 min.
• After the first run of amplification, 1 u l amounts of the PCR product is added to a fresh reaction tube containing identical reagents and the internal primers (N791 & N1129).
• Amplification is performed with a thermal profile of 94^o C for 3 min; 94^o C for 45 sec; 53^o C for 1 min and 72^o C for 1 min. This cycle profile is repeated 30 times with a final extension at 72^o C for 7 min.
• For visualization 5 ul amounts of the PCR product is electrophoresed along with 100 bp DNA marker in 2 % agarose gel. After electrophoresis, the gel is stained in ethidium bromide and viewed under UV-light.
• Amplicon size :
  • External primer : 400 bp
  • Internal primer : 380 bp