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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

REFERENCE NO: AHC/1998/3/3.4/2.2


TITLE: BVDV DETECTION BY USE OF RT-PCR


INTRODUCTION

The procedure should be performed in 3 separate locations:-

  1. Preparation of master mixes (in 'clean area')
  2. Preparation of reaction mixes involving RNA/DNA
  3. Analysis of PCR products
  • ART pipette tips must be used throughout.
  • Gloves must be worn at all times.
  • Reagent/reaction tubes should be kept on ice at all times.
  • A known positive and molecular grade water (Sigma W-4502 or equivalent) as a negative should be used as controls.
  • Each sample requires a labelled sterile 0.5ml tube.

PROCEDURE

REVERSE TRANSCRIPTION

1.1 In 'clean area' prepare the RT master mix as below and vortex.

Component per reaction

  • x5 RT buffer (Boehringer or equivalent) 2 ml
  • 100mM dNTPs (Boehringer or equivalent) 1 ml
  • RNase inhibitor (40 u/ml) (Boehringer or equivalent) 0.5 ml
  • MMuLV RT (20 u/ml) (Boehringer or equivalent) 0.25 ml
  • Molecular grade water (Sigma or equivalent) 0.75 ml

1.2 In the 'clean area' add 1 ml of anti-sense primer 326 to each reaction tube.

1.3 Transfer reaction tubes on ice to sample preparation area and add 4.5 ml of test sample.

1.4 Heat at 95C for 5 min in thermocycler (programme 11).

1.5 Chill on ice for 2 minutes and spin in microfuge for 10 seconds at 13,000 rpm.

1.6 Add 4.5 ml of RT master mix to each reaction tube using a separate ART tip for each.

1.7 Incubate at 42C for 30 minutes in thermocycler (programme 12). During this time prepare PCR master mix (see 2.1).

1.8 Denature RT reaction by heating at 95C for 5 minutes in thermocycler (programme 11).

1.9 Chill on ice for 2 min spin on microfuge for 10s at 13,000 rpm.

2. POLYMERASE CHAIN REACTION

2.1 In 'clean area' prepare PCR master mix as below, vortex then store on ice until required.

Component per reaction

  • x10 Taq buffer (Boehringer or equivalent) 10 ml
  • sense primer 324 (500 ng) (see 4 for sequence) 1 ml
  • Taq DNA polymerase (5u/ml) (Boehringer or equivalent) 0.5 ml
  • Molecular grade water (Sigma or equivalent) 78.5 ml

2.2 In sample preparation area overlay the RT reaction with 40 ml of mineral oil, using separate ART tips for each tube.
2.3 Add 90 ml PCR master mix to each reaction tube beneath the mineral oil overlay, using separate ART tips for each tube.

2.4 Place tubes in the thermocycler and run programme 13 which give 35 cycles of :

95C for 1 min

60C for 1 min

72C for 1 min, and holds at 4C.

2.5 PCR products can be stored at 2 to 8C overnight before agarose gel electrophoresis of for longer storage at <-10C.

3. AGAROSE GEL ELECTROPHORESIS

3.1 30 ml of PCR product should be run on 1 to 1.5% agarose/TBE buffer gel.

3.2 Refer to protocol AHC/1998/3/3.1/2.3 Appendix 4, "Preparation and running of minigels" OR protocol AHC/1998/3/3.4/2.3 "BVDV detection by use of a DiG labelled probe".

4. PRIMER SEQUENCES

4.1 Anti-sense primer 326 sequence

5'-TCA-ACT-CCA-TGT-GCC-ATG-TAC-3'

4.2 Sence-primer 324 sequence

5'-ATG-CCC(A/T)TA-GTA-GGA-CTA-GCA-3'
 


Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
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Copyright CABRI, 1998

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