Colorimetric detection of lagomorphs' calicivirus genomic sequences by polymerase chain reaction incorporating digoxigenin dUTP

Abstract

A method of reverse transcription followed by polymerase chain reaction (RT-PCR) has been implemented for the demonstration of the rabbit haemorrhagic disease virus (RHDV) genome in organ suspensions, leukocytes and excretions of infected rabbits. RT-PCR has been tested with 10 RHDV strains isolated at various geographic sites and times using a pair of primers coming from the gene region coding for the capsid protein VP60. The same primers were effective in the amplification of 4 of 5 European brown hare syndrome (EBHS) virus isolates. Non-radioactive labelling of PCR products with digoxigenin during the amplification and a system of colorimetric assessment of hybridization reactions between a biotin-labelled RHDV capture probe and the chains of labelled amplicons (PCR ELISA) were used for specific analyses of nucleic acid synthesis. The sensitivity of the alternative procedure of analysis of the dig-labelled PCR products with PCR ELISA was two logs 10 higher than that of conventional electrophoresis in agarose gel stained with ethidium bromide. The results of the hybridization reactions, carried out under various stringency conditions, have confirmed the presumption that the genomic similarity between the amplified and the probed areas of the capsid protein VP60 gene was not uniform within all the tested caliciviruses. A higher degree of heterogeneity was observed between the isolates of EBHSV and RHDV.