The use of ARMS PCR and RFLP analysis in identifying genetic profiles of virulent, attenuated or vaccine strains of TGEV and PRCV

Abstract

The use of ARMS (amplification refractory mutation system) PCR coupled with RFLP (restriction fragment length polymorphism) analysis has been used to identify a unique genetic marker on the Ambico oral vaccine strain. This method was also used to characterize the genetic profiles of a number of other TGEV strains. This procedure takes advantage of the nucleotide differences between the Ambico strain, and the Miller and Purdue strains. Within the S gene there are three nucleotide differences between the Ambico strain and the published Purdue sequence. There are additional nucleotide differences in the structural and non-structural gene sequences, but we have chosen to focus on the differences contained within the S gene. The Ambico strain has a closer sequence homology to the Purdue strain than to the Miller strain. The Ambico and Purdue strains contain a six nucleotide deletion at position 1122 that is not present in the Miller published sequence or the ISU-1 strain of PRCV (based on our PCR experiments). We have designed a 5' oligo whose sequence is homologous to a region located 80 nucleotides upstream of the TGEV and PRCV S gene initiation codons to be used in conjunction with either of two 3' oligos whose sequences are identical with the exception of the last six nucleotides of their 3' ends. When utilized with the appropriate PCR conditions, these oligos can differentiate between PRCV, Miller and Purdue prototype virus strains. These PCR products were then subjected to RFLP analysis using four separate restriction enzymes (BstE II, Alw26 I, Dra III, or MspA1 I). We have used this procedure to analyze six TGEV vaccine strains, intestinal derived virulent viruses, cell cultured viruses at different cell passage numbers, and field isolates of TGEV or PRCV.