Development of a pyrosequencing assay for the typing of alphaherpesviruses

Identification of herpesvirus in biological material is usually carried out by real-time PCR. With the aim to classify the strain of virus identified, real-time PCR must be often supported by time-consuming capillary electrophoresis sequencing analysis. Here we provide a protocol for the rapid and reliable identification of 5 closely related herpesviruses by PyroMark Q24 sequencing system. PyroMark performs DNA sequencing analysis using pyrosequencing, a technology based on the detection of released pyrophosphate during DNA elongation [1]. PyroMark is designed to detect changes in specified variable positions of the DNA. It can efficiently detect single nucleotide differences in sequences [2]. In the present paper we describe a protocol to pyrosequence a small polymorphic segment of the US8 gene. On the basis of the differences identified in the nucleotide sequence we could readily classify the herpesvirus as Bovine herpesvirus 1.1, Bovine herpesvirus 1.2, Bovine herpesvirus 5, Bubaline herpesvirus 1 or Caprine herpesvirus. The protocol set up offers several advantages with respect to the techniques commonly used: •it requires less than one working day to be carried;•it gives the possibility to analyze, at reasonable costs, up to 24 samples at a time; and•it allows to detect with great reliability and specificity strongly genetically correlated organisms like the herpesviruses named above. The procedure can be easily applied to other families of viruses, with opportune modifications.