Lane 1 and 18 contain a 100 bp DNA marker Lane 2 = Bovine alphaherpesvirus 1 lane 3 = Chelonid alphaherpesvirus 5, lane 4 = Macropodid alphaherpesvirus 1 lane 5 = Macropodid alphaherpesvirus 2 lane 6 = Human alphaherpesvirus 1 lane 7 = Human alphaherpesvirus 2 lane 8 = Human alphaherpesvirus 3 lane 9 = Equid alphaherpesvirus 4 lane 10 = Meleagrid alphaherpesvirus 1 lane 11 = Gallid alphaherpesvirus 2 lane 12 = Felid alphaherpesvirus 1 lane 13 = Human betaherpesvirus 5 lane 14 = Human betaherpesvirus 6 lane 15 = Human betaherpesvirus 7 lane 16 = Human gammaherpesvirus 4 lane 17 = Human gammaherpesvirus 8 The PCR products were analysed on a 1.5% agarose gel made up of 1× TBE buffer and 1× GelRed nucleic acid stain (Biotium).Įlectrophoresis of PCR products of HV DNAs obtained by STC-PCR in a 1.5% agarose gel. A Touchdown PCR protocol was carried out as outlined in Table Table3. PCR enhancers, including 5% dimethyl sulfoxide (DMSO) and tetramethylammonium chloride (15 mM TMAC), were also added to the reaction mix. The assays were successfully evaluated with ready-to-use pre-mixes including the GoTaq Hot Start Green Master Mix (Promega) and the HotStarTaq Plus master mix (Qiagen) to ensure the assays could be used across a range of PCR chemistries (data not shown). ![]() The reaction mix contained 2 μL of DNA template, 1 μM (beta-HV) or 2 μM (alpha-HV and gamma-HV) primers (Table (Table1), 1), 200 μM of each deoxynucleotide triphosphate (dNTP), 1.5 mM MgCl 2, 0.5 U of HotStarTaq polymerase and 1× PCR buffer (Qiagen). *The limit of the detection was recorded for the representative HVs testedĪViruses tested at James Cook University (JCU) laboratoryīViruses tested at The Australian Centre for Disease Preparedness (AAHL) laboratoryįollowing assay optimisation (data not shown), the STC-PCR was used to amplify HV DNA in a 20-μL reaction. This non-nested PCR assay was successfully used to detect a wide range of HVs across a broad range of herpesviruses in two independent laboratories.Īnimal clinical sample Cell culture isolate Here, we have designed singleplex touchdown consensus PCRs (STC-PCRs) that amplify regions of the DNA polymerase (DPOL) gene of alpha- and gamma-HVs and glycoprotein B (gB) gene of beta-HVs. Despite these valuable outcomes, some of the existing consensus PCR assays have variable sensitivity to different HV subfamilies and require a nested PCR format, which can be costly and prone to contamination. In fact, several new HV species of mammals, reptiles and avians have been discovered using this approach. Molecular surveys often employ consensus PCR assays to detect known and novel HVs. Therefore, there is a need for a sensitive assay that can reliably detect HV species of more than one subfamily in the same clinical samples. Mixed infections of HV species can occur in susceptible hosts leading to a variety of clinical symptoms that may be difficult to diagnose or treat. The avian and reptilian HVs have so far only been assigned to the subfamily Alphaherpesvirinae causing clinical and economic important diseases such as Marek’s disease (gallid HV2) and infectious laryngotracheitis (gallid HV1) in poultry, duck plaque enteritis (anatid HV1) in waterfowl, Pacheco’s disease (psittacid HV1) in psittacines and fibropapillomatosis (chelonid HV5) in sea turtles. ![]() The HVs of ruminants belong to the subfamilies Alpha- and Gamma- herpesvirinae, and infections are associated with rhinotracheitis (ovine HV1, caprine HV1), herpes mammalitis (bovine HV2), meningoencephalitis (bovine HV5), fatal systemic infection (caprine HV1), malignant catarrhal fever (ovine HV2, alcelaphine HV1, 2), ocular disease (cervine HV1) and fatal neurological disorder (bubaline HV1). For instance, the human HVs (HHV-1 to HHV-8) are members of Alpha-, Beta- and Gamma- herpesvirinae and have been associated with gingivostomatitis, herpetic keratitis, encephalitis, varicella, mononucleosis, lymphoproliferative malignancy, roseola and sarcoma. Clinical diseases associated with active or recrudescent HV infections vary according to the hosts and the infecting viral species. A common feature among all of the sub-groups of HVs is their ability to cause latent infection in infected hosts, which can be reactivated to cause serious illness in immunocompromised hosts. Herpesviruses are divided into three subfamilies, the Alpha-, Beta-, and Gamma- herpesvirinae on the basis of biological and molecular properties. The virus is made up of a linear, monopartite, double-stranded DNA genome that encodes up to 300 genes and ranges from 124 to 241 kbp in length. Herpesviruses (HVs) are known to have a wide host range, infecting both vertebrate and invertebrate species.
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