Rapid and simple colorimetric loop-mediated isothermal amplification (LAMP) assay for the detection of Bovine alphaherpesvirus 1
Deborah Peltzer, Kurt Tobler, Cornel Fraefel, Madeleine Maley, Claudia Bachofen
Abstract
As the causative agent of Infectious Bovine Rhinotracheitis (IBR) and Infectious Pustular Vulvovaginitis/Balanoposthitis (IPV/IPB), Bovine alphaherpesvirus 1 (BoHV-1) is responsible for high economic losses in the cattle industry worldwide. This study aimed to establish a fast, colorimetric loop-mediated isothermal amplification (LAMP) assay for the detection of viral DNA. Phenol red is used as pH-sensitive readout, relying on a distinct color change from pink to yellow in case of a positive reaction. LAMP reactions with different primers were compared and a newly designed set targeting the gene encoding the tegument protein V67 provided best results, enabling readout within 8-30 min. LAMP showed less cross-reactions with other ruminant alphaherpesviruses than qPCR but was 10-fold less sensitive. However, LAMP still detected down to 14 copies. The test performance was evaluated using 26 well-characterized nasal swabs from cattle with respiratory disease. All samples were correctly identified when using column-extracted DNA. Using a simple DNA precipitation method, only two weak-positive samples turned indeterminate. Combining this DNA precipitation with a makeshift water bath heated by a gastronomic immersion heater allowed successful application of the colorimetric LAMP assay under resource-limited conditions. This technique can therefore help in managing IBR/IPV outbreaks where sophisticated laboratory equipment is unavailable.