A Rapid Nucleic Acid Visualization Assay for Infectious Bovine Rhinotracheitis Virus That Targets the TK Gene

Abstract

ABSTRACT Infectious bovine rhinotracheitis virus (IBRV) can cause various degrees of symptoms in the respiratory system, reproductive system, and whole body of cattle. It also can lead to persistent and latent infection in cattle, posing a challenge to timely control of infectious bovine rhinotracheitis (IBR) in farms and causing large financial losses in the global cattle industry. Therefore, the goal of this study was to establish a rapid, simple, and accurate method that can detect IBRV in order to facilitate the control and eradication of IBR in cattle. We combined recombinant polymerase amplification (RPA) with a closed vertical flow visualization strip (VF) and established an RPA-VF assay that targets the thymidine kinase (TK) gene to rapidly detect IBRV. This method (reaction at 42°C for 25 min) was able to detect a minimum of 3.8 × 101 copies/μL of positive plasmid and 1.09 × 101 50% tissue culture infective dose (TCID50) of the IBRV. This assay has high specificity for IBRV and does not cross-react with other respiratory pathogens in cattle. The concordance between the RPA-VF assay and the gold standard was 100%. In addition, this assay was also suitable for the detection of DNA from clinical samples extracted by a simple method (heating at 95°C for 5 min), which can achieve the rapid detection of clinical samples in the field. Overall, the present sensitivity, specificity, and clinical applicability assessments indicated that the RPA-VF assay we developed can be utilized as a quick and accurate on-site test for IBRV detection in farms. IMPORTANCE IBRV causes different degrees of clinical symptoms in cattle and poses a great threat to the cattle industry. The infection is persistent and latent, and the elimination of IBRV in infected herds is difficult. A rapid, simple, and accurate method to detect IBRV is therefore vital to control and eradicate IBR. Combining RPA with an VF, we established an RPA-VF assay for the rapid detection of IBRV, which can complete the test of clinical samples in 35 min. The assay shows good sensitivity, specificity, and clinical applicability and can be used as an on-site test for IBRV in farms.

Keywords

• infectious bovine rhinotracheitis virus
• recombinase polymerase amplification
• TK gene
• on-site test