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

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.

difficult to eradicate the disease. New infections have emerged in recent years in some countries where IBRV infection had not previously been reported and also in countries that had previously declared that IBRV had been eradicated (9)(10)(11). A quarantine-culling strategy was adopted in a few countries (5), but it requires a huge cost and is not realistic for countries with large herds and underdeveloped economies. Furthermore, the IBR vaccine is successful in lessening the clinical effects of IBRV infection, but it does not block the establishment of the latent phase of IBRV and has the risks of abortion (4,12).
Real-time PCR, the World Organisation for Animal Health (WOAH)-recommended method for the detection of IBRV (13), is sensitive and technically mature over PCR. However, this method depends on complicated instruments and the transport of test samples, which are time-consuming and not conducive to its widespread use in small-and medium-sized farms. Another WOAH-recommended method, serological testing, is fast and requires less equipment but may be limited in the early diagnosis of IBRV infection because it takes days or weeks to generate a detectable antibody response, leading to a lag in disease prevention and control.
Therefore, this study aims to explore a novel rapid method by combining recombinant polymerase amplification (RPA) technology with a vertical closed-flow test strip (VF). We establish here a visualized IBRV RPA-VF nucleic acid assay to provide technical support for the early and rapid diagnosis of IBR that is suitable for operation in scenarios such as early screening on cattle farms or on-site quarantine at customs (Fig. 1). The latent infection which is characteristic of IBRV in cattle means that existing rapid techniques cannot distinguish between natural infection and vaccine immunity. However, the vaccine strain widely used in some countries for the immunization of cattle carries a TK gene deletion (14). Hence, the RPA-VF rapid method we established targeting the TK gene can be used for IBRV FIG 1 Schematic diagram of the RPA-VF assay. A DNA extraction kit (Axygen Company, China) was used to extract DNA from the swab-soaking solution. In addition, DNA of the swab-soaking solution also could be extracted quickly at 95°C for 5 min. The extracts were used as the templates, and an RPA reaction was run to form a double-labeled amplicon with 6-FAM and biotin in 25 min. The reaction tube was then placed in a VF, and then the results were judged in 5 min. In this experiment, the whole RPA-VF reaction took a total of 35 min. detection in cattle and has the potential to differentiate between natural infection and vaccine immunization.

RESULTS
Primers and probe design. A total of 9 TK gene sequences from different IBRV strains were aligned using MEGA 11 software. We selected the conserved fragment of the TK gene as the target (Fig. 2) and designed six sets of primers and probes for the RPA-VF method according to the targeting sequence. The reverse primer was labeled with biotin at the 59 end. The probe was labeled a 6-carboxyfluorescein (6-FAM) at the 59 end, a tetrahydrofuran (THF) residue that replaced a nucleotide at 30 bp and contained a blocking group C 3 spacer at the 39 end so that both biotin and 6-FAM can be simultaneously integrated into a doublestranded amplicon.
Optimization of the RPA-VF reaction conditions. The 10-fold dilutions of the positive plasmid pBlunt-TK containing IBRV TK gene were used as a template for the RPA-VF assay. The reaction process was performed at different temperatures (45°C, 42°C, 39°C, and 37°C) for 20 min. As shown in Table 1, the detection limit for the plasmids was 3.8 Â 10 2 copies/mL at 39 to 42°C for 20 min. In the repeated experiments, 42°C was found to be the most stable temperature. Then, the reaction was performed at different times (20,25, and 30 min) based on the above-described optimal reaction temperature, and the optimal amplification time was 25 min with a minimum detection of 3.8 Â 10 1 copies/mL pBlunt-TK DNA ( Table 2).
Sensitivity and specificity of the RPA-VF assay. The pBlunt-TK plasmids were diluted in 10-fold gradients and then utilized as the templates for the sensitivity evaluation of RPA-VF under optimized conditions. The results showed that the control line (C-line) and test line (T-line) in the VF detection device were visible when the template concentration ranged from 3.8 Â 10 5 to 3.8 Â 10 1 copies/mL, while the method gave a negative result when the concentration was #3.8 copies/mL (Fig. 3A).
Similarly, IBRV-infected cell cultures were diluted in 10-fold gradients to 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , and 10 0 tissue culture infective dose (TCID 50 ), and then DNA was extracted FIG 2 Alignment of the sequences of IBRV TK gene. A total of 9 TK gene sequences from different IBRV strains were aligned using the MEGA 11 software. TK-F and TK-R were the optimal forward primer and reverse primer for the TK gene, respectively. TK-P was the optimal probe for TK gene. The black arrow and numbers indicate the position of the primers and probe in the sequence. In addition, the region in the red box is the corresponding gene sequence of primers, and the region in the blue box is the corresponding gene sequence of the probe. The red arrows correspond to the direction of primer amplification, and the blue arrow represents the direction of probe amplification. as the template for virulence evaluation under optimized conditions, indicating that the RPA-VF assay could detect IBRV at a TCID 50 of 1.09 Â 10 1 in a minimum of 25 min (Fig. 3B). The purpose of assessing levels of IBRV DNA was to evaluate the applicability of our method to viruses.
Moreover, to evaluate the specificity of the RPA-VF method, we used this method to test nucleic acids from several respiratory pathogens or herpesviruses ( Assessment of clinical samples using the RPA-VF method. A total of 28 nasal swabs from dairy cattle and beef cattle were assessed as positive using our IBRV RPA and showed cycle threshold (C T ) values ranging from 18.14 6 0.16 to 34.58 6 0.28 using the real-time PCR assay. The remaining 92 nasal swabs were found to be negative using both methods. The coincidence rate between the RPA-VF assay and the gold standard was 100% (Table 3). Additionally, all samples, whether processed using a DNA extraction kit or simply inactivated at 95°C for 5 min, gave the same results of RPA detection (positive and negative results), and the detection coincidence rate for both DNA extraction methods remained at 100%.

DISCUSSION
Current control measures for IBR still focus on the early diagnosis of the disease and vaccination (8,15). The IBR vaccine is effective in reducing the clinical impact of IBRV infection; however, it does not block the establishment of the latent phase of IBRV (6,12). Therefore, effective methods have become a priority for the prevention and control of IBR.
In comparison to PCR and real-time PCR methods, isothermal amplification techniques are better suited for small-scale farms and on-site testing. The lowest detection limit of the loop-mediated isothermal amplification (LAMP) method constructed by researchers is 100 pg or 10 copies/mL IBRV DNA, while the results were analyzed using agarose gel electrophoresis, which cannot avoid the influence of aerosol (16,17). Another study reported an RPA method combined with a lateral flow dipstick (LFD) assay, which had a minimum detection limit of 5 copies/mL of IBRV DNA (18). Compared to the above-described methods, although our RPA-VF method can only detect 3.8 Â 10 1 copies/mL recombinant plasmid, the VFs we used have the advantage that they are sealed independently to prevent aerosol contamination. It is worth mentioning that RPA outperforms the current LAMP approach in the detection of IBRV. LAMP analysis typically needs at least four primers, but the RPA reaction uses only two pri- Concentration of positive plasmid pBlunt-TK (copies/mL): 3.8 × 10 3 3.8 × 10 2 3.8 × 10 1 3.8 × 10 0 Negative 45 0/9 a 0/9 0/9 0/9 0/9 42 9/9 9/9 0/9 0/9 0/9 39 9/9 9/9 0/9 0/9 0/9 37 0/9 0/9 0/9 0/9 0/9 a Positive number/total repeats. Concentration of positive plasmid pBlunt-TK (copies/mL): 3.8 × 10 3 3.8 × 10 2 3.8 × 10 1 3.8 × 10 0 Negative 20 9/9 a 9/9 0/9 0/9 0/9 25 9/9 9/9 9/9 0/9 0/9 30 9/9 9/9 0/9 0/9 0/9 a Positive number/total repeats. mers. Due to the high GC content of IBRV DNA, it is easier to design primers for RPA with fewer dimerization reactions. The selection of the target gene is also a critical stage in IBRV detection. The genome of IBRV is approximately 138 kb in size and comprises several significant target genes, including gB (19), gC, gD, gE (16), TK, and UL52 (18). In this study, we selected the TK gene as a target because it is a key gene in the maintenance of persistent IBRV infection and is one of the major virulence genes (20). Furthermore, the gene sequence of IBRV TK is relatively  conserved, making it a good target candidate for IBRV detection. In addition, because TK is a nonessential gene, it is one of the main target genes for developing IBR gene deletion vaccines (21)(22)(23). This means that the assay we developed can be utilized to differentiate between a naturally occurring illness and an IBRV TK deletion vaccination in cattle. Furthermore, cattle are susceptible to a variety of pathogens, of which BEFV, BPIV, BVDV, PRV, and IBRV can cause respiratory tract infections, diarrhea, and reproductive system diseases. Any method used to test clinical samples in the field must therefore have good specificity. Our assay did not show cross-reactivity with any of the above-described pathogens, demonstrating that this method is suitable for the detection of IBRV in complex clinical samples from cattle. The shorter times and fewer steps of sample processing are more beneficial for the clinical applicability of the method. There are several available rapid methods to extract DNA from samples, of which glass beads and boiling procedures have been found to be the most effective (24). Other studies have also shown that DNA extraction can be compressed to 30 s using cellulose-based filter paper (25,26). In this study, to shorten the sample processing steps and time, the clinical samples were inactivated simply by heating to 95°C for 5 min to quickly extract the viral DNA. Although the sensitivity of this pyrolysis DNA extraction method needs to be improved, the overall experimental time is very short (the whole reaction only took 35 min) in comparison with other methods, and the result is consistent with that from real-time PCR, suggesting that it is suitable for early clinical screening of IBRV in cattle farms. It cannot be denied that the sensitivity of our RPA-VF method needs to be improved. Therefore, our next research will focus on optimizing our RPA-VF method and improving the detection efficiency and sensitivity to create a more convenient, intuitive, and efficient point-of-care test.

MATERIALS AND METHODS
Animals and sampling. Experimental animal protocols were approved by the Ethics Committee for Animal Care and Use of Jilin University (permit number SY202304007; Changchun, Jilin, China). The animals received humane care following Laboratory Animal-The Guidelines for Ethical Review of Animal Welfare (GB/T 35892-2018, China).
The nasal swabs were obtained from a dairy farm in Heilongjiang Province (72 samples) and a beef farm in Jilin Province (48 samples). Dairy cattle were lactating, aged 3 to 5 years. Beef cattle were 1 to 2 years old and asymptomatic. About 30% of the samples were taken from cows with runny nose symptoms. All the cattle were kept in a clean, ventilated, warm, and comfortable enclosure and were fed a balanced diet.
Viruses. IBRV and BVDV were provided by Gao Mingchun (Northeast Agricultural University), and the cDNAs of BEFV and BPIV were provided by He Hongbin (Shandong Normal University). The DNA of PRV was stored in our laboratory. All nucleic acid samples were extracted from cell-cultured viruses.
Primers and probe design. A total of 9 TK gene sequences from different IBRV strains (GenBank accession numbers AJ004801, D00438, JN787955, JX898220, KM258883, KM258881, KU198480, MG407792, and NC_001847) were downloaded from the National Center for Biotechnology Information database (NCBI) (https://www.ncbi.nlm.nih.gov) and were aligned using MEGA 11 software. We selected the conserved fragment of the TK gene as the target (Fig. 2) and designed six sets of primers and probes for the RPA-VF method using Primer Premier 5. All primers and probes were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).
Construction of recombinant plasmids. IBRV DNA was extracted from IBRV-infected cell cultures using the AxyPrep multisource genomic DNA miniprep kit (Axygen Company, China). The primers for the conserved region of the IBRV TK gene were designed by Primer Premier 5 (Table 4) and synthesized by Sangon Biotech Co., Ltd. The TK gene was then amplified with these primers using KOD FX DNA polymerase (Toyobo, Japan) and inserted into the Blunt vector (Beijing TransGen Biotech Co., Ltd., China) to construct the recombinant plasmid pBlunt-TK. The copy number of the recombinant plasmid pBlunt-TK was calculated using the formula (6.02 Â 10 23 ) Â (ng/mL Â 10 29 )/(DNA length Â 660). The most suitable set of primers (TK-F/TK-R) and probe (TK-P) was screened with PCR (Thermo Fisher Scientific, USA) and analyzed with 3% agarose gel electrophoresis (Jun Yi, China) ( Table 5).
Establishment and optimization of the RPA reaction system. The RPA (Hangzhou ZC Bio-Sci & Tech Co. Ltd.; China) premixed reaction system (2 mM TK-F, 2 mM TK-R, 2 mM TK-P, 15.7 mL double-distilled water [ddH 2 O], and 25 mL buffer A) was formulated and transferred to lyophilized tubes containing RPA amplification enzyme and mixed thoroughly. The template (10-fold gradient dilution) was added, and then 2.5 mL buffer B ) was added to the tube cap. The tubes were wrapped with sealing film and centrifuged briefly, heated in a thermostatic apparatus to complete the RPA reaction, and then briefly placed in a 4°C refrigerator. Finally, reaction tubes were placed in the closed VF device (Ustar Biotech, Co., Ltd., China), and the results were read after 5 min. All experiments were repeated three times, and three parallels in each experiment were analyzed. A negative control was established to exclude the false-negative or false-positive results.
Testing of clinical samples using the RPA-VF method. DNA from clinical samples was processed in one of two ways. The first way was using a DNA extraction kit (AxyPrep multisource genomic DNA miniprep kit), and the second way was a crude extraction by heating at 95°C for 5 min. The DNA was then assessed using our RPA-VF method. Real-time PCR (the Chinese national standard [GB/T 27981-2011]) was used as the gold standard for this investigation. Each group included a negative and a positive control. Samples with a cycle threshold (C T ) value of ,35 were judged to be positive, and samples with a C T value of .35 and with no amplification curve in real-time PCR were judged to be negative. Each experiment was repeated three times.
Data availability. Further inquiries can be directed to the corresponding authors.