Development of a recombinase polymerase amplification assay for rapid detection of Haemophilus parasuis in tissue samples

Abstract Haemophilus parasuis is the etiological agent of Glässer's disease in swine, which associates with severe economic losses in the swine industry worldwide. A real‐time recombinase polymerase amplification assay (real‐time RPA) was developed for direct and rapid detection of H. parasuis basing on the translation‐initiation factor IF2 (infB) gene. The assay was performed successfully at 39°C for 20 min in Genie III, which is portable and chargeable by battery. The developed assay was highly specific for H. parasuis, and the limit of detection of the assay was 6.0 × 103 fg of H. parasuis genomic DNA, which was the same as that of a real‐time PCR developed previously. The assay was further evaluated on 68 pig tissue samples, and 18 (26.5%), 20 (29.4%), and 8 (11.8%) samples were positive for H. parasuis by the real‐time RPA, real‐time PCR and bacterial isolation, respectively. With the bacteria isolation as the reference method, the real‐time RPA showed a diagnostic specificity of 83.33% and a diagnostic sensitivity of 100%. The above data demonstrated the well‐potentiality and usefulness of the developed real‐time RPA assay in reliable diagnosis of swine Glässer's disease, especially in resource limited settings.

Presently, 15 serovars of H. parasuis have been identified worldwide (McCaig, Loving, Hughes, & Brockmeier, 2016), and the most common serovars in China are serovars 4 and 5 (Cai et al., 2005;Ma et al., 2016;. Haemophilus parasuis could cause a infection rate of 50%-70% and a mortality rate above 10%, thus inducing considerable production losses due to the mortality and unthrifty of pigs (McCaig et al., 2016;Zhang et al., 2014). Glässer's disease has caused severe economic losses in the swine industry worldwide.
The H. parasuis infection can be controlled by vaccination and antibiotic treatment. However, one of the key elements for controlling the H. parasuis infection is the rapid and accurate detection of the bacterium (Aarestrup, Seyfarth, & Angen, 2004;Oliveira & Pijoan, 2004). Isolation and microbiological culture of H. parasuis is the gold standard for diagnosis of Glässer's disease, but it could be ineffective due to the fastidious growth of the bacteria, easily being overgrown by other bacterial contaminants and the previous antibiotic treatment of affected animals (Angen, Oliveira, Ahrens, Svensmark, & Leser, 2007, Oliveira et al., 2001, Turni, Pyke, & Blackall, 2010. A series of molecular diagnostic methods have been described for sensitive and specific detection of H. parasuis, such as polymerase chain reaction (PCR), real-time PCR, loop-mediated isothermal amplification (LAMP) and cross-priming amplification (CPA) (Angen et al., 2007, Chen, Chu, Liu, Zhang, & Lu, 2010, Frandoloso, Martinez-Martinez, Rodriguez-Ferri, & Gutierrez-Martin, 2012, Gou et al., 2018, Oliveira et al., 2001, Turni et al., 2010, Yang et al., 2010. The requirements of high-precision thermocycler, a centralized laboratory facility and experienced technicians limit the widespread use of PCR assays in under-equipped laboratories, in clinic settings, and on farm diagnosis. Although the developed LAMP assay shows the advantages in respects to convenience and minimal equipment requirement, it still requires four primers, 60 min at 61°C or 45 min at 65°C for reaction and 10 min at 80°C to terminate the reaction (Chen et al., 2010;Yang et al., 2010). The CPA assay combines with a vertical flow visualization nucleic acid detection strip showing good specificity and sensitivity, thus reducing the requirement of specialized instrument, however, the reaction time is 1h (Gou et al., 2018).
A rapid test may assist the microbiological diagnosis for peritonitis in pigs, so a more convenient and rapid detection method is still required for detection of H. parasuis on farm and in low resource settings.
Recombinase polymerase amplification (RPA), which is an isothermal DNA amplification technique, has experienced rapid development in the field of molecular diagnostic since it was firstly reported in 2006 (Daher, Stewart, Boissinot, & Bergeron, 2016;Lei et al., 2020;Li, Macdonald, & von Stetten, 2018;Liu et al., 2018;Miao et al., 2019;Piepenburg, Williams, Stemple, & Armes, 2006;Wang, Yuan, Han, Wang, & Liu, 2017). Some studies showed that RPA may be the most applicable approach for the on farm diagnosis due to its convenience and rapidness (Amer et al., 2013;Li et al., 2018). In this study, the real-time RPA assay targeting the translation-initiation factor IF2 (infB) gene was developed for rapid and reliable detection of H. parasuis, and the performance of the assay was further evaluated and compared to the bacteria isolation and a real-time PCR by detecting the swine tissue samples.

| Bacteria strains and clinical samples
To determine the specificity of the real-time RPA, a total of 20 H. parasuis strains and 17 other bacterial strains were tested. For the H. parasuis strains, the genomic DNA of 15 serovar reference strains were kindly provided by Lanzhou Veterinary Research Institute (Lanzhou, China), two reference strains were obtained from CIVDC (China Institute of Veterinary Drug Control, Beijing, China), and three field strains were identified by species-specific PCR described previously (Oliveira et al., 2001, Turni et al., 2010.
Details of all the bacterial strains used in this study were provided in Table 1.
Thirty swine tonsils were collected at slaughterhouse, and 38 swine fresh lungs were collected from the markets for agricultural products in Shijiazhuang city, Hebei province from November to December 2018.

| DNA extraction
All the bacterial genomic DNA were extracted using the TIANamp Bacteria DNA kit (Tiangen), which were performed according to the manufacturer's instructions. The tonsil and lung samples were homogenized with phosphate-buffered saline (PBS, pH 7.4) as a 10% (w/v) suspension and centrifuged for 10 min at 10,000 g at 4°C.
After centrifugation, the supernatant was discarded and the remaining pellet was suspended in 200 μl of PBS for DNA extraction using the TIANamp Bacteria DNA kit (Tiangen). Total DNA extracted from tissue samples was finally eluted in 50 µl of nuclease-free water.
All DNA were quantified using a ND-2000c spectrophotometer (NanoDrop) and stored at −80°C until use.  Table 2
The reaction tube was vortexed briefly and spun down, and the assay was performed immediately at 39°C for 20 min in a Genie III scanner device (OptiGene Limited). Samples produced an exponential amplification curve above the threshold of the negative control were considered positive.

| Analytical specificity and sensitivity of the real-time RPA assay
The analytical specificity of H. parasuis real-time RPA assay was determined by testing the genomic DNA extracted from a panel of bacteria listed in Table 1. Three independent reactions were performed.
The analytical sensitivity of H. parasuis real-time RPA assay was determined and compared with that of real-time PCR using a 10-fold serial dilution of genomic DNA of H. parasuis ranging from 6.0 × 10 7 to 6.0 × 10 0 fg/μl. One microliter of each dilution was amplified by RPA to determine the limit of detection (LOD) of the assay, and eight independent reactions were performed.

| Real-time PCR
A real-time PCR assay for H. parasuis was performed on a ABI 7500 instrument described previously (Turni et al., 2010).
Sequences for the primers (CTinfF1 and CTinfR1) and TaqMan probe (CTinfP) are provided in

| Validation with tissue samples
The H. parasuis real-time RPA assay were assessed on 38 swine fresh lungs and 30 swine tonsils, and all samples tested by RPA assay were also tested by a real-time RT-PCR (Turni et al., 2010), which was run in parallel. Bacteria isolation of the H. parasuis was also performed for the above tissue samples, which was performed in detail as the following protocol. The surfaces of the tonsils and lungs were seared with a hot iron and cut to obtain scrapings of sample tissue for culture. Scraping of tissue was spread on TSA plates supplemented with 5% foetal bovine serum and 0.01% NAD (Sigma). The plates were incubated at 37°C for 48 hr. All suspect colonies of H. parasuis were passaged twice before identification by PCR (Oliveira et al., 2001).

| Analytical specificity of the real-time RPA assay
Specific amplification was only observed with H. parasuis, including the reference strains and the field isolates, and there was no crossdetections of other bacteria tested (Table 1). Three independent reactions were repeated and similar results were observed, which demonstrated the high specificity and good repeatability of the assay.

| Analytical sensitivity of the real-time RPA assay
Using a dilution range of 6.0 × 10 7 to 6.0 × 10 0 fg of H. parasuis genomic DNA as template, the data showed that the LOD of the real-time RPA assay was 6.0 × 10 3 fg, which was the same as that of the real-time PCR (Figure 1). The real-time RPA assay was performed eight times on the molecular standard, in which HPS-reverse GGCTTCGTCACCCTCTGT 6.0 × 10 7 -6.0 × 10 3 fg DNA molecules were detected in 8/8 runs, and 6.0 × 10 2 -6.0 × 10 0 , 0/8 (Figure 2). Two-fold serial dilutions of the genomic DNA were made from 6.0 × 10 3 to 7.5 × 10 2 fg/μl. The above twofold dilutions of the genomic DNA were further used in the RPA, and the LOD of the assay was still 6.0 × 10 3 fg per reaction.

| Validation of the real-time RPA assays on tissue samples
Of the 68 swine tissue samples, 18 (26.5%), 20 (29.4%) and 8 (11.8%) samples were positive for H. parasuis by the real-time RPA, real-time PCR and bacterial isolation, respectively (Table 3)  that the performance of the real-time RPA assay was comparable to the real-time PCR, but the real-time RPA assay is faster.

| D ISCUSS I ON
Although isolation of H. parasuis is the gold standard for diagnosis of Glässer's disease, but it is usually difficult as the bacteria is very sensitive to pH changes and heat (Morozumi & Hiramune, 1982) and it is also a slow growing, fastidious organism with specific nutritional requirements (Angen et al., 2007, Oliveira et al., 2001, Turni et al., 2010.  (Angen et al., 2007, Chen et al., 2010, Gou et al., 2018, McDowall, Slavic, MacInnes, & Cai, 2014, Oliveira et al., 2001, Turni et al., 2010, Yang et al., 2010. The PCR targeting on 16S rRNA gene has problems in specificity giving a weak positive with Actinobacillus indolicus (Turni et al., 2010), and a real-time PCR using the 16S rRNA as the target could not differentiate Pasteurella mairii from H. parasuis (Turni et al., 2010).
The infB gene codes for the two forms of the translation initiation factor IF2 -IF2 alpha and IF2 beta (Hedegaard et al., 2000). The developed real-time PCR and LAMP based on the infB gene could detect all the 15 serovars of H. parasuis and demonstrated good performance (Chen et al., 2010;Turni et al., 2010;, which shows that the infB gene is a good target for molecular detection methods for H. parasuis and could separate H. parasuis from all other closely related species. In this study, the real-time RPA primers and probe were also designed based on the infB gene in this study, just as the PCR and LAMP assays developed previously (Chen et al., 2010;Turni et al., 2010;, and the real-time RPA assay demonstrated very good specificity in the detection of H. parasuis. Of the 68 tissue samples, 18 (26.5%) and 20 (29.4%) samples were positive for H. parasuis by the real-time RPA and real-time PCR, respectively, which were much higher than the bacterial isolation (8, 11.8%).
Compared to the bacterial isolation, the PPV and NPV of real-time RPA parasuis or in situation where culture is not possible.
In summary, the developed real-time RPA assay is rapid and reliable for detection of H. parasuis, and demonstrates great promise in the diagnosis of Glässer's disease in laboratory and in the field, which is of great importance to eliminate the infected pigs from the herds.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

E TH I C A L S TATEM ENT
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. The US National Research Council's guidelines for the Care and Use of Laboratory Animals were followed.

DATA AVA I L A B I L I T Y S TAT E M E N T
The dataset analysed during this study is available from the corresponding author on reasonable request.