Development of real-time and lateral flow dipstick recombinase polymerase amplification assays for rapid detection of goatpox virus and sheeppox virus

Goatpox virus (GTPV) /AV40, GTPV/AV41, GTPV/GS-V1, sheeppox virus (SPPV) /Gulang2009, SPPV/Jingtai2011, SPPV/Hubei, Orf virus (ORFV) /Vaccine/CHA, ORFV/HB/CHA, peste des petits ruminants virus (PPRV)/Nigeria 75/1, foot-and-mouth disease virus (FMDV)/O/CHA and FMDV/A/CHA were preserved in our laboratory. To prepare reference DNA, 374 bp CaPV GPCR segments (ranging from 301 bp to 675 bp of KF661979.1) were synthesized by Genewiz (Suzhou, China) and cloned into a pUC57 vector, designated as pCaPV/RPA. The pCaPV/RPA DNA was extracted by Plasmid Mini kit I (Promega, USA) and then measured by Nanovue (GE life science). The DNA copy number was calculated using the following equation: DNA copy number = (ng × 6.02 × 10²³ × 10⁻⁹)/ (Fragment length (bp) × 660). The DNA standard was then aliquoted and stored at −80 °C until used. To prepare CaPV-spiked tissue lysates, CaPV-free tissue samples of liver, lung, stomach, kidney, lymphatic node, spleen, nasal swab and skin (n = 24, three each sample type) were collected from three healthy sheep. 10% (w/vol) tissue suspensions (one milligram tissue samples in nine volumes PBS) were then prepared by homogenizing tissue samples in PBS using MP FastPrep-24. Following a brief centrifugation, the homogenized tissue samples were spiked with SPPV/Gulang 2009 at different concentrations from 10⁷ copies to 10⁴ copies per reaction and stored at −80 °C until used. During the period of October 2014 to August 2015, one hundred and seven clinical samples (liver, lung, kidney, spleen, skin and blood) were collected from fourteen suspected sheep and six suspected goats in Gansu province which were characterized by pyrexia, excessive salivation and generalized pock lesions in the skin. Tissue samples were collected by the animal disease investigating teams based on good animal practices of the Animal Ethics Procedures and Guidelines of the People’s Republic of China (AEPGPRC), and the samples were stored at −80 °C in our laboratory until used. Nucleic acids from virus strains, spiked samples and clinical samples were extracted using the viral DNA/RNA extraction kit (TaKaRa, Dalian, China), which could only be used in the laboratory. In order to employ an energy-free assay in the field, a single innuPREP MP basic kit A (Jena Analytik, Jena, Germany) with a magnetic bead separation rack was tested using spiked samples (n = 24), and the extraction efficiency were then evaluated by real-time RPA assay, CaPV RPA LFD assay and CaPV real-time qPCR assay, respectively.

The real-time qPCR assay which could detect SPPV, GTPV and lumpy skin disease virus (LSDV) was carried out in an Agilent Technologies Stratagene Mx3005P thermocycler (Life technologies, USA) as previously reported [6]. Briefly, The PCR assay was carried out in a 25 μL reaction volume containing 2 × PCR buffer (a buffer containing 0.4 mM of each dNTP and 6 mM MgSO₄, 12.5 μL), Taq DNA polymerase (5 U/μL, 0.5 μL), the probe (5`-CAATGGGTAAAAGATTTCTA-3`) (10 μM, 0.5 μL), the forward prime (5`-GGCGATGTCCATTCCCTG-3`) (10 μM, 1 μL), reverse primer (5`-AGCATTTCATTTCCGTGAGGA-3`) (10 μM, 1 μL), the DNA template (10 pg - 0.1 μg, 2 μL) and RNase-free water (9.5 μL). The cycling proceeded at 95 °C for 5 min, followed by 40 cycles of 95 °C for 50 s, 50 °C for 50 s and 72 °C for 1 min, and an additional extension for 5 min.

After blasting GPCR gene of CaPV (number of access: KF661979.1 (SPPV), KF661976.1 (SPPV), JQ310666.1 (SPPV), FJ869364.1 (GTPV), FJ869361.1 (GTPV), KP663705.1 (GTPV), KP719918.1 (LSDV), FJ869376.1 (LSDV) and KR024780.1 (LSDV)), one probe and three different forward and reverse primers targeting the GPCR gene conserved region were designed for each assay (CaPV real-time RPA and CaPV RPA LFD assay) and synthesized by Sangon Biotech (Table 1). The RPA assay was carried out in a 50 μL freeze-dried reaction tube, using 29.5 μL rehydration buffers (TwistDx, Cambridge, UK), 2.1 μL of each primer (10 μM), 0.6 μL probe (10 μM), 11.2 μL ultrapure water, 2 μL template and 2.5 μL magnesium acetate (280 mM). The real-time CaPV RPA assay was carried out using the TwistAmp exo kit (TwistDx, Cambridge, UK), and the fluorescence signal in the FAM channel (Excitation 470 nm, Detection 520 nm) was detected in an Agilent Technologies Mx3005P thermocycler for 60 cycles at 38 °C for 20 s. The RPA reaction was completed in 20 min. A sample was deemed positive if all replicates were three and a half standard deviations (3.5SD) above the background during a defined time range (i.e. after 19 to 20 min of amplification). A threshold time range of 0 to 4 min and 30 s was used. The CaPV RPA LFD assay was performed using the TwistAmp nfo kit (TwistDx, Cambridge, UK) at 38 °C for 20 min in a water bath. The LFD strips (Milenia Biotec GmbH, Germany) were used to detect amplified products. One μL of the amplified product was diluted in 99 μL of the assay buffer (Tris-buffered saline). The LFD strip were directly inserted into the mixture and incubated at an upright position for 2 min. A test was considered positive when the detection line and the control line were visible. A test was considered negative when only the control line was visible.

To determine sensitivity of CaPV real-time RPA assay and CaPV RPA LFD assay, the pCaPV/RPA DNA (ranging from 3 × 10⁷ to 3 × 10¹ genome copies per reaction) was used in 8 replicates. RNase-free water was used as negative control reaction template in both CaPV real-time RPA assay and RPA LFD assay. The threshold time of CaPV real-time RPA assay was plotted against reference DNA molecules detected and the semi-log non-regression analysis was calculated with PRISM 5.0 software (GraphPad Software, USA). The probit analysis was performed using Statistica software (StatSoft, Hamburg, Germany). The specificity of both CaPV real-time RPA assay and RPA LFD assay was evaluated with nucleic acids extracted from different virus listed in Table 2. To determine the correlation of CaPV real-time RPA assay with CaPV real-time qPCR assay, both assays were tested with CaPV spiked samples (n = 24). The correction of CaPV real-time RPA assay threshold time (y axis) with CaPV real-time qPCR assay cycle threshold (CT) values (x axis) were generated by Excel software. To determine the optimum amplification temperature, CaPV RPA LFD assay was performed at a range of temperatures from 15 °C to 50 °C. The reaction time of CaPV RPA LFD assay was determined by terminating the RPA reaction at 0, 1, 5, 10, 15, 20, 25 and 30 min after the addition of magnesium acetate by immediate dilution and analysis on the lateral flow dipsticks.

In initial optimization, nine combinations of candidate primers (3 forward and 3 reverse, Table 1) were generated and tested for the time to fluorescence threshold with the probe. Of these, five primer pairs produced signal with the probe, and three of the primer pairs (CaPV Fe3/CaPV Re1, CaPV Fe2/CaPV Re2 and CaPV Fe3/CaPV Re2) performed similarly and produced signal faster than other pairs (CaPV Fe1/CaPV Re1 and CaPV Fe1/CaPV Re3) (Fig. 1). Due to resource constraints, the primer pairs CaPV Fe2/CaPV Re2 were selected randomly for subsequent evaluation.

To test the sensitivity of CaPV real-time RPA assay, serial dilutions of the purified reference DNA were tested for 8 replicates. As shown in Fig. 2, the dynamic detection range of the assay spans 5 logs ranging from 7 to 2 log copies per reaction, with the corresponding threshold time ranging from 3 min at 3 × 10⁷ copies per reaction to 7 min at 3 × 10² copies per reaction at 38 °C. This result indicates that CaPV real-time RPA assay has a wide dynamic range for quantifying target DNA (Fig. 2a, b). The detection limit of CaPV real-time RPA assay at 95% probability was 3 × 10² copies per reaction (probit analysis, p ≤ 0.05) (Fig. 2c). To further evaluate the sensitivity, the assay was tested with SPPV-spiked samples and compared with real-time qPCR assay. The results showed that both assays could detect viral DNA present in all the samples (Additional file 1: Table S1), and good correlation was found between threshold time (y axis) of CaPV real-time RPA assay and cycle threshold (CT) (x axis) of CaPV real-time qPCR assay (R squared 0.86, Fig. 3). In evaluation of the specificity of CaPV real-time RPA assay, consistent positive signal was only observed for GTPV strains (GTPV AV40, GTPV AV41, GTPV GS-V1) and SPPV strains (SPPV Gulang2009, SPPV Jingtai2011, SPPV Hubei), and no cross detection was observed with other viruses which can infect sheep and goats, including FMDV, ORFV and PPRV (Table 2). A simple DNA preparation method (single innuPREP MP basic kit with a magnetic bead separation rack) was also applied to spiked tissues samples. As shown in Table 3, CaPV real-time RPA assay performed well on these extracted DNA. There was no difference of the detection sensitivity between the two extraction methods when tested by CaPV real-time RPA assay (Table 3 and Additional file 1: Table S1).

In exploring the optimal temperature for amplification in CaPV RPA LFD assay, it was found that the target viral DNA gene can be amplified well from 30 °C to 45 °C and could be detected in more than 10 min (Fig. 4a, b). The sensitivity of the CaPV RPA LFD assay was determined using serial dilutions of the purified reference DNA as described above. As shown in Fig. 5a, the sensitivity of CaPV RPA LFD assay was 3 × 10² copies per reaction (Fig. 5a). The limit of detection in 95% probability was 3 × 10² copies per reaction (probit analysis, p ≤ 0.05) (Fig. 5b). To further determine its sensitivity, it was evaluated using SPPV-spiked samples (n = 24). Positive band on the LFD was observed for all spiked samples (Additional file 1: Table S1). The detection limit of CaPV RPA LFD assay was also tested on SPPV/Gulang 2009 gDNA (genomic DNA), and the results showed that it could detect as low as 10³ copies per reaction (Additional file 2: Figure S1). In investigation of the specificity of CaPV RPA LFD assay, the reactions were performed using a panel of genomes extracted from other important viruses of small ruminants as described above, which caused similar clinical signs. As shown in Table 2, the CaPV RPA LFD assay was specific for the detection of GTPV and SPPV. The CaPV RPA LFD assay also performed well on DNA extracted from spiked tissues samples using a simple DNA preparation method as described above (Table 3). And there was no difference of detection sensitivity between the two extraction methods when tested by CaPV RPA LFD assay (Table 3 and Additional file 1: Table S1).

All the clinical samples (n = 107) were detected simultaneously by CaPV real-time RPA assay, CaPV RPA LFD assay and CaPV real-time qPCR assay. Thirty-six samples were determined to be positive by CaPV real-time RPA assay (threshold time ranging from 4 to 6.6 min) and CaPV RPA LFD assay, while thirty-seven samples were positive by CaPV real-time qPCR assay (CT value ranging from 18 to 29). The clinical sensitivity and specificity of CaPV real-time RPA assay and CaPV RPA LFD assay for identification of GTPV and SPPV were 97% and 100%, respectively, when compared to CaPV real-time qPCR assay (Table 4).