Polymerase cross-linking spiral reaction (PCLSR) for detection of African swine fever virus (ASFV) in pigs and wild boars

The study reports the development of a polymerase cross-linking spiral reaction (PCLSR) for the detection of African swine fever virus (ASFV) DNA in blood collected from infected pigs and wild boars. The method uses 3 specifically designed primers. Two outer-spiral primers comprising of 3′ sequences complementary to ASFV p72 gene sequence and 5′end sequences complementary to exogenous gene of black widow alpha-latrotoxin as well as additional ASFV specific cross-linking primer. The method is specific exclusively to ASFV DNA without cross-reactions with cDNA of classical swine fever virus (CSFV), porcine reproductive respiratory syndrome (PRRSV) or porcine epidemic diarrhea virus (PEDV). The sensitivity of this technique reached 7.2 × 102 copies per μl−1 of plasmid containing p72 gene. The PCLSR was conducted at 65 °C creating cross-linked complex structures. The results of PCLSR were visualized using SYBR Green I dye, gel electrophoresis while the reaction progress was traced using real-time PCR system that resulted in registration of fluorescent curves and melting peaks at 85.3 °C. The developed PCLSR was examined using blood or tissue samples collected from selected 17 ASF cases from infected wild boars and 3 outbreaks in pigs. Further tests have been also conducted using 55 tissue samples from 23 outbreaks and 22 cases. These results showed that PCLSR might be further used for preliminary and cost-effective detection and surveillance of ASFV.

Scientific RepoRts | 7:42903 | DOI: 10.1038/srep42903 strand-displacement activity including Bst, Bsm or GspSSD. The developed assay shows some similarities with LAMP, (CPA) 15,17 , nucleic acid sequence dependent amplification (NASBA) 18 , rolling circle replication (RCR) 19 and recently polymerase spiral reaction (PSR) described for the first time by Liu et al. 20 then subsequently applied by other scientists for detection of Candida albicans 21 . All these methods have an advantage to overcome the requirement of cycling which is compulsory in case of PCR-based techniques or real-time PCR. However, the mechanism and progress of PCLSR is different and relies on formation of 3 independent prerequisite spiral products. Similarly, to CPA or PSR the final cross-linked products form complex entities which result from target DNA sequence multiplication. The final products detection is mediated after addition of double-DNA binding fluorescent dye including SYBR Green ® I. Having the possibility to trace the reaction progress, the developed PCLSR can also be performed using real-time PCR system as a semi-quantitative technique. The results are finally registered as fluorescence of samples containing ASFV DNA under UV light as greenish fluorescence, or the presence of fluorescent curves at a particular cycle of the reaction using real-time PCR system. To the best of our knowledge, this is the first report of development of the PCLSR as an alternative assay for other described isothermal detection methods. The PCLSR presents a simple alternative for real-time PCR techniques recommended by OIE or EURL for ASF diagnosis and hopefully might further be considered as an official method. The PCLSR can also be easily adapted for on-site identification of other human, animal or plant pathogens.

Results
Design of PCLSR. The PCLSR method relies on isothermal amplification of targeted DNA using DNA polymerase showing strand displacement features. The primer design was similar to those applied in other isothermal methods but included 2 outer-spiral primers with 5′ terminal ExF and ExR sequences reverse complementary to DNA of black widow (Latrodectus hesperus) alpha-latrotoxin (position 180-199) (Genbank accession: KF751511.1). The 3′ sequence of outer-spiral primers (IntF and IntR) were complementary to the p72 gene sequence (positions 104980-104999 and 105116-105135) of Georgia 2007/1 strain (Genbank accession: FR682468.1). The cross-linking primer AB (comprising of Link A and Link B) was complementary exclusively to p72 gene sequence of ASFV and included DraI restricition site for further confirmation of results specificity (positions 105014-105034 and 105054-105072). The primer location and sequence has been shown in Fig. 1.

Reaction mechanism.
The mechanism of PCLSR has been presented in Fig. 2. During the initial stage at 65 °C the double structure of DNA is displaced by GspSSD polymerase (OptiGene, Horsham, West Sussex, United Kingdom). The first stage (1) (left part of Fig. 2) shows the cross-linking primer ligating with its LinkB fragment to the targeted sequence of p72 ASFV gene, with the polymerase extending the product from 5′ to the 3′ end of the structure. At the same time, the same process occurs on the opposite 3′ -5′ strand of p72 gene (right part of the Fig. 2). The next step is the ligation of the outer primers with their 3′ ends (Int-F and Int-R) to the complementary external sequences of the targeted region (1). Within the stage (2) amplification results in displacement of the newly synthesized products - Fig. 2 left and right structure (2). Next, during the following stages the complementary strands are synthesized and this leads to multiplication of the target primary sequence (3 and 4). During the reaction progress three parallel structures with different size (115 bp, 165 bp and 185 bp) are synthesized (stages 3 and 4). The remaining 5′ ends of the products, namely ExF and ExR of outer-spiral primers which are reverse-complementary ligate to each other and form complex cross-linked spiral structures (stage 5).
Examination of PCLSR parameters. The PCLSR was conducted at 65 °C being the most optimal temperature using a water bath. The sensitivity test presented as the electrophoresis plot in 1.5% agarose gel (Invitrogen) stained with SimplySafe solution (EurX) showed the presence of ladder-like pattern of bands in serial 10-fold dilutions of a standard ASFV plasmid with p72 gene fragment (from 7.2 × 10 7 copies to 0.07 copies μl −1 ) (Fig. 3A, lane 1-9). The obtained limit of detection (LoD) reached 7.2 × 10 2 copies μl −1 (Fig. 3A, lane 6). The conducted examination of the optimal reaction time showed that the PCLSR was capable of amplifying ASFV DNA in 45 min (Fig. 3B, lane 45 min). Longer amplification time does not result in higher yield of DNA products (Fig. 3B, lanes 60 min-90 min). The specificity test showed PCLSR was specific only for ASFV DNA (Fig. 3C, lane 2). No presence of specific ladder-like products was observed in negative controls represented by DNA extracted from  of resulted products as well as using MX3005P real-time PCR system (Stratagene) which is broadly used for real-time PCR detection of ASFV DNA. The presence of relative fluorescence increase ('R'-T) during the following minutes of the reaction was considered as positive result. The increment of fluorescent signal gained during the successive PCLSR cycles was proportional to the initial concentration of ASFV DNA within the sample. The conducted PCLSR evaluation using different ASFV genotypes or strains and matrices showed the method is capable to detect DNA of strains belonging to genotype I, II, V, VIII, IX and X (Fig. 4A). The ASFV DNA was detected in bone marrow, blood, serum, spleen, kidneys, lymph nodes, tonsil, lungs or muscle fibers collected from infected pigs or wild boars (Fig. 4B).
Evaluation of PCLSR using samples from infected wild boars and pigs. Next, the developed assay was used for amplification of ASFV DNA extracted from blood of wild boars and pigs originating from selected 17 cases and 3 outbreaks (Table 1). This stage of study was conducted for examination of possible application of PCLSR as a simple method for ASFV surveillance. Additionally, a set of negative controls represented by cDNA of CSFV and PRRS was applied to test the specificity of the developed assay. The PCLSR conducted in real-time PCR system resulted in registration of fluorescent curves starting from 32 min (21 st case) up to 78 min (64 th case) (Fig. 5A). The conducted melting curve analysis showed the common temperature of products for all examined samples reached 85.3 °C (Fig. 5B). All samples containing ASFV DNA from 17 wild boar cases and 3 ASF outbreaks in pigs examined after addition of SYBR Green I showed greenish fluorescence under UV light (Fig. 5C). The conducted electrophoresis of the resulted products in 1.5% agarose gel (Invitrogen) showed the presence of specific ladder-like products in all 20 ASFV positive samples and lack of any product in negative controls (Fig. 5D). In order to confirm the obtained results PCR was conducted using a pair of outer primers. After gel electrophoresis of PCR products in 1.5% agarose gel in all ASFV positive samples the presence of band approximately 185 bp long was observed. In contrast, the presence of products was not observed in specificity controls comprising of nucleic acid extracted from CSFV and PRRS strains (Fig. 5E, lanes 21 and 22), either in negative control of reaction mixture (Fig. 4E, lane 24). Further tests conducted on 55 tissue samples collected from 23 ASF outbreaks in pigs and 22 cases in wild boars showed the ASFV DNA presence in all examined samples (data not presented). Additional tests using LAMP and CPA on field samples from 17 ASF cases in wild boar and 3 outbreaks in pigs showed that samples #5 and #12 were negative in LAMP (Fig. 6A). In case of CPA in spite of remarkable fluorescence the ladder-like products were are weak or invisible in case of samples #3-6, 12 and 20 (Fig. 6B). These results clearly indicate that data obtained by PCLSR are more robust and reliable than LAMP or CPA in comparative results on the specificity of the developed PCLSR and its potential usefulness for on-site detection of ASFV in samples collected from wild boar or pigs.

Discussion
Diagnosis of numerous infectious animal diseases remains one of the most important tool for early prevention and control 22,23 . In case of ASF, diagnosis methods are recommended by OIE as well as EURL in Spain. However, some part of these tools based on PCR methods require advanced laboratory equipment comprising of PCR thermal cycles or more expensive real-time PCR systems [6][7][8] . In fact, broad application of PCR-based methods during the last 20 years has changed and improved the current diagnosis of ASF. Next, the alternative isothermal amplification methods starting from LAMP in 2001 24 were implemented into routine detection of many pathogens. Meanwhile, isothermal amplification methods may generate some part of false positive results due to high efficiency of applied polymerases and lack of mechanism of preliminary activation of non-active or blocked enzyme 25 . Therefore, the proper selection or development of novel isothermal technique should especially take into account the possible cross-reactivity of the assay. The developed PCLSR is a novel technique of isothermal amplification that puts together the advantage of traditional PCR or real-time PCR with the relative simplicity of isothermal amplification. Application of external primers with 5′ -ends complementary to the distanced gene of black widow (Latrodectus hesperus) alpha-latrotoxin protects the initial steps of reaction. On the basis of the conducted comparison of LAMP, CPA and PCLSR it has been found that PCLSR shares some similarities with these methods but offers higher diagnostic specificity in comparison to the previously described isothermal assays 25 . The cost of all isothermal methods is comparable and reaches approximately 2 EURO per sample. Our previous experience with LAMP, CPA and PSR showed that novel design of PCLSR primers may solve the problem with specificity of isothermal methods. An advantage of this test is the possibility for its on-site ASF diagnosis as a portable assay. The observed diagnostic sensitivity reached 7.2 × 10 2 copies of plasmid containing a fragment of p72 gene which was below the value of 7.2 copies reached by CPA or real-time PCR 14 . Similarly to previously described CPA, this method might be used by veterinary practitioners, veterinary officers or hunters. The isothermal methods including PCLSR seem to be a future alternative for PCR and real-time PCR assays but are not recommended by OIE yet. Hopefully, future application of PCLSR may assist in development of better biosecurity measures in terms of transportation, destruction of ASFV infectious material storage.    The PCLSR sensitivity was evaluated using serial 10-fold dilutions of a standard ASFV plasmid with p72 gene fragment (from 7.2 × 10 7 copies to 0.07 copies, μl −1 ). The resulted PCLSR products separated in 1.5% agarose gels, stained with a SimplySafe solution (0.5 μg ml −1 ) (EURx, Gdansk, Poland) under a voltage of 100 V/50 min, to detect "ladder-like" pattern of products. Parallelly, the PCLSR was conducted in MX3005P real-time PCR system (Stratagene). The presence of relative fluorescence increase ('R'-T) during the following minutes of the reaction was considered as a positive result.

Methods
Purification and sequencing of PCLSR products. The PCLSR products were purified using  DNA extraction. The DNA from the reference virus stock as well as the whole blood from wild boars and pigs was extracted using High Pure PCR Template Preparation Kit, accordingly to the manufacturer's procedure (Roche Diagnostics, Basel, Switzerland). The extracted DNA was stored at − 20 °C for further testing.
PCR. The PCR for PCLSR results verification was performed in 25 μ l final volume using outer PCLSR primers accordingly to the procedure of MyTaq HS DNA Polymerase kit (Bioline, Gdansk, Poland). The reaction mixture contained: 12.5 μ l of MyTaq ™ HS DNA Polymerase, 9.5 μ l PCR-grade water, 1 μ l of each primer and 1 μ l (~200 ng) template DNA. The primer concentration was 40 pM of each primer. The obtained PCR products were subjected to electrophoresis in 1.5% agarose gels under voltage of 100 V/50 min. The gels were stained by addition of 5 μ l of SimplySafe solution as for PCLSR product separation. The length of PCR products were estimated on the basis of 100 bp DNA Ladder Plus GeneRuler (Thermo-scientific, Waltham, Massachusetts, USA). All PCLSR were replicated to verify reproducibility.
LAMP. LAMP has been conducted using previously described primers 13 in 15 μl reaction volume using the following concentration of reagents: 7.5 μl of Isothermal Mastermix 50 pmol of forward and backward inner primer (FIP and BIP), 10 pmol of outer primers (F3 and B3) and 25 pmol of loop primers (LF and LB), 1 μl of standard plasmid, containing a p72 gene. After incubation, 1 μl of a 1:10 stock dilution of 10,000 x DMSO concentrated SYBR Green I dye (Invitrogen) was added to the reaction vessel. The results were registered under UV illumination, to detect fluorescence.