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Detection and differentiation of classical swine fever virus strains C and Shimen by high-resolution melt analysis
Journal of Virological Methods 194 (2013) 129–131
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Detection and differentiation of classical swine fever virus strains C and Shimen by high-resolution melt analysis Pengbo Ning, Helin Li, Wulong Liang, Kangkang Guo, Xuechao Tan, Weiwei Cao, Liang Cheng, Yanming Zhang ∗ College of Veterinary Medicine, Northwest A&F University, PR China
a b s t r a c t Article history: Received 22 April 2013 Received in revised form 25 July 2013 Accepted 26 July 2013 Available online 27 August 2013 Keywords: Classical swine fever virus High-resolution melt Differential diagnosis
Differentiation of classical swine fever virus (CSFV) strains is crucial for the development of effective vaccination programs and in epidemiological investigations. Most of current detection methods do not discriminate between wild-type CSFV strains and those used in vaccines. In this study, method involving high-resolution melt (HRM) curve analysis for the simultaneous detection and differentiation of the C and Shimen strains of CSFV was developed. A specific fragment of the NS2 gene was amplified from various CSFV strains and subjected to HRM curve analysis. Analysis of the melt curve profile for the amplicons of each strain allowed the differentiation of CSFV strains in blood samples taken from the field, or from vaccinated commercial flocks. These findings indicate that HRM curve analysis is a rapid and practical technique for discriminating CSFV isolates/strains; it can contribute to epidemiological studies of CSFV and effective control of classical swine fever. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Classical swine fever (CSF), previously referred to as hog cholera, is a highly contagious disease of swine and wild boar, caused by classical swine fever virus (CSFV); it is a listed disease by the World Animal Health Organization (Zhao et al., 2008). CSF is often a fatal disease of swine that is characterized by fever, disseminated intravascular coagulation, thrombocytopenia, immunosuppression, leucopenia, and hemorrhaging (Paton et al., 2000; Summerfield et al., 1998, 2001). It is one of the most severe diseases that affect pigs worldwide, with massive economic consequences (Moennig et al., 2003). Different prevention and control strategies have been adopted to contain CSF in different parts of the world. CSF has been controlled successfully within the European Union by a stamping-out policy without prophylactic vaccination since the 1990s (Edwards et al., 2000; Moennig, 2000). For many countries outside of Europe, such as China, vaccination is a major control strategy, with attenuated vaccines used widely because they induce excellent immune responses (Suradhat et al., 2007). Most attenuated vaccines are based on the lapinized Chinese strain of hog cholera virus (HCLV), otherwise known as the C strain, which
∗ Corresponding author at: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, PR China. Tel.: +86 29 8709 2040; fax: +86 29 8709 1032. E-mail address: [email protected] (Y. Zhang). 0166-0934/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2013.07.048
are being safely and efficiently employed as prophylactics worldwide (Van Oirschot, 2003). Vaccination against CSFV has been largely successful; however, there have been some drawbacks to the mass vaccination of pig herds. Current vaccines on the market are unable to distinguish between the wild-type and HCLV strain of CSFV in vaccinated swine herds (Van Oirschot, 2003). Most of the current detection methods used, such as virus isolation, immunofluorescence tests (Bouma et al., 2001), and enzyme-linked immunosorbent assays (ELISAs) (Depner et al., 1995), are unable to distinguish between strains. Previously, it has been attempted to screen for antibodies against CSFV; unfortunately, whether the presence of these antibodies was due to a previous CSFV infection or vaccination could not be determined. This issue makes diagnosis of CSFV infections in swine very difficult and has implications on the import and export of pig products. In the past, CSF has been characterized by easily observable and typical symptoms such as fever and bleeding. Recently, however, these symptoms have been less obvious or absent in CSF cases. Therefore, it is necessary to develop a reliable and rapid method that can be used to screen pigs, especially those that are asymptomatic, and can differentiate between field and vaccine strains of CSFV. High-resolution melt (HRM) curve analysis is based on polymerase chain reaction (PCR) amplification in the presence of a fluorescent intercalating DNA dye, followed by analysis of the amplicon melting curve (Panichareon et al., 2011). Thermal cyclers are used to monitor the change in fluorescence caused by the release of the dye from the DNA duplex (the amplicon) as it is
130
P. Ning et al. / Journal of Virological Methods 194 (2013) 129–131
denatured with a stepwise increase in temperature (Wojdacz and Dobrovic, 2007). The melting profile of the DNA duplex depends on a number of factors, including GC content, length, sequence, and heterozygosity (Erali et al., 2008). In this study, a probe-free HRM method was developed in conjunction with a quantitative PCR (qPCR) technique to provide a rapid and cost-effective alternative for direct detection and differentiation of the C and Shimen strains of CSFV. The strains of CSFV (Shimen and C) used were obtained from the Control Institute of Veterinary Bioproducts and Pharmaceuticals (China). Virus strains were propagated in swine umbilical vein endothelial cells (SUVECs) as previously described (Hong et al., 2007). SUVECs were cultured in 25-cm2 tissue culture flasks at a density of 2 × 106 cells per flask. Virus was added to cultures when they were 70–80% confluent. After 1-h incubation at 37 ◦ C/5% CO2 , the medium was aspirated, and fresh medium containing 10% rectal calf serum (FCS) was added. CSFV-infected cells were collected at 72 h post-infection and stored at −70 ◦ C. Whole blood samples (n = 8) were collected from infected and vaccinated pigs across different farms in China. Infection status was diagnosed based on clinical signs and pathological findings. Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA), followed by phenol-chloroform extraction and ethanol precipitation. RNA yields were determined by measuring the absorbance of samples at 260 nm. A Poly(A) Purist kit (Ambion, Austin, TX, USA) was used to isolate the mRNA. To design oligonucleotide primers, the aligned sequences of Shimen (GenBank Accession No. AF092448) and C strain (AF091507) CSFV were used as a reference. Specific primers were designed using the DNAStar software package (DNAStar, Madison, WI, USA); their sequences were 5 -GAT CCT CAT ACT GCC CAC TTA C-3 (forward primer) and 5 -GTA TAC CCC TTC ACC AGC TTG-3 (reverse primer). cDNA was synthesized with the specific primers, and a PrimeScript RT reagent Kit containing gDNA Eraser (Takara Bio, Dalian, China) was used according to the manufacturer’s recommendations. All cDNA samples were stored at −20 ◦ C until required. The PCRs were performed in a total volume of 20 L by using a BioRad thermal cycler (BioRad Laboratories, Hercules, CA, USA). The thermal cycling profile involved an initial denaturation step at 95 ◦ C for 3 min, then 35 cycles of 94 ◦ C for 30 s, 60 ◦ C for 30 s, and 72 ◦ C for 30 s, with a final extension step at 72 ◦ C for 10 min after the 35th cycle. PCR products were detected by electrophoresis on 1.0% (w/v) agarose gels. The expected length of the PCR product was 148 bp. All PCRs were performed in triplicate. The qPCR and HRM analysis were conducted using a Type-it HRMTM PCR Kit (QIAGEN, Valencia, CA, USA). The 20-L reaction mixture contained 10 L of 2× HRM PCR Master Mix, 1 L of cDNA, 0.5 L of each primer, and 8 L of RNase-free water. The reaction was placed into a QIAGEN Rotor-GeneTM Q (QIAGEN), and the following thermal cycling profile was used: 95 ◦ C for 5 min, then 40 cycles at 95 ◦ C for 10 s, 55 ◦ C for 30 s, and 72 ◦ C for 10 s. After the amplification process was complete, HRM analysis was conducted by increasing the temperature in 0.1 ◦ C increments from 70 ◦ C to 90 ◦ C. Data generated after HRM analysis were interpreted using the Rotor-GeneTM Q software version 2.0.2. The results were confirmed by CT , melting temperature (Tm), melting curve profile, and the normalized curve profile, as recommended by the manufacturer. SUVECs infected with CSFV were analyzed by PCR, and visible bands around the expected size of 148 bp were obtained, regardless of virus strain (Fig. 1). Specific amplification curves were obtained when CSFV cDNA templates were subjected to HRM analysis (Fig. 2A). HRM analysis of amplicons from SUVECs infected with CSFV yielded 2 melt peaks, and the CSFV strains could be distinguished by HRM (Fig. 2B). Field samples were analyzed for the presence of the C and Shimen strains of CSFV by using HRM analysis (Fig. 3). These results were consistent with the PCR results
Fig. 1. Virological PCR findings in CSFV-infected. Lane 1: molecular weight marker; Lane 2: PCR product amplified from SUVEC sample infected with CSFV C strain. Lane 3: PCR product amplified from SUVEC sample infected with CSFV Shimen strain. Lane 4: PCR-negative control (no DNA); Lane 5: PCR-positive control (-actin) SUVECs.
described above (data not shown). Infection with either strain could be differentiated by HRM analysis (Fig. 3); 4 pigs were found to be positive for the C strain, and another 4 pigs were positive for the Shimen strain. Samples that were positive for either CSFV strain were not positive for BVDV, TGEV, PCV-2, or PRRSV when tested by PCR or HRM analysis (data not shown). Serial 10-fold dilutions of RNA standards were subjected to HRM analysis to evaluate the sensitivity of the method. The lowest limit of detection was found to be 10 pg of RNA. Three independent inter- and intra-assay reproducibility tests were conducted; consistent results were obtained. This is the first report presenting the successful application of HRM analysis for simultaneous, 1-step detection and differentiation of CSFV C and Shimen strains. CSF remains one of the most important swine diseases for many developing countries. In recent years, clinical manifestations of CSF epidemics and infections have markedly changed in China, with the emergence of atypical or chronic forms of CSF. One reason could be co-infections with other pathogens such as PCV2 and PRRSV (Rovira et al., 2002). It is also worth noting that mass vaccinations occur with attenuated forms of the CSFV C strain. Under these pressures, an increased pathogen burden provokes immunological tolerance mechanisms that protect the host from immune system or pathogen-related damage, resulting in mild clinical pathological symptoms (Medzhitov et al., 2012). Traditional CSFV detection methods have relied on virus isolation or serological diagnosis. These methods are often unable to differentiate between wild-type CSFV strains and those used in vaccine development. Molecular assays for the detection and differentiation of CSFV from other pathogens have been developed to replace classical assays and are in wide use. However, it has been more challenging to discriminate between the Shimen and C strains of CSFV. The present communication describes an HRM analysis used for the detection and differentiation of CSFV strains. HRM curve analysis involves precise monitoring of the change in fluorescence caused by the release of an intercalating DNA dye from a DNA duplex as it denatures at high temperatures. Therefore, primer design is a key factor for successful HRM analysis. By analyzing and comparing the full-length genome sequences of these CSFV strains, the highly conserved NS2 gene was selected as a target for the primers used in this study. By using the designed primers, a specific CSFV gene fragment that was stable with respect to variation in the amplified nucleotide fragment between the C and Shimen strains was amplified. The specificity and sensitivity of both the PCR and HRM methods showed that these methods are feasible detection tools. Further, distinctive melt curves were obtained, which allow for differentiation of the 2 CSFV strains. As expected, wild-type CSFV was
P. Ning et al. / Journal of Virological Methods 194 (2013) 129–131
131
Fig. 2. CSFV-infected SUVECs analyzed by HRM curves. (A) Normalized and (B) differential analysis of amplicons from 4 SUVEC samples infected with CSFV C and Shimen strains.
Fig. 3. CSFV-infected field samples analyzed by HRM curves. (A) Normalized and (B) differential analysis of amplicons from 8 porcine whole blood samples.
routinely detected in field samples, while the CSFV C strain was readily detected in pigs vaccinated under Chinese regimens. Reference samples, or standard controls, should be prepared to initially perform these assays, because mutations in viral genes may occur over time. The newly developed HRM assay is cost-effective, and it is more sensitive and reliable than currently available methods, because it is a single-tube, probe-free, closed system based on PCR technology. This technique is an attractive alternative to screen large numbers of clinical samples in epidemiological studies of CSFV. The HRM method can also be used in the investigation of vaccinated animals during epidemics, thereby minimizing the number of animals that are required to be culled. Competing interests The authors declare that they have no competing interests. Acknowledgments The authors thank Mr. Gang Liu, Mrs. Li Song and Mr. Jin Hao for their excellent technical assistance. This work was supported by the National Natural Science Foundation of China (No. 31172339). References Bouma, A., Stegeman, J.A., Engel, B., de Kluijver, E.P., Elbers, A.R., De Jong, M.C., 2001. Evaluation of diagnostic tests for the detection of classical swine fever in the field without a gold standard. J. Vet. Diagn. Invest. 13, 383–388. Depner, K., Paton, D.J., Cruciere, C., De Mia, G.M., Muller, A., Koenen, F., Stark, R., Liess, B., 1995. Evaluation of the enzyme-linked immunosorbent assay for the rapid screening and detection of classical swine fever virus antigens in the blood of pigs. Rev. Sci. Tech. 14, 677–689. Edwards, S., Fukusho, A., Lefevre, P.C., Lipowski, A., Pejsak, Z., Roehe, P., Westergaard, J., 2000. Classical swine fever: the global situation. Vet. Microbiol. 73, 103–119.
Erali, M., Voelkerding, K.V., Wittwer, C.T., 2008. High resolution melting applications for clinical laboratory medicine. Exp. Mol. Pathol. 85, 50–58. Hong, H.X., Zhang, Y.M., Xu, H., Su, Z.Y., Sun, P., 2007. Immortalization of swine umbilical vein endothelial cells with human telomerase reverse transcriptase. Mol. Cell. 24, 358–363. Medzhitov, R., Schneider, D.S., Soares, M.P., 2012. Disease tolerance as a defense strategy. Science 335, 936–941. Moennig, V., 2000. Introduction to classical swine fever: virus, disease and control policy. Vet. Microbiol. 73, 93–102. Moennig, V., Floegel-Niesmann, G., Greiser-Wilke, I., 2003. Clinical signs and epidemiology of classical swine fever: a review of new knowledge. Vet. J. 165, 11–20. Panichareon, B., Khawsak, P., Deesukon, W., Sukhumsirichart, W., 2011. Multiplex real-time PCR and high-resolution melting analysis for detection of white spot syndrome virus, yellow-head virus, and Penaeus monodon densovirus in penaeid shrimp. J. Virol. Methods 178, 16–21. Paton, D.J., McGoldrick, A., Belak, S., Mittelholzer, C., Koenen, F., Vanderhallen, H., Biagetti, M., De Mia, G.M., Stadejek, T., Hofmann, M.A., Thuer, B., 2000. Classical swine fever virus: a ring test to evaluate RT-PCR detection methods. Vet. Microbiol. 73, 159–174. Rovira, A., Balasch, M., Segalés, J., García, L., Plana-Durán, J., Rosell, C., Ellerbrok, H., Mankertz, A., Domingo, M., 2002. Experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type-2. J. Virol. 76, 3232–3239. Summerfield, A., Hofmann, M.A., McCullough, K.C., 1998. Low density blood granulocytic cells induced during classical swine fever are targets for virus infection. Vet. Immunol. Immunopathol. 63, 289–301. Summerfield, A., Zingle, K., Inumaru, S., McCullough, K.C., 2001. Induction of apoptosis in bone marrow neutrophil-lineage cells by classical swine fever virus. J. Gen. Virol. 82, 1309–1318. Suradhat, S., Damrongwatanapokin, S., Thanawongnuwech, R., 2007. Factors critical for successful vaccination against classical swine fever in endemic areas. Vet. Microbiol. 119, 1–9. Van Oirschot, J.T., 2003. Emergency vaccination against classical swine fever. Dev. Biol. 114, 259–267. Wojdacz, T.K., Dobrovic, A., 2007. Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation. Nucleic Acids Res. 35 (6), e41. Zhao, J.J., Cheng, D., Li, N., Sun, Y., Shi, Z., Zhu, Q.H., Tu, C., Tong, G.Z., Qiu, H.J., 2008. Evaluation of a multiplex real-time RT-PCR for quantitative and differential detection of wild-type viruses and C-strain vaccine of Classical swine fever virus. Vet. Microbiol. 126, 1–10.
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Detection and differentiation of classical swine fever virus strains C and Shimen by high-resolution melt analysis Pengbo Ning, Helin Li, Wulong Liang, Kangkang Guo, Xuechao Tan, Weiwei Cao, Liang Cheng, Yanming Zhang ∗ College of Veterinary Medicine, Northwest A&F University, PR China
a b s t r a c t Article history: Received 22 April 2013 Received in revised form 25 July 2013 Accepted 26 July 2013 Available online 27 August 2013 Keywords: Classical swine fever virus High-resolution melt Differential diagnosis
Differentiation of classical swine fever virus (CSFV) strains is crucial for the development of effective vaccination programs and in epidemiological investigations. Most of current detection methods do not discriminate between wild-type CSFV strains and those used in vaccines. In this study, method involving high-resolution melt (HRM) curve analysis for the simultaneous detection and differentiation of the C and Shimen strains of CSFV was developed. A specific fragment of the NS2 gene was amplified from various CSFV strains and subjected to HRM curve analysis. Analysis of the melt curve profile for the amplicons of each strain allowed the differentiation of CSFV strains in blood samples taken from the field, or from vaccinated commercial flocks. These findings indicate that HRM curve analysis is a rapid and practical technique for discriminating CSFV isolates/strains; it can contribute to epidemiological studies of CSFV and effective control of classical swine fever. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Classical swine fever (CSF), previously referred to as hog cholera, is a highly contagious disease of swine and wild boar, caused by classical swine fever virus (CSFV); it is a listed disease by the World Animal Health Organization (Zhao et al., 2008). CSF is often a fatal disease of swine that is characterized by fever, disseminated intravascular coagulation, thrombocytopenia, immunosuppression, leucopenia, and hemorrhaging (Paton et al., 2000; Summerfield et al., 1998, 2001). It is one of the most severe diseases that affect pigs worldwide, with massive economic consequences (Moennig et al., 2003). Different prevention and control strategies have been adopted to contain CSF in different parts of the world. CSF has been controlled successfully within the European Union by a stamping-out policy without prophylactic vaccination since the 1990s (Edwards et al., 2000; Moennig, 2000). For many countries outside of Europe, such as China, vaccination is a major control strategy, with attenuated vaccines used widely because they induce excellent immune responses (Suradhat et al., 2007). Most attenuated vaccines are based on the lapinized Chinese strain of hog cholera virus (HCLV), otherwise known as the C strain, which
∗ Corresponding author at: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, PR China. Tel.: +86 29 8709 2040; fax: +86 29 8709 1032. E-mail address: [email protected] (Y. Zhang). 0166-0934/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2013.07.048
are being safely and efficiently employed as prophylactics worldwide (Van Oirschot, 2003). Vaccination against CSFV has been largely successful; however, there have been some drawbacks to the mass vaccination of pig herds. Current vaccines on the market are unable to distinguish between the wild-type and HCLV strain of CSFV in vaccinated swine herds (Van Oirschot, 2003). Most of the current detection methods used, such as virus isolation, immunofluorescence tests (Bouma et al., 2001), and enzyme-linked immunosorbent assays (ELISAs) (Depner et al., 1995), are unable to distinguish between strains. Previously, it has been attempted to screen for antibodies against CSFV; unfortunately, whether the presence of these antibodies was due to a previous CSFV infection or vaccination could not be determined. This issue makes diagnosis of CSFV infections in swine very difficult and has implications on the import and export of pig products. In the past, CSF has been characterized by easily observable and typical symptoms such as fever and bleeding. Recently, however, these symptoms have been less obvious or absent in CSF cases. Therefore, it is necessary to develop a reliable and rapid method that can be used to screen pigs, especially those that are asymptomatic, and can differentiate between field and vaccine strains of CSFV. High-resolution melt (HRM) curve analysis is based on polymerase chain reaction (PCR) amplification in the presence of a fluorescent intercalating DNA dye, followed by analysis of the amplicon melting curve (Panichareon et al., 2011). Thermal cyclers are used to monitor the change in fluorescence caused by the release of the dye from the DNA duplex (the amplicon) as it is
130
P. Ning et al. / Journal of Virological Methods 194 (2013) 129–131
denatured with a stepwise increase in temperature (Wojdacz and Dobrovic, 2007). The melting profile of the DNA duplex depends on a number of factors, including GC content, length, sequence, and heterozygosity (Erali et al., 2008). In this study, a probe-free HRM method was developed in conjunction with a quantitative PCR (qPCR) technique to provide a rapid and cost-effective alternative for direct detection and differentiation of the C and Shimen strains of CSFV. The strains of CSFV (Shimen and C) used were obtained from the Control Institute of Veterinary Bioproducts and Pharmaceuticals (China). Virus strains were propagated in swine umbilical vein endothelial cells (SUVECs) as previously described (Hong et al., 2007). SUVECs were cultured in 25-cm2 tissue culture flasks at a density of 2 × 106 cells per flask. Virus was added to cultures when they were 70–80% confluent. After 1-h incubation at 37 ◦ C/5% CO2 , the medium was aspirated, and fresh medium containing 10% rectal calf serum (FCS) was added. CSFV-infected cells were collected at 72 h post-infection and stored at −70 ◦ C. Whole blood samples (n = 8) were collected from infected and vaccinated pigs across different farms in China. Infection status was diagnosed based on clinical signs and pathological findings. Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA), followed by phenol-chloroform extraction and ethanol precipitation. RNA yields were determined by measuring the absorbance of samples at 260 nm. A Poly(A) Purist kit (Ambion, Austin, TX, USA) was used to isolate the mRNA. To design oligonucleotide primers, the aligned sequences of Shimen (GenBank Accession No. AF092448) and C strain (AF091507) CSFV were used as a reference. Specific primers were designed using the DNAStar software package (DNAStar, Madison, WI, USA); their sequences were 5 -GAT CCT CAT ACT GCC CAC TTA C-3 (forward primer) and 5 -GTA TAC CCC TTC ACC AGC TTG-3 (reverse primer). cDNA was synthesized with the specific primers, and a PrimeScript RT reagent Kit containing gDNA Eraser (Takara Bio, Dalian, China) was used according to the manufacturer’s recommendations. All cDNA samples were stored at −20 ◦ C until required. The PCRs were performed in a total volume of 20 L by using a BioRad thermal cycler (BioRad Laboratories, Hercules, CA, USA). The thermal cycling profile involved an initial denaturation step at 95 ◦ C for 3 min, then 35 cycles of 94 ◦ C for 30 s, 60 ◦ C for 30 s, and 72 ◦ C for 30 s, with a final extension step at 72 ◦ C for 10 min after the 35th cycle. PCR products were detected by electrophoresis on 1.0% (w/v) agarose gels. The expected length of the PCR product was 148 bp. All PCRs were performed in triplicate. The qPCR and HRM analysis were conducted using a Type-it HRMTM PCR Kit (QIAGEN, Valencia, CA, USA). The 20-L reaction mixture contained 10 L of 2× HRM PCR Master Mix, 1 L of cDNA, 0.5 L of each primer, and 8 L of RNase-free water. The reaction was placed into a QIAGEN Rotor-GeneTM Q (QIAGEN), and the following thermal cycling profile was used: 95 ◦ C for 5 min, then 40 cycles at 95 ◦ C for 10 s, 55 ◦ C for 30 s, and 72 ◦ C for 10 s. After the amplification process was complete, HRM analysis was conducted by increasing the temperature in 0.1 ◦ C increments from 70 ◦ C to 90 ◦ C. Data generated after HRM analysis were interpreted using the Rotor-GeneTM Q software version 2.0.2. The results were confirmed by CT , melting temperature (Tm), melting curve profile, and the normalized curve profile, as recommended by the manufacturer. SUVECs infected with CSFV were analyzed by PCR, and visible bands around the expected size of 148 bp were obtained, regardless of virus strain (Fig. 1). Specific amplification curves were obtained when CSFV cDNA templates were subjected to HRM analysis (Fig. 2A). HRM analysis of amplicons from SUVECs infected with CSFV yielded 2 melt peaks, and the CSFV strains could be distinguished by HRM (Fig. 2B). Field samples were analyzed for the presence of the C and Shimen strains of CSFV by using HRM analysis (Fig. 3). These results were consistent with the PCR results
Fig. 1. Virological PCR findings in CSFV-infected. Lane 1: molecular weight marker; Lane 2: PCR product amplified from SUVEC sample infected with CSFV C strain. Lane 3: PCR product amplified from SUVEC sample infected with CSFV Shimen strain. Lane 4: PCR-negative control (no DNA); Lane 5: PCR-positive control (-actin) SUVECs.
described above (data not shown). Infection with either strain could be differentiated by HRM analysis (Fig. 3); 4 pigs were found to be positive for the C strain, and another 4 pigs were positive for the Shimen strain. Samples that were positive for either CSFV strain were not positive for BVDV, TGEV, PCV-2, or PRRSV when tested by PCR or HRM analysis (data not shown). Serial 10-fold dilutions of RNA standards were subjected to HRM analysis to evaluate the sensitivity of the method. The lowest limit of detection was found to be 10 pg of RNA. Three independent inter- and intra-assay reproducibility tests were conducted; consistent results were obtained. This is the first report presenting the successful application of HRM analysis for simultaneous, 1-step detection and differentiation of CSFV C and Shimen strains. CSF remains one of the most important swine diseases for many developing countries. In recent years, clinical manifestations of CSF epidemics and infections have markedly changed in China, with the emergence of atypical or chronic forms of CSF. One reason could be co-infections with other pathogens such as PCV2 and PRRSV (Rovira et al., 2002). It is also worth noting that mass vaccinations occur with attenuated forms of the CSFV C strain. Under these pressures, an increased pathogen burden provokes immunological tolerance mechanisms that protect the host from immune system or pathogen-related damage, resulting in mild clinical pathological symptoms (Medzhitov et al., 2012). Traditional CSFV detection methods have relied on virus isolation or serological diagnosis. These methods are often unable to differentiate between wild-type CSFV strains and those used in vaccine development. Molecular assays for the detection and differentiation of CSFV from other pathogens have been developed to replace classical assays and are in wide use. However, it has been more challenging to discriminate between the Shimen and C strains of CSFV. The present communication describes an HRM analysis used for the detection and differentiation of CSFV strains. HRM curve analysis involves precise monitoring of the change in fluorescence caused by the release of an intercalating DNA dye from a DNA duplex as it denatures at high temperatures. Therefore, primer design is a key factor for successful HRM analysis. By analyzing and comparing the full-length genome sequences of these CSFV strains, the highly conserved NS2 gene was selected as a target for the primers used in this study. By using the designed primers, a specific CSFV gene fragment that was stable with respect to variation in the amplified nucleotide fragment between the C and Shimen strains was amplified. The specificity and sensitivity of both the PCR and HRM methods showed that these methods are feasible detection tools. Further, distinctive melt curves were obtained, which allow for differentiation of the 2 CSFV strains. As expected, wild-type CSFV was
P. Ning et al. / Journal of Virological Methods 194 (2013) 129–131
131
Fig. 2. CSFV-infected SUVECs analyzed by HRM curves. (A) Normalized and (B) differential analysis of amplicons from 4 SUVEC samples infected with CSFV C and Shimen strains.
Fig. 3. CSFV-infected field samples analyzed by HRM curves. (A) Normalized and (B) differential analysis of amplicons from 8 porcine whole blood samples.
routinely detected in field samples, while the CSFV C strain was readily detected in pigs vaccinated under Chinese regimens. Reference samples, or standard controls, should be prepared to initially perform these assays, because mutations in viral genes may occur over time. The newly developed HRM assay is cost-effective, and it is more sensitive and reliable than currently available methods, because it is a single-tube, probe-free, closed system based on PCR technology. This technique is an attractive alternative to screen large numbers of clinical samples in epidemiological studies of CSFV. The HRM method can also be used in the investigation of vaccinated animals during epidemics, thereby minimizing the number of animals that are required to be culled. Competing interests The authors declare that they have no competing interests. Acknowledgments The authors thank Mr. Gang Liu, Mrs. Li Song and Mr. Jin Hao for their excellent technical assistance. This work was supported by the National Natural Science Foundation of China (No. 31172339). References Bouma, A., Stegeman, J.A., Engel, B., de Kluijver, E.P., Elbers, A.R., De Jong, M.C., 2001. Evaluation of diagnostic tests for the detection of classical swine fever in the field without a gold standard. J. Vet. Diagn. Invest. 13, 383–388. Depner, K., Paton, D.J., Cruciere, C., De Mia, G.M., Muller, A., Koenen, F., Stark, R., Liess, B., 1995. Evaluation of the enzyme-linked immunosorbent assay for the rapid screening and detection of classical swine fever virus antigens in the blood of pigs. Rev. Sci. Tech. 14, 677–689. Edwards, S., Fukusho, A., Lefevre, P.C., Lipowski, A., Pejsak, Z., Roehe, P., Westergaard, J., 2000. Classical swine fever: the global situation. Vet. Microbiol. 73, 103–119.
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