Development of a reverse-transcription polymerase chain reaction assay with fluorogenic probes to discriminate Korean wild-type and vaccine isolates of Classical swine fever virus

Abstract

A 1-step reverse-transcription polymerase chain reaction (RT-PCR) assay using TaqMan minor-groove-binding (MGB) probes was developed to distinguish between vaccine-type and wild-type strains of Classical swine fever virus (CSFV) in Korea. Because attenuated Korean LOM strains have been used in animal vaccination in Korea for some time but CSF remains a serious problem, there was a need for a practical approach to differentiating vaccine and field strains. We examined the fluorescence of 5 vaccine strains, 10 field strains, and 5 mixed samples. Three clusters of the samples could be distinguished: those with only fluorescence of the vaccine-type-specific probe, VIC; those with only fluorescence of the wild-type-specific probe, FAM; and those with both VIC and FAM fluorescence. The RT-PCR assay with fluorogenic probes is sensitive and accurate and is therefore useful for differentiating vaccine and field strains of CSFV in Korea.

Classical swine fever (CSF), a highly contagious and often fatal disease, affects both domestic and wild pigs. The causative agent, Classical swine fever virus (CSFV), is a member of the genus Pestivirus, which belongs to the family Flaviviridae (1). Other important animal pathogens within this genus are Bovine viral diarrhea virus (BVDV) and Border disease virus (BDV), which infects sheep (1). Both BVDV and BDV can naturally infect pigs, and the antibodies generated cross-react with CSFV in serologic assays, which makes a CSF diagnosis difficult (2,3).

In Korea, pigs have been vaccinated since the 1970s with the live attenuated strain of CSFV known as K-LOM (4). Although a CSFV eradication program (vaccination, test, and slaughter) is continuous, CSF is still a major viral problem. For this reason, distinguishing between vaccine and wild strains of the virus is important in Korea. The current method for genotyping vaccine-type and wild-type CSFV in Korea includes amplification by means of polymerase chain reaction (PCR), followed by sequencing or digestion with the restriction endonuclease Xho I (5), a process that takes more than 8 h, excluding RNA extraction.

Because serologic tests cannot distinguish between antibodies against the vaccine and field strains, and because there is no practical technique for rapid isolation and identification of CSFV, we sought to develop a molecular biologic assay as an accurate, rapid, practical approach to discriminating between vaccine and field strains and thus improve and simplify the diagnosis of CSF.

A 5′-Taq nuclease assay has identified alleles that differ by a single base (6). In this assay, probes specific for each virus strain are included in the PCR. The probe that is specific for a vaccine strain and a probe that is specific for the mutation or polymorphism are labeled with a different fluorescent reporter dye at the 5′ end. The 3′ end of each probe carries a dark quencher that suppresses the fluorescence of the reporter dye. When a probe is annealed to a perfectly matched target, the probe is cleaved by the 5′ nuclease activity of Taq polymerase. Cleavage of the reporter dye releases the dye from the constraints of the quencher, which results in increased fluorescence intensity. A mismatch between the probe and the target reduces the efficiency of probe hybridization and promotes displacement of the probe instead of cleavage, which results in decreased fluorescence intensity or an absence of fluorescence.

Conventional longer TaqMan probes (16 to 40 base pairs [bp]) cannot reliably distinguish between single mutations. Minor-groove-binder (MGB) probes can be used to analyze mutations that are in close proximity. The MGB technology is based on conjugation of nucleotides with 1,2-dihydro-(3H)-pyrrolol (3,2-e) indole 7-carboxylate (CDPI3). The conjugates bind to the minor groove of the double-stranded DNA and form stable DNA duplexes. These shorter probes (12 to 20 bp) are sensitive to single base mismatches (7). In this study, we aimed to develop a 1-step reverse-transcription PCR (RT-PCR) assay that used TaqMan MGB fluorogenic probes to distinguish vaccine strains and field isolates of CSFV in Korea.

Viral RNA was derived from 5 K-LOM vaccine strains, purchased from Choong-ang Vaccine Laboratory (Daejeon, Korea), Dae-sung Microbiological Laboratories (Seoul, Korea), Korea Microbiological Laboratories (Seoul), Green Cross (Seoul), and Bayer Korea (Seoul). Variants of the wild-type K-LOM were attenuated in bovine kidney cell lines. The CSFV-positive serum used in this study was obtained from 10 pig farms in Jeonnam province in 2002 and 2003, CSFV having been diagnosed by enzyme-linked immunosorbent assay at the Department of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea.

The CSFV RNA was extracted from the vaccine strains and serum samples with the use of a TRI reagent (Molecular Research Center, Cincinnati, Ohio, USA) according to the manufacturer’s protocol. Briefly, 200 μL of vaccine or serum was mixed with the reagent solution, vortexed vigorously for 30 s, and incubated at room temperature for 5 min. Next, 200 μL of chloroform was added. The mixture was kept at 4°C for 10 min and then was centrifuged at 14 000 × g for 15 min. The supernatant was recovered, an equal volume of isopropanol added, and the mixture incubated for 15 min at room temperature. The RNA was precipitated by centrifugation at 14 000 × g for 30 min, washed with 75% ethanol, dried, resuspended in 30 μL of RNase-free water, and stored at −20°C until used for RT-PCR.

Oligonucleotide primers for RT-PCR were designed from the published sequence of the 5′ untranslated region (UTR), 223 nt, of the CSFV genome (Alfort 187 strain, GenBank accession no. X87939) with use of a DNASIS synthesizer (version 2.5; Hitachi Software Engineering, Yokohama, Japan). Primers and probes for fluorogenic RT-PCR were designed from the published sequence of the 5′ UTR of the CSFV genome (Alfort 187 strain) and the NS9811 Korea field strain (AF521706) and purchased from Assays-by-Design Service (Applied Biosystems, Foster City, California, USA). The sequences of the primers and probes are shown in Table I.

Table I.

Sequences of the primers and minor-groove-binding (MGB) probes used for the reverse-transcription polymerase chain reaction (RT-PCR) assay and fluorogenic RT-PCR

Assay and tool	Base-pair position	Sequencea	GenBank accession number
RT-PCR
Forward primer	129–146	5′-GACTAGCCGTAGTGGCGA-3′	X87939
Reverse primer	440–423	5′-GTTCCTCCACTCCCATTG-3′	X87939
Fluorogenic RT-PCR
Forward primer	154–177	5′-GGGTGGTCTAAGTCCTGAGTACAG-3′	AF521706
Reverse primer	330–311	5′-CCTCTGCAGCACCCTATCAG-3′	AF521706
Vaccine probe	231–215	5′-VIC-CCACATAGCATATCGAG-NFQ-MGB-3′	X87939
Field probe	231–215	5′-FAM-CCACATAGCATCTCGAG-NFQ-MGB-3′	AF521706

^aUnderlined, boldface nucleotides represent the location of the polymorphism; VIC and FAM code for the reporter fluorophores, and Q codes for the nonfluorescent quencher group

For the 1-step RT-PCR and restriction-endonuclease assay, 2 μL of RNA was used with the RNA PCR Kit (Takara Bio, Otsu, Japan) according to the manufacturer’s protocol. The mixtures were incubated at 50°C for 45 min and then underwent 30 cycles of PCR at 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min in a GeneAmp PCR System 2400 (Applied Biosystems). The amplified product was visualized by ethidium bromide staining and 1% agarose gel electrophoresis. To confirm the identity of the amplicon and the digestion site of Xho I, the PCR product was sequenced with an ABI PRISM 377 DNA sequencer (Applied Biosystems) in collaboration with the Korea Basic Science Institute (Gwangju). The PCR product was then cleaved with Xho I (Takara Bio) in a reaction volume of 20 μL according to the manufacturer’s instructions. The reaction cocktail contained the PCR product (0.5 to 1.0 g of DNA), 2 μL of 10× buffer, 1 μL of Xho I (10 U), and enough sterilized distilled water to make a total volume of 20 μL. The mixture was incubated at 37°C for 2 h, and then the treated products were separated by electrophoresis on 1% agarose gel, stained with ethidium bromide, and visualized with an ultraviolet transilluminator.

For the RT-PCR with fluorogenic probes, we used a 1-step RT-PCR Master Mix kit (Applied Biosystems) containing 25 pmol of each primer set, 3.25 pmol of each probe, and 10 μL of the RNA extract. Amplification was performed with the ABI PRISM 7000 Sequence Detection System (SDS; Applied Biosystems), set at 48°C for 30 min and 95°C for 10 min, and then 40 cycles at 95°C for 15 s and 60°C for 1 min. After PCR, fluorescence of the vaccine-type-specific probe (VIC) and fluorescence of the wild-type-specific probe (FAM) was measured in each well by means of a 96-well plate reader connected to a computer with ABI PRISM 7000 SDS software (Applied Biosystems), which runs on a Windows operating system; we tested 5 vaccine strains, 10 field strains, 5 mixed samples (vaccine and field strains), and 2 samples without DNA (as controls).

A 312-bp DNA fragment was amplified with RT-PCR from the vaccine and wild-type viruses (Figure 1A). Xho I digested the fragment from the wild-type strains to produce 225-bp and 87-bp fragments (Figure 1B), but it did not cleave the product from the vaccine strains.

Figure 1.

Results of 1-step reverse-transcription polymerase chain reaction (RT-PCR) (A) and restriction-endonuclease cleavage (B) of RNA extracts from vaccine-type and wild-type strains of classical swine fever virus (CSFV). The DNA fragment of 312 base pairs (bp) from the wild-type strains was digested by Xho I into fragments of 225 and 87 bp, but the 312-bp fragment from the vaccine strains was not cleaved. Lane M, DNA ladder; lanes C, D, H, G, and B, K-LOM vaccine strains from Choong-ang Vaccine Laboratory (Daejeon, Korea), Dae-sung Microbiological Laboratories (Seoul, Korea), Korea Microbiological Laboratories (Seoul), Green Cross (Seoul), and Bayer Korea (Seoul), respectively.

When FAM fluorescence was plotted against VIC fluorescence for each sample to discriminate the 5′ UTR of CSFV, 3 clusters of samples could be distinguished for each virus type: those with only VIC fluorescence, those with only FAM fluorescence, and those with both VIC and FAM fluorescence (Figure 2).

Figure 2.

Allelic discrimination plot of RNA isolated from wild-type CSFV (circles), vaccine-type CSFV (diamonds), and mixed samples (triangles), amplified in duplicate by RT-PCR with TaqMan minor-groove-binding (MGB) probes. Controls (squares) contained no DNA. The absolute fluorescence of each reporter-dye probe was plotted.

Compared with the current method for genotyping vaccine-type and wild-type CSFV in Korea (5), which takes more than 8 h (excluding RNA extraction), the method that we have developed, a simple, rapid, inexpensive, reproducible, and reliable fluorogenic RT-PCR, is a significant improvement. The new assay can be completed in less than 2 h. In addition, it detects the virus before signs of disease appear. Nevertheless, the specificity in identifying CSFV in Korea is more important than the sensitivity.

Increasingly, 5′-Taq nuclease assays, often referred to as fluorogenic probe assays or real-time PCR assays, are used to detect viruses in clinical samples (8–10). The genotyping of single nucleotide polymorphisms has been developed using RT-PCR with TaqMan MGB probes (11,12). The results of this study show that fluorescent oligonucleotides conjugated with MGB probes can easily distinguish between vaccine-type and wild-type CSFV. The presence of the MGB group increased the annealing temperature of the matched probe–target sequence, whereas the mismatch duplex was further destabilized (7). Nonspecific probe hybridization did not occur, which eliminated the background fluorescence and enabled post-PCR measurements of the fluorescence in a plate-reader. Automated genotyping was performed with the use of spreadsheet software programmed to calculate the fluorescent cutoff values for determining the presence of alleles. In 5′-Taq nuclease assays, the use of 3′-MGB probes results in low background fluorescence and improves specificity and sensitivity compared with the more common non-MGB probes (7). Because MGB probes form extremely stable duplexes with the DNA targets (7), the probes can be shorter than those without the 3′-MGB groups (13).

In this study, the results for the 10 wild-type and 5 vaccine-type samples in genotyping of the 5′-UTR region were identical to those of conventional genotyping. Vaccine-type viruses have the T (223 nt) allele, whereas wild-type viruses have the G allele (KH2002 [AY168611], JJ9811 [AF521707], NS9811 [AF521706], YI9908 [AF521705], 96940 [AH011690], CW2002 [AF517834]). Therefore, it is possible to discriminate them by means of the MGB probes.

Also, the strains Alfort/187 (X87939), Alfort A19 (U90951), ALD (D49532), and LOA (AB019647) have the T allele, whereas the strains BRECIA (M31768), BRESCIAX (AY578687), Eystrup (AF326963), Fukuoka/72 (14), GPE (AJ605591), Kozlov (AY972477), L39 (AY761220), Shimen/HVRI (AY775178), L61 (AY761213), Ibaraki/66 (14), Ogatamura/68 (14), Switzerland 3/93/2 (14), WB82-DI (14), 96TD (AY554397), and 0406/CH/01/TWN (AY568569) have the G allele. Therefore, it is possible to discriminate them using the 2 MGB probes.

Our results show that 1-step RT-PCR using MGB probes can easily discriminate between wild-type and vaccine-type alleles. Fluorescence data can be collected directly from the 96-well PCR reaction plate by means of a plate-reader, without the need for PCR product manipulation. Common spreadsheet software can be used for the analysis. Overall, this method will facilitate automated genotyping for routine molecular diagnostics and large-scale genetic studies. Discriminating between the wild-type and vaccine-type alleles of CSFV with this method will be helpful as a screening tool in making a diagnosis of CSF due to a vaccine-type versus a wild-type virus in Korea.