much as 106 times by use of the polymerase chain reaction .... Primers 1 and 2, Outer primer pair (PCR-1); primers 3 and 4, inner primer pair (PCR-2). b Bases ...
JOURNALOF CLINICAL MICROBIOLOGY, OCt. 1990, p. 2275-2278 0095-1137/90/102275-04$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 28, No. 10
Identification of a Double-Stranded RNA Virus by Using Polymerase Chain Reaction and Magnetic Separation of the Synthesized DNA Segments ESPEN RIMSTAD,l* ERIK HORNES,"2 0RJAN OLSVIK,l AND BJ0RN HYLLSETH' Department of Microbiology and Immunology, Norwegian College of Veterinary Medicine, P.O. Box 8146, Dep, N-0033 Oslo 1,1 and Research and Development Division, Apothekernes Laboratorium A.S., Skçoyen, N-0212 Oslo 2,2 Norway Received 10 January 1990/Accepted 2 July 1990
A double-nested polymerase chain reaction assay (PCR), followed by magnetic separation of the PCRsynthesized DNA segments, was developed to detect a double-stranded RNA virus, infectious pancreatic necrosis virus from salmonid fish. Viral RNA was extracted from cell cultures and used for cDNA synthesis. The cDNA produced was used as a template in a double PCR. The sensitivity of this double PCR was approximately 0.8 pg of template double-stranded RNA. The DNA segment produced from the first PCR was also used as a template in a second PCR with a set of two 5'-labeled primers, one with biotin and the other with 32p. The PCR segment that was then synthesized was separated from the solution by using streptavidin-coated, superparamagnetic beads. The levels of radioactivity measured in the magnetically separated fractions were significantly higher in the positive samples than they were in the negative samples.
Amplification of specific nucleic acid sequences by as much as 106 times by use of the polymerase chain reaction assay (PCR), as described by Saiki et al. (14, 15), has improved the sensitivity of hybridization methods dramatically. PCR has made it possible to detect specific nucleic acid sequences from a single microorganism (13) in a mixture of nucleic acids of different origins without isolating the organism (13). The large number of PCR-produced specific DNA segments can be visualized by gel electrophoresis followed by ethidium bromide staining. Infectious pancreatic necrosis virus (IPNV) is the etiological agent of an acute, highly contagious disease in hatcheryreared salmonid fish fry (18). The virus belongs to the family of Birnaviridae (5). It is a nonenveloped, single-shelled, 60-nm virus, and its nucleic acid is bisegmented, doublestranded RNA (dsRNA). We have previously described the use of a specific synthetic oligonucleotide as a probe for identifying this virus (E. Rimstad, R. Krona, E. Homes, 0. Olsvik, and B. Hyllseth, Vet. Microbiol., in press). The sensitivity of this method was approximately 100 ng of viral
MATERIALS AND METHODS Viruses and propagation. IPNV was cultivated in CHSE214 cell cultures (Chinook salmon embryo cells; ATCC CRL 1681; American Type Culture Collection, Rockville, Md.) by using standard methods; cultures were harvested when complete cythopathic effects were observed. The viral serotypes West Buxton (strain VR-299) and Sp (strain 88B) and a field isolate (strain B2) that is closely related to the type strain of the Sp serotype were used in this study. The viral dsRNA was extracted as described by Maniatis et al. (12). Extracted total DNA (12) from CHSE-214 cells was used when testing the specificity of the primers. cDNA synthesis. The viral dsRNA was heated to 95°C for 5 min and used as a template in a cDNA synthesis with 200 U of murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) in a buffer solution consisting of 0.25 M Tris hydrochloride (pH 8.3), 0.375 M KCl, 15 mM MgCl2, 50 mM dithiothreitol, and 0.5 mM deoxynucleoside triphosphates. Both random hexamer oligonucleotides (Bethesda Research Laboratories) or a specific 16-mer oligonucleotide (identical to the plus strand of the long segment of the IPNV genome of the Jasper strain [6] bases 1794 to 1809; sequence, 5'-TCGCTCGTGGTAGTAA3') was used as primer. The reaction was performed at 42°C for 30 min. The reverse transcriptase was then inactivated by heating the mixture to 95°C for 5 min. Primers used in PCR. The primers were 24-mer DNA oligonucleotides (Table 1) synthesized in an Applied Biosystems synthesizer. The outer span, including primers 1 and 2, was 486 base pairs (bp), and the inner span, including primers 3 and 4, was 310 bp. The biotinylation of primer 3 was performed by using aminolink II (Applied Biosystems) in the synthesis, followed by purification of the primer and then reaction with Biotin X-NHS ester (Clonetech), as recommended by the supplier. Amplification of target DNA. PCR was done by using lx PCR buffer (10 mM Tris hydrochloride [pH 8.3], 50 mM KCl, 1.5 mM MgCl2, 0.01% [wt/vol] gelatin) with 200 ,uM each deoxynucleoside triphosphate, 0.2 to 0.4 ,uM each of the
RNA. PCR amplification of rotavirus nucleic acid, which is also dsRNA, from stool specimens has been described by Gouvea et al. (9) and Xu et al. (19), and they obtained a sensitivity of 20 to 100 pg of template dsRNA and 8 x 103 viral particles, respectively. Magnetic particles can be used for separating specific sequences of DNA or RNA from a mixture of nucleic acids (10, 16, 17) or for separating DNA-binding proteins (8). Covalently bound streptavidin on the surface of the particles links to biotinylated nucleic acid. The purpose of this study was to develop a diagnostic system based on the use of PCR for the amplification of dsRNA from cultivated IPNV and thereby obtain a test that is more sensitive than the specific synthetic oligonucleotide probe test.
*
Corresponding author. 2275
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RIMSTAD ET AL. TABLE 1. Two pairs of primers used in PCR
Primera
Sequence
Comment'
1 2 3 4
5'-CCAAACCAACAGGTCCTATCCTAC-3' 5'-TGATGCCGTTGTTCTCATCAGCTG-3' 5'-GAAGGAGATGACATGTGCTACACC-3' 5'-GAGAGATGTGTTTGCCACCATCTC-3'
Identical to bases 1839-1862 Complementary to bases 2301-2324 Identical to bases 1914-1937 Complementary to bases 2200-2223
a Primer 3 was biotinylated in the 5' end. Primers 1 and 2, Outer primer pair (PCR-1); primers 3 and 4, inner primer pair (PCR-2). b Bases refer to the cDNA plus strand of the long segment of the IPNV genome of the Jasper strain (6).
primers, and 1 U of thermostable DNA polymerase (Amplitaq; Perkin-Elmer Cetus, Norwalk, Conn.). cDNA from the reverse transcription was used as a template. The amplification was performed in a DNA thermal cycler (Perkin-Elmer Cetus). The cycle profile was 94°C for 1 min, then 65°C for 2 min, and finally 72°C for 1 min. When both pairs of primers were applied sequentially, 25 cycles were performed with the outer pair of primers. This was followed by 5 to 20 cycles with the inner pair of primers, in which a 100-fold dilution of the first PCR product in distilled water was used as a template. Electrophoresis. Electrophoresis was carried out in a horizontal, submarine 2.5% agarose gel in TBE buffer (0.089 M Tris borate, 0.089 M boric acid, 0.002 M EDTA [pH 8.0]) at 7.1 V/cm for 40 min, and thereafter, the gel was stained with ethidium bromide. Analysis of PCR products. To test the specificity of the PCR after having used the outer pair of primers, the PCR mixture was digested with PstI (Bethesda Research Laboratories). A DNA-RNA hybridization was performed with viral RNA as the target and by using a probe that eluted from the magnetic beads after adding 0.15 M NaOH. The hybridization procedure described earlier by Christie et al. (3) was used, with some modifications. In short, heat-denaturated dsRNAs from three strains of IPNV (VR-299, 88B, B2) were blotted onto a nylon membrane (GeneScreen; Biotechnology Systems, Boston, Mass.) and fixed to the membrane by baking at 80°C. After prehybridization of the membrane in 6x SSC (lx SSC is 0.15 M NaCI plus 0.015 M sodium citrate)-5 x Denhardt solution (12)-100 ,ug of boiled herring sperm per ml-0.08% Na2PO4-0.5% sodium dodecyl sulfate for 1 h at 42°C, hybridization was done in a solution of 6x SSC-lx Denhardt solution-0.08% Na2PO4, together with the radiolabeled probe, at 42°C for 2.5 h. The membrane was washed in 2x SSC three times for 5 min each time at room temperature and two times for 20 min each time at 48°C. The membrane was then wrapped in plastic film and exposed to X-ray film by using amplifier screens (Eastman Kodak Co., Rochester, N.Y.) at -70°C for 2 days. Magnetic separation of PCR products. When magnetic separation of the DNA segments was performed, the double PCR was done using 0.1 ,ug of dsRNA as a template in the reverse transcription. Primer 4 was 5'-end-labeled with 32p by using T4 polynucleotide kinase (Bethesda Research Laboratories) and centrifuged through a Sephadex G-50 spun column as described by Maniatis et al. (12). This primer was used together with the biotinylated primer 3 in the PCR-2 (inner pair of primers) by using 5 to 20 cycles. This 310-bp segment was bound to streptavidin-coated, superparamagnetic polystyrene beads (Dynabeads M-280 Streptavidin; Dynal, Oslo, Norway) and separated from the PCR solution with a magnet (magnetic particle concentrator; Dynal). In short, 100 ,ug of the beads was washed once in 1 M NaCl. Thereafter, 25 ,ul of the PCR solution was added and
incubated at room temperature for 20 min with gentle shaking by hand every 5 min. The beads were then washed twice for 5 min each time in distilled water at 50°C. The beads were finally suspended in scintillation fluid, and radioactivity (counts per minute) was measured in a scintillation counter (1900 CA; Packard Instrument Co., Inc., Downer Grove, Ill.). A schematic drawing of PCR of dsRNA done twice and attachment to a solid face of the second PCR product is shown in Fig. 1. RESULTS
PCR amplification. Both pairs of primers functioned in the PCR for the viral serotypes tested, giving segments of 310 and 486 bp, respectively (Fig. 2). No interference between the two pairs of primers were seen in the double PCR. Cleaving of the 486-bp PCR product originating from VR-299 with PstI yielded two fragments of 442 and 44 bp. The sensitivity of the test, i.e., the smallest amount of dsRNA that gave an amplified segment, as observed visually, was approximately 100 ng of dsRNA after the first PCR amplification (PCR-1), and the sensitivity increased to 10 pg of dsRNA after the second amplification, using 15 cycles (PCR-2). Because the amplified segment constituted approximately 12% of the viral genome, approximately 0.8 pg of template dsRNA was necessary to give a visible 310-bp band. Reverse transcription. No differences in the amount of the specific segment produced in the PCR, as observed after electrophoresis, were found when the random hexamers or the specific 16-mer was used as primer in the cDNA synthesis. Interference with the specific 16-mer reverse transcriptase primer in PCR was not observed. DNA-RNA hybridization. Hybridization between the single-stranded, 32P-labeled 310-mer probe and viral RNA is
ds RNA +Reverse Transcriptase ds or ss DNA $ PCR 1 32p -R
PCR 2 using
biotinylated (B) primer and 32P-labelled
PCR 2
primer
32pS
B -sA
Separation using streptavidin (SA)-coated magnetic beads
FIG. 1. Schematic drawing of PCR. PCR-1 and PCR-2 designate the use of the outer or inner pair of primers, respectively. ss,
Single-stranded.
IDENTIFICATION OF DOUBLE-STRANDED RNA
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E
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ce
O
FIG. 2. PCR results by using 0.1 p.g of dsRNA from IPNV as template. A 123-bp ladder (lane 1); the segment from PCR-1 (lane 2); and the inner segment after 15 cycles (lane 3), 10 cycles (lane 4), and 5 cycles (lane 5) after PCR-2 are shown.
shown in Fig. 3. This probe, which originated from the VR-299 strain (the strain used as a template in cDNA synthesis), hybridized strongly with RNA from both strain VR-299 and more weakly with strains Sp and B2. Magnetic separation of PCR products. The results of magnetic separation of PCR products are shown in Fig. 4. There was a steady increase in counts per minute in the positive samples from 5 to 20 cycles. Radioactivity from the negative control was quite low at 5, 10, and 15 cycles but became more significant after 20 cycles. The results indicate that there was a significant difference (P < 0.05; Student's t test) between the controls and the viral samples over the entire range of cycles tested, i.e., 5, 10, 15, and 20. The differences in counts per minute between the positive samples and the controls were greatest at 10 and 15 cycles. The control samples for the biotin-streptavidin reaction, in which a nonbiotinylated form of primer 3 was used, resulted in lower counts per minute after 20 cycles than the PCR control showed at 5 cycles. After treatment with 0.15% NaOH, the activities of all samples were in the same range as the background levels. DISCUSSION The sensitivity of PCR is one of the main advantages of this method (13), which is theoretically capable of detecting a single target DNA molecule (4). When the starting material is RNA, reverse transcription of the target nucleic acid is the critical point for the sensitivity of the PCR (1). Using cell culture-grown IPNV as a source of dsRNA, we obtained positive results from approximately 0.8 pg of template dsRNA. This sensitivity was better than that obtained by using a specific synthetic oligonucleotide as a probe in identifying this virus. The latter method did not detect less than approximately 100 ng of viral dsRNA (Rimstad et al., in press).
Sp
VR-299
B-2
FIG. 3. Filter hybridization (slot blot) with 10-fold dilutions (vertical) of dsRNA from IPNV strains VR-299, Sp, and B2. The probe was a 310-base, 32P-labeled single-stranded DNA obtained from magnetic beads.
5
10
15
20
Cycles FIG. 4. Average radioactivities (counts per minute) from four parallel experiments. Samples containing viral template and negative control template were tested. The average radioactivity for the PCR containing viral template, but using the nonbiotinylated primer, at cycle 20 is shown. The ratios between the counts per minute counted from samples containing the viral template and those counted from samples containing the negative control template were highest at 15 and 20 cycles.
The random hexamer primers and the specific 16-mer primer functioned equally well in cDNA synthesis. The specific 16-mer primer only contributed to single-stranded DNA synthesis, but no second-strand synthesis was necessary when the cDNA was used as a template in PCR. The high annealing temperature (65°C) that was generally used in PCR prevented hybridization of the 16-mer primer. Due to the high annealing temperature and dilution of the cDNA, no amplification of the segment between the 16-mer and the PCR primers was observed. The PCR primers were not used in cDNA synthesis, because this increased the possibility of undesirable amplifications in PCR. The use of a double-nested set of primers in PCR to enhance the specificity has been described earlier (9, 11). The inner set of primers only functions if the outer pair has been used in amplification of the correct sequence, and therefore serves as a control of the first primer set. Since the number of templates increased greatly from the first to the second PCR, a fewer number of cycles was required in the second PCR. The use of several primers and the relatively few cycles in the second PCR step are both major advantages that increase the specificity of the test. The published PstI site at bp 2281 (7) in the nucleotide sequence of IPNV (Jasper strain) was documented by using this enzyme on the PCR-1 (outer span) product. Slight differences were seen in the hybridization patterns when slot-blotted viral RNAs from strains VR-299, Sp, and B2 were hybridized with the 310-base single-stranded DNA probe (Fig. 3). The probe was obtained from the beads, and the original template was from strain VR-299. This indicates that the 310-bp fragment is different in the two serotypes. Heterogeneity in the nucleotide sequence between strains VR-299, Sp, and B2 was anticipated since they belong to different serotypes of IPNV (2), and cross-hybridization was also expected since the strains are antigenically related (2). The hybridization results also show that the dsDNA fragment of the inner span (PCR-2) indeed was captured by the streptavidin-coated beads. After magnetic separation, the ratio between counts per minute of the samples containing viral template and those containing negative control template expressed the specificity in this PCR. The counts per minute of the control gives
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a measure of nonspecific amplifications in PCR. The relatively low counts per minute observed when a nonbiotinylated primer instead of a biotinylated primer was used indicates that nonspecific binding to the beads was slight. The treatment of the beads with 0.15 M NaOH dissociated the DNA strands, but it did not interfere with biotinstreptavidin binding. The radioactivity measured after this treatment was similar to the background level and indicated that the radioactivity is associated with the DNA strand without biotin. Results from the present investigation indicate that magnetic separation of DNA may be an alternative procedure to electrophoresis to visualize PCR products. This method could possibly be used in the diagnosis of viral infections, particularly if a nonradioactive signal system is introduced. LITERATURE CITED 1. Byrne, B. C., J. J. Li, J. Sninsky, and B. J. Poiesz. 1988. Detection of HIV-1 RNA sequences by in vitro DNA amplification. Nucleic Acids Res. 16:4165. 2. Caswell-Reno, P., V. Lipipun, P. W. Reno, and B. Nicholson. 1989. Use of a group-reactive and other monoclonal antibodies in an enzyme immunodot assay for identification and presumptive serotyping of aquatic birnaviruses. J. Clin. Microbiol. 27:1924-1929. 3. Christie, K. E., L. S. Hàvarstein, H. O. Djupvik, S. Ness, and C. Endresen. 1988. Characterization of a new serotype of infectious pancreatic necrosis virus isolated from Atlantic salmon. Arch. Virol. 103:167-177. 4. Clewely, J. P. 1989. The polymerase chain reaction, a review of the practical limitations for human immunodeficiency virus diagnosis. J. Virol. Methods 25:179-187. 5. Dobos, P., B. J. Hill, R. Hallet, D. T. C. Kells, H. Becht, and D. Teninges. 1979. Biophysical and biochemical characterization of 5 animal viruses with bisegmented double-stranded RNA genomes. J. Virol. 32:593-605. 6. Duncan, R., and P. Dobos. 1986. The nucleotide sequence of infectious pancreatic necrosis virus (IPNV) dsRNA segment A reveals one large ORF encoding a precursor protein. Nucleic Acids Res. 14:5934. 7. Duncan, R., E. Nagy, P. J. Krell, and P. Dobos. 1987. Synthesis of the infectious pancreatic necrosis virus polyprotein, detec-
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