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© 1994 Oxford University Press Nucleic Acids Research, 1994, Vol. 22, No. 10 1919-1920 Specific detection of minus strand hepatitis A virus RNA by Tail-PCR following reverse transcription Ricardo L.Chaves*, Judith Graff, Andrea Normann and Bertram Flehmig Hygiene-lnstitut der Universitat Tubingen, Abteilung fur Medizinische Virologie und Epidemiologie der Viruskrankheiten, SilcherstraBe 7, Tubingen, 72076, Germany Received March 17, 1994; Accepted April 11, 1994 The biosynthesis of RNA directed by an RNA template is a reaction that is unique to RNA viruses. Hepatitis A virus (HAV) contains a single, plus strand RNA genome. Viral RNA replication occurs in the cytoplasma of infected cells, and involves first the synthesis of a minus strand RNA molecule. The minus strand HAV RNA, present in very low amounts, serves as template for the production of new copies of genomic plus strand RNA (1). Minus strand HAV RNA have been detectable for a brief period, if at all, by blot hybridization in infected cell cultures (2,3,4). Recently, McGuiness and colleagues (5) pointed out the major limitations of the specific detection of negative strand hepatitis C virus (HCV) RNA by PCR: firstly, to ensure the complete inactivation of the reverse transcriptase (RTase) and the degradation of plus strand RNA after cDNA synthesis, and secondly the RTase activity of the Taq DNA polymerase (6,7). They concluded that reliable detection of minus strand HCV RNA is not yet achievable by current methods. By designing suitable primers for a modified nested PCR (TailPCR), and also by using magnetic beads technology, we have developed a method aimed at the specific detection of singlestranded RNA. HAV strain GBM was propagated in human embryonic lung fibroblasts (8). The medium was discarded and the cells washed with phosphate buffered saline (PBS). Total RNA was then obtained from infected and noninfected cells on days 3, 7 and 10 p.i. by standard methods (9). Streptavidin-linked magnetic beads (20 /il) were prepared for binding to the biotinylated cDNA by resuspension in 40 fi\ of 2 M NaCl/TE (10 mM Tris-HCl pH 7.5/0.1 mM EDTA), as described previously (10). To detect minus strand HAV RNA, primers containing approximately 20 additional nucleotides on their 5' end were constructed so that a modified nested PCR could be performed. These additional sequences, called tail-sequences, are neither complementary nor homologous to any part of the HAV genome (11, 12). The tail-sequences can be used as the target during the amplification that follows, and were chosen using the computer program Gene Jockey (Biosoft, Cambridge). To carry out reverse transcription (RT), a biotinylated HAV positive-sense outerprimer A with a first tail-sequence, tail 1, on its 5' end was constructed (Table 1). The i00 jtl RT assay, performed at 42°C for 30 minutes, consisted of universal buffer (10 mM Tris-HCl, * To whom correspondence should be addressed pH 8.3, 50 mM KC1 and 1.5 mM MgCl2), 0.25 mM each of dATP, dCTP, dGTP, dTTP, 4.25 pmol of primer A with the first tail-sequence, 5 units of RTase from avian myoblastosis virus and 10 jig of total RNA. A 40 /tl aliquot of the cDNA was bound to the streptavidin-linked magnetic beads (Fig. 1), washed 3 times in 2 M NaCl/TE, and finally resuspended in 100 |tl of Tail-PCR mixture. The first Tail-PCR primer-set comprised a primer homologous to the first tail-sequence (primer 1) and an HAV negative-sense outer-primer B with a second tail-sequence, tail 2, on its 5' end (Fig. 1, Table 1). The Tail-PCR mixture consisted of universal buffer, 0.25 mM each of dATP, dCTP, dGTP, dTTP, 30 pmol each of the primers and 2.5 units of Taq DNA polymerase. Thirty amplification cycles were performed in a thermocycler (MWG) — 1 min denaturation at 94°C, 1 min annealing at 55°C and 2 min extension at 72°C. The resultant amplicon (817 bp long) contained the second tail-sequence on the 5' end of the newly synthesized DNA strand. A primer homologous to the second tail-sequence (primer 2) and an HAV positive-sense inner-primer C were used to carry out the final amplification (Fig. 1, Table 1). An aliquot of 1 jtl from the first amplicon was added to the second Tail-PCR mixture. Reaction conditions were the same as in the first amplification, except for a 1 min extension step. The second amplicon was 337 bp long. In order to test the specificity of the Tail-PCR, RNase A and H digestion were performed after washing the cDNA. The RNase A digestion mixture consisted of universal buffer and 0.5 units of RNase A, and was incubated for 30 min at 37°C. Thereafter, the wash steps were repeated and the PCR-mix was added. The RNase H digestion was performed for 30 min at 37°C by raising Table 1. Primer-set used for the detection of minus strand HAV RNA Reverse HAV primer A (+)(6700-6720») with the first tail-sequence': Transcription BiotinS' TGGGATTACCOAGTATGTGTCTTAGTCCATTTATGATTA 3' First Tail-PCR Primer 1 (homologous to the first tail-sequence): (817 bp product) 5' TTGGGATTAGCGAGTATG 3' HAV primer B (-)(7477-7456*) with the second tail-sequence*: 5' GACCTGGATAGGCTGTGTGATTTTACTGATAAAAGAAATAAAC 3' Second Tail-PCR Pnmer 2 (homologous to the second tail-sequence): (337 bp product) 5' GACCTGGATAGGCTGTGTGAT 3' HAV primer C (+)(7161-7183 a ): 5' GAGGATAGAATTAGACCTGCAAT 3' indicates nucleolide position in the HAV genome. Tail-sequences arc underlined. 1920 Nucleic Acids Research, 1994, Vol. 22, No. 10 ©©SIC | 8MDM0 TO STREFTAVnM-STRAWOR WHOM, WASH AMD BOLATIOM -STRAHORNA Of THE BOTMYLATED COM* | 0'"-" <—i—1 f 1 ] LTAL2J Figure 1. Scheme of the Tail-PCR. 12 3 4 5 6 7 8 793 bp 337 bp to Nylon membranes as described elsewhere (15), and finally subjected to oligonucleotide hybridization with an HAV cDNA fragment (13). HAV minus strand RNA was detected in infected cells 3, 7 and 10 days p.i. by Tail-PCR after the second amplification, but not in noninfected cells. Figure 2 shows the results of the TailPCR, RT-PCR and Southern hybridization on day 10 p.i. Neither RNase A nor RNase H digestions, done after RT, alter the results of the Tail-PCR (lanes 3 to 5). Total HAV RNA was detected by RT-PCR after the first amplification, and RNase A treatment turned RT-PCR negative, even after the second nested amplification (lanes 6, 7 and 8). If amplicons without tailsequences from the first RT-PCR (lane 6) were used as the target for the second Tail-PCR, the results of the Tail-PCR were negative (not shown). In this report, we present a method enabling the specific detection of single-stranded RNA by using PCR. After cDNA synthesis, RNase digestion does not alter the result of the TailPCR, i.e. the target for the Tail-PCR was the tailed cDNA synthesized from the HAV minus strand RNA. Plus strand RNA and RTase are eliminated by washing the biotinylated cDNA. The addition of the tail-sequences to the cDN A target molecules ensures the specificity of ssRNA detection, since they render the complementary DNA strands unsuitable for amplification by TailPCR. We could show that amplicons originating from the same HAV genomic region devoid of tail-sequences cannot serve as target for Tail-PCR. In summary, we regard Tail-PCR following RT as a new approach in detecting ssRNA and monitoring the presence of low amounts of replicative intermediates of ssRNA viruses. ACKNOWLEDGEMENTS Figure 2. Agarose gel analysis (a) of the PCR products and Southern hybridization (b). RT/Tail-PCR and RT-PCR were used to detect HAV minus strand RNA and total HAV RNA, respectively. The Southern hybrization was performed with a digoxigenin-labelled cDNA probe derived from the 3' end of HAV. Nucleic acids were obtained on day 10 p.i. Lanes: 1 — DNA marker VI (Boehringer Mannheim). 2 — RT/Tail-PCR from noninfected cells. 3 — RT/Tail-PCR from infected cells. 4 — Tail-PCR from infected cells performed after RNase A digestion of the RT product. 5 — Tail-PCR from infected cells performed after RNase H digestion of the RT product. 6 — RT-PCR from infected cells, without preceding ribonuclease A digestion. 7 — RT-PCR from infected cells, as in lane 6, performed after RNase A digestion of the viral RNA. 8 — Second (nested) PCR following that in lane 7. the buffer concentration of MgCl2 to 5 mM and adding 0.85 units of RNase H. As additional control for the specificity of the Tail-PCR, RT followed by nested PCR (RT-PCR) to detect total viral RNA was carried out with the HAV specific primers A, B and C devoid of tail-sequences. An additional HAV specific negative-sense primer (downstream to HAV primer B at genome position 7478—7492) was used, as described previously, for the first amplification (13,14). The buffer and cycling conditions were the same as described above. The first and second RT-PCR amplicons were, respectively, 793 and 317 bp long. To verify whether amplicons originating from total viral RNA turn TailPCR positive, the first amplicon of the RT-PCR was also subjected to the second Tail-PCR. The efficacy of RNase A digestion in destroying total HAV RNA was tested by performing the digestion before RT. The PCR products were electrophoresed through 2% agarose gels, visualized by staining with ethidium bromide, transferred We would like to thank Dr Jack Stapleton, University of Iowa, for helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft. R.L.C. was supported by the CAPES Scholarship (Brazilian Ministry of Education). REFERENCES 1. Cohen,J.I. 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