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Reverse transcription and subsequent DNA amplification of rubella virus RNA
Journal of Virological Methods, 25 (1989) 21-30 Elsevier
21
JVM 00889
Reverse transcription and subsequent DNA amplification of rubella virus RNA William
F. Carman,
Carolyn Williamson, Beverley Alistair H. Kidd
A. Cunliffe
and
National Institute for Virology and Department of Virology, University of the Witwatersrand, Private Bag X4, Sandringham 2131, Johannesburg, South Africa (Accepted
9 February
1989)
Summary A method is described whereby rubella virus RNA was reverse transcribed and the resulting cDNA enzymatically amplified using Tuq polymerase. The reactions were carried out in a single reaction vessel, with only minor modifications to the buffer conditions between the reverse transcription and the subsequent amplification step. Using an oligonucleotide probe to the El glycoprotein region and limited restriction endonuclease mapping, the resulting amplified products were shown to be specific for rubella virus. This method was also successfully applied to crude cell lysates, without the need for RNA purification. The possible applications of the polymerase chain reaction as applied to RNA sequences are discussed. Rubella virus; Polymerase
chain reaction; Enzymatic amplification
Introduction Rubella virus is one of the most important teratogenic viruses, still causing many cases of congenital disease worldwide. This problem is particularly apparent in the developing world, probably due to undervaccination and lack of facilities to diagnose infection in the mother. However, cases of the congenital rubella syndrome (CRS) still occur in the developed world (Hanshaw et al., 1985). It is known that maternal reinfections can occur after either vaccination or priCorrespondence to: Paddington, London 0166~0934/89/$03.50
W.F. Carman, W2 INY, U.K. @ 1989 Elsevier
Department
Science
of Medicine,
Publishers
B.V.
St. Mary’s
(Biomedical
Hospital
Division)
Medical
School,
22
mary infection with the wild virus. However, in these situations, CRS is very rare (Morgan-Capner et al., 198.5). It thus becomes important to be able to differentiate a primary infection from a reinfection. The standard method for confirming an acute rubella infection is by detection of anti-rubella IgM antibodies. Contrary to past belief, these antibodies can also arise during reinfection. For patients with anti-rubella IgM, a recent approach has been to make use of the increasing avidity over time of IgG antibodies. If the patient’s IgG is of low avidity, this implies that the present episode is a primary infection (Rousseau and Hedman, 1988). However, this approach has not yet been fully assessed. As most maternal primary infections result in fetal infection, detection of virus in amniotic fluid can be attempted. However, tissue culture methods are slow and not always successful and nucleic acid hybridization was found not to have adequate sensitivity (Ho-Terry et al., 1988). In this contribution, a method is described in which rubella RNA, from concentrated viral preparations and crude cell lysates, is enzymatically amplified (Saiki et al., 1985, 1988). The reverse transcription and subsequent amplification are carried out in a single reaction vessel, with only minor modifications to the buffer conditions between the two stages. This means that lengthy RNA purification procedures are unnecessary, thus shortening the time needed to obtain a result.
Materials
and Methods
Virus propagation
and concentration,
and RNA purification
Rubella virus (Thomas strain) was obtained from Dr. S. O’Shea of St. Thomas Hospital Medical School, U.K. The virus was grown in Vero cell monolayers (OkerBlom et al., 1983). Infected cells were lysed by repeated freeze-thawing (3 x) and clarified by centrifugation at 10000 X g for 10 min. The virus was concentrated from lysed cells and the tissue culture medium by centrifugation at 50000 x g for 3.5 h. RNA was purified by phenol:chloroform extraction as described (Maniatis et al., 1982). Infected and uninfected Vero cells were harvested by scraping and collected by centrifugation at 2000 x g for 5 min. Cells were washed twice in phosphate buffered saline (PBS) (0.05 M sodium phosphate, 0.075 M NaCl, pH 7.4), resuspended in PBS (approx. 10h cells/ml), stored at 4°C and used within 24 h. Nucleic acid template and oligonucleotide primers Fig. 1 illustrates the 124 base pair region of the rubella El glycoprotein sequence to be amplified. Primer A was chosen to anneal to the sense strand and primer B to the anti-sense strand. A third 20-mer oligonucleotide, primer C, anneals to the anti-sense strand within the target sequence. Primers were checked with published rubella sequences for homology (Frey et al., 1986; Nakhasi et al., 1986; Clarkeet et al., 1987; Vidgren et al., 1987) and against a computerised data
23
file (Genebank, National Institute of Health, MD, U.S.A.) for lack of homology with other sequences. The env sequence of human immunodeficiency virus (HIV) cloned into pSP64 and three 20-mer oligonucleotide primers to this region (Carman and Kidd, 1989) were used as a DNA control. Optimisation of reverse transcription and amplification
Three different methods for optimum reverse transcription (RT) of purified RNA and subsequent amplification of cDNA were directly compared. In the first method, the RNA was reverse transcribed in a standard RT solution (40 ~1) containing 50 mM Tris-HCl, pH 8.3; 10 mM MgCl,; 60 pmol primer A, 3.75% (v/v) dimethylsulphoxide (DMSO); 2 units RNAsin (Amersham International, U.K.); 500 FM each dNTPs and 10 units of avian myeloblastosis virus (AMV) reverse transcriptase (Boehringer Mannheim, FRG). After 1 to 3 h incubation at 42°C 2.5 units of Taq polymerase (from Thermus aquaticus, supplied by New England Biolabs, U.S.A.), 40 pmol primer A and 100 pmol primer B were added and the solution was adjusted to a final concentration of 16.6 mM ammonium sulphate. Samples were thermal cycled as described below. In the second method, RNA was reverse transcribed in optimum buffer conditions for amplification, except the solution was supplemented with KCl. Samples were reverse transcribed in a solution (40 ~1) containing: 1 x amplification buffer (67 mM Tris-HCl, pH 8.8, 6.7 mM MgCl,, 16.6 mM (NHJ2S04, 10 mM 2-mercaptoethanol); 44 mM KCl, 60 pmol of primer A; 3.75% (v/v) DMSO; 1 ~1 RNAsin; 500 p_M each dNTPs; 10 units of AMV reverse transcriptase. After 2 to 3 h incubation at 42”C, 40 pmol primer A, 100 pmol primer B were added and the solution adjusted to a concentration of 0.1 mg/ml bovine serum albumin (BSA) and 500 FM each dNTPs, giving a final volume of 100 ~1. Taq polymerase (2.5 units) was added and the sample was thermal cycled as described below. In the third method, the RNA was treated as for the second method, except DMSO was excluded. Prior to thermal cycling, samples were overlaid with 100 ~1 of liquid paraffin to prevent evaporation. Forty cycles were performed; samples were incubated at 95°C for 1 min, 60°C for 1 min and 70°C for one minute. For the first cycle only, the samples were incubated at 95°C for 5 min. Amplification of viral samples
To 8 ~1 of concentrated virus preparation was added an equal volume of RSB lysis buffer (10 mM Tris-HCl, pH 7.5; 10 mM NaCl; 5 mM MgCl* containing 2% [v/v] Triton X-100). Samples were left on ice for 5 to 10 min prior to reverse transcription and amplification by method two, described above. To 8 p,l of a cell suspension, 8 p,l RSB lysis buffer was added. After 5-10 min on ice the cellular debris was pelleted by centrifugation for 1 min in a microfuge. The supernatant was collected and reverse transcribed and amplified as described in method two.
24
Dot-blot hy~~~dizatio~ The 124 bp amplified rubella virus DNA was electroeluted from a gel and concentrated and purified as described (Carman and Kidd, 1989). The DNA was labelled with 32P-dCTP by nick translation (Nick translation kit, Amersham). Oligonucleotides (100 pmol) were dotted onto nylon membrane (Hybond-N, Amersham~ and pre-hybridized and hybridized as described (barman and Kidd, 1989). Blots were autoradiographed overnight. Restriction endonuclease digestion Ten units each of restriction endonuclease HhaI (Amersham) and M&I (New England Biolabs) were added to 40 cl1aliquots of amplified product. Samples were digested for 2 h at 37°C. The entire digested product was electrophoresed as described. Gel electrophoresis After amplification and after restriction endonuclease digestion, 40 t.~laliquots of product were electrophoresed in 3% (w/v) agarose gels in TBE buffer (0.089 M Tris-borate; 0.089 M boric acid, 0.002 M EDTA) in the presence of 1 Kg/ml ethidium bromide. DNA was visualised on a UV transilluminator (260 nm).
Results
Optimisation of conditions Reverse transcriptase and Taq polymerase differ in that the former requires potassium ions and the latter ammonium sulphate. When samples were reverse transcribed in a standard RT buffer and then amplified in a solution supplemented with ammonium sulphate, no amplified DNA was detected (method 1). However, when samples were reverse transcribed in a standard amplification solution supplemented with ISCl, and subsequently amplified, positive results were obtained (method 2). Final KC1 concentrations from 44 mM to 95 mM gave similar results. The exclusion of DMSO from the reverse transcription reaction (method 3) decreased the yield of amplified product (data not shown). Method 2 was therefore used in all subsequent experiments. HIV DNA used as a control gave the same yield of amplified product (as measured by the intensity of the band after electrophoresis) whether the reaction was performed under those conditions used for rubella, or under conditions appropriate for DNA amplification, as described previously (Car-man and Kidd, 1989). This indicated that DNA amplification was not impaired by the residual RSB, Triton X-100 or KC1 from the lysis and reverse transcription steps.
25
Amplification
Reverse transcription and amplification of concentrated viral preparations reliably resulted in the predicted 124 bp DNA fragment (Fig. 2). A 124 bp DNA fragment was also produced after reverse transcription and DNA amplification of crude cell lysates. However, the ability to detect rubella RNA in crude cell lysates appeared to decrease with an increase in cell concentration: although negative results were obtained when amplifications were performed on nucleic acid from lo4 cells, a l/10 dilution of this sample resulted in a positive result (data not shown). As RSB lysis buffer disrupts the cell membranes without disrupting nuclear membranes (Davis et al., 1989), preparations used were relatively free of nuclear material. In experiments performed in parallel using uninfected cells, no specific DNA product was detected. Identification of amplified band
The 124 bp DNA fragment was digested by restriction endonucleases Hhal and Mnll into fragment sizes predicted for rubella sequences (Figs. 1 and 2). FIGURE
0
I
/-
_//
080
773
650
‘\
,.--H
‘1
_/H/ //5”y
Prime&
Primer
rC
‘Y
El
‘\
‘.
‘\ ‘\\
Primer
3
(70
tt Bbv 1 3, 706)
glycoprotein
rA
3’
target
sequence
5’ I
t Bbvl (720)
Hha 1 (734)
Fig. 1. relevant
Illustration of the 124 bp target sequence from the rubella El glycoprotein sequence and the restriction endonuclease sites. Numbering of the bases is taken from Frey et al. (1986). Primer sequences are given and their positions on the target sequence are shown in bold.
26
124
bp
Fig. 2. Three percent agarose gel of amplified rubelta DNA stained with ethidium bromide. Concentrated rubella virus was reverse transcribed and amplified as described in method 2. Lane 1, undigested; lane 2, digested with HhaI; lane 3, digested with MnlI; lane 4, undigested; lane 5, pBR322 digested with Hue111 (fragment sizes, 587, 540, 504, 450, 434, 267, 234, 213, 192, 184, 124, 123, 104, 89, 80, 64, 57, 51 base pairs).
Primer C and three different oligonucleotides, complementary to the env region of the HIV genome, were screened for homology with the 124 bp DNA fragment by dot-blot hybridization. Hybridization was observed between the 124 bp DNA fragment and primer C, and no hyb~d~ation was observed between this probe and the HIV primers included as negative controls (results not shown). Homology between the amplified DNA and primer C and the identification of predicted endonuclease sites in the target sequence confirmed that this DNA consisted of specific rubella sequences. Sensitivity
Using gel electrophoresis to measure sensitivity, viral samples were found to be positive at a 1:125 dilution (Fig. 3) after 40 cycles. This was equivalent to approximately 2 ng rubella RNA.
27
1
2
3
4
5
6
7
124bp-
Fig. 3. Three percent agarose gel ticies were concentrated from cell and 30 ~1 of the amplified product to 250 ng
of amplified rubella DNA stained with ethidium bromide. Viral parculture supernatant, diluted (5-fold), reverse transcribed, amplified was loaded per lane. Initial input virus (lane 1) approximately equal RNA (determined spectrophotometrically).
Discussion In this paper a method is described whereby rubella virus RNA is reverse transcribed, amplified and digested, with each subsequent step requiring only the addition of reagents. The results were most reliable when free virus was used rather than infected cells. This could have been due to contaminating cellular contents that may have had an inhibitory effect on enzyme activity. It is known that crude cell lysates provide suitable material for DNA amplification using Tuq polymerase (Kogan et al., 1987), so in this situation, it is likely to be the reverse transcriptase that was affected. This may not pose a major problem when analysing clinical samples such as amniotic fluid or urine, as free virus is likely to be present. In addition, when the cells were diluted out before lysis, the inhibitory effect became less marked, and amplified products were noted. As shown in a previous report, certain restriction endonucleases function efficiently in DNA amplification buffer (Carman and Kidd, 1989). In this communication, it was shown that an additions enzyme, M&l, was also effective under these conditions. Two other reports have appeared in which viral RNA (that of rhinovirus and HIV) have been amplified after a reverse transcription step (Gama et al., 1988; Hart et al., 1988). Both methods involved lengthy RNA purification procedures prior to reverse transcription. This is the first report in which PCR is used to detect viral RNA in unpurified virus preparations and in crude cell lysates.
28
There are a number of different ways in which reverse transcription and subsequent amplification can be of benefit. It could be used to amplify up certain RNA sequences for the purpose of sequencing, thus circumventing the need to clone. It could also be useful in the detection of specific RNA transcripts in cells. With the correct primer usage, it could be employed to distinguish between sense and antisense RNA and in this way be used to determine if an RNA virus was replicating. If a reverse transcription step was not performed, but merely DNA amplification, it could also be used to investigate a possible DNA stage in the RNA virus life cycle, as occurs with retroviruses. Although the method appears to be less sensitive than dot-blot hybridization, it is less time consuming and does not involve the handling of harmful substances. The clinical application has not yet been fully assessed; however, it is hoped that reverse transcription and amplification will be of use in the management of pregnant women who have been exposed to rubella virus but in whom the diagnostic picture is unclear.
Acknowledgements
We thank Dr. S. O’Shea for donating the rubella virus. References
Carman, W.F. and Kidd, A.H. (1989) An assessment of optimal conditions for amplification of HIV cDNA using Thermus aquaticus polymerase. J. Viral. Methods 23, 277-290. Clarke, D.M., Loo, T.W.. Hui, I., Chong, P. and Gillam, S. (1987) Nucleotide sequence and in vitro expression of rubella virus 24s subgenomic messenger RNA encoding the structural proteins El, E2 and C. Nucleic Acids Res. 15, 3041-3057. Frey, T.K., Marr, L.L., Hemphill, M.L. and Dominquez, G. (1986) Molecular cloning and sequencing of the region of the rubella virus genome coding for glycoprotein El. Virology 154, 228-232. Gama, R.E., Hughes, P.J., Bruce, C.B. and Stanway, G. (1988) Polymerase chain reaction amplification of rhinovirus nucleic acids from clinical material. Nucleic Acids Res. 16, 9346. Hanshaw, J.B., Dudgeon, J.A. and Marshall~ W.C. (1985) Viral diseases of the fetus and newborn, 2nd edit. W.B. Saunders, Philadelphia. Hart, C., Schochetman, G., Spira, T., Lifson, A., Moore, J., Galphin, J., Sninsky, J. and Ou, C.-Y. (1988) Direct detection of HIVRNA expression in seropositive subjects. Lancet 2, 596-599. Ho-Terry, L., Terry, G.M., Londesborough, P., Rees, K.R., Wielaard, F. and Denissen, A. (1988) Diagnosis of fetal rubella infection by nucleic acid hybridization. J. Med. Viral. 24, 175-182. Kogan, S.C., Doherty, M. and Gitschier, J. (1987) An improved method for pre-natal diagnosis of genetic diseases by analysis of amplified DNA sequences. Application to hemophilia. N. Engl. J. Med. 317, 985-991. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York. Morgan-Capner, P., Hodgson, J., Hambling, J.M., Dulake, C., Coleman, T.J., Boswell, P.A., Watkins, R.P., Booth, J., Stern, H., Best, J.M. and Banatvala, J.E. (1985) Detection of rubella-specification IgM in subclinical rubella reinfection in pregnancy. Lancet i, 244-246. Nakhasi, H.L., Meyer, B.C. and Lin, T.-Y. (1986) Rubella virus cDNA. Sequence and expression of El envelope protein. J. Biol. Chem. 261, 16616-16621. Oker-Blom, C., Kalkkinen, N., Karriainen, L. and Pettersson, R.F. (1983) Rubella virus contains one capsid protein and three envelope glycoproteins, El, E2a and E2b. J. Virol. 46, 964-973.
29 Rousseau, S. and Hedman, K. (1988) Rubella infection and reinfection distinguished by avidity IgG. Lancet 1, 1108. Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. and Amheim, N. (1985) Enzymatic amplification of B-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 13X-13.54. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. Vidgren, G., Takkinen, K., Kalkkinen, N., Kaariainen, L. and Pettersson, R.F. (1987) Nucleotide sequence of the genes coding for the membrane glycoproteins El and E2 of rubella virus. J. Gen. Virol. 68, 2347-2357.
21
JVM 00889
Reverse transcription and subsequent DNA amplification of rubella virus RNA William
F. Carman,
Carolyn Williamson, Beverley Alistair H. Kidd
A. Cunliffe
and
National Institute for Virology and Department of Virology, University of the Witwatersrand, Private Bag X4, Sandringham 2131, Johannesburg, South Africa (Accepted
9 February
1989)
Summary A method is described whereby rubella virus RNA was reverse transcribed and the resulting cDNA enzymatically amplified using Tuq polymerase. The reactions were carried out in a single reaction vessel, with only minor modifications to the buffer conditions between the reverse transcription and the subsequent amplification step. Using an oligonucleotide probe to the El glycoprotein region and limited restriction endonuclease mapping, the resulting amplified products were shown to be specific for rubella virus. This method was also successfully applied to crude cell lysates, without the need for RNA purification. The possible applications of the polymerase chain reaction as applied to RNA sequences are discussed. Rubella virus; Polymerase
chain reaction; Enzymatic amplification
Introduction Rubella virus is one of the most important teratogenic viruses, still causing many cases of congenital disease worldwide. This problem is particularly apparent in the developing world, probably due to undervaccination and lack of facilities to diagnose infection in the mother. However, cases of the congenital rubella syndrome (CRS) still occur in the developed world (Hanshaw et al., 1985). It is known that maternal reinfections can occur after either vaccination or priCorrespondence to: Paddington, London 0166~0934/89/$03.50
W.F. Carman, W2 INY, U.K. @ 1989 Elsevier
Department
Science
of Medicine,
Publishers
B.V.
St. Mary’s
(Biomedical
Hospital
Division)
Medical
School,
22
mary infection with the wild virus. However, in these situations, CRS is very rare (Morgan-Capner et al., 198.5). It thus becomes important to be able to differentiate a primary infection from a reinfection. The standard method for confirming an acute rubella infection is by detection of anti-rubella IgM antibodies. Contrary to past belief, these antibodies can also arise during reinfection. For patients with anti-rubella IgM, a recent approach has been to make use of the increasing avidity over time of IgG antibodies. If the patient’s IgG is of low avidity, this implies that the present episode is a primary infection (Rousseau and Hedman, 1988). However, this approach has not yet been fully assessed. As most maternal primary infections result in fetal infection, detection of virus in amniotic fluid can be attempted. However, tissue culture methods are slow and not always successful and nucleic acid hybridization was found not to have adequate sensitivity (Ho-Terry et al., 1988). In this contribution, a method is described in which rubella RNA, from concentrated viral preparations and crude cell lysates, is enzymatically amplified (Saiki et al., 1985, 1988). The reverse transcription and subsequent amplification are carried out in a single reaction vessel, with only minor modifications to the buffer conditions between the two stages. This means that lengthy RNA purification procedures are unnecessary, thus shortening the time needed to obtain a result.
Materials
and Methods
Virus propagation
and concentration,
and RNA purification
Rubella virus (Thomas strain) was obtained from Dr. S. O’Shea of St. Thomas Hospital Medical School, U.K. The virus was grown in Vero cell monolayers (OkerBlom et al., 1983). Infected cells were lysed by repeated freeze-thawing (3 x) and clarified by centrifugation at 10000 X g for 10 min. The virus was concentrated from lysed cells and the tissue culture medium by centrifugation at 50000 x g for 3.5 h. RNA was purified by phenol:chloroform extraction as described (Maniatis et al., 1982). Infected and uninfected Vero cells were harvested by scraping and collected by centrifugation at 2000 x g for 5 min. Cells were washed twice in phosphate buffered saline (PBS) (0.05 M sodium phosphate, 0.075 M NaCl, pH 7.4), resuspended in PBS (approx. 10h cells/ml), stored at 4°C and used within 24 h. Nucleic acid template and oligonucleotide primers Fig. 1 illustrates the 124 base pair region of the rubella El glycoprotein sequence to be amplified. Primer A was chosen to anneal to the sense strand and primer B to the anti-sense strand. A third 20-mer oligonucleotide, primer C, anneals to the anti-sense strand within the target sequence. Primers were checked with published rubella sequences for homology (Frey et al., 1986; Nakhasi et al., 1986; Clarkeet et al., 1987; Vidgren et al., 1987) and against a computerised data
23
file (Genebank, National Institute of Health, MD, U.S.A.) for lack of homology with other sequences. The env sequence of human immunodeficiency virus (HIV) cloned into pSP64 and three 20-mer oligonucleotide primers to this region (Carman and Kidd, 1989) were used as a DNA control. Optimisation of reverse transcription and amplification
Three different methods for optimum reverse transcription (RT) of purified RNA and subsequent amplification of cDNA were directly compared. In the first method, the RNA was reverse transcribed in a standard RT solution (40 ~1) containing 50 mM Tris-HCl, pH 8.3; 10 mM MgCl,; 60 pmol primer A, 3.75% (v/v) dimethylsulphoxide (DMSO); 2 units RNAsin (Amersham International, U.K.); 500 FM each dNTPs and 10 units of avian myeloblastosis virus (AMV) reverse transcriptase (Boehringer Mannheim, FRG). After 1 to 3 h incubation at 42°C 2.5 units of Taq polymerase (from Thermus aquaticus, supplied by New England Biolabs, U.S.A.), 40 pmol primer A and 100 pmol primer B were added and the solution was adjusted to a final concentration of 16.6 mM ammonium sulphate. Samples were thermal cycled as described below. In the second method, RNA was reverse transcribed in optimum buffer conditions for amplification, except the solution was supplemented with KCl. Samples were reverse transcribed in a solution (40 ~1) containing: 1 x amplification buffer (67 mM Tris-HCl, pH 8.8, 6.7 mM MgCl,, 16.6 mM (NHJ2S04, 10 mM 2-mercaptoethanol); 44 mM KCl, 60 pmol of primer A; 3.75% (v/v) DMSO; 1 ~1 RNAsin; 500 p_M each dNTPs; 10 units of AMV reverse transcriptase. After 2 to 3 h incubation at 42”C, 40 pmol primer A, 100 pmol primer B were added and the solution adjusted to a concentration of 0.1 mg/ml bovine serum albumin (BSA) and 500 FM each dNTPs, giving a final volume of 100 ~1. Taq polymerase (2.5 units) was added and the sample was thermal cycled as described below. In the third method, the RNA was treated as for the second method, except DMSO was excluded. Prior to thermal cycling, samples were overlaid with 100 ~1 of liquid paraffin to prevent evaporation. Forty cycles were performed; samples were incubated at 95°C for 1 min, 60°C for 1 min and 70°C for one minute. For the first cycle only, the samples were incubated at 95°C for 5 min. Amplification of viral samples
To 8 ~1 of concentrated virus preparation was added an equal volume of RSB lysis buffer (10 mM Tris-HCl, pH 7.5; 10 mM NaCl; 5 mM MgCl* containing 2% [v/v] Triton X-100). Samples were left on ice for 5 to 10 min prior to reverse transcription and amplification by method two, described above. To 8 p,l of a cell suspension, 8 p,l RSB lysis buffer was added. After 5-10 min on ice the cellular debris was pelleted by centrifugation for 1 min in a microfuge. The supernatant was collected and reverse transcribed and amplified as described in method two.
24
Dot-blot hy~~~dizatio~ The 124 bp amplified rubella virus DNA was electroeluted from a gel and concentrated and purified as described (Carman and Kidd, 1989). The DNA was labelled with 32P-dCTP by nick translation (Nick translation kit, Amersham). Oligonucleotides (100 pmol) were dotted onto nylon membrane (Hybond-N, Amersham~ and pre-hybridized and hybridized as described (barman and Kidd, 1989). Blots were autoradiographed overnight. Restriction endonuclease digestion Ten units each of restriction endonuclease HhaI (Amersham) and M&I (New England Biolabs) were added to 40 cl1aliquots of amplified product. Samples were digested for 2 h at 37°C. The entire digested product was electrophoresed as described. Gel electrophoresis After amplification and after restriction endonuclease digestion, 40 t.~laliquots of product were electrophoresed in 3% (w/v) agarose gels in TBE buffer (0.089 M Tris-borate; 0.089 M boric acid, 0.002 M EDTA) in the presence of 1 Kg/ml ethidium bromide. DNA was visualised on a UV transilluminator (260 nm).
Results
Optimisation of conditions Reverse transcriptase and Taq polymerase differ in that the former requires potassium ions and the latter ammonium sulphate. When samples were reverse transcribed in a standard RT buffer and then amplified in a solution supplemented with ammonium sulphate, no amplified DNA was detected (method 1). However, when samples were reverse transcribed in a standard amplification solution supplemented with ISCl, and subsequently amplified, positive results were obtained (method 2). Final KC1 concentrations from 44 mM to 95 mM gave similar results. The exclusion of DMSO from the reverse transcription reaction (method 3) decreased the yield of amplified product (data not shown). Method 2 was therefore used in all subsequent experiments. HIV DNA used as a control gave the same yield of amplified product (as measured by the intensity of the band after electrophoresis) whether the reaction was performed under those conditions used for rubella, or under conditions appropriate for DNA amplification, as described previously (Car-man and Kidd, 1989). This indicated that DNA amplification was not impaired by the residual RSB, Triton X-100 or KC1 from the lysis and reverse transcription steps.
25
Amplification
Reverse transcription and amplification of concentrated viral preparations reliably resulted in the predicted 124 bp DNA fragment (Fig. 2). A 124 bp DNA fragment was also produced after reverse transcription and DNA amplification of crude cell lysates. However, the ability to detect rubella RNA in crude cell lysates appeared to decrease with an increase in cell concentration: although negative results were obtained when amplifications were performed on nucleic acid from lo4 cells, a l/10 dilution of this sample resulted in a positive result (data not shown). As RSB lysis buffer disrupts the cell membranes without disrupting nuclear membranes (Davis et al., 1989), preparations used were relatively free of nuclear material. In experiments performed in parallel using uninfected cells, no specific DNA product was detected. Identification of amplified band
The 124 bp DNA fragment was digested by restriction endonucleases Hhal and Mnll into fragment sizes predicted for rubella sequences (Figs. 1 and 2). FIGURE
0
I
/-
_//
080
773
650
‘\
,.--H
‘1
_/H/ //5”y
Prime&
Primer
rC
‘Y
El
‘\
‘.
‘\ ‘\\
Primer
3
(70
tt Bbv 1 3, 706)
glycoprotein
rA
3’
target
sequence
5’ I
t Bbvl (720)
Hha 1 (734)
Fig. 1. relevant
Illustration of the 124 bp target sequence from the rubella El glycoprotein sequence and the restriction endonuclease sites. Numbering of the bases is taken from Frey et al. (1986). Primer sequences are given and their positions on the target sequence are shown in bold.
26
124
bp
Fig. 2. Three percent agarose gel of amplified rubelta DNA stained with ethidium bromide. Concentrated rubella virus was reverse transcribed and amplified as described in method 2. Lane 1, undigested; lane 2, digested with HhaI; lane 3, digested with MnlI; lane 4, undigested; lane 5, pBR322 digested with Hue111 (fragment sizes, 587, 540, 504, 450, 434, 267, 234, 213, 192, 184, 124, 123, 104, 89, 80, 64, 57, 51 base pairs).
Primer C and three different oligonucleotides, complementary to the env region of the HIV genome, were screened for homology with the 124 bp DNA fragment by dot-blot hybridization. Hybridization was observed between the 124 bp DNA fragment and primer C, and no hyb~d~ation was observed between this probe and the HIV primers included as negative controls (results not shown). Homology between the amplified DNA and primer C and the identification of predicted endonuclease sites in the target sequence confirmed that this DNA consisted of specific rubella sequences. Sensitivity
Using gel electrophoresis to measure sensitivity, viral samples were found to be positive at a 1:125 dilution (Fig. 3) after 40 cycles. This was equivalent to approximately 2 ng rubella RNA.
27
1
2
3
4
5
6
7
124bp-
Fig. 3. Three percent agarose gel ticies were concentrated from cell and 30 ~1 of the amplified product to 250 ng
of amplified rubella DNA stained with ethidium bromide. Viral parculture supernatant, diluted (5-fold), reverse transcribed, amplified was loaded per lane. Initial input virus (lane 1) approximately equal RNA (determined spectrophotometrically).
Discussion In this paper a method is described whereby rubella virus RNA is reverse transcribed, amplified and digested, with each subsequent step requiring only the addition of reagents. The results were most reliable when free virus was used rather than infected cells. This could have been due to contaminating cellular contents that may have had an inhibitory effect on enzyme activity. It is known that crude cell lysates provide suitable material for DNA amplification using Tuq polymerase (Kogan et al., 1987), so in this situation, it is likely to be the reverse transcriptase that was affected. This may not pose a major problem when analysing clinical samples such as amniotic fluid or urine, as free virus is likely to be present. In addition, when the cells were diluted out before lysis, the inhibitory effect became less marked, and amplified products were noted. As shown in a previous report, certain restriction endonucleases function efficiently in DNA amplification buffer (Carman and Kidd, 1989). In this communication, it was shown that an additions enzyme, M&l, was also effective under these conditions. Two other reports have appeared in which viral RNA (that of rhinovirus and HIV) have been amplified after a reverse transcription step (Gama et al., 1988; Hart et al., 1988). Both methods involved lengthy RNA purification procedures prior to reverse transcription. This is the first report in which PCR is used to detect viral RNA in unpurified virus preparations and in crude cell lysates.
28
There are a number of different ways in which reverse transcription and subsequent amplification can be of benefit. It could be used to amplify up certain RNA sequences for the purpose of sequencing, thus circumventing the need to clone. It could also be useful in the detection of specific RNA transcripts in cells. With the correct primer usage, it could be employed to distinguish between sense and antisense RNA and in this way be used to determine if an RNA virus was replicating. If a reverse transcription step was not performed, but merely DNA amplification, it could also be used to investigate a possible DNA stage in the RNA virus life cycle, as occurs with retroviruses. Although the method appears to be less sensitive than dot-blot hybridization, it is less time consuming and does not involve the handling of harmful substances. The clinical application has not yet been fully assessed; however, it is hoped that reverse transcription and amplification will be of use in the management of pregnant women who have been exposed to rubella virus but in whom the diagnostic picture is unclear.
Acknowledgements
We thank Dr. S. O’Shea for donating the rubella virus. References
Carman, W.F. and Kidd, A.H. (1989) An assessment of optimal conditions for amplification of HIV cDNA using Thermus aquaticus polymerase. J. Viral. Methods 23, 277-290. Clarke, D.M., Loo, T.W.. Hui, I., Chong, P. and Gillam, S. (1987) Nucleotide sequence and in vitro expression of rubella virus 24s subgenomic messenger RNA encoding the structural proteins El, E2 and C. Nucleic Acids Res. 15, 3041-3057. Frey, T.K., Marr, L.L., Hemphill, M.L. and Dominquez, G. (1986) Molecular cloning and sequencing of the region of the rubella virus genome coding for glycoprotein El. Virology 154, 228-232. Gama, R.E., Hughes, P.J., Bruce, C.B. and Stanway, G. (1988) Polymerase chain reaction amplification of rhinovirus nucleic acids from clinical material. Nucleic Acids Res. 16, 9346. Hanshaw, J.B., Dudgeon, J.A. and Marshall~ W.C. (1985) Viral diseases of the fetus and newborn, 2nd edit. W.B. Saunders, Philadelphia. Hart, C., Schochetman, G., Spira, T., Lifson, A., Moore, J., Galphin, J., Sninsky, J. and Ou, C.-Y. (1988) Direct detection of HIVRNA expression in seropositive subjects. Lancet 2, 596-599. Ho-Terry, L., Terry, G.M., Londesborough, P., Rees, K.R., Wielaard, F. and Denissen, A. (1988) Diagnosis of fetal rubella infection by nucleic acid hybridization. J. Med. Viral. 24, 175-182. Kogan, S.C., Doherty, M. and Gitschier, J. (1987) An improved method for pre-natal diagnosis of genetic diseases by analysis of amplified DNA sequences. Application to hemophilia. N. Engl. J. Med. 317, 985-991. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York. Morgan-Capner, P., Hodgson, J., Hambling, J.M., Dulake, C., Coleman, T.J., Boswell, P.A., Watkins, R.P., Booth, J., Stern, H., Best, J.M. and Banatvala, J.E. (1985) Detection of rubella-specification IgM in subclinical rubella reinfection in pregnancy. Lancet i, 244-246. Nakhasi, H.L., Meyer, B.C. and Lin, T.-Y. (1986) Rubella virus cDNA. Sequence and expression of El envelope protein. J. Biol. Chem. 261, 16616-16621. Oker-Blom, C., Kalkkinen, N., Karriainen, L. and Pettersson, R.F. (1983) Rubella virus contains one capsid protein and three envelope glycoproteins, El, E2a and E2b. J. Virol. 46, 964-973.
29 Rousseau, S. and Hedman, K. (1988) Rubella infection and reinfection distinguished by avidity IgG. Lancet 1, 1108. Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. and Amheim, N. (1985) Enzymatic amplification of B-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 13X-13.54. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. Vidgren, G., Takkinen, K., Kalkkinen, N., Kaariainen, L. and Pettersson, R.F. (1987) Nucleotide sequence of the genes coding for the membrane glycoproteins El and E2 of rubella virus. J. Gen. Virol. 68, 2347-2357.