- Email: [email protected]
HCV RNA detection in heparinized blood by direct genomic RNA capture onto paramagnetic particles
Journal of Virological Methods 48 (1994) 339-341
Journal of Virological Methods
Short Communication HCV RNA detection in heparinized blood by direct genomic RNA capture onto paramagnetic particles L.J. van Dooma, V. Shyamalac, J.H. HanC and G.E.M. Kleterb “Diagnostic Centre SSDZ, Department of Molecular Biology, P.O. Box 5010. 2600 GA Delfr, The NetherIan& “Erasmus University, Department of Virology, Dr. Molewaterplein 40, Rotterdam, The Netherlands and “Chiron Corporation, 4560 Horton Street, Emeryville CA, USA
(Accepted 24 November 1993)
Hepatitis C virus (HCV), the etiological agent of parenterally transmitted non-A, non-B hepatitis, contains a positive-sense RNA genome of approximately 9.5 kb (Choo et al., 1989). Routine diagnosis of HCV infection is based on detection of antibodies against viral antigens (Kuo et al., 1989), but HCV viraemia can only be determined by specific detection of genomic RNA by the reverse transcriptase Polymerase Chain Reaction (RT-PCR; Weiner et al., 1990). The success of application of RT-PCR relies on isolation and purification of the target RNA, since biological fluids may contain potent inhibitors of Tuq DNA polymerase, such as heparin. Using the conventional isolation methods, employing extraction with organic solvents and ethanol precipitation, heparin co-purifies with the HCV RNA. Blood or heparinized plasma is therefore not suitable for RT-PCR. (Panaccio et al., 1991, Willems et al., 1992). 24 heparinized human plasma samples from patients with elevated ALT levels were sent to our diagnostic laboratory for HCV RNA determination as part of an ongoing study of a-interferon treatment of HCV infection. It was not possible to obtain EDTA-plasma or serum at the desired time-points. All samples, except one were anti-HCV positive by a second generation anti-HCV assay. Using the routine isolation method which involves guanidinium thiocyanate treatment, repeated phenol/chloroform extractions and isopropanol precipitation (Kleter et al., 1992), HCV RNA was not detected in any of the samples. Therefore the applicability of an RNA capture PCR assay as an alternative to isolation and purification of HCV RNA from these samples was investigated. The principle of the HCV RNA capture assay, as illustrated in Fig. 1, has been described previously (van Doorn 1992; 1993). Briefly, HCV RNA is released from viral particles by proteinase K digestion and hybridized to a biotinylated oligonucleotide, which is bound to streptavidin-coated paramagnetic particles (Dynabeads M-280, Dynal, Norway). The biotinylated oligonucleotide, LD57 (5’ BIO-GGCCGGGGCGGCCGCCATGiTGCACGGTCTAC0166-0934/94/%07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0166-0934(93)E0154-T
340
L.J. van Doom et d/Journal
C
El EZ/NSl
NS2
of Virological Methods 48 (1994) 339-341
NS3
NS4
NS5
core Fig. 1.
GAGACCTCCCGGGGC-3’; underlined sequence is non-complementary spacer, i is Inosine) is complementary to a highly conserved sequence of the HCV genome (positions -29 to + 3, with position 1 at the startcodon of the polyprotein). Captured RNA is washed to remove all plasma components, converted into cDNA with random primers and amplified by nested PCR. For detection of HCV viraemia, PCR primers from the highly conserved 5’ UTR region were employed. Outer primers were NCR3 (sense; 5’-GGGGCGGCCGCCACCATAGATCACTCCCCTGTGAGG-3’) and NCR4 (antisense; 5’-CACTCTCGAGCACCCTATCAGGCAGTACC-3’. Inner primers were JHC5 1 (antisense; 5’-CCCAACACTACTCGGCTA3’) and HC3 (sense: - 264 to - 238: 5’-TCTAGCCATGGCGTTAGTRYGAGTGT-3’). First and second round PCR consisted of 1 min 95°C 2 min 48°C 2 min 72°C for 40 cycles. The efficiency of this HCV RNA capture assay in EDTA-plasma samples was compared to the standard isolation method and results were very similar (van Doorn et al., 1993). The sensitivity of the current HCV capture assay has been estimated, by testing serial dilutions of a titrated chimpanzee plasma sample, to be between 150 and 1500 molecules. Using quantified in vitro transcribed RNA, covering part of the 5’ UTR, the sensitivity of our RT-PCR protocol has been estimated between 10 and 50 molecules. The results of the HCV RNA capture assay in 24 heparinized blood samples are summarized in Table 1. The capture assay allowed HCV RNA detection in 19 plasma samples. Further analysis of amplified 5’-UTR sequences (data not shown) revealed the presence of various HCV genotypes (Simmonds et al., 1993, Stuyver et al., 1993). The single anti-HCV negative sample did not contain detectable HCV RNA, and was later diagnosed as auto-immune disease. These data illustrate the
L.J. van Doorn et al./Journal of Virological Methods 48 (1994) 339-341 Table 1 HCV-RNA
and anti-HCV
detection
in 24 heparinized anti-HCV n=23
standard capture
0 19
pos
plasma
341
samples anti-HCV ?l=l
neg
0 0
capacity of the RNA capture method to detect efficiently the most common HCV genotypes known at present. In 4 samples HCV RNA was undetectable, which may be due to very low virus titres or total absence of detectable RNA. The detection rate among anti-HCV positive patients is in accordance with earlier reports. In conclusion, the RNA capture system allows HCV RNA detection even in heparinized plasma samples and experimental results indicate that heparin does not bind irreversibly to the RNA. Archived heparinized plasma samples can now be studied with RT-PCR. HCV RNA capture is a rapid and efficient method to purify RNA that is suitable for RT-PCR, even from complex biological solutions that contain potent inhibitors of Tuq DNA polymerase.
References Choo, Q.-L., Kuo, G., Weiner, A.J., Overby, L.R., Bradley, D.W. and Houghton, M. (1989) Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244, 359362. Kleter, GEM., Brouwer, J.T., Heijtink, R.A., Schalm, S.A. and Quint, W.G.V. (1993) Detection of Hepatitis C virus RNA in patients with chronic hepatitis C virus infections during and after therapy with alpha interferon. Antimicrobial Agents Chemotherapy 37, 595-597. Kuo, G., Choo, Q.-L., Alter, H.J., et al. (1989) An assay for circulating antibodies to a major etiologic agent of human non-A, non-B hepatitis. Science 244, 362-364. Panaccio, M. and Lew, A. (1991) PCR based diagnosis in the presence of 8% (v/v) blood. Nucl. AC. Res. 19, 5, 1151. Simmonds, P., McOmish, F., Yap, P.L., et al. (1993) Sequence variability in the 5’-non-coding region of hepatitis C virus: identification of a new virus type and restrictions on sequence diversity. J. Gen. Virol. 74, 661668. Stuyver, L., Rossau, R., Wyseur, A., et al. (1993) Typing of hepatitis C virus isolates and characterization of new subtypes using a line probe assay. J. Gen. Virol. 74, 6, 1093-l 102. van Doorn, L.J., van Belkum, A.F., Maertens, G., Quint, W.G.V., Kos, A. and Schellekens, H. (1992) Hepatitis C virus antibody detection by a line immunoassay and (near) full length genomic RNA detection by a new RNA-Capture polymerase chain reaction. J. Med. Virol. 38, 298-304. van Doorn, L.J., Kleter, G.E.M., Voermans, J., et al. (1993) Rapid detection hepatitis C virus RNA by direct capture from blood. J. Med. Virol. 42, 22-28. Weiner, A.J., Kuo, G., Bradley, D.W., et al. (1990) Detection of hepatitis C viral sequences in non-A, nonB hepatitis. Lancet 335, 1-3. Willems, M., Moshage, H., Nevens, F., Fevery and Yap, S.H. (1993) Plasma collected from heparinized blood is not suitable for HCV-RNA detection by conventional RT-PCR. J. Virol. Met. 43, 127-130.
Journal of Virological Methods
Short Communication HCV RNA detection in heparinized blood by direct genomic RNA capture onto paramagnetic particles L.J. van Dooma, V. Shyamalac, J.H. HanC and G.E.M. Kleterb “Diagnostic Centre SSDZ, Department of Molecular Biology, P.O. Box 5010. 2600 GA Delfr, The NetherIan& “Erasmus University, Department of Virology, Dr. Molewaterplein 40, Rotterdam, The Netherlands and “Chiron Corporation, 4560 Horton Street, Emeryville CA, USA
(Accepted 24 November 1993)
Hepatitis C virus (HCV), the etiological agent of parenterally transmitted non-A, non-B hepatitis, contains a positive-sense RNA genome of approximately 9.5 kb (Choo et al., 1989). Routine diagnosis of HCV infection is based on detection of antibodies against viral antigens (Kuo et al., 1989), but HCV viraemia can only be determined by specific detection of genomic RNA by the reverse transcriptase Polymerase Chain Reaction (RT-PCR; Weiner et al., 1990). The success of application of RT-PCR relies on isolation and purification of the target RNA, since biological fluids may contain potent inhibitors of Tuq DNA polymerase, such as heparin. Using the conventional isolation methods, employing extraction with organic solvents and ethanol precipitation, heparin co-purifies with the HCV RNA. Blood or heparinized plasma is therefore not suitable for RT-PCR. (Panaccio et al., 1991, Willems et al., 1992). 24 heparinized human plasma samples from patients with elevated ALT levels were sent to our diagnostic laboratory for HCV RNA determination as part of an ongoing study of a-interferon treatment of HCV infection. It was not possible to obtain EDTA-plasma or serum at the desired time-points. All samples, except one were anti-HCV positive by a second generation anti-HCV assay. Using the routine isolation method which involves guanidinium thiocyanate treatment, repeated phenol/chloroform extractions and isopropanol precipitation (Kleter et al., 1992), HCV RNA was not detected in any of the samples. Therefore the applicability of an RNA capture PCR assay as an alternative to isolation and purification of HCV RNA from these samples was investigated. The principle of the HCV RNA capture assay, as illustrated in Fig. 1, has been described previously (van Doorn 1992; 1993). Briefly, HCV RNA is released from viral particles by proteinase K digestion and hybridized to a biotinylated oligonucleotide, which is bound to streptavidin-coated paramagnetic particles (Dynabeads M-280, Dynal, Norway). The biotinylated oligonucleotide, LD57 (5’ BIO-GGCCGGGGCGGCCGCCATGiTGCACGGTCTAC0166-0934/94/%07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0166-0934(93)E0154-T
340
L.J. van Doom et d/Journal
C
El EZ/NSl
NS2
of Virological Methods 48 (1994) 339-341
NS3
NS4
NS5
core Fig. 1.
GAGACCTCCCGGGGC-3’; underlined sequence is non-complementary spacer, i is Inosine) is complementary to a highly conserved sequence of the HCV genome (positions -29 to + 3, with position 1 at the startcodon of the polyprotein). Captured RNA is washed to remove all plasma components, converted into cDNA with random primers and amplified by nested PCR. For detection of HCV viraemia, PCR primers from the highly conserved 5’ UTR region were employed. Outer primers were NCR3 (sense; 5’-GGGGCGGCCGCCACCATAGATCACTCCCCTGTGAGG-3’) and NCR4 (antisense; 5’-CACTCTCGAGCACCCTATCAGGCAGTACC-3’. Inner primers were JHC5 1 (antisense; 5’-CCCAACACTACTCGGCTA3’) and HC3 (sense: - 264 to - 238: 5’-TCTAGCCATGGCGTTAGTRYGAGTGT-3’). First and second round PCR consisted of 1 min 95°C 2 min 48°C 2 min 72°C for 40 cycles. The efficiency of this HCV RNA capture assay in EDTA-plasma samples was compared to the standard isolation method and results were very similar (van Doorn et al., 1993). The sensitivity of the current HCV capture assay has been estimated, by testing serial dilutions of a titrated chimpanzee plasma sample, to be between 150 and 1500 molecules. Using quantified in vitro transcribed RNA, covering part of the 5’ UTR, the sensitivity of our RT-PCR protocol has been estimated between 10 and 50 molecules. The results of the HCV RNA capture assay in 24 heparinized blood samples are summarized in Table 1. The capture assay allowed HCV RNA detection in 19 plasma samples. Further analysis of amplified 5’-UTR sequences (data not shown) revealed the presence of various HCV genotypes (Simmonds et al., 1993, Stuyver et al., 1993). The single anti-HCV negative sample did not contain detectable HCV RNA, and was later diagnosed as auto-immune disease. These data illustrate the
L.J. van Doorn et al./Journal of Virological Methods 48 (1994) 339-341 Table 1 HCV-RNA
and anti-HCV
detection
in 24 heparinized anti-HCV n=23
standard capture
0 19
pos
plasma
341
samples anti-HCV ?l=l
neg
0 0
capacity of the RNA capture method to detect efficiently the most common HCV genotypes known at present. In 4 samples HCV RNA was undetectable, which may be due to very low virus titres or total absence of detectable RNA. The detection rate among anti-HCV positive patients is in accordance with earlier reports. In conclusion, the RNA capture system allows HCV RNA detection even in heparinized plasma samples and experimental results indicate that heparin does not bind irreversibly to the RNA. Archived heparinized plasma samples can now be studied with RT-PCR. HCV RNA capture is a rapid and efficient method to purify RNA that is suitable for RT-PCR, even from complex biological solutions that contain potent inhibitors of Tuq DNA polymerase.
References Choo, Q.-L., Kuo, G., Weiner, A.J., Overby, L.R., Bradley, D.W. and Houghton, M. (1989) Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244, 359362. Kleter, GEM., Brouwer, J.T., Heijtink, R.A., Schalm, S.A. and Quint, W.G.V. (1993) Detection of Hepatitis C virus RNA in patients with chronic hepatitis C virus infections during and after therapy with alpha interferon. Antimicrobial Agents Chemotherapy 37, 595-597. Kuo, G., Choo, Q.-L., Alter, H.J., et al. (1989) An assay for circulating antibodies to a major etiologic agent of human non-A, non-B hepatitis. Science 244, 362-364. Panaccio, M. and Lew, A. (1991) PCR based diagnosis in the presence of 8% (v/v) blood. Nucl. AC. Res. 19, 5, 1151. Simmonds, P., McOmish, F., Yap, P.L., et al. (1993) Sequence variability in the 5’-non-coding region of hepatitis C virus: identification of a new virus type and restrictions on sequence diversity. J. Gen. Virol. 74, 661668. Stuyver, L., Rossau, R., Wyseur, A., et al. (1993) Typing of hepatitis C virus isolates and characterization of new subtypes using a line probe assay. J. Gen. Virol. 74, 6, 1093-l 102. van Doorn, L.J., van Belkum, A.F., Maertens, G., Quint, W.G.V., Kos, A. and Schellekens, H. (1992) Hepatitis C virus antibody detection by a line immunoassay and (near) full length genomic RNA detection by a new RNA-Capture polymerase chain reaction. J. Med. Virol. 38, 298-304. van Doorn, L.J., Kleter, G.E.M., Voermans, J., et al. (1993) Rapid detection hepatitis C virus RNA by direct capture from blood. J. Med. Virol. 42, 22-28. Weiner, A.J., Kuo, G., Bradley, D.W., et al. (1990) Detection of hepatitis C viral sequences in non-A, nonB hepatitis. Lancet 335, 1-3. Willems, M., Moshage, H., Nevens, F., Fevery and Yap, S.H. (1993) Plasma collected from heparinized blood is not suitable for HCV-RNA detection by conventional RT-PCR. J. Virol. Met. 43, 127-130.