CA2257191A1 - Method to detect hcv specific nucleic acids - Google Patents
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- CA2257191A1 CA2257191A1 CA002257191A CA2257191A CA2257191A1 CA 2257191 A1 CA2257191 A1 CA 2257191A1 CA 002257191 A CA002257191 A CA 002257191A CA 2257191 A CA2257191 A CA 2257191A CA 2257191 A1 CA2257191 A1 CA 2257191A1
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- C12Q1/706—Specific hybridization probes for hepatitis
- C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
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Abstract
A process to detect hepatitis C virus (HCV) specific nucleic acids comprising the steps of: (a) reverse transcribing the viral RNA by means of a primer having specified sequences; (b) amplifying by a single polymerase chain reaction (PCR) with a specific primer; wherein the Mg++/Taq polymerase ratio in the reaction mix is of approx. 100 nmoles/enzyme unit; (c) revealing the amplified product by means of the DEIA method using a specific probe.
Description
CA 022~7191 1998-ll-30 W O 97/46716 PCT~T97/00128 METHOD TO DETECT HCV SPECIFIC NUCLEIC ACIDS
The invention concerns a method to detect hepatitis C virus specific (HCV) nucleic acids.
In particular the invention refers to an improved method to detect HCV amplified DNA, by means of a single step polymerase chain reaction (PCR), under controlled and optimized reaction parameters, and of a revealing system of amplified products.
One of the most used methods to detect HCV specific nucleic acids is based upon the reverse transcription of viral RNA to cDNA, followed by a double amplification step (nested PCR) of the most conserved genome region (5'UTR). The amplified product of the second amplification step may be identified by means of revealing techniques as electrophoresis or enzyme mediated signals. The double amplification allows to reach a very high sensitivity able to identify even few viral RNA molecules. On the other hand the double PCR
step has many disadvantages mainly due to DNA
contamination from previous amplifications, length of time, high costs.
In order to overcome said disadvantages there is the need to set up a single step amplification protocol, which reaches similar levels of sensitivity than the nested PCR.
The authors of the instant invention have optimized the nested PCR reaction conditions in order to eliminate the second step. Moreover the system used to reveal amplified products is the DNA Enzyme Immunoassay (DEIA), which mekes the use of a specific capturing probe and of a monoclonal antibody able to recognize double strand DNA
(Mantero G. et al. Clin Chem. 37, 422-429, here incorporated by references).
.. ~ ..... . . .. .
CA 022~7191 1998-11-30 WO97/46716 PCT~T97/00128 The combination of all of optimized parameters, both of the amplification and of the revealing step, allowed to set up a method able to detect HCV specific DNA with a sensitivity equal, if not higher than the nested PCR, but with no disadvantages.
In order to optimize the PCR reaction parameters a comparison of first (not revealed by DEIA assay) and second (revealed by DEIA assay) amplification step, and an analysis to individuate the capturing probe with best features for a revealing step by DEIA assay as well, were performed.
C. Payan et al. J. Virol. Meth. (1995) 53, 167-175 describe a process to reveal HCV nucleic acids characterised by a single step for both the reverse transcription and the amplification. The amplification is a single step, no nested-PCR. Primer sequences are deduced from the HCV genome nucleotide sequence, and in particular from the high conserved 5'end region. The magnesium chloride final concentration in the PCR
solution is of 1 mM, the ratio [Mg~~]/U Taq being 35 nmol/U Taq.
C. Payan et al. Res. Virol. (1995) 146, 363-70 optimize the above referred process by modifying the MgCl2 concentration to 2 mM, the units of Mu-MLV RNAse and of Taq polymerase, to 10 U and 1 U respectively, with a ratio of 42 nmol [Mg++]/U Taq, and thus diminishing the RNA copy number to be revealed from 15 to 10.
It is therefore evident that even minima~
variations of general reaction conditions may interfere and increase significantly the yield and sensitivity.
It is an object of the instant invention a method to detect hepatitis C virus (HCV) specific nucleic acids comprising the steps of:
CA 022~7191 1998-ll-30 W O97/46716 PCT~T97/00128 a) reverse transcribing the viral RNA with a primer having a sequence substantially homologous to one of the sequences SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3;
b) amplifying by means of a single step polymerase chain reaction (PCR) wherein the primer has a sequence su~stantially homologous to sequences of SEQ ID No. 4 or SEQ ID No. 5, wherein the ratio between the Mg'+ ion concentration and of Taq polymerase in the reaction mix is of approximately of 100 nmoles/enzyme unit;
c) revealing the amplified product by means of DEIA
method using a probe having the sequence of SEQ ID No. 6, or a probe having a sequence substantially homologous or complementary thereof.
It is a further object of the invention the use of the of an oligonucleotide of SEQ ID No. 6 or o an oligonucleotide having a sequence substantially homologous or complementary thereof as probe for revealing HCV specific nucleic acids by means of the DEIA
method.
It is a further ob3ect of the invention a composition to reveal HCV specific nucleic acids by means of the DEIA method comprising an oligonucleotide of SEQ
I3 No. 6 or of sequence substantially ho~ologous or complementary thereof.
By comparing the two steps of the nested PCR, as in Table 2, it is clear that main differences are: 1) primer sequences; 2) MgCl2 concentration (which is a Taq polymerase activator); 3) buffer concentration (TRIS-~C1 pH 8.3); 4) solution ionic strength (~ainly related to the KC1 concentration)i 5) deoxinucleotidetriphosphate concentration (dNTP). Primer sequences are shown in Table la.
W O 97/46716 PCT~T97/00128 Table la Primer sequences name sequence SEQ ~D No.
1 CH 5' GGT GCA CGG TCT ACG AGA CCT 3' SEQ ID No. 1 2 CH 5' AAC TAC TGT CTT CAC GCA GAA 3' SEQ ID No. 4 1 TS 5' GCG ACC CAA CAC TAC TCG GCT 3' SEQ ID No. 2 4 CH 5' A~G GCG TTA GTA TGA GTG 3' SEQ ID No. 5 PT2 5' CGG TGT ACT CAC CGG TTC 3' SEQ ID No. 3 PKY78 5' CTC GCA AGC ACC CTA TCA GGC AGT 3 ' SEQ ID No. 7 PKY80 5' GGA GAA AGC GTC TAG CCA TGG CGT 3' SEQ ID No. 8 Probe sequences for the revealing step are shown in Table lb.
Table lb 3 CH 5' CGG TGA GTA CAC CGG AAT TGC CAG GAC CGG SEQ ID No. 9 GTC CTT TCT 3' WT 5' GCC ATA GTG GTC TGC GG 3' SEQ ID No. lO
PT 21 5' GGG AGA GCC ATA GTG GTC TGC 3' SEQ ID No. 6 KY 150 5' CAT AGT GGT CTG CGG AAC CGG TGA GT 3' SEQ ID No. ll HCV40 5' CCA TAG TGG TC~ GCG GAA CCG TGA GTA CA 3' SEQ ID No. 12 CH5 5' TAG TGG TCT GCG GAA CCG GT 3' SEQ ID No. 13 Table 2 Two step nested PCR parameters param. I step II step primerlCH~50pmol)/2CH(50pmol) lTS (~Opmol) /4CH (50pmol) [Tris-HCl] 22.5 mM 10 mM
[KCl] 62.5 mM 50 mM
[MgCl2] 4 mM 1. 5 mM
[dNTP] 260 uM ** 200 ~M
Taq pol . 2.5 u 2. 5 U
** The concentration accounts also for added dNTPs during the reverse transcription step.
CA 022~7191 1998-ll-30 W O 97/46716 PCT~T97/00128 1) SAMPLE PREPARATION
Sera to be tested by nested or single step PCR were treated to isolate RNA. Commercially available products, as RNAzol B e ULTRASPEC (Biotecx), were used following instructions of producer.
2) NESTED PROTOCOL
The nested PCR to detect HCV RNA at the 5'UTR
region was used as control. 5 ~ul of extracted RNA were reverse transcribed in 25 ~ul volume containing 22.5 mM
TRIS-HCl pH a .3, 62.5 mM KCl, 4 mM MgCl , 250 ~M dNTPs, 4U AMV-RT, 2 ~M lCH antisense primer, 25 U RNAse inhibitors (HRPI). The reverse transcription was performed at 42~C for 1 hr and the enzyme was further denatured at 100~C for 10 min. The nested PCR first step was performed in a 100 ul volume containing cDNA ~25 ~l from the reverse transcription), 22.5 mM TRIS-HCl pH 8.3, 62.5 mM KCl, 4 mM MgCl2, 200 ~M dNTPs (only for this step), 0.5 ~M 2CH sense primer, 2.5 U Taq polymerase;
thermal cycle: 94~C 1 min., 50~C 1 min., 72~C 2 min., 35 cycles. For the second step 3 ul from the first step were amplified in a 100 ul volume containing 10 mM TRIS-HCl pH
8.3, 50 mM KCl, 1.5 mM MgCl2, 200 ~M dNTPs, 0.5 ~M lTS
internal antisense primer, 0.5 ~M 4CH internal sense primer; 2.5 U Taq polymerase; thermal cycle: 94~C 1 min., 50~C 1 min., 72~C 2 min.; 25 cycles.
20 ul of each amplified product were tested by the DEIA assay using as probe the 3CH oligonucleotide.
Experimental tests are hereinbelow reported, according to different parameters of the first and second PCR steps. In all of reactions the amount of AMV-RT
enzyme was of 15 U/sample.
3) CHANGE of PRIMER SEQUENCES
Different combinations of primers were assayed in the single step RT-PCR.
CA 022~7191 1998-ll-30 W O 97/46716 PCTnT97/00128 Nested external primers: lCH (antisense) / 2CH (sense) Nested internal primers: lTS (antisense) / 4CH (sense) Nested external/internal primers: lCH (antisense) / 4CH
(sense) Nested internal/external primers: lTS (antisense) / 2CH
(sense) The antisense primer PT2 and those described in the EP 529493 application, identified as PKY78 antisense and PKY80 sense, were also assayed.
Reaction conditions were those for all of primer combinations and are as follows:
- reverse transcription was performed as for the nested protocol, but of AMV-RT units;
- PCR was performed in 100 ul final volume containing cDNA, 22.5 rnM T~IS-HCl pH 8.3, 62.5 mM KCl, 4 mM MgCl2, 200 uM dNTP (only those added in this step), O.5 ~M sense primer (according to different combinations), 2.5 U Taq polymerase; thermal cycle: 94~C
1 min., 50~C 1 min., 72~C 2 min.; 45 cycles;
- 20 ul o~ each amplified product were assayed by means of the DEIA Enzyme Immunoassay with the 3CH probe.
By only varying primer combinations lCH, 2CH, 4CH, lTS, PT2 no detectable amplified product was made in a single step PCR amplification. Even when "alternative"
sequences were use (primers as in EP 529493) no clear positive results were obtained ~positive result only in one sample), see Table 3 (positive values in bold character).
. .
, CA 02257191 1998-ll-30 W O 97/46716 PCTnT97/00128 Table 3 ne~ted~lngle 3tep PCR
PCRprlmer combination lCH/2C~ lTS/4CH lCH/4CH lTS/2CH PKY78/PKY80 ~ample O.D. 450/630 nm O RNA 0,061 0,068 0,076 0,176 0,110 0,084 N. 1 Neg0,084 0,062 0,067 0,103 0,112 0,089 N. 2 Po~2,185 0,123 0,078 0,109 0,103 l,lS9 N. 3 Pos1,979 0,066 0,081 0,109 0,109 0,092 N. 4 Po~2,566 0,054 0,084 0,105 0,113 0,069 cut-off 0,192 0,192 0,192 0,231 0,231 0,220 4) MODIFICATIONS OF MgCl~CONCENTRATION
It has been found that the MgCl- concentration (a Taq polymerase activator) has to be finely modulated to obtain the best yield of the amplification reaction.
During the amplification, MgCl concentrations of 1.5 mM, 2.5 mM, 4 mM, corresponding, respectively, to 60nmol, 100nmol, 160nmol of Mg+ per Taq unit were used. The reverse transcription reaction was performed as in the nested PCR protocol, but of enzyme units. The PCR
reaction was performed in a 100 ~l volume containing cDNA (25 ~l from the reverse transcription mix), 22.5 mM
TRIS-HCl pH 8.3, 62.5 mM KCl, MgCl at different concentrations, 200 ~M dNTPs (only dNTPs added in this step), 0,5 ~M 2CH primer and 2,5 U Taq polymerase.
Data obtained by means of the DEIA immunoenzyme assay (with the 3C~ probe), as in Table 4, show that:
a) very low MgCl2 concentrations (1.5 mM, 60 nmol/U~ reduce the yield of the single step PCR reaction;
in fact, only S0% of nested positive samples are still positive;
-b) very high MgCl~ concentrations (4 mM, 160 nmol/U) are not workable since no nested positive sample was confirmed as such in the single step reaction;
.. . . ~ . , .
W O 97/46716 PCTnT97/00128 c) a MgCl concentratlon of 2.5 mM ~100 nmol/U) is the best, confirming all of data obtained by the nested PCR.
Table 4 nested single step PCR
PCR [MgCl2]
sample 1,5 mM2,5 mM 4,0 mM
O.D. 450/630 nm N 1 2,085 2,0132,017 0,065 N 2 1,622 0,1450,913 0,092 N 3 2,107 1,7971,912 0,071 N 4 2,163 0,1150,261 0,080 N 5 2,194 0,2741,092 0,078 N 6 0,899 0,1170,560 0,071 N 7 0,107 0,1220,158 0,066 N 8 0,055 0,1150,131 0,077 N 9 0,054 0,1150,140 0,077 N10 0,051 0,1020,157 0, 063 Nll 0,084 0,1550,134 0,062 cut off 0,253 5) MODIFICATIONS OF REACTION BUFFER CONCENTRATION
As opposite to MgCl2, differences in the concentration of TRIS-HCl (12.5 mM instead of 22.5 mM) and KCl (50 mM instead of 62.5 mM) do not interfere in a significant way with the efficacy of the single step amplification, as DEIA obtained data are comparable to controls, as in Table 5.
CA 02257191 1998-ll-30 6 PCT~T97/00128 Table 5 Tris-HCl 12,5 mM Tris-HCl 22,5 mM
SAMPLEKCl 12,5 mM KCl 62,5 mM
O.D. 450/630 nm N~2368 0,900 0,912 N~2369 1,012 1,038 N~2370 1,064 1,311 N~2381 0,685 1,604 N~2397 0,549 0,508 N~2404 0,028 0,054 N~2411 1,354 1,145 N~2412 1,46~ 1,780 N~2416 1,387 1,797 N~2452 0,845 1,514 N~2464 0,872 1,175 N~2487 1,032 2,274 cut off 0,200 0,200 Reverse transcription and amplification reactions were performed as described in 4).
6) MODIFICATION OF dNTP CONCENTRATION
Experimental tests were performed in order to evaluate the nucleotide concentration during the single step amplification. Results obtained by DEIA assays (as in Table 6) show that a dNTP concentration lower than the single step optimized concentration (83 ~M instead of 200 ~M, as referred to dNTP added to the amplification step) lowers the reaction yield.
15 -~
CA 02257l9l l998-ll-30 W O 97/46716 PCT~T97/00128 Table 6 SAMPLE 83 ~M dNTP ¦ 200 ~M dNTP
O.D. 450/630 nm N~ 2364 0,351 1,140 N~ 2368 0,240 0,900 N~ 2369 0,610 1,012 N~ 2370 0,768 1,064 N~ 2383 0,131 0,580 N~ 2397 0,160 0,549 N~ 2400 0,114 0,942 N~ 2411 0,105 1,354 N~ 2412 0,126 1,465 N~ 2413 0,309 1,150 N~ 2416 0,239 1,387 cut off 0,270 0,270 Reverse transcription and amplification reactions were performed as described in 4).
The invention concerns a method to detect hepatitis C virus specific (HCV) nucleic acids.
In particular the invention refers to an improved method to detect HCV amplified DNA, by means of a single step polymerase chain reaction (PCR), under controlled and optimized reaction parameters, and of a revealing system of amplified products.
One of the most used methods to detect HCV specific nucleic acids is based upon the reverse transcription of viral RNA to cDNA, followed by a double amplification step (nested PCR) of the most conserved genome region (5'UTR). The amplified product of the second amplification step may be identified by means of revealing techniques as electrophoresis or enzyme mediated signals. The double amplification allows to reach a very high sensitivity able to identify even few viral RNA molecules. On the other hand the double PCR
step has many disadvantages mainly due to DNA
contamination from previous amplifications, length of time, high costs.
In order to overcome said disadvantages there is the need to set up a single step amplification protocol, which reaches similar levels of sensitivity than the nested PCR.
The authors of the instant invention have optimized the nested PCR reaction conditions in order to eliminate the second step. Moreover the system used to reveal amplified products is the DNA Enzyme Immunoassay (DEIA), which mekes the use of a specific capturing probe and of a monoclonal antibody able to recognize double strand DNA
(Mantero G. et al. Clin Chem. 37, 422-429, here incorporated by references).
.. ~ ..... . . .. .
CA 022~7191 1998-11-30 WO97/46716 PCT~T97/00128 The combination of all of optimized parameters, both of the amplification and of the revealing step, allowed to set up a method able to detect HCV specific DNA with a sensitivity equal, if not higher than the nested PCR, but with no disadvantages.
In order to optimize the PCR reaction parameters a comparison of first (not revealed by DEIA assay) and second (revealed by DEIA assay) amplification step, and an analysis to individuate the capturing probe with best features for a revealing step by DEIA assay as well, were performed.
C. Payan et al. J. Virol. Meth. (1995) 53, 167-175 describe a process to reveal HCV nucleic acids characterised by a single step for both the reverse transcription and the amplification. The amplification is a single step, no nested-PCR. Primer sequences are deduced from the HCV genome nucleotide sequence, and in particular from the high conserved 5'end region. The magnesium chloride final concentration in the PCR
solution is of 1 mM, the ratio [Mg~~]/U Taq being 35 nmol/U Taq.
C. Payan et al. Res. Virol. (1995) 146, 363-70 optimize the above referred process by modifying the MgCl2 concentration to 2 mM, the units of Mu-MLV RNAse and of Taq polymerase, to 10 U and 1 U respectively, with a ratio of 42 nmol [Mg++]/U Taq, and thus diminishing the RNA copy number to be revealed from 15 to 10.
It is therefore evident that even minima~
variations of general reaction conditions may interfere and increase significantly the yield and sensitivity.
It is an object of the instant invention a method to detect hepatitis C virus (HCV) specific nucleic acids comprising the steps of:
CA 022~7191 1998-ll-30 W O97/46716 PCT~T97/00128 a) reverse transcribing the viral RNA with a primer having a sequence substantially homologous to one of the sequences SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3;
b) amplifying by means of a single step polymerase chain reaction (PCR) wherein the primer has a sequence su~stantially homologous to sequences of SEQ ID No. 4 or SEQ ID No. 5, wherein the ratio between the Mg'+ ion concentration and of Taq polymerase in the reaction mix is of approximately of 100 nmoles/enzyme unit;
c) revealing the amplified product by means of DEIA
method using a probe having the sequence of SEQ ID No. 6, or a probe having a sequence substantially homologous or complementary thereof.
It is a further object of the invention the use of the of an oligonucleotide of SEQ ID No. 6 or o an oligonucleotide having a sequence substantially homologous or complementary thereof as probe for revealing HCV specific nucleic acids by means of the DEIA
method.
It is a further ob3ect of the invention a composition to reveal HCV specific nucleic acids by means of the DEIA method comprising an oligonucleotide of SEQ
I3 No. 6 or of sequence substantially ho~ologous or complementary thereof.
By comparing the two steps of the nested PCR, as in Table 2, it is clear that main differences are: 1) primer sequences; 2) MgCl2 concentration (which is a Taq polymerase activator); 3) buffer concentration (TRIS-~C1 pH 8.3); 4) solution ionic strength (~ainly related to the KC1 concentration)i 5) deoxinucleotidetriphosphate concentration (dNTP). Primer sequences are shown in Table la.
W O 97/46716 PCT~T97/00128 Table la Primer sequences name sequence SEQ ~D No.
1 CH 5' GGT GCA CGG TCT ACG AGA CCT 3' SEQ ID No. 1 2 CH 5' AAC TAC TGT CTT CAC GCA GAA 3' SEQ ID No. 4 1 TS 5' GCG ACC CAA CAC TAC TCG GCT 3' SEQ ID No. 2 4 CH 5' A~G GCG TTA GTA TGA GTG 3' SEQ ID No. 5 PT2 5' CGG TGT ACT CAC CGG TTC 3' SEQ ID No. 3 PKY78 5' CTC GCA AGC ACC CTA TCA GGC AGT 3 ' SEQ ID No. 7 PKY80 5' GGA GAA AGC GTC TAG CCA TGG CGT 3' SEQ ID No. 8 Probe sequences for the revealing step are shown in Table lb.
Table lb 3 CH 5' CGG TGA GTA CAC CGG AAT TGC CAG GAC CGG SEQ ID No. 9 GTC CTT TCT 3' WT 5' GCC ATA GTG GTC TGC GG 3' SEQ ID No. lO
PT 21 5' GGG AGA GCC ATA GTG GTC TGC 3' SEQ ID No. 6 KY 150 5' CAT AGT GGT CTG CGG AAC CGG TGA GT 3' SEQ ID No. ll HCV40 5' CCA TAG TGG TC~ GCG GAA CCG TGA GTA CA 3' SEQ ID No. 12 CH5 5' TAG TGG TCT GCG GAA CCG GT 3' SEQ ID No. 13 Table 2 Two step nested PCR parameters param. I step II step primerlCH~50pmol)/2CH(50pmol) lTS (~Opmol) /4CH (50pmol) [Tris-HCl] 22.5 mM 10 mM
[KCl] 62.5 mM 50 mM
[MgCl2] 4 mM 1. 5 mM
[dNTP] 260 uM ** 200 ~M
Taq pol . 2.5 u 2. 5 U
** The concentration accounts also for added dNTPs during the reverse transcription step.
CA 022~7191 1998-ll-30 W O 97/46716 PCT~T97/00128 1) SAMPLE PREPARATION
Sera to be tested by nested or single step PCR were treated to isolate RNA. Commercially available products, as RNAzol B e ULTRASPEC (Biotecx), were used following instructions of producer.
2) NESTED PROTOCOL
The nested PCR to detect HCV RNA at the 5'UTR
region was used as control. 5 ~ul of extracted RNA were reverse transcribed in 25 ~ul volume containing 22.5 mM
TRIS-HCl pH a .3, 62.5 mM KCl, 4 mM MgCl , 250 ~M dNTPs, 4U AMV-RT, 2 ~M lCH antisense primer, 25 U RNAse inhibitors (HRPI). The reverse transcription was performed at 42~C for 1 hr and the enzyme was further denatured at 100~C for 10 min. The nested PCR first step was performed in a 100 ul volume containing cDNA ~25 ~l from the reverse transcription), 22.5 mM TRIS-HCl pH 8.3, 62.5 mM KCl, 4 mM MgCl2, 200 ~M dNTPs (only for this step), 0.5 ~M 2CH sense primer, 2.5 U Taq polymerase;
thermal cycle: 94~C 1 min., 50~C 1 min., 72~C 2 min., 35 cycles. For the second step 3 ul from the first step were amplified in a 100 ul volume containing 10 mM TRIS-HCl pH
8.3, 50 mM KCl, 1.5 mM MgCl2, 200 ~M dNTPs, 0.5 ~M lTS
internal antisense primer, 0.5 ~M 4CH internal sense primer; 2.5 U Taq polymerase; thermal cycle: 94~C 1 min., 50~C 1 min., 72~C 2 min.; 25 cycles.
20 ul of each amplified product were tested by the DEIA assay using as probe the 3CH oligonucleotide.
Experimental tests are hereinbelow reported, according to different parameters of the first and second PCR steps. In all of reactions the amount of AMV-RT
enzyme was of 15 U/sample.
3) CHANGE of PRIMER SEQUENCES
Different combinations of primers were assayed in the single step RT-PCR.
CA 022~7191 1998-ll-30 W O 97/46716 PCTnT97/00128 Nested external primers: lCH (antisense) / 2CH (sense) Nested internal primers: lTS (antisense) / 4CH (sense) Nested external/internal primers: lCH (antisense) / 4CH
(sense) Nested internal/external primers: lTS (antisense) / 2CH
(sense) The antisense primer PT2 and those described in the EP 529493 application, identified as PKY78 antisense and PKY80 sense, were also assayed.
Reaction conditions were those for all of primer combinations and are as follows:
- reverse transcription was performed as for the nested protocol, but of AMV-RT units;
- PCR was performed in 100 ul final volume containing cDNA, 22.5 rnM T~IS-HCl pH 8.3, 62.5 mM KCl, 4 mM MgCl2, 200 uM dNTP (only those added in this step), O.5 ~M sense primer (according to different combinations), 2.5 U Taq polymerase; thermal cycle: 94~C
1 min., 50~C 1 min., 72~C 2 min.; 45 cycles;
- 20 ul o~ each amplified product were assayed by means of the DEIA Enzyme Immunoassay with the 3CH probe.
By only varying primer combinations lCH, 2CH, 4CH, lTS, PT2 no detectable amplified product was made in a single step PCR amplification. Even when "alternative"
sequences were use (primers as in EP 529493) no clear positive results were obtained ~positive result only in one sample), see Table 3 (positive values in bold character).
. .
, CA 02257191 1998-ll-30 W O 97/46716 PCTnT97/00128 Table 3 ne~ted~lngle 3tep PCR
PCRprlmer combination lCH/2C~ lTS/4CH lCH/4CH lTS/2CH PKY78/PKY80 ~ample O.D. 450/630 nm O RNA 0,061 0,068 0,076 0,176 0,110 0,084 N. 1 Neg0,084 0,062 0,067 0,103 0,112 0,089 N. 2 Po~2,185 0,123 0,078 0,109 0,103 l,lS9 N. 3 Pos1,979 0,066 0,081 0,109 0,109 0,092 N. 4 Po~2,566 0,054 0,084 0,105 0,113 0,069 cut-off 0,192 0,192 0,192 0,231 0,231 0,220 4) MODIFICATIONS OF MgCl~CONCENTRATION
It has been found that the MgCl- concentration (a Taq polymerase activator) has to be finely modulated to obtain the best yield of the amplification reaction.
During the amplification, MgCl concentrations of 1.5 mM, 2.5 mM, 4 mM, corresponding, respectively, to 60nmol, 100nmol, 160nmol of Mg+ per Taq unit were used. The reverse transcription reaction was performed as in the nested PCR protocol, but of enzyme units. The PCR
reaction was performed in a 100 ~l volume containing cDNA (25 ~l from the reverse transcription mix), 22.5 mM
TRIS-HCl pH 8.3, 62.5 mM KCl, MgCl at different concentrations, 200 ~M dNTPs (only dNTPs added in this step), 0,5 ~M 2CH primer and 2,5 U Taq polymerase.
Data obtained by means of the DEIA immunoenzyme assay (with the 3C~ probe), as in Table 4, show that:
a) very low MgCl2 concentrations (1.5 mM, 60 nmol/U~ reduce the yield of the single step PCR reaction;
in fact, only S0% of nested positive samples are still positive;
-b) very high MgCl~ concentrations (4 mM, 160 nmol/U) are not workable since no nested positive sample was confirmed as such in the single step reaction;
.. . . ~ . , .
W O 97/46716 PCTnT97/00128 c) a MgCl concentratlon of 2.5 mM ~100 nmol/U) is the best, confirming all of data obtained by the nested PCR.
Table 4 nested single step PCR
PCR [MgCl2]
sample 1,5 mM2,5 mM 4,0 mM
O.D. 450/630 nm N 1 2,085 2,0132,017 0,065 N 2 1,622 0,1450,913 0,092 N 3 2,107 1,7971,912 0,071 N 4 2,163 0,1150,261 0,080 N 5 2,194 0,2741,092 0,078 N 6 0,899 0,1170,560 0,071 N 7 0,107 0,1220,158 0,066 N 8 0,055 0,1150,131 0,077 N 9 0,054 0,1150,140 0,077 N10 0,051 0,1020,157 0, 063 Nll 0,084 0,1550,134 0,062 cut off 0,253 5) MODIFICATIONS OF REACTION BUFFER CONCENTRATION
As opposite to MgCl2, differences in the concentration of TRIS-HCl (12.5 mM instead of 22.5 mM) and KCl (50 mM instead of 62.5 mM) do not interfere in a significant way with the efficacy of the single step amplification, as DEIA obtained data are comparable to controls, as in Table 5.
CA 02257191 1998-ll-30 6 PCT~T97/00128 Table 5 Tris-HCl 12,5 mM Tris-HCl 22,5 mM
SAMPLEKCl 12,5 mM KCl 62,5 mM
O.D. 450/630 nm N~2368 0,900 0,912 N~2369 1,012 1,038 N~2370 1,064 1,311 N~2381 0,685 1,604 N~2397 0,549 0,508 N~2404 0,028 0,054 N~2411 1,354 1,145 N~2412 1,46~ 1,780 N~2416 1,387 1,797 N~2452 0,845 1,514 N~2464 0,872 1,175 N~2487 1,032 2,274 cut off 0,200 0,200 Reverse transcription and amplification reactions were performed as described in 4).
6) MODIFICATION OF dNTP CONCENTRATION
Experimental tests were performed in order to evaluate the nucleotide concentration during the single step amplification. Results obtained by DEIA assays (as in Table 6) show that a dNTP concentration lower than the single step optimized concentration (83 ~M instead of 200 ~M, as referred to dNTP added to the amplification step) lowers the reaction yield.
15 -~
CA 02257l9l l998-ll-30 W O 97/46716 PCT~T97/00128 Table 6 SAMPLE 83 ~M dNTP ¦ 200 ~M dNTP
O.D. 450/630 nm N~ 2364 0,351 1,140 N~ 2368 0,240 0,900 N~ 2369 0,610 1,012 N~ 2370 0,768 1,064 N~ 2383 0,131 0,580 N~ 2397 0,160 0,549 N~ 2400 0,114 0,942 N~ 2411 0,105 1,354 N~ 2412 0,126 1,465 N~ 2413 0,309 1,150 N~ 2416 0,239 1,387 cut off 0,270 0,270 Reverse transcription and amplification reactions were performed as described in 4).
7) SINGLE STEP PCR
According to the above described tests the best reaction conditlons to amplify HCV RNA are as follows:
A) REVERSE TRANSCRIPTION
5 ~l of RNA were reverse transcribed in a 25 ~1 volume containing 50 mM TRIS-HCl pH 8.3, 50 mM KCl, 10 mM
MgCl2, 250 ~l dNTP, 2 ~M lCH antisense primer, 25 U HRPI
and 15 U AMV-RT. The reverse transcription reaction was performed at 42~C for 1 hr, followed by an enzyme denaturation step at 100~C for 10 min.
B) AMPLIFICATION
The amplification reaction was performed in a 100 ~l volume containing the cDNA from the reverse transcription step (25 ~l), 22.5 mM TRIS-HCl pH 8.3, 62.5 mM KCl, 2.5 mM MgCl2, 0.5 ~M 2C~ sense primer, 200 ~M
CA 022~7l9l l998-ll-30 W O 97/46716 PCT~T97/00128 dNTP (referring only to dNTP added during said step, and 2.5 U Taq polymerase.
C) THERMAL CYCLE
99~C 1 min - 50~C 1 min - 72~C 2 min 45 cycles 8) REVEALING PROBE
In all of above experiments the 3CH revealing probe was used (Sorin Biomedica Diagnostics SpA PS000C
Hepatitis C). Different probes were tested, having sequences deduced from a different region of the 5' UTR
terminus. Various parameters were evaluated, as the oligonucleotide length that may allow an efficient hybridisation to the complementary sample, and a sequence excluding the formation of thermodynamically stable loops or dimers, due to the presence of internal homologous stretches, as well. In order to select the optimal sequence an analysis of thermodynamic features of some oligonucleotides, as derived from the internal region of the amplified sequence ~HCV genome 5' UTR region) was performed.
Table 7 probe ~G S~G ~G ~G
hybrid loops dimers (loops + dimers) PROBE ~cal/mol WT - 29,6 1,3 0,0 1,3 PT 21 - 35,5 1,1 0,0 l,1 KY150 - 45,7 - 0,6 - 7,4 - 8,0 HCV40 - 52,9 - 0,3 - 7,9 - 7,7 CH 5 - 35,9 - 0,3 - 7,9 - 7,7 3 CH - 83,1 - 8,1 no dimers - 8,1 Some of the selected sequences were deduced from prior art literature, others designed to satisfy the above requisltes. The thermodynamic analysis was performed with the OLIGO.EXE structure ~ (ver 3.3) program distributed by MedProbe A.S. (Norvegy), by WO97/46716 12 PCT~T97/00128 maintaining as constant two parameters: the probe (30 nM) and the salt (188 mM) concentration. These parameters are those experimentally used during the DEIA assay.
The thermodynamic analysis of sequences, as in Table 7, shows that hybridisation reactions of all of oligos with complementary sequences are thermodynamically favoured. As expected the lowest AG values are those of the longest probes (3CH, HC~40, KY150), which on the other hand favour the formation of either loops or dimers. On the other side, among the shortest probes, PT21 and WT are those that, according to their sequence typology, show the best features, whereas CH5, though having analogous dimensions to the other two probes, may give rise to undesired thermodynamically stable dimers.
The PT21 probe is able to form a more stable specific hybrid than the WT probe. The two PT21 and 3CH probes were then compared. The reaction conditions were standard conditions of the DEIA kit (Sorin Biomedica Diagnostics SpA, GEN-ETI-K-DEIA cod. PS0001) to reveal amplified HCV
and foresee a specific hybridisation at 50~C.
Experimental results, reported in Table 8, confirm that the PT21 probe allows a more efficient hybridisation with the amplified complementary strand. Approximately 96% of analyzed positive samples show absorbance values higher than 1,0 O.D., whereas with the 3CH probe only 40% of samples overcomes this threshold.
~, , .
I
CA 022~7l9l l998-ll-30 W O 97/46716 13 PCT~T97/00128 Table 8 probe sample 3CH ¦ PT21 O.D. 450/630 nm N 1 0,633 2,259 N 2 0,215 1,430 N 3 0,579 2,105 N 4 0,610 2,361 N 5 0,230 0,560 N 6 0,a33 2,051 N 7 1,734 2,926 N 8 1,872 2,852 N 9 0,574 2,546 N 10 1,147 2,829 N 11 1,562 2,788 N 12 1,206 2,897 N 13 2,002 2,876 N 14 0,499 2,253 N 16 1,600 2,715 N 17 1,522 2,656 N 18 2,281 2,682 N 19 0,690 2,238 N 20 0,275 2,274 cut off 0,190 O, 198 Finally, experimental analysis of PT21 and 3CH
probes were performed by evaluating their specificity in the DEIA conditions as above. The analytical specificity was determined with probe unrelated DNA samples, which are representative of molecular dimensions and of -~ sequence heterogeneity: different concentrations of salmon sperm DNA; different concentrations of calf thymus DNA; unrelated amplified DNA. Results, as reported in Table 9, did not show significant aspecific reactions, W O 97/46716 PCT~T97/00128 being absorbance ~alues lower than cut-off (cut-off =
average absorbance of negative samples + 0.150 O.D., as suggested in the DEIA assay).
Table 9 SPECI~ICITY PT21 - 3CH
GEN.ETI.K HCV REAGENT LOT
GEN.ETI.K DEIA #7400110 O.D. 450/630 nm PCR HIV 0,043 o 050 0,051 0,059 PCR HDV 0,057 0,105 0,078 0,11 TV 1 ~g/~l 0,079 0,058 0,058 0,058 TV 0,8 ~g/~l 0,069 0,085 0,068 0,066 TV 0,4 ~g/~l 0,061 0,053 0,062 0,056 TV 0,2 ~g/~l 0,058 0,051 0,080 0,051 TV 0,1 ~g/~l 0,061 0,053 0,059 0,048 TV 0,05 ~g/~l 0,065 0,048 0,055 0,051 SS 1 ~g/~l 0,071 0,059 , 0,069 0,058 SS 0,8 ~g/~l 0,065 0,055 0,062 0,053 SS 0,4 ~g/~l 0,054 0,053 0,055 0,052 SS 0,2 ~g/~l 0,058 0,046 - - 0,053 0,049 SS 0,1 ~g/~l 0,061 0,047 0,052 0,048 CA 02257191 1998-ll-30 W O 97/46716 15 PCT~T97/00128 SS 0,05 ~g/~l 0,061 0,049 0,045 0,059 average neg. ctrl. 0,049 0,047 average pos. ctrl. 2,103 2,331 cut off 0,199 0,197 TV: new-born calf time DNA; SS: salmon sperm DNA.
The setting of the single step DNA HCV
amplification and revealing protocol was performed by optimizing of amplification reaction conditions (in particular by changing the MgC12 concentration), and by using a new revealing probe tPT21). Said approach was able to give results comparable to the NESTED plus DEIA
method.
. , .
.. . . ... ....
W O 97146716 PCT~T97/00128 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: SORIN BIOMEDICA DIAGNOSTICS S.p.A.
(B) STREET: Via Borgonuo~o 14 (C) CITY: Milan (E) COUNTRY: Italy (F) POSTAL CODE (ZIP): 20121 (ii) TITLE OF INVENTION: Method to detect HCV specific nucleic acids (iii) NUMBER OF SEQUENCES: 13 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (2) INFO~MATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
(2) INFORMATION FOR SEQ ID NO: 2:
, . . . I _ CA 02257191 1998-ll-30 W O 97/46716 PCT~T97/00128 ~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) ~ENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
(2) INFORMATION FOR SEQ ID NO: 5:
. .
W O 97/46716 PCT~T97/00128 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
(2) INFORMATION EOR SEQ ID NO: 8:
_.
W O 97/46716 PCT~T97/00128 ~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
GCCAT~GTGG TCTGCGG 17 (2) INFORMATION FOR SEQ ID NO: 11:
.. .... . . ..
CA 02257l9l l998-ll-30 W O 97/46716 PCTnT97/00128 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
lS ~A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
(2) INFOR~ATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
~~ TAGTGGTCTG CGGAACCGGT 20
According to the above described tests the best reaction conditlons to amplify HCV RNA are as follows:
A) REVERSE TRANSCRIPTION
5 ~l of RNA were reverse transcribed in a 25 ~1 volume containing 50 mM TRIS-HCl pH 8.3, 50 mM KCl, 10 mM
MgCl2, 250 ~l dNTP, 2 ~M lCH antisense primer, 25 U HRPI
and 15 U AMV-RT. The reverse transcription reaction was performed at 42~C for 1 hr, followed by an enzyme denaturation step at 100~C for 10 min.
B) AMPLIFICATION
The amplification reaction was performed in a 100 ~l volume containing the cDNA from the reverse transcription step (25 ~l), 22.5 mM TRIS-HCl pH 8.3, 62.5 mM KCl, 2.5 mM MgCl2, 0.5 ~M 2C~ sense primer, 200 ~M
CA 022~7l9l l998-ll-30 W O 97/46716 PCT~T97/00128 dNTP (referring only to dNTP added during said step, and 2.5 U Taq polymerase.
C) THERMAL CYCLE
99~C 1 min - 50~C 1 min - 72~C 2 min 45 cycles 8) REVEALING PROBE
In all of above experiments the 3CH revealing probe was used (Sorin Biomedica Diagnostics SpA PS000C
Hepatitis C). Different probes were tested, having sequences deduced from a different region of the 5' UTR
terminus. Various parameters were evaluated, as the oligonucleotide length that may allow an efficient hybridisation to the complementary sample, and a sequence excluding the formation of thermodynamically stable loops or dimers, due to the presence of internal homologous stretches, as well. In order to select the optimal sequence an analysis of thermodynamic features of some oligonucleotides, as derived from the internal region of the amplified sequence ~HCV genome 5' UTR region) was performed.
Table 7 probe ~G S~G ~G ~G
hybrid loops dimers (loops + dimers) PROBE ~cal/mol WT - 29,6 1,3 0,0 1,3 PT 21 - 35,5 1,1 0,0 l,1 KY150 - 45,7 - 0,6 - 7,4 - 8,0 HCV40 - 52,9 - 0,3 - 7,9 - 7,7 CH 5 - 35,9 - 0,3 - 7,9 - 7,7 3 CH - 83,1 - 8,1 no dimers - 8,1 Some of the selected sequences were deduced from prior art literature, others designed to satisfy the above requisltes. The thermodynamic analysis was performed with the OLIGO.EXE structure ~ (ver 3.3) program distributed by MedProbe A.S. (Norvegy), by WO97/46716 12 PCT~T97/00128 maintaining as constant two parameters: the probe (30 nM) and the salt (188 mM) concentration. These parameters are those experimentally used during the DEIA assay.
The thermodynamic analysis of sequences, as in Table 7, shows that hybridisation reactions of all of oligos with complementary sequences are thermodynamically favoured. As expected the lowest AG values are those of the longest probes (3CH, HC~40, KY150), which on the other hand favour the formation of either loops or dimers. On the other side, among the shortest probes, PT21 and WT are those that, according to their sequence typology, show the best features, whereas CH5, though having analogous dimensions to the other two probes, may give rise to undesired thermodynamically stable dimers.
The PT21 probe is able to form a more stable specific hybrid than the WT probe. The two PT21 and 3CH probes were then compared. The reaction conditions were standard conditions of the DEIA kit (Sorin Biomedica Diagnostics SpA, GEN-ETI-K-DEIA cod. PS0001) to reveal amplified HCV
and foresee a specific hybridisation at 50~C.
Experimental results, reported in Table 8, confirm that the PT21 probe allows a more efficient hybridisation with the amplified complementary strand. Approximately 96% of analyzed positive samples show absorbance values higher than 1,0 O.D., whereas with the 3CH probe only 40% of samples overcomes this threshold.
~, , .
I
CA 022~7l9l l998-ll-30 W O 97/46716 13 PCT~T97/00128 Table 8 probe sample 3CH ¦ PT21 O.D. 450/630 nm N 1 0,633 2,259 N 2 0,215 1,430 N 3 0,579 2,105 N 4 0,610 2,361 N 5 0,230 0,560 N 6 0,a33 2,051 N 7 1,734 2,926 N 8 1,872 2,852 N 9 0,574 2,546 N 10 1,147 2,829 N 11 1,562 2,788 N 12 1,206 2,897 N 13 2,002 2,876 N 14 0,499 2,253 N 16 1,600 2,715 N 17 1,522 2,656 N 18 2,281 2,682 N 19 0,690 2,238 N 20 0,275 2,274 cut off 0,190 O, 198 Finally, experimental analysis of PT21 and 3CH
probes were performed by evaluating their specificity in the DEIA conditions as above. The analytical specificity was determined with probe unrelated DNA samples, which are representative of molecular dimensions and of -~ sequence heterogeneity: different concentrations of salmon sperm DNA; different concentrations of calf thymus DNA; unrelated amplified DNA. Results, as reported in Table 9, did not show significant aspecific reactions, W O 97/46716 PCT~T97/00128 being absorbance ~alues lower than cut-off (cut-off =
average absorbance of negative samples + 0.150 O.D., as suggested in the DEIA assay).
Table 9 SPECI~ICITY PT21 - 3CH
GEN.ETI.K HCV REAGENT LOT
GEN.ETI.K DEIA #7400110 O.D. 450/630 nm PCR HIV 0,043 o 050 0,051 0,059 PCR HDV 0,057 0,105 0,078 0,11 TV 1 ~g/~l 0,079 0,058 0,058 0,058 TV 0,8 ~g/~l 0,069 0,085 0,068 0,066 TV 0,4 ~g/~l 0,061 0,053 0,062 0,056 TV 0,2 ~g/~l 0,058 0,051 0,080 0,051 TV 0,1 ~g/~l 0,061 0,053 0,059 0,048 TV 0,05 ~g/~l 0,065 0,048 0,055 0,051 SS 1 ~g/~l 0,071 0,059 , 0,069 0,058 SS 0,8 ~g/~l 0,065 0,055 0,062 0,053 SS 0,4 ~g/~l 0,054 0,053 0,055 0,052 SS 0,2 ~g/~l 0,058 0,046 - - 0,053 0,049 SS 0,1 ~g/~l 0,061 0,047 0,052 0,048 CA 02257191 1998-ll-30 W O 97/46716 15 PCT~T97/00128 SS 0,05 ~g/~l 0,061 0,049 0,045 0,059 average neg. ctrl. 0,049 0,047 average pos. ctrl. 2,103 2,331 cut off 0,199 0,197 TV: new-born calf time DNA; SS: salmon sperm DNA.
The setting of the single step DNA HCV
amplification and revealing protocol was performed by optimizing of amplification reaction conditions (in particular by changing the MgC12 concentration), and by using a new revealing probe tPT21). Said approach was able to give results comparable to the NESTED plus DEIA
method.
. , .
.. . . ... ....
W O 97146716 PCT~T97/00128 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: SORIN BIOMEDICA DIAGNOSTICS S.p.A.
(B) STREET: Via Borgonuo~o 14 (C) CITY: Milan (E) COUNTRY: Italy (F) POSTAL CODE (ZIP): 20121 (ii) TITLE OF INVENTION: Method to detect HCV specific nucleic acids (iii) NUMBER OF SEQUENCES: 13 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (2) INFO~MATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
(2) INFORMATION FOR SEQ ID NO: 2:
, . . . I _ CA 02257191 1998-ll-30 W O 97/46716 PCT~T97/00128 ~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) ~ENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
(2) INFORMATION FOR SEQ ID NO: 5:
. .
W O 97/46716 PCT~T97/00128 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
(2) INFORMATION EOR SEQ ID NO: 8:
_.
W O 97/46716 PCT~T97/00128 ~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
GCCAT~GTGG TCTGCGG 17 (2) INFORMATION FOR SEQ ID NO: 11:
.. .... . . ..
CA 02257l9l l998-ll-30 W O 97/46716 PCTnT97/00128 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
lS ~A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
(2) INFOR~ATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
~~ TAGTGGTCTG CGGAACCGGT 20
Claims (3)
1. Process to detect hepatitis C virus (HCV) specific nucleic acids comprising the steps of:
a) reverse transcribing the viral RNA by means of a primer having a nucleotide sequence substantially homologous to SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3;
b) amplifying by a single polymerase chain reaction (PCR) wherein the primer has a nucleotide sequence substantially homologous to SEQ ID No. 4 SEQ ID No. 5;
wherein the Mg++ / Taq polymerase ratio in the reaction mix is of approx. 100 nmoles/enzyme unit;
c) revealing the amplified product by means of the DEIA method using as probe an oligonucleotide of SEQ ID
No. 6, or having a sequence substantially homologous or complementary thereof.
a) reverse transcribing the viral RNA by means of a primer having a nucleotide sequence substantially homologous to SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3;
b) amplifying by a single polymerase chain reaction (PCR) wherein the primer has a nucleotide sequence substantially homologous to SEQ ID No. 4 SEQ ID No. 5;
wherein the Mg++ / Taq polymerase ratio in the reaction mix is of approx. 100 nmoles/enzyme unit;
c) revealing the amplified product by means of the DEIA method using as probe an oligonucleotide of SEQ ID
No. 6, or having a sequence substantially homologous or complementary thereof.
2. Use of the oligonucleotide of SEQ ID No. 6 or having a sequence substantially homologous or complementary thereof as probe to reveal HCV specific nucleotides by means of the DEIA method.
3. Composition to reveal HCV specific nucleotides with the DEIA assay comprising the oligonucleotide of SEQ
ID No. 6 or an oligonucleotide of sequence substantially homologous or complementary thereof.
ID No. 6 or an oligonucleotide of sequence substantially homologous or complementary thereof.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ITRM96A000404 | 1996-06-07 | ||
| IT96RM000404A IT1284847B1 (en) | 1996-06-07 | 1996-06-07 | PROCEDURE FOR THE DETECTION OF HCV SPECIFIC NUCLEIC ACIDS |
| PCT/IT1997/000128 WO1997046716A1 (en) | 1996-06-07 | 1997-06-03 | Method to detect hcv specific nucleic acids |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2257191A1 true CA2257191A1 (en) | 1997-12-11 |
Family
ID=11404271
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002257191A Abandoned CA2257191A1 (en) | 1996-06-07 | 1997-06-03 | Method to detect hcv specific nucleic acids |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0937161A1 (en) |
| AU (1) | AU3047797A (en) |
| CA (1) | CA2257191A1 (en) |
| IT (1) | IT1284847B1 (en) |
| WO (1) | WO1997046716A1 (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19832050C2 (en) * | 1998-07-16 | 2002-10-10 | Biotest Pharma Gmbh | Procedure for the detection of hepatitis B or hepatitis C virus genomes in plasma samples and specific primers |
| US6638714B1 (en) | 1999-02-03 | 2003-10-28 | Ortho-Clinical Diagnostics, Inc. | Oligonucleotide primers for efficient detection of hepatitis C virus (HCV) and methods of use thereof |
| US6623919B1 (en) * | 1999-02-03 | 2003-09-23 | Ortho-Clinical Diagnostics, Inc | Oligonucleotide primers for efficient multiplex detection of hepatitis C virus (HCV) and human immunodeficiency virus (HIV) and methods of use thereof |
| AU2006203092B2 (en) * | 1999-02-03 | 2009-11-12 | Ortho-Clinical Diagnostics, Inc. | Oligonucleotide primers for efficient detection of hepatitis C virus (HCV) and methods of use thereof |
| KR100451049B1 (en) * | 2001-04-12 | 2004-10-02 | 바이오코아 주식회사 | Oligonucleotide chip composition for analyzing Hepatitis C virus (HCV) genotype and detecting method thereof |
| KR100658606B1 (en) | 2005-04-22 | 2006-12-15 | (주)팬바이오넷 | Method for determination of hepatitis c virus genotype |
| EP2722397B1 (en) * | 2012-10-18 | 2017-12-13 | F. Hoffmann-La Roche AG | Dual probe assay for the detection of heterogeneous amplicon populations |
| US11274998B2 (en) | 2012-12-26 | 2022-03-15 | Ventana Medical Systems, Inc. | Specimen processing systems and methods for holding slides |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100193801B1 (en) * | 1991-05-08 | 1999-06-15 | 케네스 엠. 골드만 | Hepatitis C virus (HCV) genome sequence for diagnosis and treatment |
| CA2070952A1 (en) * | 1991-06-11 | 1992-12-12 | Makoto Seki | Gene of hepatitis c virus or fragment thereof, polypeptide encoded by the same |
| DK0529493T3 (en) * | 1991-08-27 | 1998-08-24 | Hoffmann La Roche | Methods and Reagents for Hepatitis C Detection |
| JPH06311885A (en) * | 1992-08-25 | 1994-11-08 | Mitsubishi Kasei Corp | Anti-sense compound complementary with gene of hepatitis c virus |
| AU7718594A (en) * | 1993-09-03 | 1995-03-22 | United States Of America As Represented By The Secretary Department Of Health And Human Services | Method and compositions for primer specific solid-phase detection of pcr products |
| JPH07250700A (en) * | 1994-03-15 | 1995-10-03 | Tonen Corp | Simple quantification of nucleic acid by competitive pcr process |
-
1996
- 1996-06-07 IT IT96RM000404A patent/IT1284847B1/en active IP Right Grant
-
1997
- 1997-06-03 EP EP97925277A patent/EP0937161A1/en not_active Withdrawn
- 1997-06-03 AU AU30477/97A patent/AU3047797A/en not_active Abandoned
- 1997-06-03 WO PCT/IT1997/000128 patent/WO1997046716A1/en not_active Application Discontinuation
- 1997-06-03 CA CA002257191A patent/CA2257191A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| WO1997046716A1 (en) | 1997-12-11 |
| EP0937161A1 (en) | 1999-08-25 |
| ITRM960404A1 (en) | 1997-12-07 |
| ITRM960404A0 (en) | 1996-06-07 |
| IT1284847B1 (en) | 1998-05-22 |
| AU3047797A (en) | 1998-01-05 |
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