ITRM960404A1 - PROCEDURE FOR THE DETECTION OF HCV SPECIFIC NUCLEIC ACIDS - Google Patents

Description

DESCRIPTION

accompanying a patent application for industrial invention entitled:

"Procedure for the detection of specific HCV nucleic acids"

The present invention relates to a process for the detection of specific nucleic acids of the hepatitis C virus (HCV).

More specifically, the invention relates to an improved HCV-specific amplified DNA detection process, which uses the polymerase chain reaction (PCR) in a single step, with controlled and optimized reaction conditions, and a product detection system. amplified.

One of the most popular methods for detecting HCV-specific nucleic acids is based on the reverse transcription of the viral RNA into cDNA, followed by a double-step amplification (nested PCR) of the most conserved region (5'UTR). The amplified product of the second step can be identified by detection methods such as electrophoretic ones or those using enzymatic signals. The double amplification allows to reach a sensitivity such as to identify even a few copies of viral RNA. On the other hand, the double PCR step has considerable drawbacks due above all to problems of contamination of specific DNA generated in previous amplifications, operating times, costs.

To overcome these drawbacks, it is necessary to have a one-step amplification protocol capable of achieving the same sensitivity levels as nested PCR.

The authors of the present invention have, with this aim, optimized the PCR reaction conditions of the nested method, so as to eliminate the second step. The system used for the detection of the amplified products is the DNA Enzyme Immunoassay (DEIA), which involves the use of a specific capture probe and a monoclonal antibody capable of selectively recognizing double-stranded DNA (Maniero G. et al. Clin Chem. 37, 422-429).

The combination of all the optimized parameters, both for PCR amplification and for detection, has made it possible to develop a procedure capable of detecting HCV-specific DNA, with a sensitivity comparable if not greater than the double-pass method, but without inconveniences.

The approach followed for PCR optimization is based on the comparison of the reaction conditions used for the first nested step (not detectable in DEIA) and for the second step (detectable in DEIA), as well as on a study for the identification of a capture probe with optimal characteristics for the detection of the amplified by the DEIA method.

C. Payan et al. J. Virol. Meth. (1995) 53,167-75 describe a procedure for the detection of HCV nucleic acids characterized by the single-step joining of the reverse transcription and amplification reactions. The amplification process is also characterized by a single step but is not nested-PCR. The primers used for the detection were deduced from the nucleotide sequence of the HCV virus genome and precisely from the highly conserved 5'-UTR region among the different variants of the virus. The final concentration of magnesium chloride in the PCR solution is 1 mM, the ratio [Mg <++>] / U Taq is 35 nmol / U Taq.

C. Payan et al. Res. Virai. (1995) 146, 363-70 optimize the procedure described in the previous document, modifying the concentration of MgCl2 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 decreasing the number of RNA copies detectable by the assay from 15 to 10.

It is therefore evident that even minimal changes in the general reaction conditions can significantly improve the yield and sensitivity of the reaction.

Therefore, the subject of the present invention is a process for the detection of specific nucleic acids for the hepatitis C virus (HCV) comprising the steps of:

a) reverse transcription of viral RNA by means of primer having a sequence substantially homologous to that of one of the following oligonucleotides:

1CH: 5 'GGT GCA CGG TCT ACG AGA CCT 3',

1TS; 5 'GCG ACC CAA CAC TAC TCG GCT 3',

PT2: 5 'CGG TGT ACT CAC CGG TTC 3';

b) amplification with a single-step polymerase chain reaction (PCR) method in which the primer is of a sequence substantially homologous to that of one of the following oligonucleotides: 2CH: 5'AAC TAC TGT CTT CAC GCA GAA 3 ',

4CH: 5 'ATG GCG TTA GTA TGA GTG 3;

and the ratio between the concentration of Mg <++> ions and Taq polymerase in the reaction mixture is about 100 nmoles / unit of enzyme;

c) detection of the amplified product with the DEIA method by using the sequence probe PT21 oligonucleotide:

5 'GGG AGA GCC ATA GTG GTC TGC 3', or of an oligonucleotide of substantially homologous or complementary sequence to it.

A further object of the present invention is the use of the oligonucleotide PT21 or an oligonucleotide of substantially homologous or complementary sequence to it as a probe for the detection with the DEIA method of nucleic acids specific for HCV.

A further object of the present invention is a composition for the detection with the DEIA method of nucleic acids specific for HCV comprising the oligonucleotide PT21 or an oligonucleotide of substantially homologous or complementary sequence to it.

From the comparison between the two steps of the nested PCR, as in Table 2, it emerges that the main differences are: 1) sequences of the primers used; 2) concentration of MgCl2 (activator of

polymerase Taq); 3) buffer concentration (TRIS-HCl pH 8.3); 4)

ionic strength of the solution (especially as a function of the

concentration of KCl); 5) concentration of deoxynucleotid triphosphate

(dNTP). The sequences of the primers used are illustrated in

Table 1a below.

Table 1a

Sequence of primers used

The probes sequences used for the detection are

shown in Table 1b.

Table 1b

Table 2

Parameters of the two steps of nested PCR

The concentration also takes into account the dNTPs added in the reverse transcription step.

1) PREPARATION OF THE SAMPLE

The sera to be tested with nested PCR and single pass PCR methods were treated to extract any RNA present. For the extraction, currently marketed products such as RNAzol B and ULTRASPEC (Biotecx) were used, following the instructions of the house.

2) NESTED PROTOCOL

Nested PCR for the detection of HCV RNA in the 5'UTR region was used as a reference. 5 µl of extracted RNA was back-transcribed in a volume of 25 μl containing TRIS-HCl pH 8.3 final 22.5 mM, final KCl 62.5 mM, final MgCl2 4 mM, dNTP 250 μΜ, AMV-RT 4U, 2 μΜ antisense primer 1CH, 25 U of RNase inhibitor enzyme (HRPI). The reverse transcription was carried out at 42 ° C for 1 hour and then the enzyme was denatured at 100 ° C for 10 minutes. The first step delta nested PCR was performed in a volume of 100 µl containing cDNA (25 μl from the reverse transcription), TRIS-HCl pH 8.3, 22.5 mM, KCl 62.5 mM, MgCl2 4 mM, dNTP 200 μΜ (concentration referred only to dNTP present in this step), trigger sense 2CH 0.5 μΜ; 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 μl of the first step were re-amplified in a volume of 100 μl containing TRIS-HCl pH 8.3 10 mM, KCl 50 mM, MgCl2 1.5 mM, dNTP 200 μΜ, internal antisense trigger 1TS 0.5 μΜ, internal sense trigger 4CH 0.5 μΜ; 2.5 U Taq polymerase; thermal cycle: 94 ° C 1 min., 50 ° C 1 min., 72 ° C 2 min .; 25 cycles.

20 μl of each amplified product were tested by the DEIA assay using a 3CH sequence oligonucleotide as a probe.

The experimental tests carried out taking into account the parameters that differentiate the first and second pass are reported below. For all the operating conditions tested, the quantity of AMV-RT enzyme used in the reverse transcription was 15 U / sample.

3) SEQUENCE VARIATION of the primers used Different combinations of primers used in the nested PCR were tested in the single step RT-PCR reaction.

Nested external trigger: 1CH (antisense) / 2CH (sense) Nested internal trigger: 1TS (antisense) / 4CH (sense) External / internal trigger nested: 1CH (antisense) / 4CH (sense) Internal / external trigger nested: 1TS (antisense) ) / 2CH (sense) The antisense trigger PT2 was also tested. The triggers described in patent application EP 529493, identified as PKY78 antisense and PKY80 sense, were also tested.

For all the combinations of primers used the reaction conditions were the following:

- the reverse transcription took place as described in the nested protocol (with the exception of the AMV-RT units);

- PCR was carried out in a final volume of 100 µl containing cDNA, TRIS-HCl pH 8.3 final 22.5 mM, final KCl 62.5 mM, final MgCl2 4 mM, dNTP 200 μΜ (only dNTP added in this step), priming sense ( depending on the combinations) 0.5 μΜ. 2.5 U Taq polymerase; thermal cycle: 94 ° C 1 min., 50 ° C 1 min., 72 ° C 2 min .; 45 cycles;

- 20 μl of each amplified product were assayed by the DNA Enzyme Immunoassay using the 3CH sequence as probe.

The variation of the combinations of primers 1CH, 2CH, 4CH, 1TS, PT2 alone was not able to allow an amplification so efficient as to be able to detect the amplified product after a single PCR step. The same applies to the use of "alternative" sequences to those used in the nested protocol (primer EP 529493) which gave a positive result only in one of the samples (Table 3, positive values in bold).

Table 3

4) CHANGE IN THE CONCENTRATION of MgCl2

It has been found that the concentration of MgCl2 (activator of Taq polymerase) must be suitably modulated to obtain the maximum yield of the amplification reaction. Concentrations of MgCl2 equal to 1.5 mM, 2.5 mM, 4 mM were used in the amplification step, corresponding, respectively, to the following nmoles of Mg <++> per unit of Taq: 60nmol; 100nmol; 160nmol. The reverse transcription reaction was conducted as per the nested protocol (with the exception of the enzyme units). The PCR was carried out in a final volume of 100 μl containing the cDNA (25 μl coming from the reverse transcription), TRIS-HCl pH 8.3 final 22.5 mM, final KCl 62.5 mM, MgCl2 at different concentrations, dNTP 200 μΜ (only the added dNTPs in this step), priming 2CH 0.5 μΜ and 2.5 U Taq polymerase.

The data obtained through the enzyme immunoassay (DEIA with 3CH probe), shown in Table 4, indicate that:

a) too low MgCl2 concentrations (1.5 mM, 60 nmol / U) make PCR not particularly efficient; in fact, only 50% of positive nested samples are confirmed as such with this type of approach;

b) too high concentrations of MgCl2 (4 mM, 160 nmol / U) represent a particularly unfavorable reaction condition since no positive nested sample has been confirmed as such;

c) the concentration of MgCl2 2.5 mM (100 nmol / U) was found to be optimal since the data obtained confirmed the totality of the nested data.

Table 4

5) CHANGES in the CONCENTRATION of the REACTION BUFFER

Unlike MgCl2, variations in the concentrations of TRIS-HCl (12.5 mM final instead of 22.5 mM final) and KCl (50 mM final instead of 62.5 mM final) do not significantly affect the single-pass amplification efficiency, as the data obtained in DEIA are comparable to the reference ones, as shown in Table 5.

Table 5

The reverse transcription and amplification reactions were performed as described in point 4.

6) CHANGE in CONCENTRATION of dNTPs

Experimental tests were performed in order to evaluate the influence of nucleotide concentration on the single-step amplification protocol. The results obtained in DEIA (see Table 6) demonstrate that a dNTP concentration lower than that optimized for the one-step protocol (83 μΜ instead of 200 μΜ, referring to the dNTPs added in the amplification step) makes the amplification reaction less efficient. .

Table 6

The reverse transcription and amplification reactions were performed as described in point 4.

7) SINGLE PASS PCR

From the tests performed and described above, it appears that the best conditions for the amplification of single-pass HCV RNA are the following:

A) RETROTRASCRIPTION

5 µl of extracted RNA is back-transcribed in a volume of 25 µl containing TRIS-HCl pH 8.3 50 mM, KCl 50 mM, MgCl2 10 mM, dNTP 250 μl, antisense primer 2 μΜ (1CH), 25 U HRPI and 15 U AMV- RT. The reverse transcription reaction is carried out at 42 ° C for 1 hour, followed by the denaturation of the enzyme at 100 ° C for 10 minutes.

B) AMPLIFICATION

The amplification reaction is carried out in a final volume of 100 μl containing the cDNA coming from the reverse transcription (25 μl), TRIS-HCl pH 8.3 final 22.5 mM, final KCl 62.5 mM, final CClI2 2.5 mM, sense primer 0.5 μΜ (2CH) , dNTP 200 μΜ (concentration referred only to the dNTPs added in this step) and 2.5 U Taq polymerase.

C) THERMAL CYCLE

94 ° C 1 min - 50 ° C 1 min - 72 ° C 2 min 45 cycles

8) CHOICE OF THE PROBE USED for DETECTION In setting up the single-pass amplification method for HCV, the capture probe 3CH (Sorin Biomedica Diagnostics SpA PS000C Hepatitis C) was used for the detection of PCR products. Different probes were used, with sequence deduced from a different zone of the 5 'UTR region. The choice was made by evaluating a series of parameters, such as the length of the oligonucleotide which must allow an efficient hybridization with the complementary sample and at the same time present a type of sequence such as to exclude the formation of thermodynamically stable loops or dimers, due to presence of homologous traits within the sequence itself. To identify the optimal sequence, an analysis of the thermodynamic characteristics of some oligonucleotides obtained within the amplified zone (5 'UTR region of the HCV genome) was performed.

Table 7

Some of the sequences are taken from the literature, while others have been appropriately "designed" in order to satisfy the above requirements. The thermodynamic analysis was done using the OLIGO.EXE structure © program (ver 3.3) distributed by MedProbe A.S. (Norway) 2 and choosing to keep two parameters constant: the probe concentration (30 nM) and the saline concentration (188 mM). These two conditions are those that are experimentally employed in the DEIA test.

The thermodynamic analysis of the sequences, reported in Table 7, shows that the hybridization reactions of all the oligos with their respective complementary sequences are thermodynamically favored. As expected, the lowest AG values are given by the longer probes (3CH, HCV40. KY150), for which, however, the formation of loops or dimers is favored. Among the shorter probes, on the other hand, PT21 and WT are the ones that, by type of sequence, have the best characteristics, while CH5, despite having the same dimensions as the other two, can give rise to the undesired formation of thermodynamically stable dimers. However, PT21 is able to form a more stable specific hybrid than WT. Experimentally, therefore, a comparison was made between the PT21 sequences, chosen for the characteristics described above, and the 3CH which, as previously mentioned, was used for the detection of the amplified substances in setting up the one-step method. The operating conditions used are the standard ones of the DEIA kit (Sorin Biomedica Diagnostics SpA GEN-ETI-K-DEIA code PS0001) for the detection of amplified HCV and require a specific hybridization at 50 ° C. The experimental results, reported in Table 8, confirm that the PT21 probe allows a more efficient hybridization with the complementary amplified tract. In fact, about 96% of the positive samples analyzed have absorbance values greater than 1,000 O.D., while with the 3CH probe only 40% of the samples exceed this threshold.

Table 8

The experimental analyzes on the PT21 and 3CH probes were finally completed by evaluating their specificity in the same operating conditions as DEIA as above. Analytical specificity was determined on non-probe related DNA samples, representative for molecular size and sequence heterogeneity: sonicated salmon sperm DNA at various concentrations; Digested DNA of calf thyme at various concentrations; Unrelated amplified DNA. The results,

reported in Table 9, do not show any non-specific reactions

significant, since the absorbances of the analyzed samples are lower than

cut-off {cut-off = mean absorbance of negatives 0.150 O.D., calculated

as suggested on DEIA specifications).

Table 9

COMPARISON OF SPECIFICITY PT21 - 3CH

TV: Veal thyme DNA; SS: Salmon sperm DNA.

The set-up of the described single-pass HCV DNA amplification and detection protocol was achieved both by optimizing the amplification reaction conditions (by modulating in particular the MgCI2 concentration) and by using a new probe for the detection. (PT21). This approach was able to provide results comparable to the current NESTED plus DEIA method.

Claims (3)

CLAIMS 1. Procedure for the detection of specific nucleic acids for the hepatitis C virus (HCV) including the steps of: a) retro-transcription of viral RNA by means of a primer having a sequence substantially homologous to that of one of the following oligonucleotides: 1CH: 5 'GGT GCA CGG TCT ACG AGA CCT 3', 1TS: 5 'GCG ACC CAA CAC TAC TCG GCT 3', PT2: 5 'CGG TGT ACT CAC CGG TTC 3'; b) amplification with a single step polymerase chain reaction (PCR) method in which the primer is of substantially homologous sequence to that of one of the following oligonucleotides: 2CH: 5 'AAC TAC TGT CTT CAC GCA GAA 3', 4CH: 5'ATG GCG TTA GTA TGA GTG 3 '; wherein the ratio between the concentration of Mg <++> ions and Taq polymerase in the reaction mixture is about 100 nmoles / unit of enzyme; c) detection of the amplified product with the DEIA method by using the sequence probe PT21 oligonucleotide: 5 'GGG AGA GCC ATA GTG GTC TGC 3', or of substantially homologous or complementary sequence oligonucleotide. 2. Use of the PT21 oligonucleotide or an oligonudeotide of substantially homologous or complementary sequence to it as a probe for the detection by the DEIA method of nucleic acids specific for HCV. 3. Composition for the detection by the DEIA method of nucleic acids specific for HCV comprising the oligonucleotide PT21 or an oligonucleotide of substantially homologous or complementary sequence to it.