JPH10248579A - Measurement of hcv gene by real time detection pcr method, and primer and probe to be used therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new primer for highly sensitive and accurate detection of hepatitis C virus(HCV), etc., useful for measurement of HCV gene by a rear time detection PCR method, etc., consisting of an oligonucleotide having a specific base sequence. SOLUTION: The primer is a new forward-side primer as an oligonucleotide having the base sequence consisting of consecutive 15-40 bases of the base sequence expressed by formula I, to be used for measurement of hepatitis C virus(HCV) gene by a real time detection PCR method and a new reverse-side primer as a second oligonucleotide having the base sequence consisting of consecutive 15-40 bases of the base sequence expressed by formula II to be used for measurement of HCV gene by a real time detection PCR method, and can measure HCV in a highly sensitive, accurate and easy way. This primer is obtained by chemically synthesizing forward-side and reverse-side DNAs of the base sequence of HCV genome.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring hepatitis C virus (hereinafter referred to as "HCV" in the present specification) by a real-time detection PCR method and primers and probes used therefor.

[0002]

2. Description of the Related Art Conventionally, detection of HCV in a sample such as serum has been performed by a reverse transcription PCR (RT-PCR) method. This method includes (1) extraction of RNA (HCV gene is RNA) from serum, (2) cDNA synthesis using the extracted RNA as a template, (3) amplification by 1st PCR, (4)
Amplification by ndPCR (amplification is performed twice to increase sensitivity (nested PCR)), (5) agarose gel electrophoresis,
(6) Data processing is performed in six steps.
In addition, the test RNA can be quantified by the RT-PCR Southern method which combines the RT-PCR method and the Southern analysis.

[0003]

[0008] Quantitatively measuring the viral load of HCV with high sensitivity is important for monitoring the progress of treatment in addition to simply knowing the degree of viral infection. However, the above-mentioned RT-PCR method can detect HCV in a test sample but cannot quantify it. In addition, the RT-PCR Southern method requires complicated steps and requires much time and effort. The measurement of HCV is mainly performed at clinical test centers and the like, and it is necessary to process a large number of samples within a certain time. Therefore, it would be very advantageous if the test could be performed efficiently and the test time could be reduced. is there.

Accordingly, an object of the present invention is to provide a means for measuring HCV with high sensitivity, accurately and easily.

[0005]

Means for Solving the Problems The present inventors have developed a so-called real-time detection PCR method (Proc. Natl. Acad. Sci.
USA, Vol. 88, pp. 7276-7280, August 1991, Biochemi
stry; Japanese Patent Publication No. 6-500021).
It was thought that the above object could be achieved by performing the measurement. However, depending on the settings of primers and probes used, the sensitivity and / or reproducibility of measurement cannot always be satisfied. Therefore, as a result of earnest research, they have found that HCV can be accurately quantified with very high sensitivity and high reproducibility using a real-time detection PCR method by using specific primers and probes, and completed the present invention. .

[0006] That is, the present invention relates to SEQ ID NO: 1 in the sequence listing.
15 to 40 consecutive nucleotides in the nucleotide sequence represented by
Provided is a forward primer which is an oligonucleotide having a base sequence consisting of bases and which is used for measuring an HCV gene by a real-time detection PCR method. Further, the present invention is used for measuring an HCV gene by a real-time detection PCR method, which is an oligonucleotide having a nucleotide sequence consisting of 15 to 40 consecutive nucleotides among the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing. The reverse side primer is provided. Further, the present invention provides a method in which a reporter fluorescent dye and a quencher fluorescent dye are bound to an oligonucleotide having a base sequence consisting of consecutive 15 to 46 bases among the base sequence shown in SEQ ID NO: 5 in the sequence listing. The reporter fluorescent dye, when the reporter fluorescent dye is bound to the same probe as the quencher fluorescent dye, its fluorescence intensity is suppressed by fluorescence resonance energy transfer, and the same as the quencher fluorescent dye Provided is a probe used for measurement of an HCV gene by a real-time detection PCR method, the fluorescence intensity of which is not suppressed when not bound to the probe. Furthermore, the present invention uses the forward primer of the present invention, the reverse primer of the present invention, and the probe of the present invention to perform PCR using the HCV gene to be measured in the test sample as a template, Provided is a method for measuring an HCV gene in a test sample, comprising measuring fluorescence from a reaction solution in real time.

[0007] The forward primer of the present invention comprises 15 consecutive nucleotides of the base sequence represented by SEQ ID NO: 1 in the sequence listing.
It is an oligonucleotide having a base sequence consisting of bases to 40 bases, preferably 18 bases to 22 bases. Of these, particularly preferred are those having a base sequence of 20 bases shown in SEQ ID NO: 2. The nucleotide sequence represented by SEQ ID NO: 2 corresponds to the 58th nucleotide of the HCV genome (hereinafter, described as “58 nt”) to 77 nt. The entire nucleotide sequence of the HCV genome is known, and is described in Kato N. et al., Proc. Natl. Acad. Sci.
A 1990; 87: 9524-9528.

The reverse primer of the present invention is an oligonucleotide having a nucleotide sequence consisting of 15 to 40, preferably 18 to 22 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing.
Of these, particularly preferred are those having a base sequence of 20 bases shown in SEQ ID NO: 4. The base sequence represented by SEQ ID NO: 4 hybridizes to 294 nt to 313 nt of the HCV genome.

The probe of the present invention is a probe in which a reporter fluorescent dye and a quencher fluorescent dye described later are bound to an oligonucleotide. The oligonucleotide part of the probe has a base sequence consisting of 15 to 46 consecutive bases, preferably 24 to 28 bases, of the base sequence represented by SEQ ID NO: 5 in the sequence listing. Preferred examples of the oligonucleotide portion include those comprising 26 bases represented by SEQ ID NO: 6.
The nucleotide sequence represented by SEQ ID NO: 6 corresponds to 84 nt to 109 nt of the HCV genome. The region of the HCV gene to which the oligonucleotide portion of the probe hybridizes is close to and partially overlaps with the region to which the forward primer hybridizes. However, when the reaction is actually performed, the forward primer It is necessary to select a combination in which the regions where the probe and the probe hybridize do not overlap each other.

When the reporter fluorescent dye is bound to the same probe as the quencher fluorescent dye, the fluorescence intensity of the reporter fluorescent dye is suppressed by fluorescence resonance energy transfer, and the same as the quencher fluorescent dye. When the probe is not bound to the probe, the fluorescence intensity is not suppressed. As the reporter fluorescent dye, a fluorescein-based fluorescent dye such as FAM (6-carboxy-fluorescein) is preferable, and as the quencher fluorescent dye, TAMRA (6-carboxy-fluorescein) is used.
Rhodamine-based fluorescent dyes such as tetramethyl-rhodamine) are preferred. These fluorescent dyes are known and can be used since they are contained in a commercially available kit for real-time detection PCR. The binding position of the reporter fluorescent dye and the quencher fluorescent dye is not particularly limited,
Usually, a reporter fluorescent dye is bound to one end (preferably the 5 'end) of the oligonucleotide portion of the probe, and a quencher fluorescent dye is bound to the other end. Incidentally, a method of binding a fluorescent dye to an oligonucleotide is known, for example, Nobl
e et al., (1984) Nuc. Acids Res. 12: 3387-3403 and I
yer et al., (1990) J. Am. Chem. Soc. 112: 1253-1254.
It is described in.

In the method of the present invention, the forward primer of the present invention, the reverse primer of the present invention, and the probe of the present invention are used to reverse the HCV gene to be measured in a test sample as a template. Copy PCR (RT-
PCR) is performed, and the fluorescence from the reaction solution is measured in real time. The real-time detection PCR method itself is publicly known, and apparatuses and kits for the real-time detection PCR method are also commercially available. Therefore, the PCR can be performed using such commercially available apparatuses and kits.

The reaction is carried out using a solution containing the test HCV RNA, the above-mentioned forward primer, the reverse-side primer and the above-mentioned probe, a thermostable DNA polymerase (which also has reverse transcriptase activity), dATP, dGTP, dCTP and dTTP. Prepare and perform. Using dUTP instead of dTTP and adding uracil-N-glycosylase (UNG) is preferable because contaminating DNA from the previous PCR product can be degraded. Specific conditions for the reaction are described in detail in the Examples below. The test sample was HCV.
May be included, for example, a body fluid such as serum. Preparation of HCV RNA can be performed in the same manner as in the case of conventional RT-PCR, and is also specifically described in the following Examples.

In the reaction, first, cDNA is synthesized using HCV RNA as a template, and then amplification of DNA is performed by PCR using the cDNA as a template. Amplified DNA
Contains a region complementary to the probe, so that the probe hybridizes to the single-stranded amplified DNA. In the state where the probe is completely hybridized, when the elongation using the single-stranded DNA to which the probe is hybridized as a template occurs, the probe is hydrolyzed from the 5 ′ terminal side by the exonuclease activity of DNA polymerase. As a result of this decomposition, the reporter fluorescent dye and the quencher fluorescent dye bound to the oligonucleotide portion of the probe are separated, and the fluorescence from the reporter fluorescent dye suppressed by the fluorescence resonance energy transfer caused by the quencher fluorescent dye is reduced. The fluorescence intensity increases. On the other hand, when no HCV RNA is present in the test sample, amplification of the DNA does not occur, so that the probe does not hybridize to the DNA, and is therefore not hydrolyzed by the DNA polymerase. Therefore, the fluorescence from the reporter fluorescent dye remains suppressed by the quencher fluorescent dye, and the fluorescence intensity does not increase. Therefore, by measuring the fluorescence intensity, the HCV R
It is possible to detect NA.

In the method of the present invention, the fluorescence intensity is measured in real time. That is, while measuring the fluorescence intensity, the PC
Perform the R reaction. The measured fluorescence intensity exceeds the detection limit after a certain number of cycles and increases rapidly. Then, as the amount of HCV RNA in the test sample increases, the fluorescence intensity increases rapidly with a small number of cycles. Therefore, it is possible to perform a quantitative measurement of HCV RNA in the test sample by examining the number of cycles after which the rapid increase in the fluorescence intensity starts. More specifically, for example, HC
By setting a threshold value of 10 times the standard deviation of the fluorescence intensity of each cycle (for example, 3 to 15 cycles) in the negative control containing no VRNA as a threshold value and examining the number of cycles in which the fluorescence intensity exceeds this threshold value, the test sample can be accurately detected. HCV RNA in the sample can be quantitatively measured. That is, when the common logarithm of the number of HCV RNAs in the test sample is plotted on the horizontal axis, and the cycle number when the threshold value is exceeded is plotted on the vertical axis,
Since the measurement results are almost completely on a straight line, if a calibration curve is prepared, the amount of HCV RNA in the test sample can be quantitatively measured by examining how many cycles the threshold value is exceeded. Therefore, according to the method of the present invention, the conventional R
As in T-PCR, there is no need to perform an operation to check the amplification by performing electrophoresis after PCR, which is very simple.

[0015]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below more specifically based on embodiments. However, the present invention is not limited to the following examples.

Example 1 (1) Synthesis of Primer A 20-mer oligo DNA having a base sequence consisting of 20 bases shown in SEQ ID NO: 2 was chemically synthesized and used as a forward primer. In addition, 20 shown in SEQ ID NO: 4
20-mer oligo DN having a base sequence consisting of bases
A was chemically synthesized and used as a reverse primer.

(2) Preparation of Probe A 26-mer oligo DNA having a base sequence consisting of 26 bases shown in SEQ ID NO: 6 was chemically synthesized. FAM is added to the 5 'end of this oligo DNA and TAMRA is added to the 3' end.
Was bound by the method described in the above-mentioned literature to obtain a probe.

(3) Preparation of Synthetic RNA pCII, a plasmid containing the 5 'region of the HCV gene
5 (J. Virol. Vol. 66, p.1476-1483, 1992) with T7 polymerase as a template to synthesize a 1762 base RNA, a 10-fold dilution series from 5.04 x 10 ⁷ to 5.04 x 10 ⁰ copies / [mu] l Produced.

(4) Preparation of Reaction Solution The above primer, probe and Perkin Elmer TaqMan EZ
Using an RT-PCR Core Kit (trade name, manufactured by Perkin Elmer), reaction solutions shown in Table 1 below were prepared per reaction tube.

[0020]

[Table 1]

(5) Real-time detection PCR Perkin Elmer MicroAmp Optical Tube (trade name, Perkin
48 μl of the above reaction solution was added per one (Elmer),
2 μl of the above-mentioned RNA solution derived from the serum to be tested was added thereto.
ABI after cap (Perkin Elmer Optical Cap)
PRISM 7700 Sequence Detection System (trade name, Perk
in Elmer) and reacted under the following conditions. The fluorescence intensity was measured at each cycle of PCR and data was collected. Degradation reaction of contaminated DNA by UNG 50 ° C, 2 minutes Reverse transcription step 60 ° C, 30 minutes Inactivation of UNG 95 ° C, 5 minutes PCR 95 ° C, 20 seconds 60 ° C, 1 minute This PCR cycle was repeated 53 times.

(6) Results FIG. 1 shows the results of plotting the number of cycles on the horizontal axis and the change in fluorescence intensity on the vertical axis. In FIG. 1, near each line, an index portion of the copy number of the HCV RNA in the sample (two times the concentration (copy / μl) since the sample used 2 μl as described above) is shown. FIG. 1 shows that the fluorescence intensity of each test sample does not change until after a certain number of cycles, but the fluorescence intensity sharply increases after a certain number of cycles. It can be seen that the cycle number at which the rapid increase in the fluorescence intensity starts decreases as the HCV RNA copy number in the test sample increases. In the real-time PCR, the threshold value is set to 10 times the standard deviation of the fluorescence intensity in 3 to 15 cycles of the negative control performed on the sample containing no HCV RNA, and the number of cycles exceeding the threshold value (that is, the fluorescence intensity (The number of cycles when it began to increase rapidly). The common logarithm of the RNA copy number is plotted on the horizontal axis,
FIG. 2 is a diagram in which the number of cycles exceeding the threshold is plotted on the vertical axis. As shown in FIG. 2, there is a linear relationship between the common logarithm of the RNA copy number and the number of cycles exceeding the threshold (correlation coefficient 0.990), and the number of cycles in the test sample is determined by measuring the number of cycles. It was revealed that HCV RNA can be quantitatively measured.

Example 2 (1) Preparation of test HCV RNA To 200 μl of each serum from a hepatitis C patient, 600 μl of ISOGEN-LS (trade name, Nippon Gene guanidine thioisocyanate nucleic acid extraction reagent) (3 μl of 2ME, tRN)
A (addition of 10 μg A) was added, and the mixture was allowed to stand on ice for 15 minutes. 1
After adding 60 μl of chloroform, the mixture was allowed to stand on ice for 15 minutes, and then centrifuged at 12,000 × g for 15 minutes. To the aqueous phase was added an equal volume of isopropanol and 1 μl of glycogen,
After standing at 1 ° C. for 1 hour, the mixture was centrifuged at 12000 × g for 10 minutes. Then, wash twice with 1 ml of 75% ethanol,
The solution was dissolved in 1 l of DTT-RNase inhibitor in DEPC water and stored at -80 ° C until measurement.

Using the same probes and primers as in Example 1, a reaction solution having the same composition as in Example 1 was prepared. Perkin E
lmer MicroAmp Optical tube
45.5 μl was added, and the test serum-derived RNA solution 4.5
The reaction was performed in the same manner as in Example 1, and the fluorescence was measured at each cycle. On the other hand, the conventional RT-PCRSout
Commercial products using the hern method (Ampricor (trade name), Wolfe
L., et al., New diagnostic tools, pp.83-94, 1994)
And bDNA method (Lau JYN, et al., Lancet 1993; 341: 1501-
1504), the copy number of HCV RNA was measured for the same test sample. The results are shown in Table 2 below.

[0025]

As shown in Table 2, it was found that the measurement results obtained by the method of the present invention correlated well with the results obtained by the conventional method, and that the measurement could be performed with high sensitivity.

[0027]

According to the present invention, it has become possible to measure HCV with high sensitivity, accurately and easily.

[0028]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 40 Sequence type: Nucleic acid Sequence TGAGGAACTA CTGTCTTCAC GCAGAAAGCG TCTAGCCATG 40

SEQ ID NO: 2 Sequence length: 20 Sequence type: nucleic acid sequence CTGTCTTCAC GCAGAAAGCG 20

SEQ ID NO: 3 Sequence length: 40 Sequence type: nucleic acid sequence CCTCCCGGGG CACTCGCAAG CACCCTATCA GGCAGTACCA 40

SEQ ID NO: 4 Sequence length: 20 Sequence type: nucleic acid sequence CACTCGCAAG CACCCTATCA 20

SEQ ID NO: 5 Sequence length: 46 Sequence type: nucleic acid sequence AGCGTCTAGC CATGGCGTTA GTATGAGTGT CGTGCAACCT CCAGGA 46

SEQ ID NO: 6 Sequence length: 26 Sequence type: nucleic acid sequence CATGGCGTTA GTATGAGTGT CGTGCA 26

[Brief description of the drawings]

FIG. 1. HCV in various test sera by the method of the present invention.
FIG. 4 is a diagram showing the relationship between the number of PCR cycles and the change in fluorescence intensity when RNA was measured.

FIG. 2 shows the common logarithm of the copy number of HCV RNA for various test samples and the HC in the test sample by the method of the present invention.
FIG. 9 is a diagram showing the relationship between the change in fluorescence intensity and the number of cycles in which the change in fluorescence intensity exceeds a threshold value when measuring RNA of V.

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁶ Identification code FI G01N 33/576 G01N 33/576 Z (72) Inventor Asao Katsume 6-6 Higashinakacho, Urawa-shi, Saitama 1001 Belm Urawa 1001 (72) Inventor Tomoko Takeuchi Parkside Nara 303, 2-45-11 Izumi, Suginami-ku, Tokyo (72) Inventor Ryuji Kawaguchi 51 Komiyacho, Hachioji-shi, Tokyo Inside SRL Hachioji Laboratory Co., Ltd.

Claims (8)

[Claims] 1. An oligonucleotide having a nucleotide sequence consisting of 15 to 40 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing, which is used for measuring the HCV gene by real-time detection PCR. Forward side primer. 2. The primer according to claim 1, wherein the oligonucleotide comprises 20 bases having a base sequence represented by SEQ ID NO: 2 in the sequence listing. 3. An oligonucleotide having a nucleotide sequence consisting of 15 to 40 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing, which is used for measuring the HCV gene by real-time detection PCR. Reverse side primer. 4. The primer according to claim 3, wherein the oligonucleotide comprises 20 bases having a base sequence represented by SEQ ID NO: 2 in the sequence listing. 5. A reporter fluorescent dye and a quencher fluorescent dye are bound to an oligonucleotide having a continuous base sequence of 15 to 46 bases in the base sequence represented by SEQ ID NO: 5 in the sequence listing. The reporter fluorescent dye, when the reporter fluorescent dye is bound to the same probe as the quencher fluorescent dye, the fluorescence intensity is suppressed by fluorescence resonance energy transfer, the same probe as the quencher fluorescent dye A probe used for measurement of an HCV gene by a real-time detection PCR method, wherein the fluorescence intensity is not suppressed in a state where it is not bound to the DNA. 6. The probe according to claim 5, wherein the oligonucleotide comprises 26 bases having a base sequence represented by SEQ ID NO: 6 in the sequence listing. 7. The probe according to claim 5, wherein the reporter fluorescent dye is a fluorescein-based fluorescent dye, and the quencher fluorescent dye is a rhodamine-based fluorescent dye. 8. A method for measuring a test sample using the forward primer according to claim 1 or 2, the reverse primer according to claim 3 or 4, and the probe according to claim 5, 6, or 7. Reverse transcription using the HCV gene to be used as a template
A method for measuring an HCV gene in a test sample, comprising performing CR and measuring fluorescence from a reaction solution in real time.