TWI295321B - - Google Patents

Description

1295321 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a virus detection technique, and more particularly to an assay technique for quantifying a virus by an instant polymerase chain reaction. [Prior Art] Hepatitis C is a globally prevalent liver disease. In the epidemiological survey of chronic hepatitis C in Taiwan, it was found that the number of chronic hepatitis C patients in the northern region accounted for 1.5% to 2% of the total population, but in the south-central region. Some prevailing rates in towns and villages in coastal areas are as high as 20 to 60%. The route of infection of hepatitis C is closely related to the use of blood transfusion and non-disposable needles, and hepatitis C is also an important factor leading to cirrhosis and even liver cancer. The hepatitis C with the original is slightly different from the hepatitis B with the original: about 80% of the hepatitis B with the original is healthy, but only about 20% of the hepatitis C with the original liver function is normal of. Therefore, chronic hepatitis C requires treatment. In recent years, there has been a major breakthrough in the treatment of hepatitis C, such as the treatment of interferon combined with ribavirin. Current studies have shown that if the modified interferon treated with polyethylene glycol is used (polymerized) Ethylene glycolated interferon, Peg-IFN plus triazole nucleoside, the cure rate for chronic hepatitis C can be increased to five to eighty percent. Therefore, hepatitis C prevention and treatment should be directed at the treatment and treatment of patients with hepatitis C, and actively treat them, and can protect these people from liver disease, and reduce the source of infection. It can be said to kill two birds in one fell swoop. Expenses. At present, the diagnosis of chronic hepatitis C is a liver index (such as alanine amino transferase (ALT) abnormality for more than half a year, and serum anti-hepatitis C antibody is positive. However, the true virus reproduction status depends only on poly 94203. .doc 1295321 Polymerase Chain Reaction to detect the presence of hepatitis C virus RNA in blood. Determine the infection of hepatitis C virus, before treatment (such as interferon or antiviral drugs), type C The detection of hepatitis virus RNA (Viral load) allows physicians to evaluate the efficacy of hepatitis C virus RNA every three months throughout the course of treatment. Hepatitis C virus RNA detection can also reflect whether a patient has sustained response. Therefore, continuous monitoring of hepatitis C virus is considered as the best indicator for evaluating the efficacy of medication and whether to continue medication. The method for measuring the amount of virus, including the competitive nucleic acid polymerase chain reaction and the instant detection of polymerase chain reaction Methods such as Amplicor monitor (RocheTM Diagnostics, Basel, Switzerland), Branched-chain DNA (BayerTM Diagnostics, Emeryville, USA), and NASBA (Organon TeknikaTM, Boxtel, The Netherlands). However, the above-mentioned goods have the following disadvantages: (1) time-consuming operation, cumbersome steps; (2) poor linear range of detection; (3) no internal sample quality control. For the sake of the job, development is easy and sensitive. A highly reliable hepatitis C detection technique is required by the industry. SUMMARY OF THE INVENTION An object of the present invention is to provide an primer for quantifying hepatitis C virus, which can amplify a hepatitis C virus cDNA, the primer Is selected from the group consisting of the following primers and variants thereof: having the sequence shown in sequence identification number 1 94203.doc 1295321 The upstream primer has one upstream sequence as shown in sequence identification number 4... as shown by sequence identification number 2 One of the downstream primers of the sequence has a good downstream of one of the sequences indicated by the sequence identification number 3, and has a downstream primer such as sequence identification, one of the five sequences of the singular number, and There is a downstream primer which is one of the sequences shown in Sequence Identification No. 6. Another object of the present invention is to provide a kit for quantifying hepatitis C virus, which comprises the primers as described above. A further object of the present invention is to provide A method for quantifying hepatitis C virus by using the above primer for immediate polymerase chain reaction. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a primer for quantifying hepatitis C virus, which can be applied to various instant polymerase chain reactions And to quantify hepatitis C virus. p According to the primer of the present invention, the hepatitis C virus CDNA can be amplified, and the primer is selected from the group consisting of the following primers and variants thereof, and has an upstream primer such as sequence identification, which is not a sequence of number 1, and has An upstream primer of one of the sequences indicated by sequence identification number 4, a downstream primer having one of the sequences as shown in sequence identification number 2, a downstream primer having one of the sequences as shown in sequence identification number 3, and having a sequence as shown in sequence identification number 5. A downstream primer and a downstream primer having one of the sequences as shown in SEQ ID NO: 6. Preferably, the primers according to the present invention form the following primer pair: (a) an upstream primer having one of the sequences shown in Sequence Identification Number 1 and a downstream primer having a sequence such as the sequence identification number 2 or 3; b) an upstream primer having one of the sequences as shown in SEQ ID NO: 4 and a downstream primer having one of the sequences as shown in SEQ ID NO: 5 or 6. 94203.doc 1295321 ..., the "polymerase chain reaction" of the medium contains four steps: (1) making n 'reverse enthalpy to form two single strands; (2) making the two primers separately with the two steps Adhesing; (7) extending the _ two (4) with the polymerase to obtain two pairs of DNA. The above steps are repeated, and the DNA fragment of the particular one can be amplified. "Instant Polymerase Chain Reaction": To use fluorescence detection. The occurrence of a "polymerase chain reaction" is made to quantify: and thus the amount of template involved in the reaction can be inferred. At present, the immediate development of the enzyme, the enzyme chain reaction in a closed reaction tube, in addition to the primers and test enzymes required for the polymerase chain reaction, _ and the fluorescent signal is added to the reaction tube and then σ instrument cycle by cycle By detecting, it is possible to simultaneously amplify DNA and simultaneously control the function. Therefore, the instant polymerase chain reaction has the advantages of direct amplification and no post-processing, which not only saves time and effort, but also avoids the reprocessing of reprocessing (4). At the same time, because of the narration record of the dna glory Λ 5 tiger, while depicting the complete pattern of gene duplication, it has better sensitivity and reproducibility. The concentration range of the reagent of the present invention can reach up to (7) 8 copies, which is the largest of the current methods. The term "variant" as used herein refers to an oligonucleotide which can replace the oligonucleotide molecule of the primer of the present invention and which can still amplify the sequence of the same specific fragment. Due to the nature of the polymerase chain reaction itself, even if there is variability in the sequence between the primer and the template to be amplified, a specific DNA fragment can be synthesized by adjusting the reaction temperature of the adhesion step in the polymerase chain reaction. For example, if the sequence variability between the primer and the template to be amplified is larger, the reaction temperature of the adhesion step is lower; in other words, the smaller the sequence variability between the template and the template to be amplified is, The reaction temperature of the adhesion step. Therefore, according to the disclosure of the present invention, a person having general knowledge in the specific field 94203.doc 1295321 can design different primers according to the DN A fragment to be amplified, and any base substitution, addition or reduction for the primer of the present invention, The introduction of a particular fragment, as it is still possible with the primers of the present invention, is intended to be within the scope of the invention. The primer according to the present invention can be applied to various instant polymerase chain reactions, for example, can be used for DNA-binding fluorophore (DNA_binding flu〇r〇Ph〇res), adjacent linear oligo probe (adjacent Unear 〇Ug) 〇pr〇bes), 5 nuclease olig0pr〇bes, hairpin 〇ligoprobes, and self-fluorescent amplicon (self_fh^(4)} ton of amplicons) . The invention further provides a kit of hepatitis C virus comprising the primer or primer pair described above. In one embodiment of the invention, a TaqMan8 instant polymerase chain anti-Qin system is used. The system uses a target probe to quantify the polymerase chain reaction product. The target probe has two fluorescent groups as a reporter and a quencher at the 5 and 3, respectively. The fluorescent base is the fluorescent portion of the reported protein, and the fluorescent base usually used is FAM. If the target probe is not hybridized with the template, it is located at the 3rd end of the quencher fluorescent base (usually a long wavelength color, such as red) by fluorescence resonance energy transfer (Flu〇rescence Res〇nanee Energy Transfer) , fret) and reduce the fluorescence of the reporter fluorescent base (usually a short wavelength color, such as green) at the end of the 5th. If the target probe is denatured to the template and adhered to the single-strand template, the polymerase can add the core % S text according to the sequence of the template, and remove the target probe from the 5 end, thereby making the 3, terminal quencher The fluorescent base is separated from the reporter at the 5' end, so that the reporter can diverge its fluorescent energy. 94203.doc 1295321 If the number of times the polymerase chain reaction occurs, the more light is diverged, so by measurement Reporting the amount of light in the camp, you can quantify the product of the poly-lock reaction. Therefore, according to the kit of the present invention, the target probe system further comprises hybridization with the hepatitis virus sac A fragment according to the present invention, and the 51 and 3 ends respectively have The reporter and the first-stage quencher used in the present invention may be conventional fluorescent groups, such as μμ as a director. Preferably, when the (4) primer pair is used, the target probe has a sequence as shown by the sequence identification number 7 or 8. The signal is determined to reflect the actual experimental conditions or operational errors, and the kit according to the present invention is tasted by another person, and further includes an internal control mechanism, and 1 includes an internal control mass having an insert. The insert fragment comprises a hepatitis C virus CDNA fragment amplified by a primer or primer pair according to the present invention, which is substituted with a hepatitis C virus or a human genome sequence; and an internal control probe, It may be hybridized with the insert of the internal control plastid, the 5th and 3rd ends respectively having a second reporter and a second quencher, and the second reporter is different from the first reporter. The instant polymerase chain reaction of the internal control plastid and the internal control probe is operated simultaneously with the sample and the target probe during operation to facilitate observation. The second reporter and the second quencher used in the present invention may be conventional fluorescent groups, such as the use of hydrazine as a second reporter. Preferably, the insert 35 has a sequence as shown in SEQ ID NO: 12 or its complementary strand, and the internal control probe has a sequence as indicated by the sequence identification number 3-1 or its complementary strand. In another embodiment of the invention, a hybrid probe is used to immediately polymerize the 94203.doc 1295321 synthase chain reaction system. The system quantifies the polymerase chain reaction product by means of a certain anchor probe and a sensor probe. The sensing probe has a donor fluorescent light at the 3 end, and the 5' end of the anchoring probe has an acceptor fluorescent light. The donor fluorescence can be excited by a suitable wavelength and can transfer energy to the acceptor's fluorescent light, while the stimulated fluorescent light can emit a longer wavelength. If the error-producing probe and the sensing probe are not hybridized with the template, the donor fluorescence can be emitted under the excitation of the appropriate wavelength, but since it is not adjacent to the receptor fluorescence, the energy cannot be transferred, so it can only be observed. Have to apply the amount of body fluorescence. If the error-producing probe and the sensing probe are attached to the single-strand template when the template is denatured, the donor glory can transfer the energy to the acceptor fluorescence, so that the amount of receptor fluorescence, such as polymerase, can be observed. The more the number of chain reactions occurs, the more products are used as templates, the more the fluorescence is transferred to the receptor fluorescence, and the receptor glory is observed, so the receptor fluorescence is measured by measurement. The amount of the polymerase chain reaction can be quantified2. Therefore, the kit according to the present invention further comprises an inductive probe and a certain wrong needle. The 3' end of the inductive probe has a -first-body fluorescent, the wrong probe has 5, and the end has a -1 Receptor fluorescence, and the sensing probe and the (four) probe can be hybridized with the amplified hepatitis C virus cDNA fragment of the primer or the primer pair according to the present invention, respectively, and when the (8) probe and the sensing probe are At the same time, hybridization to the amplified hepatitis C virus cDNA fragment according to the primer or primer pair of the present invention can shift the energy of the first donor fluorescence to the first receptor fluorescence. The first acceptor fluorescent light and the first donor fluorescent light used in the present invention may be conventional light-emitting groups: if FL is used as the first donor fluorescent light, and LC Red 64 is used as the first-receptive fluorescent light. Light. Preferably, when the (b) primer pair is used, wherein the sensing probe has a sequence as shown in SEQ ID NO: 9 or its complementary strand: the 94203.doc 1295321 error-correcting probe has a sequence identification number of 1 or序列3iul or its complementary strands. Similarly, the embodiment also includes an internal control mechanism for the above-mentioned internal control plastid and an internal control error-fixing probe, the fifth end of which: the second receptor is fluorescent and can be internally The intervening fragment of the plastid is controlled to be heterozygous: and when the inner (four) probe and the sensing probe are simultaneously hybridized to the internal control plastid, the energy of the first donor fluorescent light can be shifted to the second acceptor glory. During operation, the internal polymerization plastid and the internal _needle and the sensing probe are simultaneously coupled with the sample and the error-producing probe and the sensing probe for easy observation. Preferably, the internal control anchor probe has a sequence as shown by sequence identification number 15 or its complementary strand. Preferably, the kit according to the invention further comprises a standard plastid for the reference curve '纟, the standard plastid has an additional fragment, the additional patch 2 comprising a pair of primers or primers according to the invention The amplified hepatitis C virus cDNA fragment. The design of the other parts of the standard plastid is well known to those of ordinary skill in the art to which this invention pertains. In an embodiment of the preferred embodiment, the additional fragment has a sequence as shown in SEQ ID NO: 14 or its complementary strand. The kit according to the present invention is compared with the nucleic acid sequence of all full-length hepatitis C. The syndrome according to the present invention can identify existing known genotypes and avoid the occurrence of pseudotypes due to differences in viral genotypes or species. Negative, the detection rate is the highest. The invention further relates to a method for quantifying hepatitis C virus using an instant polymerase chain reaction using a bow tweezers or kit according to the invention. 94203.doc -12- 1295321 In particular, when using the TaqMan® Instant Polymerase Chain Reaction System, the method according to the invention comprises the following steps: (1) Hybridization with a target probe at different concentrations of standard plastids In response, the standard plastid has an additional fragment comprising a hepatitis C virus cDNA fragment amplified by a primer or primer pair according to the present invention, and the target probe is compatible with the primer or primer according to the present invention Hybridization of the amplified hepatitis C virus cDNA fragment, wherein the 5, and 3 ends of the target probe respectively have a first reporter and a first quencher; (U) = different concentrations in the step (1) The amount of the first reported V agent in the standard plastid reaction to obtain a standard curve of the standard plastid concentration versus the first reported dose; ((1)) is obtained from the sample of hepatitis C virus; preferably, The sample is derived from blood, such as serum or plasma; () X primer or primer pair, target probe, and CDNA in step (iii) are immediately polymerase chain reaction; (): first report in 4 steps (lv) The strength of the agent, and the standard of the second step in step (9) Curve comparisons to quantify the concentration of the sample in the sample. ^ _ The method according to the invention comprises - an internal control step, an addition, a cadence (1V) and (7), and a measurement of the second reporter's detailed it number, indicating that the reaction conditions are successful. According to the present day ^=hybrid probe instant polymerase chain reaction system, the method includes the following steps: 94203.doc -13- 1295321 (i) Standard plastids with different concentrations and a sensing probe and certain anchoring The needle is subjected to a heterozygous reaction; the standard plastid has an additional fragment comprising a cDNA fragment of hepatitis C virus amplified by the primer or primer pair according to the present invention; the third end of the sensing probe has _first The body is fluorescent, and the 5th end of the anchor probe has a first receptor camping light. The 5 Hai induction picking needle and the error correcting probe can respectively enlarge the type C hepatitis with the primer or the primer pair according to the present invention. The viral cDNA fragment is heterozygous, and when the anchor probe and the sensing probe are simultaneously hybridized to the amplified hepatitis C virus cDNA fragment according to the primer or the primer pair of the present invention, the energy of the first donor fluorescent light can be made Transfer to the first receptor glory; (ii) measure the amount of the first receptor fluorescence in the standard plastid reaction at different concentrations in the step (1) to obtain a standard curve of the standard plastid concentration versus the receptor fluorescence (iii) cDNA obtained from the hepatitis C virus derived from the sample Preferably, the sample alpha is derived from blood 'such as serum or plasma; (iv) is immediately imbibed with the primer or primer pair, the sensing probe, the anchor probe, and the cDNA in step (iii) according to the present invention. Polymerase chain reaction; (v) Measure the intensity of the receptor fluorescence in step (iv) and compare it to the standard curve obtained in step (丨丨) to quantify the concentration of cDNA in the sample. Preferably, the method according to the invention comprises an internal control step of adding an internal control mass and an internal control anchor probe, and performing steps (iv) & with the hepatitis C cDNA and the anchor probe. v), and measuring the amount of second receptor fluorescence 'if positive signal is detected, it means that the reaction conditions are successful. In the present invention, the extraction of the viral cDNA in the sample, the immediate polymerization of the enzyme, the detection of the 94203.doc -14-1295321, and the analysis of the signal are achieved by methods well known to those skilled in the art, preferably in the sample. The extraction of viral cDNA, the immediate polymerase chain reaction, and the analysis of the signal are illustrated in the following examples. The invention is illustrated by the following examples, which are not intended to be construed as limiting the invention. [Examples] Example 1: Preparation of Hepatitis C cDNA Preparation of serum samples: - The blood of the patient is collected using a blood vessel containing no anticoagulant, and the blood is coagulated for about 1 to 15 minutes; - Centrifugation at 3000 rpm 10 Minutes (completed within two hours at room temperature) - Pipetting the upper serum • Dispense 50 pL in a microcentrifuge tube

- Stored in -80°CC hepatitis RNA extraction step (using Gentra®, R_5000 reagent group) - Add 50 pL of serum to a microcentrifuge tube and add 250 pL of Cell Lysis solution - After shaking for 10 seconds, 65QC Heat for 5 minutes - shake for 10 seconds, then centrifuge for 5 to 10 seconds at low speed to add 100 pL of Protein-DNA precipitation solution - shake the microcentrifuge tube upside down 10 times and mix well, place on ice for 5 minutes - Centrifuge at 15,000 rpm for 3 minutes at low temperature (4 ° C) - pipette the supernatant to another microcentrifuge tube, add 300 μL isopropanol and 0 · 5 94203.doc -15-1295321 μι glycosides (Roche®, 901393) - Invert the microcentrifuge tube upside down 50 times to mix and shake - low temperature (4QC) high speed centrifugation 15000 rpm for 3 minutes - pour off the supernatant - add 300 pL 70% alcohol and reverse the microcentrifuge tube 10 times - low temperature (4 ° C) high speed centrifugation 15000 rpm 3 minutes - pour off the supernatant - low temperature (4 ° C) high speed centrifugation 15000 rpm 1 minute - try to absorb residual alcohol - open the microcentrifuge tube cover, air dry the residual liquid for about 5 minutes - join 10 μΐ DEPC water, after the solution is completely dissolved, the oscillation is even - Lines of RNA reverse transcribed cDNA was placed on ice to prepare reverse transcription steps: - preparing the test solution as ··

RNA 10 pL

5 mM dNTP 4 pL

5x RT buffer 8 pL

1% BSA 4 pL

Rnasin (TaKaRa®, Cat. Νο·23 10A) 0.5 pL

20 μΜ RT primer (SEQ ID NO: 16) 2 pL H2〇10.5 μΐ, -70 ° C, heating for 10 minutes - 5 minutes on ice, low speed centrifugation - Add 1 pL of MMLV-RT (Promega®, Cat. Νο· Μ1701 ), 94203.doc -16- 1295321 0.5 pL of Rnasin and 0.5 μί of sterile water, and mix well - 42 ° C water bath for 1 hour - 95 ° C for 10 minutes - ice for 10 minutes - low speed centrifugation, shake and mix Slow centrifugation again

- placed on ice or frozen at -20 ° C Example 2: TaqMan® instant polymerase chain reaction to put the reversely transcribed hepatitis C cDNA 1 pL with the prepared polymerase chain reaction reagent 24 μΐ^ into 96 In the well plate, the 96-well plate was placed in a ΑΒΙ® 7900 machine, and the hydrolysis probe was used to detect the fluorescence of the polymerase chain reaction and quantify the fluorescence value. The steps are detailed as follows: Step 1. Take a tube of standard (101G virus / microliter) or 10 times serial dilution with the standard plastid shown in Figure 1 (1〇8, 1〇6, 1〇4) 1, 2, 101 viruses / microliters); Step 2: Prepare reagents and consumables, PCR reagents are ABI TaqMan® PCR Core Reagents Kit, Cat· No. N808-0228; take 13.125 pL sterile water, 3.5 μL 25 mM MgCl2, 2_5 μί A buffer, each 0_25 pL dATP/dUTP/dGTP/dCTP, 0.125 pL enzyme, 0.25 pL UNG, 1.5 gL 10 μΜ with an upstream primer of sequence shown in sequence number 1, 1.5 pL 10 μΜ a downstream primer having a sequence as shown in SEQ ID NO: 2, a 0.5 μί 10 μΜ 5' end having a FAM fluorophore and having a sequence of the sequence shown in SEQ ID NO: 7 and 1 pL of cDNA mixed in a 96-well reaction vessel; The internal quality control probe is internally controlled by the internal control mass shown in Figure 2 and the VIC fluorescent base at the 5' end and having the identification number 13 of the 94203.doc • 17-1295321 column identification number; Optical adhesive cover Part number 4311971 Covered with a uniform heating plate, placed ABI8 79〇〇 machine (a well should be placed in the upper left corner); Step 4. The machine has a polymerase chain reaction condition of 5 〇 (>c 2 minutes, % 〇c 10 minutes, then 50 cycles of 95 〇c 15 seconds, 6〇γ丨 minutes; Step 5. Complete the reading of the data, remove the plate and discard the machine power off and then turn off the computer; Step 6. Dilute the standard concentration of the known concentration, concentration It is 1 〇 8 to 1 〇 1 virus / microliter, according to the standard product (^ value data can be extracted from the standard curve, the results are not shown in Figure 3, and its internal quality control detection diagram is shown in Figure 4; The Ct value is obtained and compared with the standard curve to know the concentration. Example 3: Hybrid probe instant polymerase chain reaction The reverse transcription of the hepatitis C cDNA 2 pL and the ready-to-use polymerase chain reaction reagent Add 18 μΕ to a glass capillaries, mix it by centrifugation, put it into a LightCycler® machine for PCR, and use a hybrid probe to replicate the polymerase chain reaction and instantly detect the fluorescence value and quantify it. Step 1 · Take one tube of standard (1〇10 virus/μl) or standard The plastid is diluted 1 fold (1〇8, 1〇6, 1〇4, 1〇2, 101 viruses/μl); Step 2. Prepare the Centrifuge adapter, capillaries and reagents, Take 12.96 jliL sterile water, 1·6 μΐ^ 25 mM MgCl2, 0.5 is called 2〇μΜ. The upstream primer with the sequence shown in sequence identification number 4, 0.5 20 μΜ, the downstream primer with the sequence shown in sequence identification number 5, 0.22 pL 20 μΜ 94203.doc -18- 1295321 5f-end anchor probe with LC Red 640 fluorophore and sequence as shown in sequence identification number 1〇, 0.22 μί 20 μΜ 3, end with FL fluorophore and with The sensing probe of the sequence of sequence identification number 9 and the enzyme called 2 are formulated into reagents; together with the internal control plastid having the sequence shown in sequence identification number 12, the sensing probe and the 5' end LC Red 720 fluorescent base and internal control anchor probe with sequence as shown in sequence identification number 15 for internal quality inspection; Step 3: Take the required number of capillary placement centrifugal adapters in order, take 18 pL The reagent is added to the capillary, and the standard is 2 The product is sequentially added to the capillary tube, and the 2 吣 sample (c hepatitis C cna) is attached to the capillary after the standard is added; Step 4. Cap the capillary and centrifuge 3 rpm for 3 sec. The officer placed the turntable (LCcarousel) in turn, and put the turntable into the LightCycler® machine; Step 5·On the machine, the polymerase chain reaction condition was 95 () (: 1 〇 minutes, then 50 cycles 95 〇 C 5 seconds, 62 γ 15 seconds, 72. 〇 13 seconds, then cool down to 40QC for 30 seconds; Step 6. Complete the reading of the data, remove the turntable to discard the capillary, turn off the power of the machine, and then turn off the computer; Step 7. Dilute the standard concentration of the known concentration, concentration It is 1〇, 1〇1 and sick mother/3⁄4 liter. According to the Ct value data obtained from the standard product, the standard curve can be extracted. The results are shown in Figure 5, and the internal quality control debt test chart is shown in Figure 6. The q value of the sample is compared with the standard curve to know its concentration. Example 4: Correspondence between standard serum and immediate polymerase chain reaction 94203.doc -19- 1295321 The correspondence between the standard serum ordered by the World Health Organization (WHO) and the quantitative result obtained by the method according to the present invention is shown in Table 1, and The conversion equation is shown in Figure 7. Table 1: Standard serum (IU) log-instant polymerase chain reaction (replica number) logarithm 50000 4.69897 20760 4.317227 12500 4.09691 3067.6 3.486799 3125 3.49485 431.76 2.635242 781.25 2.89279 30.56 1.485153 The above examples are merely illustrative of the principles and efficacy of the invention, and The invention is not limited. Modifications and variations of the embodiments described above will be apparent to those skilled in the art without departing from the spirit of the invention. The scope of the invention should be as set forth in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic view showing the construction of a standard mass body of the present invention. Fig. 2 is a schematic view showing the construction of the internal control mass of the present invention. Figure 3 is a standard curve of the TaqMan® instant polymerase chain reaction. Figure 4 shows an internal quality control map with a TaqMan® instant polymerase chain reaction. Figure 5 is a standard curve of an instant polymerase chain reaction with a hybrid probe. Figure 6 is an internal quality tube detection map of a hybrid polymerase chain reaction with a hybrid probe. Figure 7 is a graph showing the correspondence between the standard serum and the instant polymerase chain reaction. 94203.doc -20- 1295321 Sequence Listing <110> National Cheng Kung University <120> Quantitative Hepatitis C Virus Initiator, Set and Method <130>None<160> 16 <170> Patentln version 3.2 <;210> 1 <211> 17 <212> DNA <213> Artificial sequence <400> 1 cgtgcagcct ccaggac 17 <210> 2 <211> 21 <212>DN A <213> Artificial sequence <400> 2 tggcaattcc ggtgtactca c 21 <210> 3 <211> 21 <212> DNA <213> Artificial sequence <400> 3 gtgagtacac cggaattgcc a 21 <210> 4 <211> 26 <;212> DNA <213> Artificial sequence <400> 4 ccctgtgagg aactactgtc ttcacg 26 <210> 5 <211> 19 <212> DNA 94203.doc 1295321 <213> Artificial sequence <400> 5 ggtgtactca Ccggttccg 19 <210>6 <211> 19 <212> DNA <213> Artificial sequence <400>6 cggaaccggt gagtacacc 18 <210>7 <211> 17 <212> DNA <213> Artificial sequence <400〉 7 accactatgg ctctccc 17 <210〉 8 <211> 17 <212>DNA ≪ 213 > artificial sequence <400> 8 gggagagcca tagtggt 17 <210> 9 <211> 19 <212> DNA <213> Artificial sequence <400> 9 gcagcctcca ggacccccc 19 <210> 10 <211> 23 <212> DNA <213>Artificial sequence<400> 10 cccgggagag ccatagtggt ctg 23 94203.doc -2- 1295321 <210> 11 <211> 22 <212> DNA <213> Sequence <400> 11 ccgggagagc catagtggtc tg 22 <210> 12 <211> 244 <212> DNA <213> Artificial sequence <400> 12 actccaccat agatcactcc cctgtgagga actactgtct tcacgcagaa agcgtctagc 60 catggcgtta gtatgagtgt cgtgcagcct ccaggacccc ccctcttgct gaagcttctg 120 Actacgactc ggaaccggtg agtacaccgg aattgccagg acgaccgggt cctttcttgg 180 atcaacccgc tcaatgcctg gagatttggg cgtgcccccg cgagactgct agccgagtag 240 tgtt 244 <210> 13 <211> 17 <212> DNA <213>Artificial sequence<400> 13 ctcttgctga agcttct 17 <210> 14 <211> 377 <212> DNA <213>Artificial sequence<400> 14 actccaccat agatcactcc c ctgtgagga actactgtct tcacgcagaa agcgtctagc 60 catggcgtta gtatgagtgt cgtgcagcct ccaggtcccc ccctcccggg agagccatag 120 tggtctgcgg aaccggtgag tacaccggaa ttgccaggac gaccgggtcc cttcttggat 180 caacccgctc aatgcctgga gatttgggcg tgcccccgcg agaccgctag ccgagtagtg 240 ttgggtcgcg agaggccttg tggtactgcc tgatagggtg cttgcgagtg ccccgggagg 300 94203.doc 1295321 tctcgtagac cgtgcatcat gagcacaaat cctaaacctc aaagaaaaac caaacgtaac accaaccgcc gcccaca < 210 > 15 <211> 25 <212> DNA <213> Artificial sequence <400> 15 cttgctgaag cttctgacta cgact <210> 16 <211> 20 <212> DNA <213> Artificial sequence <400> 16 Aacactactc ggctagcag 4- 94203.doc

Claims (1)

, 曰修(more) 正本I29发益36949会翁蠢 [Year, I Chinese patent application scope replaced by $6, ten, patent application scope: 1. A primer for quantifying hepatitis C virus, which can be enlarged Hepatitis C virus CDNA, the primer is selected from the group consisting of an upstream primer having one of the sequences shown in SEQ ID NO: 1, an upstream primer having a sequence as shown in SEQ ID NO: 4, and having sequence recognition a downstream primer of one of the numbered ice sequences, a downstream primer having one of the sequences as shown in sequence identification number 3, a downstream primer having one of the sequences as shown in sequence identification number 5, and a downstream primer having one of the sequences as shown in sequence identification number 6. . 2. A pair of primers for quantifying hepatitis C virus, which can be amplified. Hepatitis virus cDNA, the primer pair is selected from the group consisting of the following primer pairs: (a) has an upstream primer as shown in sequence identification number 1 and has one of the sequences shown as sequence identification number 2 or 3. a downstream primer; and (b) an upstream primer having one of the sequences shown in Sequence Identification No. 4 and a downstream primer having a sequence as shown in Sequence Identification No. 5 or 6. 3. A kit for quantifying hepatitis C virus, comprising a primer pair as claimed in claim 1 or a primer pair in claim 2. 4. The kit according to claim 3, further comprising a target probe, wherein the target probe is a heterozygous hepatitis C virus cDna fragment amplified by the primer or the primer pair, and the 5 and 3' ends respectively have a first reporter and a first quencher. 5. 5. The kit according to claim 4, having the pair of primers as claimed in item (4), and the target needle having the sequence identification number 7 or 8 The sequence. 6. The kit according to claim 4, further comprising - an internal control plastid having an insert, the insert package 94203oal replacement version. d〇c 129 issue 136949 patent application Chinese patent application scope replacement (September 96) Hepatitis C virus CDNA fragment amplified by a primer pair or a primer pair of claim 2, which has a similarity to a hepatitis C virus cDNA or a human genome sequence. And an internal control probe, which can be hybridized with the insert of the internal control plastid, and has a second reporter and a second quencher at the 5' and 3' ends, respectively, and the reporter and the reporter The first reporter is different. 7. The kit of claim 6, wherein the insert has a sequence as set forth in Sequence Identification No. 12 or its complementary strand, and the internal control probe has a sequence as set forth in Sequence Identification Number 13 or its complementary strand. 8. The kit according to claim 3, further comprising a sensing probe and a certain anchor probe, wherein the third end of the sensing probe has a first donor fluorescent light, and the 5' end of the fixed anchor probe has a first receptor is fluorescent, and the sensing probe and the anchoring probe are respectively hybridized with the amplified hepatitis C virus cDNA fragment of the primer or the primer, and when the anchor probe and the sensing probe are simultaneously The energy of the first donor fluorescence can be transferred to the first receptor fluorescence when hybridized to the amplified hepatitis C virus 〇〇1^8 fragment, such as the primer of the request item or the primer of the request item 2. . 9. The set according to claim 8 having a pair of primers as claimed in (1) of claim 2, and having the sequence of sequence identification number 9 or its complementary strands and the anchoring The needle has a sequence as indicated by the sequence identification number 丨〇 or 丨丨 or its complementary strand. 1 . The kit of claim 8 further comprising an internal control plastid having an insert comprising a fragment that is non-similar to the hepatitis C virus cDNA or to the human genome sequence Replacing the version of the patent application by the introduction of the request item 1 or the introduction of the item 2, the code of the doc -2- Ι29@31ΐ36949 Patent Application Replacement (September 96) The amplified hepatitis C virus cDNA fragment And an internal control (four) needle having a second receptor fluorescence at the 5' end and hybridized with the insert of the internal control plastid, and when the internal error probe is hybridized to the induction probe simultaneously When the plastid is internally controlled, the energy of the first donor fluorescent light can be transferred to the second acceptor fluorophore. η. The kit according to claim 10, wherein the insert has a sequence as shown in sequence identification number 12 or its complementary strand, and the internal control error-determining probe has a sequence as shown in sequence identification number 15 or its complementary strand sequence. 12. The kit according to claim 3, further comprising - a standard plastid, wherein the standard plastid has - an additional fragment - the additional fragment comprises a hepatitis C virus cDNA fragment amplified by the primer or primer pair. 13. The sequence according to claim 12: i1, whistle, and wherein the additional segment has a sequence as shown by sequence identification number 14 or its complementary strand. 14. A method for quantifying hepatitis C virus &<>> using an immediate polymerase chain reaction using a primer such as a request or a primer pair of claim 2. 15. The method of claim 14, comprising the steps of: (1) using a standard plastid of different concentrations, the standard plastid having an additional fragment, the additional fragment = being derived from the request item a or the claim 2 The amplified hepatitis C virus sac A fragment is introduced, and the target probe is hybridized with the amplified hepatitis C virus cDNA fragment of the primer or the primer, and the target probe 5, and 3, respectively Having a -the first reporter and a first quencher; (ϋ) in the measurement step (1) in the standard plastid reaction at different concentrations, the first report 94203oal replacement version. d〇c 129 ¥ along the 136949 patent application The scope of the patent application is replaced by this (September 1996) y y ΐ 'to obtain a standard plastid concentration versus the first reported dose standard curve; (iii) to obtain cDNA derived from the sample of hepatitis C virus; (IV) Performing an instant polymerase chain reaction with the primer or primer pair, the target probe, and the cDNA in step (iii); (V) the intensity of the first reporter in the step (1V), and in step (ii) Compare the standard curve of the sample to quantify the cDN in the sample The concentration of A. 16. The method according to claim 15, wherein the sample of step (4) is derived from blood. 17. The method according to claim 16, wherein the sample is derived from serum or plasma. 18. The method according to claim 15, which has the (4) primer pair as in claim 2, and the target probe has a sequence as shown in sequence identification number 7 or 8. 19. The method of claim 15, wherein the additional fragment has a sequence as shown by sequence identification number 14 or its complementary strand. 20. In accordance with the method of claim 15, further comprising an internal control step of adding an internal control mass and an internal control probe, and performing step (iv) & with the hepatitis C virus cDNA and the target probe ( v), wherein the internal control plastid has an insert comprising a fragment that is replaced with a hepatitis C virus cDNA or a fragment having no similarity to a human genome sequence, such as the primer of claim 1 or claim 2 The amplified c-type hepatitis C virus cDNA fragment, and the internal control probe may be heterozygous with the insert of the internal control plastid, and the 5th and 3rd ends respectively have a second reporter and a a second quencher, and the second reporter is different from the first reporter. The method according to claim 20, wherein the insert has a sequence as shown in sequence identification number 12 or its complementary strand, and the internal control probe has a 94203oal replacement version. doc 1293⁄43⁄4^36949 Patent Application Chinese Patent Application Range Replacement (September 1996) Sequence as indicated by Sequence Identification Number 13 or its complementary strand. 22. The method of claim 14, comprising the steps of: (1) performing a heterozygous reaction with a sensing probe and a certain anchor probe with standard plasmons of different velocities; the standard plastid having an additional fragment, The additional fragment comprises a hepatitis C virus cDNA fragment amplified by a primer pair such as the primer of claim 1 or the primer of claim 2; the sensor probe 3 has a first donor fluorescent light, and the anchor probe 5, the end has a first stylistic fluorescence. The sensing probe and the anchoring probe can be hybridized with the amplified hepatitis C virus 片段 into the fragment, respectively, and the anchor When the probe and the sensing probe are simultaneously hybridized to the amplified hepatitis C virus cDNA fragment of the primer or the primer pair, the energy of the first donor fluorescent light can be transferred to the first receptor fluorescent light; In the measuring step (1), the amount of the first receptor fluorescence in the standard plastid reaction at different concentrations is used to obtain a standard curve of the standard plastid concentration to the first receptor fluorescence amount; (iii) obtaining the type C derived from the sample. Hepatitis virus 2CDNA; (1^) with the primer or primer pair, the sensing probe, the anchor probe, and The cDNA in 丨(1) is subjected to an instant polymerase chain reaction; (v) the intensity of the receptor fluorescence in the step (iv) is measured and compared with the standard curve obtained in the step (Η) to quantify the cDNA in the sample. The method of claim 22, wherein the sample of step (iii) is derived from blood. 24. The method according to claim 23, wherein the sample is derived from serum or plasma. a method having a pair of primers as in item (b) of claim 2, the probe having a sequence of 94203oal as shown in sequence identification number 9 or its complementary strands. doc -5 - I29@ (34i36949 Patent Application Chinese The scope of the patent application is replaced by this (September 1996) column 'and the anchor probe has a sequence as indicated by the sequence identification number 丨〇 or 丨丨 or its complementary strand. 26) The method according to claim 22, wherein The additional fragment has a sequence as shown by sequence identification number 14 or its complementary strands. 27. The method of claim 22, further comprising an internal control step of adding an internal control mass and an internal control anchor probe, Hepatitis C virus cDNA Anchoring the probe to perform a step of ambiguity; wherein the internal control plastid has an insert comprising a fragment such as a request for replacement with a hepatitis C virus cDNA or a fragment having no similarity to a human genome sequence The amplified hepatitis C virus cDNA fragment of the primer of claim 1 or the primer of claim 2; the 5th end of the internal control anchor probe has a second receptor fluorescence and can be interspersed with the insert of the internal control plastid And, when the internal anchor probe and the sensing probe are simultaneously hybridized to the internal plastid, the energy of the first donor fluorescent light can be transferred to the second acceptor fluorescent. 2 8 · According to claim 2 The method of 7 wherein the insert has a sequence as shown in sequence identification number 12 or its complementary strand and the internal control anchor probe has a sequence identification number of 15 or its complementary strand The sequence shown. 29. A method of quantifying (3) a hepatitis virus, the money of which is subjected to an instant polymerase chain reaction according to any of the sets of claims 3 to 13. 94203oal replacement version.d〇c 1295321 VII. Designated representative map: (1) The representative representative of the case is: (7). (2) A brief description of the symbol of the symbol of the representative figure: (No description of the symbol of the component) 8. If there is a chemical formula in this case, please disclose the chemical formula that best shows the characteristics of the invention: (none) 94203.doc