CN117947214A - Composition for detecting hepatitis C and parting, kit and application thereof - Google Patents
Composition for detecting hepatitis C and parting, kit and application thereof Download PDFInfo
- Publication number
- CN117947214A CN117947214A CN202410073604.2A CN202410073604A CN117947214A CN 117947214 A CN117947214 A CN 117947214A CN 202410073604 A CN202410073604 A CN 202410073604A CN 117947214 A CN117947214 A CN 117947214A
- Authority
- CN
- China
- Prior art keywords
- hcv
- composition
- detecting
- probe
- hepatitis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 52
- 208000005176 Hepatitis C Diseases 0.000 title description 8
- 238000001514 detection method Methods 0.000 claims abstract description 24
- 239000000523 sample Substances 0.000 claims description 62
- 108020004707 nucleic acids Proteins 0.000 claims description 15
- 150000007523 nucleic acids Chemical class 0.000 claims description 15
- 102000039446 nucleic acids Human genes 0.000 claims description 15
- 125000006853 reporter group Chemical group 0.000 claims description 15
- 238000011144 upstream manufacturing Methods 0.000 claims description 13
- 208000006454 hepatitis Diseases 0.000 claims description 12
- 231100000283 hepatitis Toxicity 0.000 claims description 12
- 238000003753 real-time PCR Methods 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 230000003321 amplification Effects 0.000 claims description 6
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 4
- SGTNSNPWRIOYBX-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-{[2-(3,4-dimethoxyphenyl)ethyl](methyl)amino}-2-(propan-2-yl)pentanenitrile Chemical group C1=C(OC)C(OC)=CC=C1CCN(C)CCCC(C#N)(C(C)C)C1=CC=C(OC)C(OC)=C1 SGTNSNPWRIOYBX-UHFFFAOYSA-N 0.000 claims description 3
- 238000010791 quenching Methods 0.000 claims description 2
- 230000000171 quenching effect Effects 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 claims 1
- 238000000605 extraction Methods 0.000 claims 1
- 241000711549 Hepacivirus C Species 0.000 abstract description 74
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 238000000034 method Methods 0.000 abstract description 7
- 238000007689 inspection Methods 0.000 abstract description 4
- 238000010222 PCR analysis Methods 0.000 abstract description 2
- 241000711557 Hepacivirus Species 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 15
- 108090000790 Enzymes Proteins 0.000 description 15
- 238000011282 treatment Methods 0.000 description 11
- 239000002299 complementary DNA Substances 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 244000052769 pathogen Species 0.000 description 4
- 238000003908 quality control method Methods 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- 102100034343 Integrase Human genes 0.000 description 3
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 206010061818 Disease progression Diseases 0.000 description 2
- 239000003443 antiviral agent Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 230000005750 disease progression Effects 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000011895 specific detection Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 description 1
- WNDDWSAHNYBXKY-UHFFFAOYSA-N ATTO 425-2 Chemical compound CC1CC(C)(C)N(CCCC(O)=O)C2=C1C=C1C=C(C(=O)OCC)C(=O)OC1=C2 WNDDWSAHNYBXKY-UHFFFAOYSA-N 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 241000710781 Flaviviridae Species 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 241000709721 Hepatovirus A Species 0.000 description 1
- 241000700588 Human alphaherpesvirus 1 Species 0.000 description 1
- 241000701074 Human alphaherpesvirus 2 Species 0.000 description 1
- 241000701024 Human betaherpesvirus 5 Species 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- 241000712431 Influenza A virus Species 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 241001112090 Pseudovirus Species 0.000 description 1
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 241000589884 Treponema pallidum Species 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229940095731 candida albicans Drugs 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 238000011284 combination treatment Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 238000007403 mPCR Methods 0.000 description 1
- -1 mgCl 2 Chemical compound 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 229960000329 ribavirin Drugs 0.000 description 1
- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 230000009265 virologic response Effects 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of molecular biology detection, and particularly relates to a composition and application for detecting and distinguishing hepatitis C viruses HCV 4, HCV 1b, HCV 1, HCV 2 and HCV 3. The joint inspection composition provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different sites and detect the hepatitis C virus, so that the detection and the distinction of HCV 4, HCV 1b, HCV 1, HCV 2 and HCV 3 are simultaneously realized in a single-tube reaction system. Meanwhile, the composition has higher detection sensitivity reaching 400 copies/mL and more accurate detection.
Description
Technical Field
The invention belongs to the field of molecular biology detection, and particularly relates to a composition and application for detecting and distinguishing hepatitis C HCV 4, HCV 1b, HCV 1, HCV 2 and HCV 3.
Background
Hepatitis C Virus (HCV) is a single-stranded positive strand RNA virus with an envelope, and is classified into the Flaviviridae, the genus hepatitis virus. As a globally circulating virus, HCV infection is over 7000 ten thousand, and at present, HCV viruses are divided into 6 main genotypes and more than 80 subtypes, and the difference of nucleic acids between different genotypes can reach 30%. Because of the different genotypes and subtypes respond differently to treatment, accurate typing of HCV is of great importance for the formulation of treatment regimens.
The most common types of HCV viruses are types 1,2 and 3, with different types having different epidemic conditions in different regions. Currently, HCV type 1 is the most common type worldwide, accounting for about 46% of total infected people, followed by HCV 3 (22%), HCV 2 (13%) and HCV type 4 (13%), and in addition, other unusual HCV genotypes, including HCV types 5, 6 and 7, 8, 9 are novel, mainly found in lower industrialized countries (india, southeast asia and middle east countries). In China, the most common are HCV 1 and 2 genotypes, with type 1b being the dominant (56.8%).
Currently, the treatment of hepatitis c is mainly dependent on antiviral Drugs (DAAs), while HCV of different genotypes has a significant difference in the efficacy of therapeutic drugs. For example, DAAs is less effective in HCV type 1 and type 4 patients, while type 2 and type 3 drugs are better. Furthermore, the significance of the different HCV subtypes for guiding subsequent treatments is mainly represented by the following points: 1. the sensitivity of the different subtypes to drug treatment is different. For example, type 2 and type 3 antiviral drugs have better therapeutic effects. Combination treatment with pegylated interferon and ribavirin resulted in sustained virologic responses in 80% of untreated HCV type 2/3 non-liver cirrhosis patients, whereas this proportion of type 1 patients was approximately 50%; the patients of type 2 or 3 hepatitis C virus can obtain the maximum therapeutic benefit only by 6 months of treatment, and the patients of type 1 can be treated by 12 months. For these reasons, it is necessary to identify the hepatitis C virus gene in all infected patients who are considered to be receiving antiviral treatment. 2. The different subtypes are related to the rate and severity of disease progression. Some studies have found that the disease progression is faster for genotype 1b and 3a, and relatively slower for genotype 2 and 4. 3. The different subtypes are associated with risk of recurrence after treatment. Studies have shown that genotypes 1a, 1b and 3a have a higher risk of recurrence, while genotypes 2 and 4 have a lower risk of recurrence. Since HCV is different in response to treatment, and is closely related to the expected response and duration of treatment, understanding HCV typing is of great importance for the therapeutic effect of hepatitis c.
Therefore, there is a need in the art for a product that can simply and rapidly detect the above pathogens, has high sensitivity and good specificity, provides a basis for a clinician to diagnose and eliminate infection by different pathogens more fully and rapidly, shortens the time for the clinician to diagnose the disease condition of the patient, and accelerates the implementation of therapeutic measures to the patient.
Disclosure of Invention
In view of this, in a first aspect, the present invention provides a composition for detecting and typing of hepatitis c comprising:
An upstream primer and a downstream primer and a probe for detecting HCV 1b as shown in SEQ ID NO. 1-3;
An upstream primer and a downstream primer for detecting the HCV 1 probe as shown in SEQ ID NO. 4-6;
an upstream primer and a downstream primer and a probe for detecting HCV 2 as shown in SEQ ID NO. 7-9;
An upstream primer and a downstream primer and a probe for detecting HCV 3 as shown in SEQ ID NO. 10-12; and
The upstream and downstream primers and probes for detecting HCV 4 are shown as SEQ ID NOS.13-15.
The joint inspection composition provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect and type the hepatitis C by detecting different sites, so that the detection and the distinction of HCV 4, HCV 1b, HCV 1, HCV 2 and HCV 3 are realized in a single-tube reaction system at the same time, and a targeted strategy is provided for subsequent treatment. Meanwhile, the composition has higher detection sensitivity reaching 400 copies/mL, is more accurate in detection, provides a relatively sufficient and rapid diagnosis basis for a clinician, shortens the time of diagnosing the disease condition of a patient for the clinician, and quickens the implementation of treatment measures for the patient.
Further, the composition includes an upstream and downstream primer and a probe for detecting an internal standard.
In some specific embodiments, the internal standard is a human internal standard gene. In a specific embodiment, the internal standard is a human housekeeping gene.
Further, the fluorophores of the probes of the compositions of the invention are different from each other and do not interfere with each other.
As used herein, "distinct and non-interfering with each other" means that the fluorophores used for each probe in the composition are different and do not affect each other's detection, i.e., can be performed using different channels. For example, ATTO 425, quasar705, FAM, HEX, ROX, CY, CY5.5, etc. can be used, and these groups do not have close absorbance values, and different channels can be selected, so that they do not interfere with each other.
Further, in some embodiments, the compositions of the present invention may include one or more of the above-described primer and probe pairs simultaneously. In the present invention, "pair" refers to matched upstream and downstream primers and probes that detect a target.
The compositions of the invention can be combined in any combination to detect 5 targets. Those skilled in the art can combine the primers and probe pairs as necessary to detect which targets are the corresponding targets. These combinations are included in the present invention.
For example, any 4 pairs of the above 5 pairs of primers and probes may be included, any 3 pairs of the above 5 pairs of primers and probes may be included, any 2 pairs of the above 5 pairs of primers and probes may be included, or any 1 pair of the above 5 pairs of primers and probes may be included.
In some specific embodiments, the compositions of the invention are used in fluorescent PCR.
In a specific embodiment, the fluorescent reporter group of the HCV 1b probe is FAM; the fluorescence reporter group of the HCV 1 probe is CY5; the fluorescent reporter group of the HCV 2 probe is Quasar 705; the fluorescent reporter group of the probe of HCV 3 is HEX; the fluorescent reporter group of the HCV 4 probe is ROX.
Further, the 3' end of the probe also has a non-fluorescent quencher.
Further, the 3' -end of the probe also has a quenching group, such as MGB, BHQ1 or BHQ2.
In a specific embodiment, the 3' end of the probe is an MGB.
Further, the amount of the primer in the composition is 0.2 to 0.5. Mu.M; the amount of probe in the composition is 0.1 to 0.2. Mu.M.
In a particular embodiment, the ingredients of the composition of the invention are present in separate packages.
In a particular embodiment, the ingredients of the composition of the invention are present in the same package.
Further, the components of the composition of the present invention are present in a mixed form.
In a second aspect, the invention provides the use of the composition of the invention described above for the preparation of a kit for detecting and typing and distinguishing hepatitis C, wherein the type of hepatitis C is HCV 4, HCV 1b, HCV 1, HCV 2, HCV 3.
In a third aspect, the present invention provides a kit for detecting and typing and distinguishing hepatitis C, the kit comprising a composition of the invention as described above.
Further, the kit also comprises a negative quality control and a positive quality control.
In a specific embodiment, the negative quality control is at least one of DEPC H 2 O, physiological saline, and an internal standard gene. The positive quality control agent is at least one of pseudovirus or fragment genes of HCV 4, HCV 1b, HCV 1, HCV 2 and HCV 3.
Further, the kit further comprises nucleic acid amplification reagents.
Still further, the amplification reagents include at least one of dNTPs, PCR buffer, reverse transcriptase, DNA polymerase, and Mg 2+.
Still further, the kit further comprises: nucleic acid releasing reagent and nucleic acid extracting reagent.
Further, the concentration of the DNA polymerase is 3U/reaction to 15U/reaction, for example, the DNA polymerase may be Taq enzyme. The concentration of the reverse transcriptase is 0.1U/reaction to 5U/reaction. For example, the reverse transcriptase may be a Tth enzyme.
In a specific embodiment, the kit of the invention comprises: taq enzyme, mg 2+、Mn2+, dNTPs, primers, probes and PCR buffer.
Common PCR buffers consist of Tris-HCl, mgCl 2, KCl, triton X-100 and other buffer systems. The total volume in a typical single PCR reaction tube is 20. Mu.l to 200. Mu.l.
In a fourth aspect, there is provided a method for detecting and typing hepatitis c, the method comprising the steps of:
1) Extracting nucleic acid of a sample to be detected;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;
3) The results were obtained and analyzed.
In the present invention, the sample for detection may be serum, blood, or the like, but is not limited thereto.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
Activating enzyme at 90-99 deg.c for 5-120 sec for 1 circulation; reverse transcription of cDNA at 50-65 deg.c for 20-40 min and 1 circulation; the cDNA is pre-denatured, the temperature is 90-99 ℃, the time is 5-120 seconds, and 1 cycle is performed; denaturation at 95 ℃ for 5-20 seconds, annealing at 55-60 ℃ for 10-60 seconds, 30-50 cycles, and fluorescence collection.
In a specific embodiment, a method for detecting and typing hepatitis c for non-diagnostic purposes is provided, the method comprising the steps of:
1) Extracting nucleic acid of a sample to be detected;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;
3) The results were obtained and analyzed.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
Activating enzyme at 90-99 deg.c for 5-120 sec for 1 circulation; reverse transcription of cDNA at 50-65 deg.c for 20-40 min and 1 circulation; the cDNA is pre-denatured, the temperature is 90-99 ℃, the time is 5-120 seconds, and 1 cycle is performed; denaturation at 95 ℃ for 5-20 seconds, annealing at 55-60 ℃ for 10-60 seconds, 30-50 cycles, and fluorescence collection.
In a specific embodiment, there is provided the use of a composition for the preparation of a composition for detecting and typing of hepatitis c, said use detecting the steps of:
1) Extracting nucleic acid of a sample to be detected;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;
3) The results were obtained and analyzed.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
Activating enzyme at 90-99 deg.c for 5-120 sec for 1 circulation; reverse transcription of cDNA at 50-65 deg.c for 20-40 min and 1 circulation; the cDNA is pre-denatured, the temperature is 90-99 ℃, the time is 5-120 seconds, and 1 cycle is performed; denaturation at 95 ℃ for 5-20 seconds, annealing at 55-60 ℃ for 10-60 seconds, 30-50 cycles, and fluorescence collection.
Drawings
FIG. 1 is a graph showing the detection results of the composition of the present invention;
FIG. 2 is a graph showing the sensitivity test results of the composition of the present invention;
FIG. 3 is a graph showing the results of specific detection of the composition of the present invention;
FIG. 4 is a graph showing the results of the triple test of comparative example 1 of the present invention;
FIG. 5 is a graph showing the results of five-joint inspection of comparative example 1 of the present invention;
FIG. 6 is a graph showing the results of the triple test of comparative example 2 of the present invention;
FIG. 7 is a graph showing the results of five-joint inspection in comparative example 2 of the present invention.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Example 1, primers and probes used in the present invention
TABLE 1
Wherein, the fluorescence reporter group of the HCV 1b probe is FAM; the fluorescence reporter group of the HCV 1 probe is CY5; the fluorescent reporter group of the HCV 2 probe is Quasar 705; the fluorescent reporter group of the probe of HCV 3 is HEX; the fluorescent reporter group of the HCV 4 probe is ROX.
Example 2 method for detecting and typing hepatitis C
The detection sample of the invention is serum and blood. The real-time fluorescent PCR reaction system was configured as follows in table 2:
TABLE 2
| The components | Volume/concentration in each reaction |
| PCR buffer | 32.6μL |
| Primer (50 pmol/. Mu.L) | 0.20μL |
| Probe (50 pmol/. Mu.L) | 0.10μL |
| dNTPs | 1.2μL |
| Mn2+ | 1.2μL |
Preparing an enzyme mixed solution:
the enzyme mixed solution consists of Tth enzyme and Taq enzyme. Tth enzyme (5U/. Mu.L) and Taq enzyme (5U/. Mu.L) were mixed in a certain ratio (2. Mu.L of Tth enzyme per person was mixed with 0.5. Mu.L of Taq enzyme). The enzyme mixture was used in an amount of 2.5. Mu.L per person.
The PCR amplification procedure was set up as follows table 3:
TABLE 3 Table 3
If FAM, CY5, quasar705, HEX and ROX channels have obvious S-shaped amplification curves and Ct value is less than or equal to 38, judging positive; if FAM, CY5, quasar705, HEX and ROX channels have No amplification curve (No Ct) or Ct value > 38, then the result is judged as negative; specifically, the results are shown in Table 4.
TABLE 4 interpretation of test results
Example 3 detection results of test samples of the inventive composition
The primers and probes shown in example 1 were used to verify samples of HCV 4, HCV 1b, HCV 1, HCV 2 and HCV 3 in the same manner as in example 2, and the results showed that HCV 4, HCV 1b, HCV 1, HCV 2 and HCV 3 of the hepatitis C virus could be detected and distinguished, and the detection results are shown in FIG. 1.
Example 4 sensitivity of the composition of the invention
Using the composition of example 1 of the present invention, LOD (sensitivity) detection was performed on each target to simulate a clinical sample, and multiplex PCR detection was performed on a fluorescent quantitative PCR instrument. The results are shown in Table 5 and FIG. 2, and demonstrate that each channel can still be accurately detected for samples as low as 400 copies/mL, indicating a sensitivity of 400 copies/mL for the compositions of the present invention.
TABLE 5
EXAMPLE 5 specificity of the composition of the invention
The composition of the invention has no cross reaction with other pathogens (such as hepatitis B virus, human cytomegalovirus, hepatitis A virus, EB virus, treponema pallidum, herpes simplex virus type 1, herpes simplex virus type 2, staphylococcus aureus, candida albicans, human immunodeficiency virus, influenza A virus and the like) with similar infection symptoms. The results are shown in FIG. 3, which shows that the composition of the present invention has good specificity.
Comparative example 1, remaining poorly performing primers and probes designed according to the invention
Because of the base-pairing rules, dimers are formed between the primer and/or probe, but with little probability, this can be eliminated at the beginning of the design. However, when multiple pathogens are jointly detected, a plurality of primers and probes are arranged, dimers are easy to occur between the primers and the primers, between the probes and the probes or between the primers and the probes, so that the conservation of design (which is crucial to the accuracy of detection) is ensured, and the mutual interference among different primer probes is considered, so that the primer probes need to be carefully designed.
Therefore, the inventors have designed the other primers and probes (the sequences are shown in tables 6 and 7) to form different detection systems, and the detection systems are also used for detecting HCV 4, HCV 1b, HCV 1, HCV 2 and HCV 3, for example, specific detection results are shown in figures 4-7, and the amplification graphs of figures 4 and 5 show that the effect of HCV typing (triple detection) is better, but the phenomenon of low fluorescence signal and Ct trailing exists when five genotypes are combined; similarly, the detection performance of the combined 2HCV typing gene (five-joint detection) combined target is reduced compared with that of a three-joint detection combined part, and after Ct value is dragged, fluorescence increment is reduced.
TABLE 6
TABLE 7
Claims (10)
1.A composition for detecting and typing of hepatitis c comprising:
An upstream primer and a downstream primer and a probe for detecting HCV 1b as shown in SEQ ID NO. 1-3;
the upstream and downstream primers and probes for detecting HCV 1 are shown as SEQ ID NO. 4-6;
an upstream primer and a downstream primer and a probe for detecting HCV 2 as shown in SEQ ID NO. 7-9;
An upstream primer and a downstream primer and a probe for detecting HCV 3 as shown in SEQ ID NO. 10-12; and
The upstream and downstream primers and probes for detecting HCV 4 are shown as SEQ ID NOS.13-15.
2. The composition of claim 1, further comprising upstream and downstream primers and probes for detecting an internal standard.
3. The composition of claim 2, wherein the fluorophores of the composition probes are different from each other and do not interfere with each other.
4. The composition of claim 3, wherein the fluorescent reporter group of the HCV 1b probe is FAM; the fluorescence reporter group of the HCV 1 probe is CY5; the fluorescent reporter group of the HCV 2 probe is Quasar 705; the fluorescent reporter group of the probe of HCV 3 is HEX; the fluorescent reporter group of the HCV 4 probe is ROX.
5. The composition of claim 4, wherein the 3' end of the probe further has a quenching group.
6. The composition according to any one of claims 1 to 5, wherein the components of the composition are present in a mixed form.
7. Use of the composition of any one of claims 1-6 in the preparation of a kit for detecting and typing of hepatitis c, wherein the type of hepatitis c is HCV 4, HCV 1b, HCV 1, HCV 2, HCV 3.
8. A kit for detecting and typing of hepatitis c, the kit comprising a composition as claimed in any one of claims 1 to 6.
9. The kit of claim 8, further comprising at least one of a nucleic acid release reagent, a nucleic acid extraction reagent, and a nucleic acid amplification reagent.
10. Use of a composition for the preparation of a composition for detecting and typing and distinguishing hepatitis c, said detection comprising the steps of:
1) Extracting nucleic acid of a sample to be detected;
2) Performing a fluorescent quantitative PCR analysis on the nucleic acid obtained in step 1) using the composition of any one of claims 1 to 6 or the kit of claim 8 or 9;
3) The results were obtained and analyzed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410073604.2A CN117947214A (en) | 2024-01-18 | 2024-01-18 | Composition for detecting hepatitis C and parting, kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410073604.2A CN117947214A (en) | 2024-01-18 | 2024-01-18 | Composition for detecting hepatitis C and parting, kit and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117947214A true CN117947214A (en) | 2024-04-30 |
Family
ID=90791710
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410073604.2A Pending CN117947214A (en) | 2024-01-18 | 2024-01-18 | Composition for detecting hepatitis C and parting, kit and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117947214A (en) |
-
2024
- 2024-01-18 CN CN202410073604.2A patent/CN117947214A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114752711B (en) | Composition, kit and method for detecting and parting monkeypox virus and application thereof | |
| CN114369688B (en) | Compositions, kits, methods and uses for detecting a variant of SARS-CoV-2 Oncuronte | |
| CN113881812B (en) | Composition, kit and method for detecting SARS-CoV-2 mutant strain and use thereof | |
| CN113981152B (en) | Composition, kit and method for detecting SARS-CoV-2 variant strain and its use | |
| CN114410848B (en) | Composition, kit and method for detecting SARS-CoV-2 and application thereof | |
| WO2023279042A2 (en) | Compositions and methods for detection of severe acute respiratory syndrome coronavirus 2 variants | |
| CN116024386B (en) | Primer probe combination and kit for detecting novel coronaviruses and distinguishing Omicron different mutant strains | |
| CN116240312A (en) | Novel composition for combined detection of coronavirus, HIV and monkey pox virus | |
| CN111378787A (en) | Novel coronavirus detection method | |
| CN111100924B (en) | Quality control product for detecting repetition number of FMR1 gene CGG, application of quality control product and kit containing quality control product | |
| CN117947214A (en) | Composition for detecting hepatitis C and parting, kit and application thereof | |
| CN117106970A (en) | Composition, kit, method and application for detecting novel coronavirus | |
| CN115725794B (en) | Compositions, methods and uses for novel coronavirus and monkey pox virus joint detection | |
| CN115807129B (en) | Novel coronavirus and monkey pox virus joint inspection and typing composition | |
| CN116042918B (en) | Five virus joint inspection compositions, kit, method and application thereof | |
| CN115838836B (en) | Composition, kit, method and application of different types of virus joint inspection | |
| CN116516037A (en) | Composition and kit for detecting mycoplasma pneumoniae drug-resistant gene and application of composition and kit | |
| CN116411133A (en) | Compositions, methods and uses for novel coronavirus and monkey pox virus joint detection | |
| KR102499185B1 (en) | Composition comprising primers and probe for detecting COVID-19 by TaqMan real-time polymerase chain reaction | |
| CN117344044A (en) | Composition, kit and application for detecting pathogen related to lung infection | |
| CN116287436A (en) | Compositions, methods and uses for novel coronavirus and monkey pox virus joint detection | |
| CN118086584A (en) | Composition and kit for detecting neonatal enterovirus and application of composition and kit | |
| CN116732152A (en) | Composition, kit, method and application for detecting different types of interferons | |
| CN117802253A (en) | Composition, kit and application for detecting infection-related pathogens | |
| CN116042921A (en) | Composition, kit and method for detecting viral encephalitis virus and application of composition |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |