Composition and kit for detecting hepatitis c and typing same, and use thereof

Abstract

The present invention relates to a composition for detecting hepatitis C viruses HCV 4, HCV 1b, HCV 1, HCV 2 and HCV 3 and differentiating same, and the use thereof.

Description

Composition, kit and use thereof for detecting and typing hepatitis C

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the Chinese patent application with application number 202410073604.2, application date January 18, 2024, and invention name “Composition, kit and use for detecting and typing hepatitis C”, and claims the priority of these Chinese patent applications. The entire content of the Chinese patent application is hereby incorporated into this application by introduction. Technical Field

[0003] The present invention belongs to the field of molecular biological detection, and in particular, relates to a composition and use for detecting and distinguishing hepatitis C HCV 4, HCV 1b, HCV 1, HCV 2, and HCV 3. Background Art

[0004] Hepatitis C virus (HCV) is an enveloped, single-stranded, positive-sense RNA virus classified in the Flaviviridae family and the Hepacivirus genus. A globally prevalent virus, HCV infects over 70 million people. Currently, HCV is classified into six major genotypes and over 80 subtypes, with nucleic acid differences between genotypes reaching up to 30%. Because different genotypes and subtypes respond differently to treatment, accurate HCV typing is crucial for developing treatment plans.

[0005] The most common HCV genotypes are 1, 2, and 3, with different prevalence rates varying across different regions. Currently, HCV genotype 1 is the most common genotype globally, accounting for approximately 46% of all infections, followed by HCV 3 (22%), HCV 2 (13%), and HCV 4 (13%). Other less common HCV genotypes, including HCV genotypes 5 and 6, and newer genotypes 7, 8, and 9, are primarily found in less industrialized countries (India, Southeast Asia, and the Middle East). In my country, HCV genotypes 1 and 2 are the most prevalent, with genotype 1b being the predominant genotype (56.8%).

[0006] Currently, treatment for hepatitis C primarily relies on antiviral drugs (DAAs), but the efficacy of these drugs varies significantly between HCV genotypes. For example, DAAs are less effective for patients with HCV genotypes 1 and 4, while drugs for types 2 and 3 are more effective. Furthermore, the significance of different HCV subtypes in guiding subsequent treatment lies in the following key aspects: 1. Different subtypes exhibit varying susceptibility to drug therapy. For example, types 2 and 3 respond better to antiviral drugs. Combination therapy with pegylated interferon and ribavirin achieves a sustained virologic response in 80% of untreated non-cirrhotic patients with HCV genotypes 2 / 3, compared to approximately 50% for genotype 1. Patients with HCV genotypes 2 or 3 achieve maximum therapeutic benefit with only six months of treatment, while those with genotype 1 require 12 months. For these reasons, it is essential to determine the HCV genomic profile in all infected patients being considered for antiviral therapy. 2. Different subtypes are associated with the rate of disease progression and severity. Some studies have found that individuals infected with genotypes 1b and 3a have faster disease progression, while those infected with genotypes 2 and 4 progress more slowly. 3. Different subtypes are associated with the risk of relapse after treatment. Studies have shown that genotypes 1a, 1b, and 3a have a higher risk of relapse, while genotypes 2 and 4 have a lower risk of relapse. Because different HCV subtypes respond differently to treatment and are closely related to the expected treatment response and duration, understanding HCV subtypes is crucial for evaluating the effectiveness of hepatitis C treatment.

[0007] Therefore, this field requires a product that can simply and quickly detect the above pathogens with high sensitivity and good specificity, providing clinicians with a more sufficient basis for rapid diagnosis and exclusion of different pathogen infections, shortening the time it takes for clinicians to diagnose the patient's condition, and speeding up the implementation of treatment measures for the patient. Summary of the Invention

[0008] In view of this, in a first aspect, the present invention provides a composition for detecting and typing hepatitis C, comprising:

[0009] Upstream and downstream primers and probes for detecting HCV 1b as shown in SEQ ID NOs: 1 to 3;

[0010] HCV 1 detection probes and upstream and downstream primers as shown in SEQ ID NOs: 4 to 6;

[0011] Upstream and downstream primers and probes for detecting HCV 2 as shown in SEQ ID NOs: 7 to 9;

[0012] Upstream and downstream primers and probes for detecting HCV 3 as shown in SEQ ID NOs: 10 to 12; and

[0013] The upstream and downstream primers and probes for detecting HCV 4 are shown in SEQ ID NOs: 13-15.

[0014] The combined detection composition provided by the present invention primarily utilizes a multiplex fluorescent PCR analysis method to detect and subtype hepatitis C by detecting different sites. This allows for the simultaneous detection and differentiation of HCV 4, HCV 1b, HCV 1, HCV 2, and HCV 3 in a single-tube reaction system, providing a targeted strategy for subsequent treatment. Furthermore, the composition of the present invention offers a higher sensitivity of up to 400 copies / mL, resulting in more accurate detection. This provides clinicians with a more comprehensive basis for rapid diagnosis, shortening the time it takes to diagnose a patient's condition and expediting the implementation of treatment measures.

[0015] Furthermore, the composition includes upstream and downstream primers and probes for detecting the internal standard.

[0016] 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.

[0017] Furthermore, the fluorescent groups of the probes in the composition of the present invention are different from each other and do not interfere with each other.

[0018] In this context, "different and non-interfering" means that the fluorescent groups used in each probe in the composition are different and do not affect each other's detection, that is, they can be detected using different channels. For example, ATTO 425, Quasar 705, FAM, HEX, ROX, CY5, and CY5.5 can be used. These groups have different absorbance values ​​and can be used in different channels, thus preventing interference.

[0019] Furthermore, in some embodiments, the composition of the present invention may include one or more pairs of the above primer and probe pairs. In the present invention, a "pair" refers to upstream and downstream primers and probes that match each other for detecting a target.

[0020] The compositions of the present invention can be combined in any combination to detect any of the five targets. Those skilled in the art can determine the desired combination by combining primer and probe pairs corresponding to the targets to be detected. These combinations are encompassed by the present invention.

[0021] For example, it can include any 4 pairs of the above 5 pairs of primers and probes, any 3 pairs of the above 5 pairs of primers and probes, any 2 pairs of the above 5 pairs of primers and probes, or any 1 pair of the above 5 pairs of primers and probes.

[0022] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0023] In a specific embodiment, the fluorescent reporter group of the HCV 1b probe is FAM; the fluorescent 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 HCV 3 probe is HEX; and the fluorescent reporter group of the HCV 4 probe is ROX.

[0024] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0025] Furthermore, the 3' end of the probe also has a quencher group, such as MGB, BHQ1 or BHQ2.

[0026] In a specific embodiment, the 3' end of the probe is MGB.

[0027] Furthermore, the amount of the primer in the composition is 0.2 to 0.5 μM; the amount of the probe in the composition is 0.1 to 0.2 μM.

[0028] In a specific embodiment, the components of the composition of the present invention are each present in a separate package.

[0029] In a particular embodiment, the components of the composition of the present invention are present in the same package.

[0030] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0031] In a second aspect, the present invention provides use of the composition of the present invention in preparing a kit for detecting and typing hepatitis C, wherein the types of hepatitis C are HCV 4, HCV 1b, HCV 1, HCV 2, and HCV 3.

[0032] In a third aspect, the present invention provides a kit for detecting and typing hepatitis C, the kit comprising the composition of the present invention as described above.

[0033] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0034] In a specific embodiment, the negative control is at least one of DEPC H2O, physiological saline, and an internal standard gene. The positive control is at least one of pseudoviruses or fragment genes of HCV 4, HCV 1b, HCV 1, HCV 2, and HCV 3.

[0035] Furthermore, the kit also includes a nucleic acid amplification reagent.

[0036] Furthermore, the amplification reagents include dNTP, PCR buffer, reverse transcriptase, DNA polymerase, and Mg 2+ At least one of .

[0037] Furthermore, the kit also includes: a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0038] Furthermore, the concentration of the DNA polymerase is 3 U / reaction to 15 U / reaction, for example, the DNA polymerase may be Taq enzyme. The concentration of the reverse transcriptase is 0.1 U / reaction to 5 U / reaction, for example, the reverse transcriptase may be Tth enzyme.

[0039] In a specific embodiment, the kit of the present invention comprises: Taq enzyme, Mg 2+ 、Mn 2+ , dNTPs, primers, probes and PCR buffer.

[0040] Common PCR buffers are composed of Tris-HCl, MgCl2, KCl, Triton X-100, etc. The total volume of a single PCR reaction tube is generally 20μl to 200μl.

[0041] In a fourth aspect, a method for detecting and typing hepatitis C is provided, the method comprising the following steps:

[0042] 1) Extracting nucleic acid from the sample to be tested;

[0043] 2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention or the kit of the present invention as described above;

[0044] 3) Obtain and analyze the results.

[0045] In the present invention, the sample used for detection can be serum, blood, etc., but is not limited thereto.

[0046] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0047] Enzyme activation, temperature 90-99°C, time 5-120 seconds, 1 cycle; cDNA reverse transcription, temperature 50-65°C, time 20-40 minutes, 1 cycle; cDNA pre-denaturation, temperature 90-99°C, time 5-120 seconds, 1 cycle; denaturation, temperature 95°C, time 5-20 seconds, annealing, temperature 55°C-60°C, time 10-60 seconds, 30-50 cycles, and fluorescence collection.

[0048] In a specific embodiment, a method for detecting and typing hepatitis C for non-diagnostic purposes is provided, the method comprising the following steps:

[0049] 1) Extracting nucleic acid from the sample to be tested;

[0050] 2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention or the kit of the present invention;

[0051] 3) Obtain and analyze the results.

[0052] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0053] Enzyme activation, temperature 90-99°C, time 5-120 seconds, 1 cycle; cDNA reverse transcription, temperature 50-65°C, time 20-40 minutes, 1 cycle; cDNA pre-denaturation, temperature 90-99°C, time 5-120 seconds, 1 cycle; denaturation, temperature 95°C, time 5-20 seconds, annealing, temperature 55°C-60°C, time 10-60 seconds, 30-50 cycles, and fluorescence collection.

[0054] In a specific embodiment, a composition is provided for preparing a composition for detecting and typing hepatitis C, wherein the composition comprises the following steps:

[0055] 1) Extracting nucleic acid from the sample to be tested;

[0056] 2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention or the kit of the present invention;

[0057] 3) Obtain and analyze the results.

[0058] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0059] Enzyme activation, temperature 90-99°C, time 5-120 seconds, 1 cycle; cDNA reverse transcription, temperature 50-65°C, time 20-40 minutes, 1 cycle; cDNA pre-denaturation, temperature 90-99°C, time 5-120 seconds, 1 cycle; denaturation, temperature 95°C, time 5-20 seconds, annealing, temperature 55°C-60°C, time 10-60 seconds, 30-50 cycles, and fluorescence collection. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] FIG1 is a graph showing the test results of the composition of the present invention;

[0061] FIG2 is a diagram showing the sensitivity test results of the composition of the present invention;

[0062] FIG3 is a graph showing the specificity detection results of the composition of the present invention;

[0063] FIG4 is a diagram showing the triple test results of Comparative Example 1 of the present invention;

[0064] FIG5 is a diagram showing the five-joint test results of Comparative Example 1 of the present invention;

[0065] FIG6 is a diagram showing the triple test results of Comparative Example 2 of the present invention;

[0066] FIG7 is a diagram showing the five-joint test results of Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0067] The present disclosure will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present disclosure will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present disclosure, rather than to limit the present disclosure.

[0068] Example 1: Primers and probes used in the present invention

[0069] Table 1

[0070] Among them, the fluorescent reporter group of the HCV 1b probe is FAM; the fluorescent 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 HCV 3 probe is HEX; and the fluorescent reporter group of the HCV 4 probe is ROX.

[0071] Example 2: Method for detecting and typing hepatitis C

[0072] The samples tested in the present invention are serum and blood. The real-time fluorescence PCR reaction system is configured according to the following Table 2:

[0073] Table 2

[0074] Preparation of enzyme mixture:

[0075] The enzyme mixture consists of Tth enzyme and Taq enzyme. Mix Tth enzyme (5 U / μL) and Taq enzyme (5 U / μL) in a specific ratio (2 μL of Tth enzyme and 0.5 μL of Taq enzyme per person). Use 2.5 μL of the enzyme mixture per person.

[0076] The PCR amplification program is set as shown in Table 3 below:

[0077] Table 3

[0078] If the FAM, CY5, Quasar705, HEX, and ROX channels have obvious S-shaped amplification curves and Ct values ​​≤ 38, it is judged as positive; if the FAM, CY5, Quasar705, HEX, and ROX channels have no amplification curves (No Ct) or Ct values ​​> 38, it is judged as negative; the details are shown in Table 4.

[0079] Table 4. Interpretation of test results

[0080] Example 3: Test results of test samples of the composition of the present invention

[0081] The primers and probes shown in Example 1 were verified on samples of HCV 4, HCV 1b, HCV 1, HCV 2, and HCV 3 according to the method of Example 2. The results showed that HCV 4, HCV 1b, HCV 1, HCV 2, and HCV 3 of hepatitis C virus could be detected and differentiated. The detection results are shown in Figure 1.

[0082] Example 4: Sensitivity of the composition of the present invention

[0083] Using the composition of Example 1 of the present invention, LOD (Loss of Detection) analysis was performed for each target, simulating clinical samples, using a multiplex PCR instrument. The results, shown in Table 5 and Figure 2, demonstrate that accurate detection was achieved across all channels even at concentrations as low as 400 copies / mL, indicating that the composition of the present invention has a sensitivity of 400 copies / mL.

[0084] Table 5

[0085] Example 5: Specificity of the composition of the present invention

[0086] The composition of the present invention showed no cross-reactivity with common pathogens and other pathogens with similar infection symptoms (e.g., hepatitis B virus, human cytomegalovirus, hepatitis A virus, Epstein-Barr virus, Treponema pallidum, herpes simplex virus type 1, herpes simplex virus type 2, Staphylococcus aureus, Candida albicans, human immunodeficiency virus, influenza A virus, etc.). The results, shown in Figure 3, demonstrate that the composition of the present invention has excellent specificity.

[0087] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0088] Due to the principle of complementary base pairing, dimers can form between primers and / or probes, but this probability is very low and can be eliminated at the beginning of the design. However, when testing for multiple pathogens together, there are many primers and probes, and dimers are likely to form between primers, probes, or even between primers. To ensure the conservative design (conservatism is crucial to the accuracy of the test) and to consider the mutual interference between different primers and probes, the primers and probes must be carefully designed.

[0089] Therefore, the inventors also designed some other primers and probes (sequences are shown in Tables 6 and 7) to form different detection systems, which are also used to detect HCV 4, HCV 1b, HCV 1, HCV 2, and HCV 3. For example, the specific detection results are shown in Figures 4 to 7. It can be seen from the amplification curves in Figures 4 and 5 that the effect is better when HCV typing (triplet detection) is performed, but when the five genotypes are detected together, there is a phenomenon of low fluorescence signal and delayed Ct; similarly, the combination of 2 HCV typing genes (quintuple detection) has lower detection performance for some targets than the triple detection combination, the Ct value is delayed, and the fluorescence increment decreases.

[0090] Table 6

[0091] Table 7

Claims

1. A composition for detecting hepatitis C and typing, comprising: The upstream and downstream primers and probes for detecting HCV 1b as shown in SEQ ID NO: 1-3; The upstream and downstream primers and probes for detecting HCV 1 as shown in SEQ ID NO: 4-6; The upstream and downstream primers and probes for detecting HCV 2 as shown in SEQ ID NO: 7-9; The upstream and downstream primers and probes for detecting HCV 3 as shown in SEQ ID NO: 10-12; and The upstream and downstream primers and probes for detecting HCV 4 as shown in SEQ ID NO: 13-15.

2. The composition according to claim 1, wherein, The composition further comprises upstream and downstream primers and probes for detecting an internal standard.

3. The composition according to claim 2, wherein The fluorescent groups of the probes in the composition are different from each other and do not interfere with each other.

4. The composition according to claim 3, wherein, The fluorescent reporter group of the HCV 1b probe is FAM; the fluorescent 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 HCV 3 probe is HEX; the fluorescent reporter group of the HCV 4 probe is ROX.

5. The composition according to 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 exist in a mixed form.

7. Use of the composition according to any one of claims 1 to 6 in the preparation of a kit for detecting hepatitis C and typing, wherein, The types of hepatitis C are HCV 4, HCV 1b, HCV 1, HCV 2, HCV 3.

8. A kit for detecting hepatitis C and typing, the kit comprising the composition according to any one of claims 1-6.

9. The kit according to claim 8, wherein, The kit further comprises at least one of a nucleic acid release reagent, a nucleic acid extraction reagent, and a nucleic acid amplification reagent.

10. A method for detecting a composition for hepatitis C and typing, the method comprising the following steps: 1) Extract the nucleic acid of the sample to be tested; 2) Perform fluorescence quantitative PCR analysis on the nucleic acid obtained in step 1) using the composition according to any one of claims 1-6 or the kit according to claim 8 or 9; 3) Obtain and analyze the results.

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