Nucleic acid combination, hepatitis delta virus nucleic acid detection reagent and application thereof

Abstract

The invention relates to a nucleic acid composition, a hepatitis delta virus nucleic acid detection reagent and application thereof. The nucleic acid combination comprises a detection primer and a detection probe, the nucleotide sequence of the detection primer is as shown in SEQ ID NO: 1-3, and the nucleotide sequence of the detection probe is as shown in SEQ ID NO: 4. The kit is used for detection and content determination of 1-8 genotypes of hepatitis D viruses, and has the advantages of strong inclusiveness, good accuracy, high sensitivity and strong specificity.

Description

Technical Field

[0001] The present application relates to the technical field of molecular biology in vitro diagnosis, and specifically to a nucleic acid combination, a hepatitis D virus nucleic acid detection reagent and applications thereof. Background Art

[0002] Hepatitis D virus (HDV) was discovered by Italian scholars Rizzetto et al. from liver tissue specimens of patients with severe hepatitis B and was first reported in 1977. HDV is a defective virus whose envelope protein is the surface antigen (HBsAg) from other hepatotropic DNA viruses such as hepatitis B virus (HBV), and it relies on HBV for viral replication and packaging. The HDV virus is spherical with a diameter of about 36nm. The genome is a covalently closed circular single negative-strand RNA with a total length of about 1672 bp~1697 bp. It is the smallest virus known to infect humans so far and can cause severe viral hepatitis. To date, HDV can be divided into 8 genotypes (HDV type 1 to HDV type 8), with differences within the same genotype of less than 20%, and differences between different genotypes of up to 40%. HDV type 1 is prevalent worldwide, accounting for about 90%. According to phylogenetic analysis, HDV type 1 has 4 subtypes, HDV 1a is mainly distributed in Africa, and HDV type 1b is mainly distributed in Africa, South America, and western Asia. HDV type 2 is mainly distributed in Asia, of which HDV type 2a is distributed in eastern and southern Asia, such as China and Japan. HDV type 3 is quite different from other genotypes and is only distributed in the Amazon Basin in South America. HDV type 4 is common in eastern Asia, such as China and Japan. Other genotypes are mainly concentrated in central and northern Africa. The HDV genotypes in my country are mainly HDV type 1, HDV type 2, and HDV type 4, of which HDV type 1a is represented by Henan strain and Taiwan strain, and HDV type 1b is represented by Sichuan strain and Guangxi strain. Different genotypes may be associated with the progression of liver disease.

[0003] Although it has been more than 40 years since HDV was discovered, the scientific community and the public have paid limited attention to HDV. In recent years, large-scale meta-analyses have shown that the total number of HDV-infected people in the world is currently about 12 million to 72 million, and may even reach 74 million. The prevalence of HDV is 0.16% to 0.8% in the general population and 4.5% to 13% in HBsAg-positive people, which overturns the previous belief that the incidence of hepatitis D is low. HDV shows obvious regional differences in my country, mainly occurring in Inner Mongolia and Xinjiang Uygur Autonomous Region. Studies have shown that the prevalence of HDV antibodies in HBsAg carriers in my country is 2.1% to 5.57%. There are two main forms of HDV infection. One is the first exposure of patients to HBV and HDV at the same time, called HBV / HDV co-infection. Most patients can recover spontaneously, and a few develop severe hepatitis. The other is exposure to HDV on the basis of previous HBV infection, called superinfection, of which 70% to 90% will develop into chronic HDV infection. Compared with HBV monoinfection, combined HDV infection accelerates the progression of liver inflammation to cirrhosis, liver cancer, and other end-stage liver diseases, progressing to cirrhosis within an average of 5 years and hepatocellular carcinoma within 10 years. There is a lack of effective treatment for HDV. Both EASL (2017) and AASLD (2018) recommend PegIFNα as the only drug currently used to treat HDV infection, but the sustained virological response rate is only 20-31%, and the recurrence rate can be as high as 40%. In recent years, the research and development of HDV targeted drugs has achieved remarkable results. The prodrug Myrcludex B has been shown in a Phase IIa clinical study to be used as a single treatment or in combination with PEG-IFNα-2a for patients infected with HDV. After 24 weeks, HDV RNA has significantly decreased, indicating that Myrcludex B is well tolerated. Similarly, the entry inhibitor Bulevirtide has been shown to have a good anti-HDV viral effect in a Phase II clinical study. The drug has been approved by the European Medicines Agency in July 2020 for conditional antiviral treatment of chronic HDV infection. Currently, Bulevirtide is conducting Phase III clinical studies for antiviral treatment in chronic HDV-infected patients in many places around the world. In addition, drugs used for antiviral treatment of hepatitis D, such as Lonafarnib, a farnesyl transferase inhibitor used to intervene or inhibit the prenylation modification of L-HDAg to prevent viral assembly, nucleotide polymer REP 2139-Ca, and small siRNA interference molecule JNJ-3989, have also entered Phase III and Phase II clinical trials, respectively, and have shown good antiviral treatment effects, and are expected to be approved for use soon.

[0004] The diagnosis of HDV infection can be confirmed by detecting HDV antigen (HDAg), anti-HDV IgM, anti-HDV IgG antibodies, or HDV RNA. HDAg is rarely detected in serum except in cases of acute infection (before antibody seroconversion) or in severely immunosuppressed chronically infected patients. However, HDAg cannot be detected in the late acute infection and mid-chronic infection because it forms immune complexes with homologous antibodies. Therefore, it is difficult to confirm HDV infection clinically by detecting HDAg. Anti-HDV IgM reflects persistent viral replication and can be detected within the first 2-3 weeks of acute HDV infection. It may persist in some patients when the infection progresses to chronicity, but the sensitivity of anti-HDV IgM detection is poor and it is not suitable for large-scale screening of HDV infection. Large-scale screening for HDV infection relies on the detection of specific anti-HDV IgG antibodies. All individuals infected with HDV will produce anti-HDV IgG antibodies, and anti-HDV IgG will persist even weeks or years after the HDV virus has been cleared. However, the detection of anti-HDV antibodies cannot distinguish between past infection and current infection. HDV RNA is direct evidence of viral replication. Blood HDV RNA detection is recognized as the best indicator for diagnosing active HDV infection because of its sensitivity and practicality. It is also the gold standard for current hepatitis D infection. In 2023, the European Association for the Study of the Liver (EASL) clinical practice guidelines on HDV recommended that individuals with positive HDV antibodies undergo HDV RNA testing to diagnose active HDV infection; in 2024, the WHO guidelines for the prevention and diagnosis and treatment of chronic hepatitis B recommended total anti-HDV antibody screening for all HBsAg-positive patients, and HDV RNA testing for individuals with positive total anti-HDV antibodies to distinguish active HDV infection from past infection. In clinical practice, the absence of HDV-RNA in serum / plasma is a necessary condition for diagnosing the cure of hepatitis D. HDV RNA is the best indicator for active HDV infection and can be used for the diagnosis of current HDV infection and monitoring of antiviral treatment response. After treating patients with hepatitis D with high-dose IFNα-2a, the HDV RNA titer in the patient's serum continued to decrease significantly, and the liver tissue pathology, clinical outcomes and survival rates after drug withdrawal during the same period improved to varying degrees; the HDV RNA level at 24 weeks of peg-IFN treatment was an independent predictor of virological response at 24 weeks after the end of the treatment course. The clinical practice guidelines point out that HDV RNA quantitative testing should be performed every 6 months during treatment of HDV-infected patients or when clinical features are present, and HDV RNA testing should be performed at different time points after drug withdrawal according to the type of drug treatment. In the case of Bulevirtide discontinuation, viral recurrence should be monitored after 1, 3, 6 and 12 months, and annually thereafter.Therefore, quantitative detection of HDV RNA in human serum samples can be used as evidence of current HDV infection and as a primary indicator for evaluating antiviral efficacy.

[0005] As a genetic detection technology, qualitative reverse transcription-polymerase chain reaction (RT-PCR) can be used to detect HDV RNA in serum and effectively evaluate the replication of HDV, but it cannot accurately evaluate the efficacy of antiviral treatment like quantitative PCR. Quantitative RT-PCR technology can provide a sensitive and accurate method for quantifying viral genomes, effectively reducing or avoiding contamination caused by post-processing of PCR products. In 2005, Le et al. used RT-PCR quantitative technology to develop a method for quantitative determination of HDV RNA in serum, which can quantitatively analyze HDV RNA infected with any subtype of the eight HDV genotypes for the evaluation of antiviral treatment response in HDV-infected patients. However, due to the high genetic variation of HDV, the difference between HDV types is as high as 37% in the entire nucleotide sequence of the genome, and the "rod-like" structure formed by intramolecular base pairing of more than 70% of the HDV RNA sequence often impairs the efficiency of cDNA synthesis and PCR efficiency. In recent years, many developers have reported some reagents related to HDV detection: for example, the conventional PCR method is used to detect HDV viral RNA. This method requires a reverse transcription operation to reverse transcribe the RNA into cDNA before conventional PCR amplification. The operation is cumbersome and requires sample transfer during the process, with a high risk of aerosol contamination. The amplified product is detected by agarose gel electrophoresis, and the detection sensitivity is poor. This method is only for HDV viral gene 1 and genotype 2 of the Han people and has poor inclusiveness. In addition, as a qualitative detection method, this method can only perform qualitative detection of HDV RNA in patient plasma, but cannot be used for antiviral treatment monitoring like quantitative methods. For example, the digital PCR method is used to detect HDV viral RNA, but it also has many shortcomings: 1. The published primer probes only cover 6 HDV genotypes, namely HDV-1, HDV-2, HDV-4, HDV-5, HDV-6, and HDV-7, and cannot detect two genotypes, namely HDV-3 and HDV-8, and its inclusiveness is insufficient; 2. The claimed detection limit of this method is only 1000 copies / mL, and the detection sensitivity is not high. It cannot detect HDV viruses with lower loads, and the missed detection rate is high; 3. The test results are expressed in copies / mL, without traceability, and the test results are inaccurate; 4. Digital PCR detection has the disadvantages of expensive instruments and reagents, cumbersome operations such as droplet generation, long detection time (at least 2-3 hours), and high cost, and is not suitable for large-scale promotion and use.For example, the kit for quantitative detection of HDV viral RNA developed by the RT-qPCR method uses purified RNA lyophilized powder as the calibrator in the kit, which cannot participate in the nucleic acid extraction process, resulting in the actual quantitative result being biased high; the extraction reagent that comes with the kit packages the internal reference control and captured RNA together with the detection kit, and the sample volume is as high as 400 μL, making the nucleic acid extraction operation very cumbersome, and the use of a high sample volume will increase the pain of blood drawing for patients; in addition, the kit uses non-specific random primers, which will interfere with the detection and pose a risk of false positives. The reagent is only for antiviral drug treatment monitoring and cannot be used for the diagnosis of HDV infection. It has currently only obtained CE approval and has not obtained NMPA certification.

[0006] Given that there is currently no method in China that can detect and quantify all eight genotypes of hepatitis D virus with high sensitivity and specificity, and serological testing often has a window period and poses biosafety risks; furthermore, the sensitivity of serological diagnosis is not high, and the high genetic variation of hepatitis D virus easily leads to antigenic drift and low detection rate. Therefore, the development of a hepatitis D virus nucleic acid quantification method with higher inclusiveness, high sensitivity, and high specificity, which can be used in the clinical diagnosis of current HDV virus infection and monitoring of antiviral drug treatment, has important economic and social significance for virus detection, virus prevention, and patient treatment of hepatitis D patients. Summary of the invention

[0007] Based on this, the present application provides a nucleic acid combination, a detection reagent and a detection kit for the detection and content determination of 1-8 genotypes of hepatitis D virus, which has strong inclusiveness, good accuracy, high sensitivity and strong specificity.

[0008] The technical solutions include the following:

[0009] A nucleic acid combination for detecting hepatitis D virus, comprising a detection primer and a detection probe; the nucleotide sequence of the detection primer is shown in SEQ ID NO: 1-3, and the nucleotide sequence of the detection probe is shown in SEQ ID NO: 4.

[0010] In one embodiment, the two ends of the detection probe are connected to a fluorescent reporter group and a fluorescent quencher group respectively.

[0011] In one embodiment, the fluorescent reporter group is located at the 5' end of the detection probe, and the fluorescent quencher group is located at the 3' end of the detection probe.

[0012] A hepatitis D virus nucleic acid detection reagent, the reagent comprising the nucleic acid combination.

[0013] In one embodiment, the reagents also include detection primers and detection probes for internal standard quality control;

[0014] In one embodiment, the nucleotide sequence of the detection primers for the internal standard quality control is shown in SEQ ID NO: 5-6, and the nucleotide sequence of the detection probe for the internal standard quality control is shown in SEQ ID NO: 7.

[0015] In one embodiment, the reagent also includes PCR reaction solution.

[0016] In one embodiment, the PCR reaction solution includes one or more of Tris buffer, potassium chloride, magnesium chloride, TritonX-100, bovine serum albumin, trehalose, betaine, dATP, dTTP, dGTP, dCTP, dUTP, Heat-UDG, RNasin and nuclease-free distilled water.

[0017] In one embodiment, the PCR reaction solution includes the following components at working concentrations: 10mM-100mM Tris buffer, 10mM-100mM potassium chloride, 1mM-4mM magnesium chloride, 0.1%v / v-1.0%v / v Triton X-100, 0.1mg / mL-1.0mg / mL bovine serum albumin, 100mM-300mM trehalose, 400mM-1000mM betaine, 0.15mM-0.25mM dATP, 0.15mM-0.25mM dTTP, 0.15mM-0.25mM dGTP, 0.15mM-0.25mM dCTP, 0.15mM-0.25mM dUTP, 0.01U / μL-0.1U / μL Heat-UDG and 0.8U / μL-1.0U / μL RNasin.

[0018] In one embodiment, the reagent also includes PCR enzyme solution;

[0019] In one embodiment, the PCR enzyme solution includes one or both of reverse transcriptase and Taq DNA polymerase.

[0020] In one embodiment, the PCR enzyme solution includes 0.1U / μL-0.2U / μL reverse transcriptase and 0.1U / μL-0.2U / μL Taq DNA polymerase.

[0021] A hepatitis D virus nucleic acid detection kit, the kit comprising the nucleic acid combination or the hepatitis D virus nucleic acid detection reagent.

[0022] In one embodiment, the kit further comprises one or more of a negative control, a positive control and a calibrator.

[0023] The detection method of the hepatitis D virus nucleic acid detection kit comprises the following steps:

[0024] Using the extracted RNA of the sample to be tested as a template, amplification is performed using the hepatitis D virus nucleic acid detection kit; and

[0025] Whether the sample to be tested contains hepatitis D virus and / or the content of hepatitis D virus in the sample to be tested is determined according to the amplification result.

[0026] In one embodiment, the detection method includes one-step multiplex fluorescence quantitative PCR.

[0027] The use of the nucleic acid combination or the hepatitis D virus nucleic acid detection reagent in any of the following:

[0028] (1) Application in the preparation of products for screening, prevention, control and management of hepatitis D virus infection;

[0029] (2) Use in the preparation of products for diagnosis or auxiliary diagnosis of hepatitis B surface antigen-positive patients with concurrent or superinfection with hepatitis D virus;

[0030] (3) Use in the preparation of products for diagnosing or distinguishing between current and past hepatitis D virus infection;

[0031] (4) Use in the preparation of products for diagnosing or confirming active HDV infection in patients with total anti-HDV antibody positive;

[0032] (5) Use in the preparation of products for evaluating or monitoring the efficacy of antiviral drug treatment and antiviral immune response in patients with hepatitis D; and

[0033] (6) Application in the preparation of products for monitoring the cure and post-recovery recurrence of hepatitis D patients.

[0034] In one embodiment, the hepatitis D virus includes one or more hepatitis D viruses of genotypes HDV-1 to HDV-8.

[0035] Compared with the traditional technology, this application has the following beneficial effects:

[0036] The nucleic acid combination, reagents and kit of the present application can be effectively used for the detection of 1-8 genotypes of hepatitis D virus nucleic acid, with high detection sensitivity and strong specificity.

[0037] The detection method of the present application effectively solves the problems of a "window period", low detection sensitivity, and easy false negatives and missed detections in the detection of HDV antibodies and HDV antigens by serological methods.

[0038] The detection method of the present application effectively solves the problems of poor timeliness, long cycle, complex operation and easy biosafety risks caused by virus identification through virus isolation and culture.

[0039] The kit and detection method of the present application can be effectively used for monitoring the hepatitis D virus content and changes in hepatitis D patients, preventing and controlling viral infections, and evaluating the efficacy of antiviral drug treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0041] Figure 1 Schematic diagram of HDV genome structure and primer probe design;

[0042] Figure 2 The results of positive coincidence rate analysis (A) and negative coincidence rate analysis (B);

[0043] Figure 3 It is the result of HDV reagent sensitivity and calibration linearity analysis, where A is the dose response curve, and B is the standard curve fitted for HDV calibrator determination;

[0044] Figure 4 To test the results of inclusiveness and linear range analysis, AH represents genotypes 1-8;

[0045] Figure 5 Pearson correlation coefficient (r) was calculated for Deming regression analysis to determine the linear relationship between the reagent of this application and the CE reagent of Jena, Germany (A) and the consistency results between the assay results evaluated by Bland-Altman method (B);

[0046] Figure 6 is the stability analysis result;

[0047] Figure 7 Comparative Example 1 is a comparative analysis of the HDV-qPCR-F1 primer in the HDV-7 genotype HDV virus gene sequence;

[0048] Figure 8This is the comparison analysis of the HDV-F1 primer of this application in the HDV-7 genotype HDV virus gene sequence;

[0049] Fig. 9 The amplification efficiency of the primer-probe combination described in Example 1 of the present application and the primer-probe combination described in Comparative Example 1 was analyzed by qPCR. DETAILED DESCRIPTION

[0050] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0052] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0053] In this article, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and the like, when used in other ways to mean "one or more", are also understood in the same way unless otherwise specified.

[0054] Herein, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0055] In this article, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally include", etc. in this application, taking "optionally include" as an example, mean "may include or not include".

[0056] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0057] Existing HDV RNA detection technology has the disadvantages of poor inclusiveness, failure to cover 1 to 8 genotypes; low sensitivity, high missed detection rate; cumbersome operation, high cost; and limited clinical application. Currently, there is a lack of a method with high sensitivity and high specificity to detect and quantify all 8 genotypes of hepatitis D virus. Therefore, the development of a hepatitis D virus nucleic acid quantification method with higher inclusiveness, high sensitivity, and high specificity, which can be applied in the clinical diagnosis of current HDV virus infection and the evaluation of antiviral drug treatment response and efficacy, has important economic and social significance for virus detection, virus prevention, and patient treatment of hepatitis D patients.

[0058] Since RT-qPCR based on TaqMan technology has good sensitivity and specificity, and can quantify DNA or cDNA within a large dynamic range, in order to provide a standard HDV RNA viral load quantification method for antiviral drug treatment monitoring and strengthen the clinical diagnosis and treatment management of patients.

[0059] Based on this, one embodiment of the present application provides a nucleic acid combination for detecting hepatitis D virus, the nucleic acid combination comprising a detection primer and a detection probe; the nucleotide sequence of the detection primer is shown in SEQ ID NO: 1-3, and the nucleotide sequence of the detection probe is shown in SEQ ID NO: 4.

[0060] The present application designs specific primers and probes for the conserved nucleic acid sequence of the ribozyme domain of the HDV gene, which are used to detect 1-8 genotypes of hepatitis D virus, with an amplification efficiency of about 100%, high sensitivity, good specificity and strong tolerance.

[0061] In a specific example, the two ends of the detection probe are connected to a fluorescent reporter group and a quencher group respectively.

[0062] In a specific example, the fluorescent reporter group is located at the 5' end of the detection probe, and the fluorescent quencher group is located at the 3' end of the detection probe.

[0063] In a specific example, the fluorescent reporter group includes one or more of FAM, Cy5, VIC, JOE, TET, HEX, Cy3 and ROX.

[0064] In a specific example, the fluorescence quenching group includes one or more of MGB, TAMRA, BHQ1 and BHQ2.

[0065] One embodiment of the present application also provides a hepatitis D virus nucleic acid detection reagent, which includes the nucleic acid combination. The reagent can be used to quantify the viral load of all 8 genotypes of HDV, and is used in clinical diagnosis of hepatitis D virus infection and monitoring of antiviral drug treatment.

[0066] In a specific example, the reagents further include a detection primer and a detection probe for the HDV internal standard.

[0067] In a specific example, the nucleotide sequence of the detection primer of the HDV internal standard is shown in SEQ ID NO: 5-6, and the nucleotide sequence of the detection probe of the HDV internal standard is shown in SEQ ID NO: 7.

[0068] In a specific embodiment, the HDV internal standard is a pseudovirus containing the amplified target sequence.

[0069] The present application designs a synthetic non-competitive sequence as an internal standard control to monitor the entire process of nucleic acid extraction and amplification detection in a fully closed reaction system, which can effectively monitor the occurrence of false negatives. In other specific examples, for the detection primers and detection probes of the internal standard quality control, those skilled in the art can perform conventional designs based on common knowledge in the art.

[0070] In a specific example, the reagent also includes a PCR reaction solution.

[0071] In a specific example, the PCR reaction solution includes one or more of Tris buffer, potassium chloride, magnesium chloride, Triton X-100, bovine serum albumin, trehalose, betaine, dATP, dTTP, dGTP, dCTP, dUTP, Heat-UDG, RNasin, and nuclease-free distilled water.

[0072] In a specific example, the PCR reaction solution includes 10mM-100mM Tris buffer, 10mM-100mM potassium chloride, 1mM-4 mM magnesium chloride, 0.1%-1.0% Triton X-100, 0.1mg / mL-1.0mg / mL bovine serum albumin, 100mM-300mM trehalose, 400mM-1000mM betaine, 0.15mM-0.25mM dATP, 0.15mM-0.25mM dTTP, 0.15mM-0.25mM dGTP, 0.15mM-0.25mM dCTP, 0.15mM-0.25mM dUTP, 0.01U / μL-0.1U / μL Heat-UDG and 0.8U / μL-1.0U / μL RNasin. Thermosensitive uracil-N-glycosylase (Heat-UNG) can effectively prevent PCR contamination and avoid the occurrence of false positive results.

[0073] In a specific example, the detection primers and detection probes for detecting HDV RNA and the detection primers and detection probes for internal standard quality control are in the PCR reaction solution. In a specific example, in the PCR reaction solution, the concentration of the detection primers and detection probes is 100nM / each - 200nM / each.

[0074] In a specific example, the reagent also includes a PCR enzyme solution. In a specific example, the PCR enzyme solution includes one or both of a reverse transcriptase and a Taq DNA polymerase. In a specific example, the PCR enzyme solution includes a reverse transcriptase and a Taq DNA polymerase. In a specific example, the PCR enzyme solution includes 0.1U / μL-0.2U / μL reverse transcriptase and 0.1U / μL-0.2U / μL Taq DNA polymerase.

[0075] One embodiment of the present application also provides a hepatitis D virus nucleic acid detection kit, which includes the nucleic acid combination or the hepatitis D virus nucleic acid detection reagent.

[0076] The test results of the reagents and kits in this application can be traced back to the WHO international standard (PEI: 7657 / 12), making the quantitative results of HDVRNA more accurate. The kit has high sensitivity and can detect samples with low viral loads. It can be used for the diagnosis of HDV current infection and monitoring of antiviral drug treatment. This application has high sensitivity and can detect 6 IU / mL WHO HDV RNA international standard; it has good specificity and does not cross-react with hepatitis B virus, hepatitis C virus, hepatitis E virus, etc.; this application designs detection targets based on the conserved structural domain of the HDV gene, which is highly inclusive and can not only detect the popular domestic genotypes such as HDV1, HDV2 and HDV4, but also detect genotypes of HDV such as HDV3, HDV5, HDV6, HDV7, and HDV8.

[0077] In a specific example, the kit further includes one or more of a HDV positive control, a HDV negative control, and a HDV calibrator.

[0078] In a specific example, the HDV negative control is a negative plasma / serum sample. In a specific example, the HDV positive control and the HDV calibrator are pseudoviruses containing HDV-1 amplified target sequence fragments.

[0079] In a specific example, the HDV positive control includes a HDV strong positive control and a HDV weak positive control. In a specific example, the HDV calibrator includes four calibrators of different magnitudes.

[0080] The HDV-1 amplified target sequence fragment was connected to the pLV-CMV-MCS-EF1-ZsGreen vector through genetic engineering, and then co-transfected into 293T cells with the lentiviral packaging auxiliary vector psPAX2 vector and pMD2G vector. After virus culture, concentration and purification, the HDV-1 pseudovirus was obtained, and then diluted with negative plasma / serum at a certain ratio to prepare HDV strong positive quality control products, HDV weak positive quality control products, and HDV calibrators.

[0081] One embodiment of the present application also provides a detection method of a hepatitis D virus nucleic acid detection kit for the purpose of screening, diagnosis or monitoring the efficacy of drug treatment for patients, comprising the following steps:

[0082] The extracted RNA of the sample to be tested is used as a template and amplified using the hepatitis D virus nucleic acid detection kit; and, based on the amplification result, it is determined whether the RNA of the sample to be tested contains hepatitis D virus and / or the content of hepatitis D virus in the sample to be tested is determined.

[0083] In one specific example, the detection method includes one-step multiplex fluorescence quantitative PCR.

[0084] In a specific example, the amplification system includes a PCR reaction solution, a PCR enzyme solution, and a nucleic acid sample (RNA).

[0085] In a specific example, the amplification program includes 53°C-58°C for 12min-18min; 94°C-96°C for 1min-3min; 94°C-96°C for 8s-12s, 56°C-60°C for 28s-32s, cycles 8-12; 94°C-96°C for 8s-12s, 56°C-60°C for 28s-32s, 70°C-74°C for 28s-32s, cycles 38-42.

[0086] In a specific example, the method further includes extracting HDV RNA from the sample to be tested.

[0087] In one specific example, the sample to be tested includes plasma or serum.

[0088] In a specific example, a method for determining whether a sample to be tested contains hepatitis D virus and / or determining the content (load) of hepatitis D virus in the sample to be tested according to the amplification result is as follows:

[0089] 1) If the sample amplification curve shows an "S" shape, and the sample Ct value is ≤34, and the internal standard Ct value is ≤38, the result is judged to be valid and can be directly reported as positive; if the sample test result shows that the HDV target has no Ct value, and the internal standard Ct value is ≤38, the reported result is negative.

[0090] 2) A standard curve is constructed based on the Ct value and the Log value of the HDV calibrator concentration to obtain a standard curve formula, and the Ct value of the sample to be tested is entered into the standard curve formula to calculate the content of the hepatitis D virus in the sample to be tested, or the hepatitis D virus load is automatically read by the instrument.

[0091] One embodiment of the present application further provides the use of the nucleic acid combination or the hepatitis D virus nucleic acid detection reagent in any of the following:

[0092] (1) Application in the preparation of products for screening, prevention, control and management of hepatitis D virus infection;

[0093] (2) Use in the preparation of products for diagnosis or auxiliary diagnosis of hepatitis B surface antigen-positive patients with concurrent or superinfection with hepatitis D virus;

[0094] (3) Use in the preparation of products for diagnosing or distinguishing between current and past hepatitis D virus infection;

[0095] (4) Use in the preparation of products for diagnosing or confirming active HDV infection in patients with total anti-HDV antibody positive;

[0096] (5) Use in the preparation of products for evaluating or monitoring the efficacy of antiviral drug treatment and antiviral immune response in patients with hepatitis D; and

[0097] (6) Application in the preparation of products for monitoring the cure and post-recovery recurrence of hepatitis D patients.

[0098] In a specific example, the hepatitis D virus includes one or more hepatitis D viruses of genotypes HDV-1 to HDV-8.

[0099] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0100] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0101] In the following examples, the test samples, reagents and instruments involved are mainly:

[0102] 1. The healthy human serum / plasma, human HBV-positive serum / plasma, human HCV-positive serum / plasma, and human HDV-positive serum / plasma used in the experiment were clinical specimens collected by the Beijing-Yahuilong Joint Laboratory for Molecular Diagnosis of Infectious Diseases.

[0103] 2. Reference or quality control products include national reference products for hepatitis E virus RNA detection reagents (China National Institute for Inspection and Quarantine: 340044-202201), HBV DNA / HCV RNA / HIV1 RNA mixed (liquid) indoor quality control (Guangzhou Bondesheng: BDS-IQC-223), dengue virus type I RNA liquid indoor quality control (Guangzhou Bondesheng: BDS-IQC-088), Epstein-Barr virus deoxyribonucleic acid (EBV DNA) liquid indoor quality control (Guangzhou Bondesheng: BDS-IQC-050), human cytomegalovirus deoxyribonucleic acid (HCMV DNA) liquid indoor quality control (Guangzhou Bondesheng: BDS-IQC-025), human immunodeficiency virus type 1 RNA (HIV-1 RNA) serum (liquid) standard substance (Guangzhou Bondesheng: BDS-BW-015), AMPLIRUN TREPONEMAPALLIDUM DNA CONTROL (Syphilis Treponema DNA quality control) (vircell: MBC109), HHV-6 DNA CONTROL (Human herpes virus type 6 nucleic acid quality control) (vircell: MBC025-R), herpes simplex virus type I deoxyribonucleic acid (HSV I DNA) liquid indoor quality control product (Guangzhou Bondesheng: BDS-IQC-031), herpes simplex virus type II deoxyribonucleic acid (HSV IIDNA) liquid indoor quality control product (BDS-IQC-034), Staphylococcus aureus deoxyribonucleic acid (SA DNA) liquid indoor quality control product (Guangzhou Bondesheng: BDS-IQC-345), Candida albicans deoxyribonucleic acid liquid indoor quality control product (Guangzhou Bondesheng: BDS-IQC-347), etc. were purchased or obtained from outside.

[0104] 3. WHO HDV RNA international standard was purchased from Paul-Ehrlich Institute, Germany (PEI code: 7657 / 12, 5.75×10 5 IU / mL), which is a lyophilized product of HDV-1 plasma. Before use, add 500 μL of nuclease-free water for reconstitution.

[0105] 4. The virus cultures of five HDV genotypes, including HDV-1, HDV-2, HDV-3, HDV-4, and HDV-8, were provided by Professor Xiang Kuanhui of the Peking University Health Science Center.

[0106] 5. The inventors provided sequences of four pseudoviruses, including HDV-1 pseudovirus (sequence extracted from NCBI database: gene accession number OQ200691), HDV-5 pseudovirus (sequence extracted from NCBI database: gene accession number LT604956), HDV-6 pseudovirus (sequence extracted from NCBI database: gene accession number LT604964), and HDV-7 pseudovirus (sequence extracted from NCBI database: gene accession number LT604969), and entrusted Sangon Biotech (Shanghai) Co., Ltd. to carry out gene synthesis, pseudovirus packaging and digital PCR quantification.

[0107] 6. Reagents used in the experiment

[0108] The nucleic acid extraction reagent used in the experiment is a nucleic acid extraction or purification reagent produced by Shenzhen Yahuilong Biotechnology Co., Ltd. (Guangdong Shenzhen Medical Equipment No. 20240285).

[0109] The methodological comparison reagents were the commercial HDV RNA quantification reagent RoboGene HDV RNA Quantification Kit 2.0 produced by CE Jena, Germany, and its matching extraction reagent INSTANT Virus RNA / DNA Kit for comparative analysis.

[0110] 7. The instruments used in the experiment include:

[0111] The fluorescence quantitative PCR instrument used in the experiment was the QuantStudio 5 real-time fluorescence quantitative PCR analysis system produced by Applied Biosystems.

[0112] Example 1

[0113] A hepatitis D virus nucleic acid detection kit, comprising the following primers and probes:

[0114] like Figure 1 As shown in the schematic diagram, the HDV RNA detection primers include a primer pair consisting of an upstream primer and a downstream primer, the upstream primer is designed in the conserved segment of the ribozyme domain of the HDV genome, the downstream primer is designed in the I segment of the ribozyme domain of the HDV antigen genome, the probe is designed in the conserved sequence segment of the ribozyme domain of the HDV genome, and is in the same direction as the upstream primer, and is used for annealing and hybridization with the ribozyme domain in the HDV antigen genome, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a quencher group.

[0115] The upstream primer has a high GC content and is paired with the downstream primer to amplify a 223 bp HDV RNA target sequence. The 5' end of the probe is labeled with a 6-FAM fluorescent reporter group and the 3' end is labeled with a BHQ1 quencher group. Specifically, the primer and probe sequences are as follows:

[0116] HDV-F1: 5'-TGGCCGGCATGG T CCCAGC-3' (SEQ ID NO: 1),

[0117] HDV-R1: 5'-TCCTCTTCGGGTCGGCATGG-3' (SEQ ID NO: 2),

[0118] At the same time, considering that there is a base mismatch between the HDV-F1 primer and the HDV3 genotype virus sequence, HDV-F2 was specifically designed: 5'-TGGCCGGCATGG C CCCAGC-3' (SEQ ID NO: 3), paired with HDV-R1, for detection of HDV genotype 3 isolates;

[0119] HDV-P: 5'-FAM-CGGATGCCCAGGTCGGACCGCG-BHQ1-3' (SEQ ID NO: 4).

[0120] The above primers and probes were used simultaneously to amplify all eight genotypes of HDV.

[0121] In addition, in order to realize the monitoring of the detection process in a fully closed reaction system, the present application also introduces a non-competitive sequence that does not interfere with the target gene of hepatitis D virus as an internal standard quality control, wherein the internal standard quality control target sequence is selected from the Escherichia coli MS2 phage gene (gene accession number: LC710218.1), and the internal standard quality control test primer combination amplifies an amplified fragment of 71 bp in size, and the 5' end of the internal standard probe is labeled with a VIC fluorescent reporter group, and the 3' end is labeled with a BHQ1 quenching group. Specifically, the internal standard primer and probe sequences are as follows:

[0122] ICMS2-F: 5'-TCCAGTATGGAACCACGCTATG-3' (SEQ ID NO: 5);

[0123] ICMS2-R: 5'-TCGCTCGAGAACGCAAGTT-3' (SEQ ID NO: 6);

[0124] ICMS2-P: 5'-VIC-CGACCACTGTCGTGCTTTTCGCTG-BHQ1-3' (SEQ ID NO: 7).

[0125] The kit may also include HDV PCR reaction solution, HDV PCR enzyme solution, HDV negative quality control product, HDV strong positive quality control product, HDV weak positive quality control product, HDV internal standard and 4 HDV calibrators of different magnitudes.

[0126] Exemplarily, the HDV calibrator is prepared by diluting a quantitatively high-concentration HDV-1 pseudovirus with negative plasma in a certain proportion to obtain HDV calibrator 1 (2.5×10 6 IU / mL), HDV calibrator 2 (2.5×10 5 IU / mL), HDV calibrator 3 (2.5×10 4 IU / mL), and HDV calibrator 4 (2.5×10 3 IU / mL). The preparation of the HDV PCR reaction solution and enzyme solution is carried out as follows.

[0127] Table 1 HDV PCR reaction solution

[0128]

[0129] Table 2 HDV PCR enzyme solution

[0130]

[0131] Example 2 One-step fluorescence quantitative PCR for detecting hepatitis D virus nucleic acid

[0132] Based on one-step fluorescence quantitative PCR, the hepatitis D virus nucleic acid detection reagent in Example 1 is used to detect hepatitis D virus nucleic acid.

[0133] 1. Viral nucleic acid extraction

[0134] The nucleic acid extraction of the sample to be tested is carried out using a nucleic acid extraction or purification reagent (Yueshenxiebei 20240285) produced by Shenzhen YHLO Biotech Co., Ltd. 200 μL of each sample is taken and 5 μL of HDV internal standard is added for extraction. The HDV negative control, HDV weak positive control, HDV strong positive control, and HDV calibrators (1-4) in the kit are all processed according to the sample extraction method. The extraction process is carried out strictly according to the product instruction manual. Finally, 65 μL of nuclease-free water is added to elute the nucleic acid and it is reserved for use.

[0135] 2. Reaction system preparation

[0136] Calculate the volume of the reaction mixture PCR-Mix according to the number of samples to be tested, HDV negative control, HDV weak positive control, HDV strong positive control, and HDV calibrator (1-4). Prepare PCR-Mix according to the table below, mix well, centrifuge briefly, and dispense into 8 fluorescent quantitative PCR tubes at 20 μL / well. Add 30 μL of nucleic acid in the order of HDV negative control, sample to be tested, HDV weak positive control, HDV strong positive control, HDV calibrator 4, HDV calibrator 3, HDV calibrator 2, and HDV calibrator 1, cover the tube caps, mix well, centrifuge briefly, and transfer to the fluorescent quantitative PCR instrument.

[0137] Table 3 Reaction system preparation table

[0138]

[0139] 3. Amplification program setting and PCR detection

[0140] Amplification program setting: Place each reaction tube into the amplification detection instrument, set the HDV negative quality control, HDV positive quality control, HDV calibrators 1 to 4 and the sample to be tested in the corresponding order, set the sample name and calibrator concentration, and perform PCR amplification according to the following procedure.

[0141] Table 4 Amplification procedures

[0142]

[0143] 4. Result acquisition and interpretation

[0144] 1) The threshold setting principle is that the threshold line just exceeds the highest point of the normal negative quality control or is automatically set by the instrument;

[0145] 2) Selection of fluorescence detection channels: FAM channel detects HDV RNA, and VIC / HEX channel detects internal standard.

[0146] 3) If the sample amplification curve shows an "S" shape, and the sample Ct value is ≤34, and the internal standard Ct value is ≤38, the result is judged to be valid and can be directly reported as positive; if the sample detection result shows that the HDV target has no Ct value, and the internal standard Ct value is ≤38, the reported result is negative.

[0147] 4) constructing a standard curve based on the Ct value and the Log value of the HDV calibrator concentration to obtain a standard curve formula, and calculating the hepatitis D virus content in the sample to be tested by substituting the Ct value of the sample to be tested into the standard curve formula, or automatically determining the hepatitis D virus load by the instrument.

[0148] 5. Statistical analysis

[0149] Statistical analyses were performed using MedCalc version 18.2.1 software (MedCalc, Ostend, Belgium). Sensitivity was assessed by Probit regression analysis to estimate the limit of detection (LOD), and the coefficient of determination was assessed by linear regression analysis. In addition, the Pearson correlation coefficient (r) was calculated by Deming regression analysis to determine the linear relationship between the two assays, and the agreement between the assays was assessed using the Bland-Altman method.

[0150] Example 3 Analysis of the detection performance of HDV quantitative method

[0151] 1. Analysis of positive compliance rate

[0152] 10 positive reference samples were tested with the reagent described in Example 1, each reference sample was tested once, and the positive coincidence rate was calculated. Figure 2 As shown in A, the reagent described in Example 1 tested 10 positive reference samples, all of which were positive. The positive compliance rate was 100%.

[0153] 2. Negative compliance rate analysis

[0154] Use the reagent described in Example 1 to test 10 negative reference samples, test each reference sample once, and calculate the negative compliance rate. The negative reference sample test results are shown in Figure 2 As shown in B, the reagent described in Example 1 tested 10 negative reference samples, all of which were negative. The negative compliance rate was 100%.

[0155] 3. Sensitivity analysis

[0156] Take 1 bottle of HDV RNA international standard and add 500 μL nuclease-free water to reconstitute it. Then dilute it with negative plasma to 11 concentrations, including 575 IU / mL, 287.5 IU / mL, 143.75 IU / mL, 57.5 IU / mL, 28.75 IU / mL, 14 IU / mL, 10 IU / mL, 8 IU / mL, 6 IU / mL, 4 IU / mL, and 2 IU / mL. Each concentration was tested 20 times. The concentration with a detection rate of 95% was taken as the sensitivity of this reagent. The dose-response curve is shown in the figure below. Figure 3 As shown in Figure A, the positive detection rate of samples with a concentration greater than or equal to 8 IU / mL is 100%, the positive detection rate of samples with a concentration of 6 IU / mL is 95%, and the detection rate of samples with a concentration of 2 IU / mL is 50%. Therefore, the sensitivity of the reagent is 6 IU / mL. Figure 3 (B) The slope of the equation is -3.306, the square of the linear equation correlation coefficient (R2) is 0.999, the amplification efficiency is 100.656%, and the calibration linearity is good.

[0157] 4. Specificity analysis

[0158] Specific analysis is performed using pathogens or pathogens that have homology in nucleic acid sequences or are likely to cause the same or similar clinical symptoms. The pathogens or pathogens used in specific analysis are described in the clinical specimens and materials section. Each sample is tested once.

[0159] The results of specific analysis are shown in Table 5. The kit described in Example 1 has no cross reaction with hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, dengue virus, Epstein-Barr virus, human cytomegalovirus, human immunodeficiency virus 1, Treponema pallidum, human herpes virus type 6, herpes simplex virus type 1, herpes simplex virus type 2, influenza A virus, Staphylococcus aureus, Candida albicans and human genome.

[0160] Table 5 Specificity analysis

[0161]

[0162] Undetermined: indicates that the signal has not been detected.

[0163] 5. Linear range analysis

[0164] The linear range of the kit was analyzed using pseudovirus or virus culture traceable to international standards. Five HDV genotype virus cultures, including HDV-1, HDV-2, HDV-3, HDV-4, and HDV-8, and three pseudoviruses, including HDV-5, HDV-6, and HDV-7, were cultured from 2 × 10 8 IU / mL was diluted 10-fold to 2×10 7 IU / mL, 2×10 6 IU / mL, 2×10 5 IU / mL, 2×10 4 IU / mL, 2×10 3 IU / mL, 2×10 2 IU / mL, 2×10 1 IU / mL and other 8 concentrations, each concentration was tested 3 times, the average value of the test concentration logarithm and the theoretical concentration logarithm of each concentration were calculated, the test concentration logarithm was used as the Y axis and the theoretical concentration logarithm was used as the X axis, the least squares method was used for linear fitting, and the square of the linear correlation coefficient R was calculated. 2 , the linear correlation coefficient of the linear regression equation satisfies R 2 ≥0.960 (or |R|≥0.980) is the linear range of the conditional analysis kit. The linear range analysis results are as follows Figure 4 As shown in the figure, the linear range of the detection of 8 genotypes of HDV can reach 20~2×10 8IU / mL, R 2 The linear range is between 0.9933 and 0.9992, which is relatively wide. Meanwhile, it is also proved that the reagent described in Example 1 can detect 1 to 8 genotypes of HDV.

[0165] 6. Accuracy analysis

[0166] The WHO HDV RNA international standard was diluted to 1×10 5 IU / mL, 1×10 4 IU / mL, 1×10 3 IU / mL, 1×10 2 IU / mL and 20 IU / mL, and 5 concentration levels were tested 3 times for each level, and the absolute deviation between the logarithm of the test concentration and the logarithm of the theoretical concentration was calculated. The accuracy analysis results (Table 6) showed that in the 3 tests, the absolute values ​​of the absolute deviations between the logarithm of the test concentration and the logarithm of the theoretical concentration were all less than 0.5.

[0167] Table 6 Accuracy test results

[0168]

[0169] 7. Precision analysis

[0170] For precision analysis, select high (1~5)×10 4 IU / mL), low (100-1000 IU / mL) and negative samples were tested 10 times for each sample, and the logarithm, mean, standard deviation and coefficient of variation of the 10 measured concentrations of each sample were calculated. The test results are shown in Table 7. All 10 negative samples were negative, the coefficient of variation of the high value sample was 2.2%, and the coefficient of variation of the low value sample was 6.6%, and the reagent showed good repeatability.

[0171] Table 7

[0172]

[0173] Undetermined: indicates that the signal has not been detected.

[0174] Example 4 Comparative analysis with foreign commercial reagents

[0175] The HDV RNA concentration of 26 HDV IgG positive plasma samples was measured using the reagent in Example 1 and the commercial HDV RNA quantitative reagent RoboGene HDV RNA Quantification Kit 2.0 from Jena CE, Germany. The results were analyzed by Deming regression to calculate the Pearson correlation coefficient (r) to determine the linear relationship between the two assays, and the Bland-Altman method was used to evaluate the consistency between the assay results. The results are shown in Figure 5 As shown, the reagent described in this application and the commercial reagent kit in Jena, Germany both detected 22 samples of HDV RNA positive, and the detection rate was consistent. Deming regression analysis showed that the Pearson correlation coefficient was 0.9170, and the test results of the kit described in this application and the commercial reagent in Jena, Germany had a good correlation. Bland-Altman analysis showed that the test results of the kit described in this application and the commercial reagent in Jena, Germany had a good consistency.

[0176] Example 5 Accelerated stability analysis

[0177] In order to evaluate the stability of the hepatitis D virus nucleic acid assay reagent of the present application, the reagents of Example 1 were divided into four groups, each with 48 portions. One group was stored at -15 to -35°C, one group was stored at 2 to 8°C, one group was stored at room temperature, and one group was stored in a 37°C constant temperature box. After processing for 1 day, 3 days, 5 days, and 7 days, the reagents were taken out to test the HDV strong positive quality control product and the weak positive quality control product, and each was repeated 3 times. The logarithmic values ​​of the concentration values ​​of the quality control products measured by the reagents treated at different temperature conditions for different times were calculated, and the accelerated stability of the reagent was evaluated based on the absolute value of the absolute deviation of the logarithmic value being no more than 0.5 logarithmic order of magnitude. The results of the accelerated stability analysis are as follows. Figure 6 As shown, the hepatitis D virus nucleic acid determination kit described in the present application is stored at room temperature or 37°C for 7 days, and the concentration of the strong positive and weak positive quality control products is not significantly changed. This fully demonstrates that the kit described in the present application can still maintain its good detection effect and good stability when stored at room temperature or 37°C for 7 days.

[0178] In summary, the present application has developed a highly sensitive and specific HDV RNA quantitative determination kit based on multiplex fluorescence quantitative PCR, which can quantitatively determine the patient's HDV viral load. Compared with qualitative reagents that can only qualitatively detect HDV RNA, this kit can be used to monitor the HDV RNA content and changes in the plasma or serum of hepatitis D patients, and to evaluate the response and treatment effect of antiviral treatment.

[0179] In addition, by designing a synthetic non-competitive sequence as an internal standard control, the entire process of nucleic acid extraction and amplification detection can be monitored in a fully closed reaction system, which can effectively monitor the occurrence of false negatives; furthermore, by taking advantage of the design of heat-sensitive uracil-N-glycosylase (Heat-UDG) treatment, it can effectively prevent secondary contamination of aerosols and avoid the occurrence of false positive results. The test results can be traced back to WHO international standards, making the HDV RNA quantification results more accurate. The high sensitivity, good repeatability and wide dynamic range make this kit suitable for detecting and quantifying HDV RNA in clinical samples for diagnosing HDV infection, monitoring the therapeutic effects of antiviral drugs and evaluating disease progression.

[0180] Comparative Example 1

[0181] Comparative Example 1 provides a hepatitis D virus nucleic acid detection kit, which differs from the kit in Example 1 in that it includes the following primers and probes:

[0182] HDV-qPCR-F1: CTCCCTTAGCCATCCGAG (SEQ ID NO: 8), HDV-qPCR-F2: CTCCCTTTGCCATCCGAG (SEQ ID NO: 9), HDV-qPCR-R1: TCCTCTCGGGTCGGCATGG (SEQ ID NO: 10), HDV-qPCR-probel: FAM-CGGATGCCCAGGTCGGACC-MGB (SEQ ID NO: 11).

[0183] Comparison of the primer and probe combinations of Example 1 and Comparative Example 1:

[0184] 1. The primers described in the present application and the primers described in Comparative Document 1 can both detect 1 to 8 HDV genotype viruses, but Example 1 of the present application has a higher sensitivity. In Example 1, a large number of published HDV gene sequences were homologously compared and analyzed to find that the primers HDV-qPCR-F1 and HDV-qPCR-F2 described in Comparative Example 1 have variations in the HDV-7 genotype ( Figure 7 ), which may easily lead to missed detection of HDV variant strains of the HDV-7 genotype; while the primers HDV-F1 and HDV-F2 described in Example 1 of the present application are more conservative in the gene sequences of all 8 genotypes of HDV viruses including HDV-7 ( Figure 8 ), compared with the primers HDV-qPCR-F1 and HDV-qPCR-F2 described in Comparative Example 1, it has higher detection tolerance and has detection reliability for potential variants of HDV virus.

[0185] 2. The probe described in this application is a common TaqMan probe, which is cheaper than the TaqMan-MGB probe used in Comparative Example 1. The common TaqMan probe is longer than the TaqMan-MGB probe, and is easier to bind to the target sequence and has a higher amplification efficiency. Fig. 9 .

[0186] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0187] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. A nucleic acid combination, characterized in that For detecting hepatitis D virus, the nucleic acid combination comprises a detection primer and a detection probe; The nucleotide sequences of the detection primers are shown in SEQ ID NOs: 1-3, and the nucleotide sequence of the detection probe is shown in SEQ ID NO:

4.

2. The nucleic acid combination according to claim 1, characterized in that The two ends of the detection probe are respectively connected with a fluorescent reporter group and a fluorescent quencher group; Optionally, the fluorescent reporter group is located at the 5' end of the detection probe, and the fluorescent quencher group is located at the 3' end of the detection probe.

3. A hepatitis D virus nucleic acid detection reagent, characterized in that: The reagent comprises the nucleic acid combination according to claim 1 or 2.

4. The hepatitis D virus nucleic acid detection reagent according to claim 3, characterized in that: The reagents also include detection primers and detection probes for internal standard quality control; Optionally, the nucleotide sequence of the detection primers for the internal standard quality control is shown in SEQ ID NO: 5-6, and the nucleotide sequence of the detection probe for the internal standard quality control is shown in SEQ ID NO:

7.

5. The hepatitis D virus nucleic acid detection reagent according to claim 3, characterized in that: The reagents also include PCR reaction solution; Optionally, the PCR reaction solution includes one or more of Tris buffer, potassium chloride, magnesium chloride, Triton X-100, bovine serum albumin, trehalose, betaine, dATP, dTTP, dGTP, dCTP, dUTP, Heat-UDG, RNasin and nuclease-free distilled water; Optionally, the PCR reaction solution includes the following components at working concentrations: 10mM-100mM Tris buffer, 10mM-100mM potassium chloride, 1mM-4mM magnesium chloride, 0.1%v / v-1.0%v / v Triton X-100, 0.1mg / mL-1.0mg / mL bovine serum albumin, 100mM-300mM trehalose, 400mM-1000mM betaine, 0.15mM-0.25mM dATP, 0.15mM-0.25mM dTTP, 0.15mM-0.25mM dGTP, 0.15mM-0.25mM dCTP, 0.15mM-0.25mM dUTP, 0.01U / μL-0.1U / μL Heat-UDG and 0.8U / μL-1.0U / μL RNasin.

6. The hepatitis D virus nucleic acid detection reagent according to any one of claims 3 to 5, characterized in that: The reagents also include PCR enzyme solution; Optionally, the PCR enzyme solution includes one or both of reverse transcriptase and Taq DNA polymerase; Optionally, the PCR enzyme solution includes 0.1U / μL-0.2U / μL reverse transcriptase and 0.1U / μL-0.2U / μL Taq DNA polymerase.

7. A hepatitis D virus nucleic acid detection kit, characterized in that: The kit comprises the nucleic acid combination according to claim 1 or 2, or the hepatitis D virus nucleic acid detection reagent according to any one of claims 3 to 6.

8. The hepatitis D virus nucleic acid detection kit according to claim 7, characterized in that: The kit also includes one or more of a negative quality control product, a positive quality control product and a calibrator.

9. A detection method of a hepatitis D virus nucleic acid detection kit, characterized in that: The steps include: Using the extracted RNA of the sample to be tested as a template, amplification is performed using the hepatitis D virus nucleic acid detection kit according to any one of claims 7 to 8; and Determining whether the sample to be tested contains hepatitis D virus and / or determining the content of hepatitis D virus in the sample to be tested according to the amplification result; Optionally, the detection method comprises one-step multiplex fluorescence quantitative PCR.

10. Use of the nucleic acid combination according to claim 1 or 2 or the hepatitis D virus nucleic acid detection reagent according to any one of claims 3 to 6 in any of the following: (1) Application in the preparation of products for screening, prevention, control and management of hepatitis D virus infection; (2) Use in the preparation of products for diagnosis or auxiliary diagnosis of hepatitis B surface antigen-positive patients with concurrent or superinfection with hepatitis D virus; (3) Use in the preparation of products for diagnosing or distinguishing between current and past hepatitis D virus infection; (4) Use in the preparation of products for diagnosing or confirming active HDV infection in patients with total anti-HDV antibody positive; (5) Use in the preparation of products for evaluating or monitoring the efficacy of antiviral drug treatment and antiviral immune response in patients with hepatitis D; and (6) Application in the preparation of products for monitoring the cure and post-cure recurrence of hepatitis D patients; Optionally, the hepatitis D virus includes one or more hepatitis D viruses of genotypes HDV-1 to HDV-8.

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