Primer group and method for quantitative amplification and detection of HBV RNA in serum or plasma

Abstract

The invention relates to the technical field of biology, in particular to a primer group and a method for quantitatively amplifying and detecting HBV RNA in serum or plasma. The invention provides a blocking primer, a primer group, a reagent and a kit for RNA amplification of hepatitis B virus and application of the blocking primer, the primer group, the reagent and the kit. The blocking primer for hepatitis B virus RNA amplification is obtained through screening, the optimal primer group for hepatitis B virus RNA amplification is obtained through further optimization, the primer group is used for hepatitis B virus RNA amplification, it can be guaranteed that specific amplification is selectively carried out only on HBV RNA in HBV DNA and RNA genome mixed nucleic acid, time and labor are saved, and the detection efficiency is improved. The method is good in repeatability, good in accuracy, high in sensitivity and low in detection limit, can be matched with an automatic device for use, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a primer set and a method for quantitatively amplifying and detecting HBV RNA in serum or plasma. Background Art

[0002] Hepatitis B virus (HBV) is a DNA virus belonging to the family Hepadnaviridae. The full genome of HBV is about 3.2kb. HBV has multiple genotypes AH (ref). HBV covalently closed circular DNA (cccDNA) is the replication template of HBV; and HBV RNA is the direct transcript of cccDNA in hepatocytes. Existing studies have shown that HBV RNA can be used as a reference indicator for the clinical cure of HBV, and its level and changes can predict the activity status of HBV cccDNA to a certain extent.

[0003] In 2022, China published an expert consensus on the detection and clinical application of serum HBV RNA in chronic HBV infection, which mentioned serum HBV RNA as an indicator for the presence and transcriptional activity of cccDNA. The detection of HBV RNA is different from that of RNA viruses such as the new coronavirus. The serum samples of hepatitis B patients contain both DNA and RNA. The key point of its detection is how to eliminate DNA interference in the sample without losing RNA.

[0004] In the prior art, the RT-qPCR method is used to quantify HBV RNA. Because it cannot distinguish between cccDNA and DNA, it is necessary to use DNase I to digest DNA before the reaction to eliminate the effect of DNA on the quantification of HBV RNA. However, this artificial intervention treatment easily leads to RNA degradation, resulting in reduced system sensitivity and inaccurate test results. At the same time, the digested samples cannot be used for joint analysis with HBV DNA. The RNA real-time fluorescence constant temperature amplification detection technology (SAT) method is used to quantify HBV RNA, which uses specific target capture magnetic bead extraction technology to specifically extract and amplify HBV RNA in the sample, thereby solving the problem of DNA+RNA mixed samples. However, this method requires the use of a specific all-in-one machine to achieve automation, which is costly, and the extracted samples still cannot meet the requirements of joint analysis with HBV DNA (samples need to be re-extracted). Therefore, it is urgent to develop a one-step detection technology that selectively amplifies RNA from HBV whole nucleic acid (DNA+RNA), with simple steps, low cost, high sensitivity and good specificity. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a primer set and method for quantitatively amplifying and detecting HBV RNA in serum or plasma.

[0006] The present invention provides a blocking primer for detecting hepatitis B virus RNA, wherein the nucleotide sequence of the blocking primer is at least one of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0007] The total HBV nucleic acid extracted from the patient's serum includes DNA and RNA. The RNA is 3.5kb in length and contains the entire sequence of the genotype DNA (3.2kb). The PCR primers currently used can bind to both RNA and DNA at the same time and cannot selectively amplify HBV RNA. Therefore, in the detection process, the HBV virus total nucleic acid (DNA+RNA) is usually extracted first, and then the DNA is removed by DNase digestion, and then RNA extraction and detection are performed. It can be seen that the existing detection process involves multiple steps, and its disadvantages are that it is time-consuming, costly, and requires setting and strict control. Based on this, the present invention provides a blocking primer for hepatitis B virus RNA detection (which competitively binds to HBV DNA), and screens the blocking primer for hepatitis B virus RNA detection. The results show that among the blocking primers shown in SEQ ID NO:1 to SEQ ID NO:10, the blocking primers of the nucleotide sequences shown in SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 have a better ability to block the amplification of hepatitis B virus DNA, among which the blocking primers of the nucleotide sequences shown in SEQ ID NO:4 or SEQ ID NO:5 have the best blocking effect.

[0008] The present invention provides a primer set, which comprises the blocking primer of the present invention and a primer set for amplifying hepatitis B virus RNA.

[0009] The primer set for amplifying hepatitis B virus RNA includes an upstream amplification primer and a downstream amplification primer;

[0010] The nucleotide sequence of the upstream amplification primer is at least one of SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29 or SEQ ID NO: 32;

[0011] The nucleotide sequence of the downstream amplification primer is at least one of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30 or SEQ ID NO:33.

[0012] Furthermore, the primer set of the present invention also includes a probe targeting hepatitis B virus RNA.

[0013] The nucleotide sequence of the probe is at least one of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31 or SEQ ID NO:34.

[0014] The 5' end of the probe is coupled to a fluorescent group, and the 3' end is coupled to a fluorescent quenching group.

[0015] The fluorescent group is selected from any one of FAM, VIC, ROX or CY-5.

[0016] The fluorescence quenching group is selected from any one of BHQ1, MGB, MGB or BHQ2.

[0017] Furthermore, the primer set of the present invention includes at least one of the following combinations:

[0018] An upstream primer having a nucleotide sequence as shown in SEQ ID NO: 14, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 15, a blocking primer having a nucleotide sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 16; and / or

[0019] An upstream primer having a nucleotide sequence as shown in SEQ ID NO: 17, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 18, a blocking primer having a nucleotide sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 16; and / or

[0020] An upstream primer having a nucleotide sequence as shown in SEQ ID NO: 17, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 18, a blocking primer having a nucleotide sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 19; and / or

[0021] An upstream primer having a nucleotide sequence as shown in SEQ ID NO: 20, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 21, a blocking primer having a nucleotide sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 22; and / or

[0022] An upstream primer having a nucleotide sequence as shown in SEQ ID NO: 23, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 24, a blocking primer having a nucleotide sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 25; and / or

[0023] An upstream primer having a nucleotide sequence as shown in SEQ ID NO: 26, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 27, a blocking primer having a nucleotide sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 28; and / or

[0024] An upstream primer having a nucleotide sequence as shown in SEQ ID NO:29, a downstream primer having a nucleotide sequence as shown in SEQ ID NO:30, a blocking primer having a nucleotide sequence as shown in SEQ ID NO:4 and SEQ ID NO:5, and a probe having a nucleotide sequence as shown in SEQ ID NO:31; and / or

[0025] An upstream primer having a nucleotide sequence as shown in SEQ ID NO:32, a downstream primer having a nucleotide sequence as shown in SEQ ID NO:33, a blocking primer having a nucleotide sequence as shown in SEQ ID NO:4 and SEQ ID NO:5, and a probe having a nucleotide sequence as shown in SEQ ID NO:34; and / or

[0026] An upstream primer having a nucleotide sequence as shown in SEQ ID NO: 14, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 18, a blocking primer having a nucleotide sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 22; and / or

[0027] An upstream primer having a nucleotide sequence as shown in SEQ ID NO: 23, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 18, a blocking primer having a nucleotide sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 25; and / or

[0028] An upstream primer having a nucleotide sequence as shown in SEQ ID NO: 23, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 21, a blocking primer having a nucleotide sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 22; and / or

[0029] An upstream primer having a nucleotide sequence as shown in SEQ ID NO:26, a downstream primer having a nucleotide sequence as shown in SEQ ID NO:18, a blocking primer having a nucleotide sequence as shown in SEQ ID NO:4 and SEQ ID NO:5, and a probe having a nucleotide sequence as shown in SEQ ID NO:34.

[0030] The present invention screened the combination of blocking primers and primer probes, and the results showed that among the above 12 groups of screening, SEQ ID NO: 4 and SEQ ID NO: 5 had the best hepatitis B virus DNA blocking effect and the best hepatitis B virus RNA amplification effect.

[0031] The present invention takes the conserved region of the hepatitis B virus genome as a target and designs specific primer probes. The fluorescent quantitative PCR HBV RNA detection method constructed based on the primer probes of the present invention has a sensitive and accurate effect on detecting serum HBV RNA of patients with low viral load. Based on this method, accurate detection of hepatitis B virus is achieved, and the whole process can be fully automated, saving time and labor and having good repeatability.

[0032] Furthermore,

[0033] The present invention provides a reagent combination, which includes a template, Tricine, KOAc, glycerol, DMSO, Taq enzyme, reverse transcriptase, UDG enzyme, dNTP and / or Mn 2+ At least one of and at least one of the following A) to B):

[0034] A), the blocking primer of the present invention;

[0035] B) The primer set of the present invention.

[0036] The present invention provides a kit, characterized in that it comprises at least one of a positive quality control product and / or a negative quality control product and at least one of the following I) to III):

[0037] 1), the blocking primer of the present invention;

[0038] II), the primer set of the present invention;

[0039] III), the reagent combination described in the present invention.

[0040] The present invention provides the use of at least one of the following a) to d) in the detection of hepatitis B virus RNA:

[0041] a), the blocking primer of the present invention;

[0042] b) the primer set of the present invention;

[0043] c) the reagent combination of the present invention;

[0044] d) The kit of the present invention.

[0045] The present invention provides a method for detecting hepatitis B virus RNA for non-diagnostic purposes, which is to detect a sample using at least one of the following i) to iii):

[0046] i), the blocking primer of the present invention;

[0047] ii), the primer set of the present invention;

[0048] iii), the reagent combination of the present invention;

[0049] iv), the kit of the present invention.

[0050] Furthermore, the samples include HBV RNA national standard products and hepatitis B clinical high-value samples.

[0051] The present invention provides blocking primers, primer sets, reagent combinations, kits and applications for amplifying hepatitis B virus RNA. The present invention obtains blocking primers for amplifying hepatitis B virus RNA by screening, and obtains the optimal primer set for amplifying hepatitis B virus RNA by further optimization. The primer set is used for amplifying hepatitis B virus RNA, and can ensure that only HBV RNA is selectively amplified in a mixed nucleic acid of HBV DNA and RNA genome, and it is time-saving and labor-saving, has good repeatability, good accuracy, high sensitivity, low detection limit, can be used in conjunction with an automated device, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The screening results of blocking primers are shown;

[0053] Figure 2 The screening results of blocking primers and primer set combinations are shown;

[0054] Figure 3 The effect of adding blocking primers on the detection results is shown in Figure 2.

[0055] Figure 4 The national standard was diluted to a concentration of 15 IU / mL, and all 20 replicate wells were detected;

[0056] Figure 5 Shown is the comparison between DNase digested and undigested;

[0057] Figure 6 Indicates HBV virus RNA standard detection;

[0058] Figure 7 The results showed that the specificity of RNA detection in serum of HBV infected patients was 0. DETAILED DESCRIPTION

[0059] The present invention provides a primer set and method for quantitative amplification and detection of HBV RNA in serum or plasma. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0060] The present invention relates to a method for rapidly detecting the presence of HBV RNA in a biological sample for use in monitoring HBV disease status and treatment effects;

[0061] In the present invention, in the presence of homologous DNA, in HBV patients, both viruses containing DNA and RNA circulate in the blood, and in untreated patients, DNA levels are generally higher than RNA levels. These two forms can also be found together in other sample types. Conventional PCR techniques cannot distinguish HBV RNA (mainly pgRNA) from DNA because they have a common sequence (only a subset of HBV RNA forms is spliced). DNA removal techniques (such as DNA enzymes or other sample processing methods that may be beneficial to RNA or depleted DNA) may also affect RNA titer, may not be completely effective, or have variable effectiveness, and have stability, contamination or other disadvantages that prevent it from being used in automated or high-throughput workflows.

[0062] One sequence difference between HBV DNA and RNA is the poly A tail of pgRNA and other mRNAs; however, methods using oligo d(T) primers can detect non-target RNA or other sequences with poly A fragments. However, this is a trade-off strategy that results in some binding to HBV DNA. To improve the assay performance (sensitivity and specificity) of targeted RNA in the presence of DNA, the present invention utilizes blocking primers that specifically match the DNA sequence. The binding of the blocking primer to the homologous HBV DNA prevents the binding of primers (e.g., RT primers) to it, thereby reducing the amplification of the homologous genomic HBV DNA.

[0063] The primer set for amplifying hepatitis B virus RNA targets HBV RNA, especially HBV RNA transcribed from cccDNA, and is directed to the poly A tail of the transcript (such as pgRNA, but also other mRNA and spliced ​​RNA); and the primer set (blocking primer, amplification primer and probe) of this kit is not specially modified, its cost is low, and the reagents can be used directly on a fully automatic instrument, which is convenient and fast.

[0064] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0065] Example 1 Obtaining the Primers and Kits of the Present Invention

[0066] 1. Screening of blocking primers and primer-probe combinations

[0067] (i) Screening of blocking primers;

[0068] 1. Screen the blocking primers. The blocking primer sequences are shown in Table 1.

[0069] Table 1. Blocking primer sequences to be screened

[0070]

[0071] The blocking primers shown in Table 1 were respectively matched with DNA high-efficiency amplification primers for DNA amplification, that is, only one blocking primer was added to each reaction system, and the blocking primers were screened; specifically, HBV DNA was used as a template (in the present invention, the optical detection label of all probes was FAM, which was connected to the 3' end of the probe sequence shown);

[0072] 2. Sample testing:

[0073] The reaction system was, with a total volume of 50 μL, including: 30 μL Mix, 20 μL template HBV DNA, with a total volume of 50 μL;

[0074] Mix components include: Tricine (1M) 3μL, KOAc (10M) 0.5μL, glycerol (100%) 0.5μL, DMSO 2.5μL, upstream primer (100μM) 0.1μL, downstream primer (100μM) 0.1μL, probe (100μM) 0.05μL, blocking primer (100μM) 0.1μL, Taq enzyme (1U / μL) 1μL, UDG enzyme (1U / μL) 0.2μL, dNTP (dATP+dGTP+dUTP+dCTP) (20mM) 0.8μL, Mn 2+ (25mM) 6μL, H2O 15.05μL.

[0075] The reaction procedure was 42℃2min; 94℃2min; (94℃10s, 58℃30s, 45Cycles); 3. Test results

[0076] Table 2. Blocking primer screening results

[0077]

[0078] The results are shown in Table 2, where the control was a high-value clinical HBV DNA sample (1×10 7 IU / mL, without adding blocking primers, only adding DNA high-efficiency amplification primer probes to determine the DNA content in the sample); from Table 2 and Figure 1 It can be seen that blocking primer 5 and blocking primer 4 have the best blocking effect on HBV DNA amplification (where Z-1 to Z-10 are the detection results of experimental groups 1 to 10, respectively, Z represents blocking primer, and one group of two results is the parallel experimental results of duplicate well detection).

[0079] (II) Screening of blocking primer and primer probe combinations

[0080] Table 3. Blocking primer and primer probe combinations

[0081]

[0082]

[0083] 1. The combination forms and sequences of the blocking primer and primer probe combination screening are shown in Table 3 (in Table 3, w represents base A or T, and n represents base A, T, C, G or U);

[0084] 2. Sample testing;

[0085] Reaction system: total volume is 50 μL, including: 30 μL Mix, 20 μL template HBV nucleic acid (a mixture of HBV RNA and HBV DNA), total volume is 50 μL;

[0086] Mix1 ingredients include: Tricine (1M) 3μL, KOAc (10M) 0.5μL, glycerol (100%) 0.5μL, DMSO 2.5μL, upstream primer (100μM) 0.1μL, downstream primer (100μM) 0.1μL, probe (100μM) 0.05μL, blocking primer 4 (100μM) 0.1μL, blocking primer 5 (100μM) 0.1μL, Taq enzyme (1U / μL) 1μL, reverse transcriptase (1U / μL) 1μL, UDG enzyme (1U / μL) 0.2μL, dNTP (dATP+dGTP+dUTP+dCTP) (20mM) 0.8μL, Mn 2+ (25mM) 6μL, H2O 15.05μL.

[0087] Reaction procedure: reverse transcription at 52°C for 5 min; pre-denaturation at 95°C for 2 min; reverse transcription at 65°C for 15 min; denaturation at 95°C for 10 s, annealing / extension at 60°C for 20 s, for a total of 45 cycles;

[0088] 3. Test results

[0089] The sample test results are shown in Table 5 and Figure 2 , the results show that the primer-probe combination of combination 1 is the best (one set of two results is the parallel experimental results of duplicate well detection);

[0090] Table 5. Screening results of blocking primer and primer probe combination

[0091]

[0092]

[0093] (III) Optimization of blocking primers

[0094] 1. The method of adding blocking primers was optimized as shown in Table 7. The sequences of DNA high-efficiency amplification and blocking primers used were shown in Table 6;

[0095] Table 6. Primers used for optimization of blocking primer addition method

[0096]

[0097] Table 7. Optimization of adding blocking primers

[0098]

[0099]

[0100] 8. Blocking primer addition method to optimize results

[0101]

[0102] 2. Sample testing

[0103] Detection system: The reaction system has a total volume of 50 μL, including: 30 μL Mix, 20 μL template HBV nucleic acid (a mixture of HBV RNA and HBV DNA), and a total volume of 50 μL;

[0104] Mix components include: Tricine (1M) 3μL, KOAc (10M) 0.5μL, glycerol (100%) 0.5μL, DMSO 2.5μL, upstream primer (100μM) 0.1μL, downstream primer (100μM) 0.1μL, probe (100μM) 0.05μL, blocking primer (100μM) 0.1μL, Taq enzyme (1U / μL) 1μL, UDG enzyme (1U / μL) 0.2μL, dNTP (dATP+dGTP+dUTP+dCTP) (20mM) 0.8μL, Mn 2+ (25mM) 6μL, H2O 15.05μL.

[0105] The reaction procedure was 42℃2min; 94℃2min; (94℃10s, 58℃30s, 45 cycles); 3. Results

[0106] The results are as follows Figure 3 It can be seen that the addition of two blocking primers in group D can completely block the amplification of DNA.

[0107] 2. Detection of performance after optimization

[0108] 1. Detection of HBV RNA

[0109] Using combination 1 and the addition of both blocking primers (blocking primer 4 and blocking primer 5), the reaction system and reaction procedure were as follows: the national standard was diluted to a concentration of 15 IU / mL, and 20 replicate wells were tested:

[0110] Reaction system: total volume 50 μL, including: 30 μL Mix, 20 μL nucleic acid template diluted to 15 IU / mL concentration by national standard, total volume 50 μL;

[0111] Mix1 ingredients include: Tricine (1M) 3μL, KOAc (10M) 0.5μL, glycerol (100%) 0.5μL, DMSO 2.5μL, upstream primer (100μM) 0.1μL, downstream primer (100μM) 0.1μL, Taqman probe (100μM) 0.05μL, blocking primer 4 (100μM) 0.1μL, blocking primer 5 (100μM) 0.1μL, Taq (1U / μL) 1μL, reverse transcriptase (1U / μL) 1μL, UDG enzyme (1U / μL) 0.2μL, dNTP (dATP+dGTP+dUTP+dCTP) (20mM) 0.8μL, Mn 2+ (25 mM) 6 μL, H2O 14.15 μL;

[0112] Reaction procedure: reverse transcription at 52°C for 5 min; pre-denaturation at 95°C for 2 min; reverse transcription at 65°C for 15 min; denaturation at 95°C for 10 s, annealing / extension at 60°C for 20 s, for a total of 45 cycles.

[0113] The results are as follows Figure 4 As shown, all 20 replicate wells were detected, and the lowest detection concentration was 15 IU / mL.

[0114] 2. Comparison of HBV RNA selective RT-qPCR in DNase digested and undigested

[0115] The HBV genome was extracted using nucleic acid extraction or purification reagents (registration number: Yuzheng Xiebei 20180037, Yuzheng Xiebei 20220461) as a template for RT-qPCR. The RT-qPCR reaction system is as above; the RT-qPCR results are as follows: Figure 5 As shown, Group A: HBV DNA detection (DNA concentration was quantified using the commercially available Shengxiang kit and then detected; the samples used in Groups A, B, C, and D were consistent, and Group A measured the concentration of the sample before testing); Groups B and C: duplicate well detection, after the samples were digested with DNA enzyme, HBV RNA was detected using the reagents and methods of the present invention; Group D: HBV RNA was detected using the reagents and methods of the present invention without DNA enzyme digestion (this kit cannot amplify HBV DNA); The experimental results show that: the system can only amplify and analyze HBV RNA, and there is no signal for HBV DNA and negative control (nuclease-free water).

[0116] 3. HBV virus RNA standard detection (determination of detection limit)

[0117] Preparation of HBV RNA standard. The HBV RNA national standard was diluted in a gradient manner to A: 2×10 5 IU / mL, B: 2×10 4IU / mL, C: 2×10 3 IU / mL, D: 2×10 2 IU / mL, E: 2×10 1 IU / mL, F: 2IU / mL.

[0118] The quantitative PCR reaction program was as follows: reverse transcription at 2°C for 5 min; pre-denaturation at 95°C for 2 min; reverse transcription at 65°C for 15 min; denaturation at 95°C for 10 s, annealing / extension at 60°C for 20 s, for a total of 45 cycles;

[0119] The total volume of the reaction system was 50 μL, including 30 μL Mix and 20 μL template, with a total volume of 50 μL.

[0120] 30 μL Mix includes: Tricine (1M) 3 μL, KOAc (10M) 0.5 μL, glycerol (100%) 0.5 μL, DMSO 2.5 μL, upstream primer (100 μM) 0.1 μL, downstream primer (100 μM) 0.1 μL, Taqman probe (100 μM) 0.05 μL, blocking primer 4 (100 μM) 0.1 μL, blocking primer 5 (100 μM) 0.1 μL, Taq (1 U / μL) 1 μL, reverse transcriptase (1 U / μL) 1 μL, UDG enzyme (1 U / μL) 0.2 μL, dNTP (dATP+dGTP+dUTP+dCTP) (20 mM) 0.8 μL, Mn 2+ (25mM) 6μL, H2O 15.05μL.

[0121] from Figure 6 The results show that this kit has good detection sensitivity ( Figure 6 A) in the figure can effectively amplify 2IU / reaction, and the detection curve is relatively standard.

[0122] 4. Specificity of RNA detection in serum of HBV infected patients

[0123] HBV total nucleic acid extraction: HBV patient serum with known DNA concentration (6 copies) was selected. 500μL HBV patient serum was extracted with nucleic acid extraction or purification reagent (registration number: Yu Zheng Xie Bei 20180037, Yu Zheng Xie Bei 20220461) to extract HBV viral nucleic acid. The elution volume was 100μL, and 20μL of eluted nucleic acid was retained. Quantitative PCR detection: Using the same detection kit (Antu HBV RNA Nucleic Acid Detection Kit), this experiment used 6 different patient serum samples. Referring to the standard curve, the obtained viral RNA was calculated. The test results are shown in Figure 7 .

[0124] like Figure 7As shown, in the mixed DNA+RNA template, if reverse transcriptase is not added, no signal is detected; only in the reaction wells where reverse transcriptase is added, there is an amplification signal. This result shows that downstream primer 1 (SEQ ID NO: 2) can selectively amplify HBV RNA, but cannot amplify using DNA as a template.

[0125] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A blocking primer for detecting hepatitis B virus RNA, characterized in that: The nucleotide sequence is at least one of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:

5.

2. A primer set, characterized in that: The invention comprises the blocking primer according to claim 1 and a primer set for amplifying hepatitis B virus RNA.

3. The primer set according to claim 2 and claim 1, characterized in that: The primer set for amplifying hepatitis B virus RNA includes an upstream amplification primer and a downstream amplification primer; The nucleotide sequence of the upstream amplification primer is at least one of SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29 or SEQ ID NO: 32; The nucleotide sequence of the downstream amplification primer is at least one of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30 or SEQ ID NO:

33.

4. The primer set according to claim 2 or 3, characterized in that: Also included are probes targeting hepatitis B virus RNA.

5. The primer set according to claim 4, characterized in that: The nucleotide sequence of the probe is at least one of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31 or SEQ ID NO:

34.

6. The primer set according to any one of claims 2 to 5, characterized in that: Include at least one of the following combinations: An upstream primer having a nucleotide sequence as shown in SEQ ID NO: 14, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 15, a blocking primer having a nucleotide sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 16; and / or An upstream primer having a nucleotide sequence as shown in SEQ ID NO: 17, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 18, a blocking primer having a nucleotide sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 16; and / or An upstream primer having a nucleotide sequence as shown in SEQ ID NO: 17, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 18, a blocking primer having a nucleotide sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 19; and / or An upstream primer having a nucleotide sequence as shown in SEQ ID NO: 20, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 21, a blocking primer having a nucleotide sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 22; and / or An upstream primer having a nucleotide sequence as shown in SEQ ID NO: 23, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 24, a blocking primer having a nucleotide sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 25; and / or An upstream primer having a nucleotide sequence as shown in SEQ ID NO: 26, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 27, a blocking primer having a nucleotide sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 28; and / or An upstream primer having a nucleotide sequence as shown in SEQ ID NO:29, a downstream primer having a nucleotide sequence as shown in SEQ ID NO:30, a blocking primer having a nucleotide sequence as shown in SEQ ID NO:4 and SEQ ID NO:5, and a probe having a nucleotide sequence as shown in SEQ ID NO:31; and / or An upstream primer having a nucleotide sequence as shown in SEQ ID NO:32, a downstream primer having a nucleotide sequence as shown in SEQ ID NO:33, a blocking primer having a nucleotide sequence as shown in SEQ ID NO:4 and SEQ ID NO:5, and a probe having a nucleotide sequence as shown in SEQ ID NO:34; and / or An upstream primer having a nucleotide sequence as shown in SEQ ID NO: 14, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 18, a blocking primer having a nucleotide sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 22; and / or An upstream primer having a nucleotide sequence as shown in SEQ ID NO: 23, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 18, a blocking primer having a nucleotide sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 25; and / or An upstream primer having a nucleotide sequence as shown in SEQ ID NO: 23, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 21, a blocking primer having a nucleotide sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 22; and / or An upstream primer having a nucleotide sequence as shown in SEQ ID NO:26, a downstream primer having a nucleotide sequence as shown in SEQ ID NO:18, a blocking primer having a nucleotide sequence as shown in SEQ ID NO:4 and SEQ ID NO:5, and a probe having a nucleotide sequence as shown in SEQ ID NO:

34.

7. A reagent combination, characterized in that include: Template, Tricine, KOAc, glycerol, DMSO, Taq enzyme, reverse transcriptase, UDG enzyme, dNTP and / or Mn 2+ At least one of and at least one of the following A) to B): A), the blocking primer according to claim 1; B) The primer set according to any one of claims 2 to 6.

8. A kit, characterized in that Including at least one of the positive quality control and / or negative quality control and at least one of the following I) to III): 1), the blocking primer according to claim 1; II), a primer set according to any one of claims 2 to 6; III) The reagent combination according to claim 7.

9. Use of at least one of the following a) to d) in the detection of hepatitis B virus RNA: a), the blocking primer according to claim 1; b) a primer set according to any one of claims 2 to 6; c) The reagent combination according to claim 7; d) The kit according to claim 8.

10. A method for detecting hepatitis B virus RNA for non-diagnostic purposes, characterized in that: The sample is tested using at least one of the following i) to iii): i), the blocking primer according to claim 1; ii), the primer set according to any one of claims 2 to 6; iii) the reagent combination according to claim 7; iv) The kit according to claim 8.

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