CN107058623A - A kind of High sensitivity and special blood HBV pgRNA fluorescence quantitative PCR detections systems and detection method - Google Patents

Abstract

The invention discloses a blood hepatitis B virus (HBV) pregenome RNA (pregenome RNA, pgRNA) fluorescent quantitative PCR detection system and a method for detecting the HBV pgRNA by using the detection system. The detection system includes a forward primer, whose nucleotide sequence is shown in SEQ NO:1; a reverse primer, whose nucleotide sequence is shown in SEQ NO:2; a reverse transcription primer with a random anchor sequence, Its nucleotide sequence is shown in SEQ NO:3; and the probe, its nucleotide sequence is shown in SEQ NO:4. The detection system and method of the invention can be used to prepare HBV diagnostic reagents or kits. The invention eliminates the possible interference caused by HBV DNA or other RNA during the detection process, and ensures the specificity of blood HBV pgRNA detection.

Description

A highly sensitive and specific blood HBV pgRNA fluorescent quantitative PCR detection system and detection method

technical field

The present invention generally relates to the diagnosis and treatment of viral hepatitis B, as well as the fields of infectious disease, molecular biology, cell biology and the like. More specifically, the present invention relates to a fluorescent quantitative PCR detection method for hepatitis B virus (Hepatitis B virus, HBV) pgRNA in blood.

Background technique

Hepatitis B virus (HBV) infection is prevalent in the world, but the epidemic intensity of HBV infection varies greatly in different regions. my country belongs to the high prevalence area of HBV infection. The national HBV blood epidemiological survey in 2006 showed that the HBsAg carrier rate among the general population aged 1-59 in my country was 7.18%. Based on this calculation, there are about 93 million chronic HBV-infected people in my country, including about 20 million chronic hepatitis B (chronic hepatitis B) patients. In my country, chronic HBV infection is closely related to the occurrence of end-stage liver diseases such as liver cirrhosis and liver cancer. Currently, there are two main types of drugs for the treatment of chronic hepatitis B: interferon and nucleoside (acid) analogues. Both types of drugs can efficiently inhibit HBV replication, but due to the persistence of covalently closed circular DNA (cccDNA) in liver cells, the current clinical cure rate of chronic hepatitis B treatment is very low (<5%), stop The recurrence rate of chronic hepatitis B after taking medicine is high, so patients with chronic hepatitis B need to take medicine for a long time or even for life. Long-term medication will bring great burden to patients and even the society, and may lead to the emergence of HBV drug-resistant strains in the course of treatment. Based on this, there is an urgent need for a more sensitive and accurate hematological index for monitoring the efficacy of CHB treatment and safe drug withdrawal.

Many articles have confirmed the presence of HBV RNA in blood, specifically, HBV RNA can exist in serum and/or plasma, and its level is closely related to the curative effect and prognosis of chronic hepatitis B treatment. However, the sensitivity and specificity of the current fluorescent quantitative PCR method for detecting blood HBV RNA need to be improved. The inventor systematically confirmed the existence form and production mechanism of HBV RNA in the blood in previous studies, which is that the HBV Pregenome RNA (pgRNA) present in the nucleocapsid is secreted to the extracellular. Thus, the HBV RNA in the blood is actually pgRNA. Quantitative detection of the level of HBV pgRNA in blood can better monitor the level of HBV cccDNA in hepatocytes, which is a good predictor for detecting the curative effect of chronic hepatitis B treatment and safe drug withdrawal. The development of diagnostic reagents or kits for the quantitative detection of pgRNA in blood has important guiding significance for the effective treatment of chronic hepatitis B and safe drug withdrawal in clinical practice.

Contents of the invention

One aspect of the present invention provides a blood HBV pgRNA fluorescence quantitative PCR detection system comprising: a forward primer, its nucleotide sequence is shown in SEQ NO:1; a reverse primer, its nucleotide sequence is shown in SEQ NO:2 and the reverse anchor primer, its nucleotide sequence is shown in SEQ NO:3.

Wherein, the forward primer SEQ NO:1 is AGACCACCCAAATGCCCCT; the reverse primer SEQ NO:2 is (N)16-30. (N) 16-30 refers to the nucleotide sequence as a random anchor sequence, N can be A, T, C or G, and the subscript 16-30 represents the number of bases. The reverse anchor primer SEQ NO:3 is (N)16-30-AGGCGAGGGAGTTTCTTCTTCTA. (N)16-30 in SEQ NO:2 and SEQ NO:3 represent the same random anchor sequence. Those skilled in the art know that although (N)16-30 is a random sequence, in order to ensure the efficiency of amplification, it should be designed to avoid forming a primer dimer with HR-F or Probe-HR. In addition, in order to increase the amplification efficiency, the primer sequence formed by adding random bases at both ends of the primer is also included in the present invention.

In some embodiments of the present invention, the blood HBV pgRNA fluorescent quantitative PCR detection system further includes a probe, the nucleotide sequence of which is shown in SEQ NO:4. Wherein, the probe SEQ NO:4 is CAACACTTCCGGARACTACTGTTGTTAGACG.

In some specific embodiments of the present invention, in the blood HBV pgRNA fluorescence quantitative PCR detection system, the probe is one of TaqMan probe, MGB probe and hybridization probe. Labeling the 5' end of the probe is a fluorescent luminescent group, which is one of FAM, VIC, TET, JOE, ROX, CY3, CY5, HEX; and labeling the 3' end of the probe is a The fluorescence quenching group is one of BHQ1, BHQ2, BHQ3, TAMRA, DABCYL, and NFQ.

Another aspect of the present invention also provides the application of the aforementioned blood HBV pgRNA fluorescent quantitative PCR detection system in the preparation of HBV diagnostic reagents or kits.

Another aspect of the present invention also provides a blood HBV pgRNA fluorescent quantitative PCR detection method, comprising:

Step 1. Using the blood HBV pgRNA to be tested as a template, reverse transcription is performed with a primer having a sequence such as SEQ NO: 3;

Step 2. The obtained reverse transcription product is used as a template, and the primers with sequences such as SEQ NO: 1 and 2 and the probe with sequence such as SEQ NO: 4 are used to amplify to quantitatively detect HBV pgRNA in blood.

In some embodiments of the present invention, step 2 can also use the obtained reverse transcription product as a template, use primers with sequences such as SEQ NO: 1 and 2 to amplify, incorporate fluorescent dyes, and use the SYBR Green method for quantitative detection HBV pgRNA in blood.

Beneficial effects of the present invention

According to the characteristics of HBV pgRNA, the present invention designs highly conserved specific primers and probes for different genotypes of HBV at the 5' end of pgRNA for the first time, thus eliminating possible interference caused by other HBV RNAs. In addition, the present invention also adds an artificially designed anchor sequence to the reverse transcription primer to eliminate possible interference caused by HBV DNA during the detection process, thereby ensuring the specificity of blood HBV pgRNA detection.

The specific primers and probes of the present invention can detect all genotypes of HBV existing in my country, including type B and type C HBV mainly existing in China, type D HBV distributed in Xinjiang, Tibet and other places in my country, and type A sporadically distributed HBV.

The primers, probes and anchor sequences of the present invention are sequences with optimal specificity and sensitivity determined after multiple rounds of screening and optimization on the basis of initially designed multiple sequences.

Description of drawings

Fig. 1 shows the design of forward primer, reverse transcription primer and corresponding probe, wherein Fig. 1A analyzes the conserved sequences of different genotypes of HBV by MegAlign software, Fig. 1B and 1C show respectively and utilizes Primer Premier 5 software to analyze the anchor sequence (reverse direction primer) and forward direction primer hairpin structure, dimer and mismatch analysis.

Fig. 2 shows a flow chart of detecting blood HBV pgRNA by using the serum HBV pgRNA fluorescent quantitative PCR detection system implemented in the present invention.

Fig. 3 A shows the melting curve when using HR-F and HR-R amplification; Fig. 3 B shows using non-patent literature 1 (van F, et al.Serum Hepatitis B Virus RNA Levels as an Early Predictor of Hepatitis B Envelope Antigen Seroconversion During Treatment With Polymerase Inhibitors, Hepatology.2015; 61:66-76.) The melting curve during primer amplification; Figure 3C shows The influence of the amount of probes in the detection system on the fluorescent quantitative PCR reaction, where 1 is the addition of 0.6 μl of probes with a concentration of 10 μM in the system, and 2 is the addition of 0.8 μl of probes with a concentration of 10 μM in the system, and the amplification curves of the two basically overlap ; Figure 3D shows the impact of the annealing temperature on the fluorescent quantitative PCR reaction in the detection system, where the annealing temperatures of 56, 58, 60, and 62 degrees Celsius were used for quantitative PCR reactions, and the obtained amplification curves basically overlapped.

Figure 4 shows the sequencing results of diagnostic reagent standard products.

Fig. 5 shows the sensitivity detection of the fluorescent quantitative PCR detection method of the present invention, wherein Fig. 5A shows the Ct values after the detection of different final concentrations of plasmids, Fig. 5B shows a standard curve constructed using concentrations and Ct values, and Fig. 5C shows The amplification curve finally obtained by fluorescent quantitative PCR.

Fig. 6 shows the sensitivity evaluation of the serum HBV RNA fluorescence quantitative detection method reported in Non-Patent Document 1, wherein, Fig. 6A shows the Ct values after the detection of different final concentration plasmids, and Fig. 6B shows the concentration and Ct values constructed using As for the standard curve, FIG. 6C shows the final amplification curve obtained by fluorescent quantitative PCR.

Figure 7 shows the detection of different genotypes of HBV pgRNA levels, wherein Figure 7A shows the detection results of B genotype HBV in the serum of chronic hepatitis B patients, and Figure 7B shows the detection results of C genotype HBV in the serum of chronic hepatitis B patients, Fig. 7C shows the detection results of the supernatant of Hep AD38 cells, which secrete and produce D genotype HBV.

Figure 8 shows the specificity evaluation of the fluorescent quantitative PCR detection method of the present invention to HBV pgRNA, wherein, Figure 8A shows the use of the quantitative PCR system of the present invention to detect different templates, wherein 1 is that the extracted HBV nucleic acid mixture is subjected to DnaseI After treatment, the product after reverse transcription using primer HR-RT; 2 is the extracted, untreated HBV nucleic acid mixture; 3 is the HBV nucleic acid mixture after DNase I treatment. Figure 8B shows that normal human blood has been extracted from nucleic acid , the result of quantitative detection after reverse transcription treatment, wherein 4 is the positive control of serum of patients with chronic hepatitis B; 5 and 6 are the detection results of normal human serum.

Figure 9 shows the repeatability detection results of the plasmid concentration of 1 × 103copies/ml standard.

detailed description

Unless otherwise specified, terms in the present invention have meanings commonly used in the art.

The term "nucleotide" is intended to include those moieties that contain not only the known purine and pyrimidine bases but also modified other heterocyclic bases. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated ribose sugars or other heterocyclic compounds. Furthermore, the term "nucleotide" includes those moieties which contain haptens or fluorescent labels and which may contain not only conventional ribose and deoxyribose sugars but also other sugars. Modified nucleosides or nucleotides may also comprise modifications on the sugar moiety, for example, wherein one or more hydroxyl groups are replaced with halogen atoms or aliphatic groups, functionalized as ethers, amines, and the like.

The term "primer" refers to a short, chemically synthesized oligonucleotide, typically about 20 to 30 bases in length. They hybridize to target DNA, which is then replicated by DNA polymerase to produce a complementary DNA strand. The "Forward Primer" and "Reverse Primer" make up the "PCR Primer Set" used in PCR, where they hybridize to complementary DNA strands and direct replication towards each other, creating the upper and lower strands, respectively, resulting in the target DNA fragment in the index increase.

The method according to the invention comprises the last 1 to 10 nucleotides upstream of the (preferably 3' polyA) tail of the RNA and the (polyA) tail for reverse transcription, preferably 1 to 5 nucleotides, more preferably 3 to 5 nucleotides specific primers and/or an amplification primer specific to the corresponding (5' polythymidylic acid) sequence of the cDNA that is specific to the RNA tail (3 The nearest 1 to 10 nucleotides, preferably 1 to 5 nucleotides, more preferably 3 to 5 nuclei downstream of the 'polyA) tail and the corresponding (5' polyT) sequence of the cDNA nucleotide complementation. Accordingly, in addition to the aforementioned disclosed primers, it is possible to increase the selectivity of primer pairs and thus the number of cDNA populations defined by a particular nucleotide closest to the anchor point of the terminal sequence.

The term "anchor primer" refers to the addition of a modified sequence (including restriction sites, tag sequences and artificially designed random sequences, etc.) to the 5' end of the gene-specific primer, and then the modified sequence is amplified by reverse transcription or PCR The sequence is anchored on the target gene, and the anchor sequence is finally used as a primer for subsequent detection. To some extent, the specificity of specific gene detection can be improved.

The term "probe" refers to the labeling of a primer with a capture label or a detection label and the detection of the primer product. The probe sequence is used to hybridize to the sequence generated by the primer sequence, and typically to a sequence that does not include the primer sequence. Similar to the primer sequence, the probe sequence is also labeled with a capture label or a detection label. It should be noted that when the primer is labeled with a capture label, the probe is labeled with a detection label, and vice versa. In the present invention, the probe can be one of TaqMan probe, MGB probe and hybridization probe. For example, the probe is a TaqMan probe, and the 5' end of the labeled probe is a fluorescent luminescent group, which is one of FAM, VIC, TET, JOE, ROX, CY3, CY5, and HEX; the labeled probe The 3' end is a fluorescent quenching group, which is one of BHQ1, BHQ2, BHQ3, TAMRA, DABCYL, and NFQ.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods; the materials used, unless otherwise specified, were purchased from conventional biochemical reagent companies.

1. Design of primers and probes

At present, HBV is divided into at least 10 genotypes, and there are 4 genotypes A to D in my country. According to the HBV genotypes existing in my country, the inventors downloaded multiple A-D genotype HBV reference sequences from GenBank, and analyzed the conserved regions of HBV genomes of different genotypes by MegAlign software. Following the design principles of primers and probes, specific primers and probe sequences for pgRNA detection were designed in these conserved regions by Primer Premier 5 software. In order to exclude the interference of HBV DNA, the inventors added a random anchor sequence to the 5' end of the reverse transcription primer. During the design process of the above-mentioned primers, probes and random anchor sequences, try to avoid the formation of hairpin structures, internal dimers of primers, dimers between primers and mismatches. In addition, the inventors used the NCBI Blast online database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to compare and analyze the HBV-specific primer and probe sequences and random anchor sequences designed above to avoid Non-specific binding to other viruses or human genes. Through multiple rounds of screening and optimization, a set of primers, probes and random anchor sequences with optimal sensitivity and specificity were finally determined, and the sequences are shown in Table 1. HBV-specific sequences are highly conserved sequences of HBV genotypes A to D (see Figure 1A).

Table 1. Primer sequences

Among them, the 5' fluorescent probe of Probe-HR is FAM, and the 3' fluorescent probe is BHQ1. The underlined parts of the HR-R1 and HR-RT1 sequences are the same random anchor sequences, and the underlined parts of the HR-R2 and HR-RT2 sequences are the same random anchor sequences. As known to those skilled in the art, the inventors have further confirmed through experiments that the random anchor sequence does not affect the detection efficiency, as long as it does not form primer dimers with HR-F or Probe-HR as much as possible.

2. Establishment of detection method and system

The principle of the TaqMan probe method used for the quantitative detection of blood HBV pgRNA is: first, the extracted RNA is reverse-transcribed into cDNA with an anchor sequence by using the specific reverse transcription primer HR-RT added with the anchor sequence.

The specific operation is as follows, the extracted nucleic acid mixture is treated with DNase I (Thermo Fisher Scientific, Waltham, MA, USA).

DNase I treatment system (10μl) is:

Incubate the mixture at 37°C for 30 minutes before adding

EDTA 1 μl

HR-RT (10μM) lμl

Incubate at 65°C for 10 minutes

It was then reverse transcribed into cDNA by RevertAid First Strand DNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA).

The reverse transcription reaction system (20μl) is:

The reaction conditions were: 5 minutes at 25°C, 60 minutes at 42°C, and 5 minutes at 70°C.

Then, the HBV-specific forward sequence and anchor sequence were used as the forward and reverse primers for quantitative PCR detection, respectively, for amplification, and the TaqMan probe sequence was next to the forward primer (Figure 2). In order to determine the detection system of the primers and probes identified in the present invention, the inventors first tested the specificity of the primers using the method of SYBR Green, and compared their amplified melting curves with the primers reported in Non-Patent Document 1. The results showed that the specificity of the primers of the present invention ( FIG. 3A ) was better than that of the primers used in Non-Patent Document 1 ( FIG. 3B ). In addition, the inventors respectively tried the effects of two key factors in the detection system, the amount of probe and the annealing temperature, on the fluorescent quantitative PCR reaction. The results showed that different concentrations of probes (Figure 3C) and different annealing temperatures (Figure 3D) had no significant effect on the amplification intensity and sensitivity of the fluorescent quantitative PCR reaction, and the obtained amplification curves basically overlapped.

Finally, the reaction system (30 μl) of the fluorescent quantitative PCR adopted is as follows:

The amplification was carried out on the ABI Step One Plus fluorescent quantitative PCR instrument, and the amplification cycle parameters were 50°C, 5 minutes, 94°C, 2 minutes; then 94°C, 15 seconds, 58°C, 45 seconds, 45 cycles; Fluorescence was collected multiple times simultaneously during the extension phase (58°C) of each cycle.

Alternatively, SYBR-Green I can be used for fluorescent staining. The reaction system (20μl) is as follows:

The amplification was carried out on the ABI Step One Plus fluorescent quantitative PCR instrument, and the amplification cycle parameters were 50°C, 5 minutes, 94°C, 2 minutes; then 94°C, 15 seconds, 58°C, 45 seconds, 45 cycles; Fluorescence was collected multiple times simultaneously during the extension phase (58°C) of each cycle. Add melting curve steps: 95°C for 15 seconds, 60°C for 1 minute, then gradually increase the temperature from 60°C, collect fluorescence signals every 0.3°C increase until the temperature rises to 95°C, and maintain 95°C for 15 seconds.

3. Construction of standard diagnostic reagents

Extract the HBV pgRNA from the culture supernatant of the HBV stable replicating cell line HepAD38, perform reverse transcription with HR-RT reverse transcription primers, amplify with HR-F and HR-R, and cut the amplified target fragment into gel to recover Then ligated to the pEASY-Blunt cloning vector. After being verified as the loaded target fragment by sequencing, it is used as a standard for testing blood HBVpgRNA in the laboratory.

The final destination fragment sequence is:

AGACCACCAAATGCCCCTATCCTATCAACACTTCCGGAAACTACTGTTGTTAGACGACGAGGCAGGTCCCTAGAAGAAGAACTCCCTCGCCTGTGTGTCGTGTGTTACGGTGTGA (SEQ NO: 9)

The peak map of the sequencing verification is shown in Figure 4.

4. Detection of sensitivity

Dilute the constructed standard 10 times, the concentrations are 1.00E+08, 1.00E+07, 1.00E+06, 1.00E+05, 1.00E+04, 1.00E+03, 1.00E+02 , 1.00E+01 copies/ml. Quantitatively detect the doubly diluted standard substance with the detection system and cycle parameters determined above. The results show that the fluorescent quantitative detection method of the present invention has higher sensitivity, and the linear range can be as low as 1.00E+01copies/ml; the linear regression equation of the standard curve is: y=-3.2645x ⁺ 40.393, the square R of the correlation coefficient = 0.9993 (Fig. 5).

Compared with the blood HBV RNA fluorescence quantitative detection method reported in non-patent literature 1, the results show that the linear regression equation of the amplification standard curve of the fluorescence quantitative PCR method established in non-patent literature 1 is: y=-3.4315x+45.463 , the square of the correlation coefficient R ² =0.9952 (Fig. 6). With the standard substance of the same concentration, the ct value amplified by the fluorescent quantitative PCR detection method of the present invention is lower than that of the method established in Non-Patent Document 1 by more than 3 cycles, indicating that the detection method of the present invention is more sensitive.

On the basis of detecting plasmid standard products, the level of HBV pgRNA of different genotypes (B, C and D genotypes) was detected by using the fluorescent quantitative PCR method established by the present invention, and its sensitivity was evaluated.

Wherein B and C types are derived from the patient's blood, and the concentrations of HBV pgRNA measured by the fluorescent quantitative PCR detection method of the present invention are respectively 1.33E+06, 1.50E+06 copies/ml, diluted by 10 times, and the diluted The concentrations were 1.33E+05, 1.33E+04, 1.33E+03, 1.33E+02copies/ml, 1.50E+05, 1.50E+04, 1.50E+03, 1.50E+02copies/ml.

The source of type D is the supernatant of HepAD38 cells, and the concentration of HBV pgRNA measured by the fluorescent quantitative PCR detection method of the present invention is 3.26E+05 copies/ml, which is diluted by 10 times, and the concentration after dilution is 3.26E+04, 3.26E+03, 3.26E+02, 3.26E+01 copies/ml.

The results show that in the quantitative detection of HBV pgRNA of B, C and D genotypes, the fluorescent quantitative PCR method established by the present invention still shows high sensitivity ( FIG. 7 ).

5. Detection of specificity

Nucleic acid (including DNA and RNA) in the blood of HBV-infected patients was extracted using the EasyPure Viral RNA Kit (TransGen, catalog number: ER201-01) nucleic acid extraction kit. The HBV DNA load in the blood was 9.45E+07copies/ml, and the load of HBV pgRNA measured by the fluorescent quantitative PCR detection method of the present invention was 1.48E+06copies/ml. After the nucleic acid was extracted, it was divided into 3 groups. After the DNase I treatment, the reverse transcription reaction was carried out, the second group was not treated, and the third group was treated with DNase I; the three groups were detected by the fluorescent quantitative PCR method established by the present invention at the same time.

The results showed that in the blood with HBV DNA level (9.45E+07copies/ml) higher than that of HBV pgRNA (1.48E+06copies/ml), even if the extracted nucleic acid was not treated with DNase, it had little interference to the detection of HBV pgRNA (2 in FIG. 8A ), and the interference disappeared after being treated with DNase (3 in FIG. 8A ).

In addition, the blood of two normal persons was also detected, and the results were all lower than the detection limit (5 and 6 in Fig. 8B).

The above results show that the blood HBV pgRNA fluorescent quantitative detection method established by the present invention has high specificity, even without DNase pretreatment, the interference of HBV DNA on pgRNA detection is insignificant.

6. Repeatability detection

A standard product with a plasmid concentration of 1.00E+03copies/ml was selected for detection and repeated 30 times. The results showed that the standard deviation (SD value) of the ct value of 30 repeated detections was 0.275537, and the repeatability was good (Fig. 9).

7. Detection of clinical samples

The fluorescent quantitative PCR method of the present invention was used to detect the blood HBV pgRNA of 5 cases of chronic hepatitis B patients without antiviral treatment. At the same time, the blood HBV DNA level was detected by a commercial HBV DNA quantitative detection kit (Hunan Shengxiang Biotechnology Co., Ltd.).

The results showed that there was a certain level of HBV pgRNA in the blood of chronic hepatitis B patients without antiviral treatment, which was lower than the level of HBV DNA (Table 2).

Subsequently, the above-mentioned blood is diluted 10 times, and then the blood samples with different dilutions are detected by the fluorescent quantitative PCR detection method of the present invention. The results are shown in Table 3, the fluorescence quantitative PCR detection method of the present invention should show higher sensitivity for the detection of clinical samples.

Table 2. HBV DNA and HBV pgRNA levels in the blood of chronic hepatitis B patients without antiviral treatment

Table 3. HBV pgRNA levels in the blood of patients with chronic hepatitis B after serial dilution

The SYBR-Green method can also be used to quantitatively detect the level of serum HBV pgRNA. First, use the SYBR-Green method to detect the standard substance, and draw a standard curve, and then follow the above steps to detect the clinical samples to quantitatively detect the level of HBVpgRNA in the blood.

SEQUENCE LISTING

<110> Peking University

<120> A highly sensitive and specific blood HBV pgRNA fluorescent quantitative PCR detection system and detection method

<130> 1700051CN

<150> CN201610202753.X

<151> 2016-04-01

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<170> PatentIn version 3.3

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Claims (10)

1. A blood HBV pgRNA fluorescent quantitative PCR detection system, comprising: A reverse anchor primer, the reverse anchor primer includes a gene-specific primer part and a modified sequence part, the gene-specific primer part is AGGCGAGGGAGTTTCTTCTTCTA, located at the 3' end of the reverse anchor primer; preferably, the The modified sequence part includes a restriction site, a tag sequence or an artificially designed random sequence; further preferably, the nucleotide sequence of the reverse anchor primer is shown in SEQ NO: 3; further preferably, the detection system is also Includes forward and reverse primers. 2. blood HBV pgRNA fluorescent quantitative PCR detection system as claimed in claim 1, is characterized in that: Forward primer, its nucleotide sequence is shown in SEQ NO:1; Reverse primer, its nucleotide sequence is shown in SEQ NO:2; And Reverse anchor primer, its nucleotide sequence is shown in SEQ NO:3; Preferably, the detection system also includes: a probe, its nucleotide sequence is shown in SEQ NO:4; For example, the probe The needle is one of TaqMan probe, MGB probe, and hybridization probe; preferably, one of FAM, VIC, TET, JOE, ROX, CY3, CY5, and HEX is labeled at the 5' end of the probe; And label one of BHQ1, BHQ2, BHQ3, TAMRA, DABCYL, NFQ at the 3' end of the probe. 3. the application of blood HBV pgRNA fluorescent quantitative PCR detection system as claimed in claim 1 or 2 in the preparation of HBV diagnostic reagent or test kit. 4. A blood HBV pgRNA fluorescent quantitative PCR detection method, comprising: Step 1. Using the blood HBV pgRNA to be tested as a template, carry out reverse transcription with a reverse anchor primer, the reverse anchor primer includes a gene-specific primer part and a modified sequence part, and the gene-specific primer part is AGGCGAGGGAGTTTCTTCTTCTA, Located at the 3' end of the reverse anchor primer; preferably, the modified sequence part includes a restriction site, a tag sequence or an artificially designed random sequence; preferably, the nucleotide sequence of the reverse anchor primer is as follows Shown in SEQNO:3; Also preferably, described detection method also comprises: Step 2. The obtained reverse transcription product is used as a template, amplified with primers such as SEQ NO: 1 and 2 and a probe with a sequence such as SEQ NO: 4, to quantitatively detect HBV pgRNA in blood; or obtained The reverse transcription product was used as a template, amplified by primers with sequences such as SEQ NO: 1 and 2, and incorporated with fluorescent dyes, and the HBV pgRNA in the blood was quantitatively detected by the SYBR Green method. 5. The application of the reverse anchor primer in the preparation of HBV diagnostic reagents or test kits, characterized in that the reverse anchor primer comprises a gene-specific primer part and a modified sequence part, and the gene-specific primer part is AGGCGAGGGAGTTTCTTCTTCTA , located at the 3' end of the reverse anchor primer; preferably, the modified sequence part includes a restriction site, a tag sequence or an artificially designed random sequence; also preferably, the nucleotide of the reverse anchor primer The sequence is shown in SEQ NO:3. 6. A blood HBV pgRNA fluorescent quantitative PCR detection kit, comprising: A reverse anchor primer, the reverse anchor primer includes a gene-specific primer part and a modified sequence part, the gene-specific primer part is AGGCGAGGGAGTTTCTTCTTCTA, located at the 3' end of the reverse anchor primer. 7. The blood HBV pgRNA fluorescence quantitative PCR detection kit as claimed in claim 6, wherein, the modified sequence part comprises an enzyme cutting site, a tag sequence or an artificially designed random sequence. 8. blood HBV pgRNA fluorescent quantitative PCR detection kit as claimed in claim 6, wherein, the nucleotide sequence of described reverse anchor primer is as shown in SEQ NO:3. 9. The blood HBV pgRNA fluorescent quantitative PCR detection kit according to any one of claims 6-8, further comprising: a forward primer and a reverse primer. 10. blood HBV pgRNA fluorescent quantitative PCR detection kit as claimed in claim 9, is characterized in that: Forward primer, its nucleotide sequence is shown in SEQ NO:1; Reverse primer, its nucleotide sequence is shown in SEQ NO:2; And Reverse anchor primer, its nucleotide sequence is shown in SEQ NO:3; Preferably, the kit also includes: a probe, its nucleotide sequence is shown in SEQ NO:4; Preferably, the The probe is one of TaqMan probe, MGB probe, and hybridization probe; further preferably, one of FAM, VIC, TET, JOE, ROX, CY3, CY5, and HEX is labeled at the 5' end of the probe species; and one of BHQ1, BHQ2, BHQ3, TAMRA, DABCYL, NFQ is labeled at the 3' end of the probe.