CN107058623B

CN107058623B - High-sensitivity and specific blood HBV pgRNA fluorescent quantitative PCR detection system and detection method

Info

Publication number: CN107058623B
Application number: CN201710213191.3A
Authority: CN
Inventors: 鲁凤民; 王杰; 陈然
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2016-04-01
Filing date: 2017-04-01
Publication date: 2020-04-14
Anticipated expiration: 2037-04-01
Also published as: CN105734173A; CN107058623A

Abstract

The invention discloses a fluorescence quantitative PCR detection system for Hepatitis B Virus (HBV) pregenome RNA (pgRNA) in blood and a method for detecting the HBV pgRNA by using the detection system. The detection system comprises a forward primer, the nucleotide sequence of which is shown as SEQ NO. 1; the reverse primer has a nucleotide sequence shown as SEQ NO. 2; the reverse transcription primer with random anchoring sequence has the nucleotide sequence as shown in SEQ NO 3; and a probe, the nucleotide sequence of which is shown as SEQ NO. 4. The detection system and the method can be used for preparing HBV diagnostic reagent or kit. The invention eliminates the possible interference brought by HBV DNA or other RNA in the detection process and ensures the specificity of the detection of blood HBV pgRNA.

Description

High-sensitivity and specific blood HBV pgRNA fluorescent quantitative PCR detection system and detection method

Technical Field

The present invention relates generally to the diagnosis and treatment of viral hepatitis b, and to the fields of infectious pathology, molecular biology, cell biology, and the like. More specifically, the invention relates to a fluorescence quantitative PCR detection method of Hepatitis B Virus (HBV) pgRNA in blood.

Background

Hepatitis B Virus (HBV) infection is prevalent worldwide, but the prevalence of HBV infection varies widely in different regions. China belongs to a region with high prevalence of HBV infection. The national HBV blood epidemiological survey in 2006 shows that the HBsAg carrying rate in general people of 1-59 years old in China is 7.18%. According to the calculation, about 9300 million people are infected by chronic HBV in China, and about 2000 million patients with chronic hepatitis B (chronic hepatitis B) are infected by the chronic HBV. In China, chronic HBV infection is closely related to the occurrence of end-stage liver diseases such as liver cirrhosis, liver cancer and the like. The existing medicines for treating chronic hepatitis B mainly comprise two types of interferon and nucleoside (acid) analogues. Both drugs can effectively inhibit HBV replication, but due to the continuous existence of covalently closed circular DNA (cccDNA) in hepatocytes, the clinical cure rate of the existing chronic hepatitis B treatment is very low (< 5%), and the recurrence rate of chronic hepatitis B after drug withdrawal is high, so that patients with chronic hepatitis B need to take the drugs for a long time or even for life. The long-term administration of the drug can bring great burden to patients and even society, and can cause the emergence of HBV drug-resistant strains in the treatment process. Based on this, there is an urgent need for a more sensitive and accurate hematological index for monitoring the efficacy of chronic hepatitis B treatment and for safe withdrawal.

Several papers have demonstrated the presence of HBV RNA in blood, in particular, HBV RNA may be present in serum and/or plasma, and its levels are closely related to the efficacy and prognosis of chronic hepatitis b treatment. However, the sensitivity and specificity of the current fluorescent quantitative PCR method for detecting HBV RNA in blood are still to be improved. The present inventors have systematically confirmed the existence form and production mechanism of HBV RNA in blood in a preliminary study, which is that HBV Pregenome RNA (pgRNA) present in nucleocapsid is secreted extracellularly without reverse transcription or incomplete reverse transcription. It can be seen that HBV RNA in blood is actually pgRNA. The quantitative detection of the blood HBV pgRNA level can better monitor the HBV cccDNA level in the liver cells, and is a good prediction index for detecting the treatment curative effect and safe drug withdrawal of chronic hepatitis B. The development of a diagnostic reagent or a kit for quantitatively detecting pgRNA in blood has important guiding significance for effective treatment and safe withdrawal of chronic hepatitis B in clinic.

Disclosure of Invention

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

Wherein, the forward primer SEQ NO. 1 is AGACCACCAAATGCCCCT; the reverse primer SEQ NO. 2 is (N) 16-30. (N)16-30 refers to a nucleotide sequence as a random anchor sequence, N may be A, T, C or G, and 16-30 indicates the number of bases at the subscript. The reverse anchored primer SEQ NO. 3 is (N) 16-30-AGGCGAGGGAGTTCTTCTTCTA. (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, it should be avoided to design to form primer dimers with HR-F or Probe-HR in order to ensure the efficiency of amplification. In addition, in order to increase the amplification efficiency, a primer sequence formed after adding random bases at both ends of the primer is also encompassed in the present invention.

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

In some 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 with a fluorescent luminescent group, wherein the fluorescent luminescent group is one of FAM, VIC, TET, JOE, ROX, CY3, CY5 and HEX; and the 3' end of the probe is marked with a fluorescence quenching group which is one of BHQ1, BHQ2, BHQ3, TAMRA, DABCYL and NFQ.

The invention also provides the application of the blood HBV pgRNA fluorescent quantitative PCR detection system in the preparation of HBV diagnostic reagent or kit.

The invention also provides a blood HBV pgRNA fluorescent quantitative PCR detection method, which comprises the following steps:

step 1, taking HBV pgRNA of blood to be detected as a template, and using a gene sequence shown as SEQ NO:3, carrying out reverse transcription on the primer;

and 2, taking the obtained reverse transcription product as a template, and performing reverse transcription by using a reverse transcription product with a sequence shown as SEQ NO:1 and 2 are shown as SEQ NO:4 to quantitatively detect HBV pgRNA in blood.

In some embodiments of the present invention, step 2 may further be performed by using the obtained reverse transcription product as a template, and using a sequence shown in SEQ NO:1 and 2, and a fluorescent dye is doped, and quantitative detection of HBVpgRNA in blood is carried out by a SYBR Green method.

The invention has the advantages of

The invention designs specific primers and probes highly conserved for HBV of different genotypes at the 5' end of pgRNA for the first time according to the characteristics of the HBV pgRNA, thereby excluding possible interference brought by other HBV RNA. In addition, the reverse transcription primer is added with an artificially designed anchor sequence to eliminate the possible interference brought by HBV DNA in the detection process and ensure the specificity of the blood HBV pgRNA detection.

The specific primer and the probe of the invention can detect all genotypes of HBV in China, including B-type HBV and C-type HBV which mainly exist in China, D-type HBV which is distributed in Xinjiang, Tibet and the like in China, and A-type HBV which is distributed sporadically.

The primer, the probe and the anchoring sequence are sequences with optimal specificity and sensitivity determined by multiple rounds of screening and optimization on the basis of multiple initially designed sequences.

Drawings

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

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

FIG. 3A shows the lysis curves when using HR-F and HR-R amplification; FIG. 3B shows utilization of non-patent document 1 (van)

F,et al.Serum Hepatitis B Virus RNA Levels as an Early Predictor ofHepatitis B Envelope Antigen Seroconversion During Treatment With PolymeraseInhibitors,Hepatology.2015; 61:66-76.) by amplification of the primers; FIG. 3C shows the effect of the probe amount in the detection system on the fluorescent quantitative PCR reaction, wherein 1 is the addition of 0.6. mu.l probe with a concentration of 10. mu.M to the system, and 2 is the addition of 0.8. mu.l probe with a concentration of 10. mu.M to the system, and the amplification curves of the two are substantially overlapped; FIG. 3D shows the effect of annealing temperature on the fluorescent quantitative PCR reaction in the detection system, wherein the quantitative PCR reaction was performed using annealing temperatures of 56, 58, 60, and 62 degrees Celsius, respectively, and the resulting amplification curves substantially overlap.

Figure 4 shows diagnostic reagent standard sequencing results.

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

FIG. 6 shows the sensitivity evaluation of the fluorescence quantitative detection method for serum HBV RNA reported in non-patent document 1, wherein FIG. 6A shows Ct values of plasmids with different final concentrations after detection, FIG. 6B shows a standard curve constructed by using the concentrations and the Ct values, and FIG. 6C shows an amplification curve finally obtained by fluorescence quantitative PCR.

FIG. 7 shows the measurement of the levels of pgRNA of HBV of different genotypes, wherein FIG. 7A shows the measurement of B genotype HBV in serum of chronic hepatitis B patient, FIG. 7B shows the measurement of C genotype HBV in serum of chronic hepatitis B patient, and FIG. 7C shows the measurement of supernatant of Hep AD38 cells secreting D genotype HBV.

FIG. 8 shows the specific evaluation of HBV pgRNA by the fluorescent quantitative PCR detection method of the present invention, wherein FIG. 8A shows the detection of different templates by the quantitative PCR system of the present invention, wherein 1 is the product of the extracted HBV nucleic acid mixture after treatment with DnaseI and reverse transcription with primer HR-RT; 2 is an extracted, untreated HBV nucleic acid mixture; FIG. 8B shows the result of quantitative detection of normal human blood after nucleic acid extraction and reverse transcription treatment, wherein 4 is the positive control of chronic hepatitis B patient serum; 5 and 6 are the detection results of normal human serum.

FIG. 9 shows the results of repeated assays for plasmid concentrations of 1X 103copies/ml standard.

Detailed Description

The terms of the present invention have meanings commonly used in the art unless otherwise specified.

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

The term "primer" refers to a short, chemically synthesized oligonucleotide of typically about 20 to 30 bases in length. They hybridize to the target DNA, which is then replicated by a DNA polymerase to produce a complementary DNA strand. The "forward primer" and "reverse primer" constitute a "PCR primer set" used in PCR, where they hybridize to complementary DNA strands and direct replication toward each other, resulting in the production of upper and lower strands, respectively, resulting in exponential growth of the target DNA fragment.

The method according to the invention comprises a primer specific for the (preferably 3 'poly a) tail of RNA and the nearest 1 to 10 nucleotides, preferably 1 to 5 nucleotides, more preferably 3 to 5 nucleotides upstream of the (poly a) tail for reverse transcription and/or an amplification primer specific for the corresponding (5' poly thymidylate) sequence of cDNA, which is complementary to the (3 'poly a) tail of RNA and the nearest 1 to 10 nucleotides, preferably 1 to 5 nucleotides, more preferably 3 to 5 nucleotides downstream of the corresponding (5' poly thymidylate) sequence of cDNA. Accordingly, in addition to the primers disclosed above, the selectivity of the primer pair and thus the number of cDNA populations defined by the particular nucleotide closest to the anchor of the terminal sequence can be increased.

The term "anchor primer" refers to a modified sequence (including cleavage site, tag sequence and artificially designed random sequence) added to the 5' end of a gene-specific primer, and then the modified sequence is anchored to the target gene by reverse transcription or PCR amplification, and finally the anchor sequence is used as a primer for subsequent detection. The specificity of detecting a specific gene can be improved to a certain extent.

The term "probe" refers to the detection of a primer product by labeling the primer with a capture label or a detection label. The probe sequence is used to hybridize to a 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 labeled with a capture label or a detection label, indicating that when the primer is labeled with a capture label, the probe is labeled with a detection label, and vice versa. The probe in the invention 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 3' end of the labeled probe is a fluorescence quenching group which is one of BHQ1, BHQ2, BHQ3, TAMRA, DABCYL and NFQ.

The invention will now be further illustrated by reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified; the materials used, unless otherwise specified, were purchased from conventional biochemicals.

1. Design of primers and probes

Currently, HBV is classified into at least 10 genotypes, and A to D4 genotypes exist in our country. According to the HBV genotypes existing in China, the inventor downloads a plurality of HBV reference sequences of the genotypes A to D from GenBank and analyzes the conserved regions of HBV genomes of different genotypes by MegAlign software. Following the principle of Primer and probe design, specific Primer and probe sequences for detection of pgRNA were designed in these conserved regions by Primer Premier 5 software. To exclude the interference of HBV DNA, the inventors added a random anchor sequence to the 5' end of the reverse transcription primer. In the process of designing the primers, the probes and the random anchoring sequences, the formation of hairpin structures, primer internal dimers, primer-to-primer dimers and mismatches is avoided as much as possible. Furthermore, the inventors performed an alignment analysis of the above designed HBV specific primer and probe sequences and random anchor sequences by NCBI Blast online database (http:// Blast. NCBI. nlm. nih. gov/Blast. cgi) to avoid non-specific binding to other viral or human genes. Through multiple rounds of screening and optimization, a set of primers, probes and random anchoring sequences with optimal sensitivity and specificity are finally determined, and the sequences are shown in Table 1. The HBV specific sequences are highly conserved sequences of A-D genotype HBV (see FIG. 1A).

TABLE 1 primer sequences

Wherein, the 5 'end fluorescent Probe of the Probe-HR is FAM, and the 3' end fluorescent Probe is BHQ 1. The underlined parts of the HR-R1 and HR-RT1 sequences are identical random anchor sequences, and the underlined parts of the HR-R2 and HR-RT2 sequences are identical random anchor sequences. As known to those skilled in the art, the inventors have further confirmed by experiments that the random anchor sequence does not affect the efficiency of detection, as long as it does not form primer dimer with HR-F or Probe-HR as much as possible.

2. Establishment of detection method and system

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

Specifically, the extracted nucleic acid mixture was treated with DNase I (Thermo Fisher Scientific, Waltham, MA, USA).

DNase I treatment system (10. mu.l) was:

the mixture was incubated at 37 ℃ for 30 minutes and then added

EDTA lμl

HR-RT(10μM) lμl

Incubation at 65 ℃ for 10 min

This was subsequently used for reverse transcription into cDNA by the RevertAID First Strand DNA Synthesis Kit (Thermo Fisher scientific, Waltham, Mass., USA).

The reverse transcription reaction system (20. mu.l) was:

the reaction conditions are as follows: 5 minutes at 25 ℃, 60 minutes at 42 ℃ and 5 minutes at 70 ℃.

Then, HBV specific forward sequence and anchoring sequence are used as forward and reverse primers for quantitative PCR detection respectively for amplification, and a TaqMan probe sequence is close to the forward primer (figure 2). In order to determine the detection system of the primer and the probe determined by the present invention, the inventors first detected the specificity of the primer by the method of SYBR Green, and compared the amplified melting curve with the primer reported in non-patent document 1. As a result, the specificity of the primer of the present invention (FIG. 3A) is superior to that of the primer used in non-patent document 1 (FIG. 3B). In addition, the inventors respectively tried the influence of two key factors, namely the probe amount and the annealing temperature in the detection system, on the fluorescent quantitative PCR reaction. The results show that different concentrations of probe (FIG. 3C) and different annealing temperatures (FIG. 3D) have no significant effect on the amplification intensity and sensitivity of the fluorescent quantitative PCR reaction, and the resulting amplification curves substantially overlap.

Finally, the reaction system (30. mu.l) for the fluorescent quantitative PCR was as follows:

performing amplification on an ABI Step One Plus fluorescent quantitative PCR instrument, wherein the amplification cycle parameters are 50 ℃, 5 minutes, 94 ℃ and 2 minutes; then, 45 cycles of 94 ℃, 15 seconds, 58 ℃, 45 seconds are carried out; fluorescence was collected multiple times simultaneously during the extension phase (58 ℃) of each cycle.

In addition, fluorescent staining can be performed using SYBR-Green I. The reaction system (20. mu.l) was as follows:

performing amplification on an ABI Step One Plus fluorescent quantitative PCR instrument, wherein the amplification cycle parameters are 50 ℃, 5 minutes, 94 ℃ and 2 minutes; then, 45 cycles of 94 ℃, 15 seconds, 58 ℃, 45 seconds are carried out; fluorescence was collected multiple times simultaneously during the extension phase (58 ℃) of each cycle. Adding a dissolution curve: 95 ℃ for 15 seconds, 60 ℃ for 1 minute, then increasing the temperature from 60 ℃ gradient, collecting a fluorescence signal at 0.3 ℃ per liter until the temperature rises to 95 ℃ and the temperature is maintained at 95 ℃ for 15 seconds.

3. Construction of diagnostic reagent standards

Extracting HBV pgRNA in the culture supernatant of a HBV stable replication cell line HepAD38 cell, carrying out reverse transcription by using an HR-RT reverse transcription primer, amplifying by using HR-F and HR-R, cutting gel of an amplified target fragment, recovering, and connecting to a pEASY-Blunt cloning vector. After the target fragment was confirmed by sequencing, it was used as a standard for detecting HBVpgRNA in blood in a laboratory.

The sequence of the final target fragment is as follows:

AGACCACCAAATGCCCCTATCCTATCAACACTTCCGGAAACTACTGTTGTTAGACGACGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTGTGTCGTGTGTTACGGTGTGA(SEQ NO:9)

the peak pattern of sequencing validation is shown in FIG. 4.

4. Detection of sensitivity

The constructed standard substance is diluted by 10 times, and the concentration is respectively 1.00E +08, 1.00E +07, 1.00E +06, 1.00E +05, 1.00E +04, 1.00E +03, 1.00E +02 and 1.00E +01 copies/ml. And (4) quantitatively detecting the standard substance diluted by times by using the detection system and the circulation parameters determined above. The result shows that the fluorescence quantitative detection method has higher sensitivity, and the linear range can be as low as 1.00E +01 copies/ml; the linear regression equation for the standard curve is: y-3.2645 x +40.393, square R of the correlation coefficient²0.9993 (picture)5)。

As a result of comparison with the fluorescence quantitative detection method for HBV RNA in blood reported in non-patent document 1, the linear regression equation of the amplification standard curve of the fluorescence quantitative PCR method established in non-patent document 1 is: y-3.4315 x +45.463, square R of the correlation coefficient²0.9952 (fig. 6). With the same concentration of standard, the ct value amplified by the fluorescence quantitative PCR detection method is lower by more than 3 cycles than that of the method established in non-patent document 1, which indicates that the detection method is more sensitive.

On the basis of detecting plasmid standard, the fluorescence quantitative PCR method established by the invention is used for detecting the level of HBV pgRNA of different genotypes (B, C and D genotype) and evaluating the sensitivity of the HBV pgRNA.

Wherein the B, C type is derived from the blood of a patient, the concentrations of HBV pgRNA measured by the fluorescence quantitative PCR detection method of the invention are 1.33E +06 and 1.50E +06copies/ml respectively, and the diluted concentrations are 1.33E +05, 1.33E +04, 1.33E +03 and 1.33E +02copies/ml respectively, and 1.50E +05, 1.50E +04, 1.50E +03 and 1.50E +02copies/ml respectively.

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

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

5. Detection of specificity

Nucleic acids (including DNA and RNA) in blood of HBV-infected persons were extracted using the EasyPure Viral RNA Kit (TransGen, cat # ER201-01) nucleic acid extraction Kit. The HBV DNA loading capacity in the blood is 9.45E +07copies/ml, the HBV pgRNA loading capacity measured by the fluorescence quantitative PCR detection method of the invention is 1.48E +06copies/ml, the HBV pgRNA is divided into 3 groups after nucleic acid extraction, the 1 st group is treated by DNase I and then carries out reverse transcription reaction, the 2 nd group is not treated at all, and the 3 rd group is treated by DNase I; the three groups simultaneously utilize the fluorescent quantitative PCR method established by the invention to detect.

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 acids were not treated with DNase, the interference with the detection of HBV pgRNA was small (2 in FIG. 8A), and the interference disappeared after DNase treatment (3 in FIG. 8A).

In addition, blood was examined from two normal persons, and the results were below the lower limit of detection (5 and 6 in fig. 8B).

The results show that the fluorescence quantitative detection method for the HBV pgRNA in blood established by the invention has higher specificity, and even if DNase pretreatment is not carried out, the HBV DNA has negligible interference on the pgRNA detection.

6. Repeatability detection

The standard substance with the plasmid concentration of 1.00E +03copies/ml is selected for detection and repeated for 30 times. The results showed that the standard deviation (SD value) of ct values detected in 30 replicates was 0.275537, with better reproducibility (fig. 9).

7. Detection of clinical samples

The fluorescence quantitative PCR method of the invention is used for detecting HBV pgRNA in blood of 5 patients with chronic hepatitis B who do not have antiviral treatment. Meanwhile, the HBV DNA level in blood is detected by a commercial HBV DNA quantitative detection kit (Shengxiang Biotechnology Co., Ltd., Hunan).

The results showed that HBV pgRNA was present at a level lower than HBV DNA in the blood of patients with chronic hepatitis B who had not been treated with antiviral treatment (Table 2).

Subsequently, the blood is diluted by 10 times, and then the blood samples with different dilutions are detected by the fluorescent quantitative PCR detection method of the invention. As shown in Table 3, the fluorescence quantitative PCR detection method of the present invention should exhibit high sensitivity for detection of clinical samples.

TABLE 2 HBV DNA and HBV pgRNA levels in blood of patients with chronic hepatitis B who have not been treated with antiviral therapy

TABLE 3 HBV pgRNA levels in blood of patients with chronic hepatitis B after gradient dilution

Serum HBV pgRNA levels can also be quantitatively determined by the SYBR-Green method. Firstly, detecting a standard substance by using a SYBR-Green method, drawing a standard curve, and then detecting a clinical sample according to the steps, namely quantitatively detecting the HBVpgRNA level in blood.

SEQUENCE LISTING

<110> Beijing university

<120> a highly sensitive and specific blood HBV pgRNA fluorescence quantitative PCR detection system and detection method

<130>1700051CN

<150>CN201610202753.X

<151>2016-04-01

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Claims

1. A reagent set for blood HBV pgRNA fluorescent quantitative PCR detection, comprising:

(1) a reverse anchor primer comprising a gene specific primer portion of AGGCGAGGGAGTTCTTCTTCTA located at the 3' end of the reverse anchor primer and a modification sequence portion; the modified sequence part comprises enzyme cutting sites, tag sequences or artificially designed random sequences; the nucleotide sequence of the reverse anchored primer is shown as SEQ NO. 3;

(2) a forward primer and a reverse primer; and

(3) and the template is HBV pgRNA of blood to be detected.

2. The set of reagents for the fluorescent quantitative PCR detection of HBV pgRNA in blood according to claim 1, wherein:

the nucleotide sequence of the forward primer is shown as SEQ NO. 1;

the nucleotide sequence of the reverse primer is shown as SEQ NO. 2.

3. The set of reagents for the fluorescent quantitative PCR detection of HBV pgRNA in blood according to claim 2, characterized in that: the reagent group also comprises a probe, and the nucleotide sequence of the probe is shown as SEQ NO. 4.

4. The set of reagents for the fluorescent quantitative PCR detection of HBV pgRNA in blood according to claim 2, characterized in that: the reagent set also comprises a probe, wherein the probe is one of a TaqMan probe, an MGB probe and a hybridization probe.

5. The set of reagents for the fluorescent quantitative PCR detection of HBV pgRNA in blood according to claim 2, characterized in that: the reagent set also comprises a probe, and one of FAM, VIC, TET, JOE, ROX, CY3, CY5 and HEX is marked at the 5' end of the probe; and marking one of BHQ1, BHQ2, BHQ3, TAMRA, DABCYL and NFQ at the 3' end of the probe.

6. The application of the reverse anchored primer in the preparation of blood HBV diagnostic reagent or kit is characterized in that the reverse anchored primer comprises a gene specific primer part and a modification sequence part, wherein the gene specific primer part is AGGCGAGGGAGTTCTTCTTCTA and is positioned at the 3' end of the reverse anchored primer; the modified sequence part comprises enzyme cutting sites, label sequences or artificially designed random sequences, and the nucleotide sequence of the reverse anchoring primer is shown as SEQ NO. 3.

7. A kit for blood HBV pgRNA fluorescent quantitative PCR detection comprises:

the reverse anchoring primer comprises a gene specific primer part and a modified sequence part, wherein the gene specific primer part is AGGCGAGGGAGTTCTTCTTCTA and is positioned at the 3' end of the reverse anchoring primer, the nucleotide sequence of the reverse anchoring primer is shown as SEQ NO. 3, and the modified sequence part comprises an enzyme cutting site, a tag sequence or an artificially designed random sequence; and

a forward primer and a reverse primer.

8. The kit for the fluorescent quantitative PCR detection of HBV pgRNA in blood according to claim 7, characterized in that:

the nucleotide sequence of the forward primer is shown as SEQ NO. 1;

the nucleotide sequence of the reverse primer is shown as SEQ NO. 2; and

the kit also comprises a probe, and the nucleotide sequence of the probe is shown as SEQ NO. 4.

9. The kit for the fluorescent quantitative PCR detection of HBV pgRNA in blood according to claim 8, characterized in that: the probe is one of a TaqMan probe, an MGB probe and a hybridization probe.

10. The kit for the fluorescent quantitative PCR detection of HBV pgRNA in blood according to claim 8, characterized in that: labeling one of FAM, VIC, TET, JOE, ROX, CY3, CY5 and HEX at the 5' end of the probe; and marking one of BHQ1, BHQ2, BHQ3, TAMRA, DABCYL and NFQ at the 3' end of the probe.