CN106916905B - Invisible hepatitis B detection kit and its detection method - Google Patents

Abstract

The present invention provides a kind of occult hepatitis B virus detection kit and detection method thereof, and primer group comprises upstream primer CA, downstream primer CB, upstream primer CC, downstream primer CD in this reagent kit; The sequence of described upstream primer CA is as SEQ ID NO: shown in 1; the sequence of the downstream primer CB is shown in SEQ ID NO: 2; the sequence of the upstream primer CC is shown in SEQ ID NO: 3; the sequence of the downstream primer CD is shown in SEQ ID NO: 4 shown. The present invention redesigns the primers in the Core/pre-core region by analyzing the existing nested PCR primers for diagnosing occult hepatitis B virus infection, thereby improving the detection efficiency, thereby laying the foundation for future detection of occult hepatitis B virus infection in Chinese populations .

Description

Occult hepatitis B virus detection kit and its detection method

technical field

The invention relates to the field of biotechnology, in particular to a detection kit and a detection method for occult hepatitis B virus.

Background technique

Hepatitis B virus (HBV) infection has become a serious global public health problem. The total number of people who have been infected with hepatitis B virus in the world is as high as 2 billion, and about 1 million people die each year from liver cirrhosis caused by hepatitis B. There are obvious regional differences in the prevalence of diseases such as primary liver cancer and liver failure. Since 1992, my country has included hepatitis B vaccine in the children's immunization program, and remarkable achievements have been made in neonatal hepatitis B immunization prevention. According to the 2006 national serum epidemiological survey of viral hepatitis B in the population, the HBsAg carrier rate of the population was 7.18%, which was 26.35% lower than that of 9.75% in 1992. 0.96%.

Occult hepatitis B virus infection (Occult Hepatitis B Virus Infection, OBI) refers to the persistent presence of hepatitis B virus DNA in the liver, and the level of HBV DNA in serum is low (<200IU/ml) or negative, and HBsAg cannot be detected at the same time. People in areas with high hepatitis B prevalence are prone to occult HBV infection, and occult HBV infection can also occur in children after hepatitis B vaccination.

Since the 1970s, occult hepatitis B virus infection has received more and more attention. Shahmoradi S et al. reported that nested PCR was used to detect occult hepatitis B virus infection among children vaccinated against hepatitis B in Iran, and a total of 21 children with occult hepatitis B virus infection were detected. In the previous experiments, the nested PCR method reported in the literature was verified, and it was found that the amplification results of the Core/pre-core segment were not satisfactory. Improve.

Contents of the invention

In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide a detection kit for concealed hepatitis B virus and its detection method, which is used to solve the problem of the detection method of concealed hepatitis B virus in the prior art for the diagnosis of Chinese population. Unsatisfactory results, poor sensitivity and other issues.

In order to achieve the above purpose and other related purposes, the first aspect of the present invention provides a primer set for occult hepatitis B virus detection, including upstream primer CA, downstream primer CB, upstream primer CC, and downstream primer CD;

The sequence of the upstream primer CA is shown in SEQ ID NO: 1;

The sequence of the downstream primer CB is shown in SEQ ID NO: 2;

The sequence of the upstream primer CC is shown in SEQ ID NO: 3;

The sequence of the downstream primer CD is shown in SEQ ID NO:4.

The second aspect of the present invention provides a kit for detecting occult hepatitis B virus containing the above primer set.

Further, the upstream primer CA and the downstream primer CB are the first-round specific primers, and the upstream primer CC and the downstream primer CD are the second-round specific primers.

Further, the kit also includes one or more combinations of PCR master mix, HBV DNA, and water.

Preferably, the PCR master mix contains one or more combinations of Taq DNA polymerase, dNTPs, standard Taq enzyme reaction buffer, and enzyme stabilizer.

More preferably, the PCR master mix is 2×Taq PCR Master Mix.

The third aspect of the present invention provides the application of the above primer set or kit in detecting occult hepatitis B virus.

Further, at least include the following steps:

1) extract the HBV DNA in the sample to be tested;

2) Nested PCR reaction: using the upstream primer CA and the downstream primer CB to carry out the first round of PCR amplification of the HBV DNA obtained in step 1), and then using the amplification product of the first round of PCR as a template, using the upstream Primer CC, downstream primer CD for the second round of PCR amplification;

3) According to the gel electrophoresis results of the PCR amplification products, it is judged whether the target band appears.

Further, the sample to be tested in step 1) is selected from serum, plasma, whole blood, pure virus culture and vector organisms carrying such viruses.

Further, in step 2), the first round of PCR amplification reaction conditions are as follows: pre-denaturation: temperature is 94°C, time is 3min; denaturation: temperature is 94°C, time is 30s; annealing: temperature is 55°C, time is 30s ;Extension: the temperature is 72°C, and the time is 1min; after 34 cycles of denaturing annealing and extension, the temperature is 72°C, and the time is 5min.

Further, in step 2), the conditions of the second round of PCR amplification reaction are as follows: pre-denaturation: temperature is 94°C, time is 3min; denaturation: temperature is 94°C, time is 30s; annealing: temperature is 55°C, time is 30s ;Extension: the temperature is 72°C, and the time is 1min; after 34 cycles of denaturing annealing and extension, the temperature is 72°C, and the time is 5min.

As mentioned above, a occult hepatitis B virus detection kit and its detection method of the present invention have the following beneficial effects: the present invention redesigns the Core/pre The primers in the -core region improve the detection efficiency, thereby laying the foundation for the detection of occult hepatitis B virus infection in the Chinese population in the future.

Description of drawings

Figure 1 shows the gel electrophoresis images of HBsAg-negative children's specimens detected by primers C1, C3, and C4 in the Core/pre-core region of the embodiment of the present invention.

Fig. 2a shows the gel electrophoresis image detected by the primers C1, C3 and C4 of Example P4 of the present invention.

Fig. 2b shows the alignment of nucleotide sequences detected by primers C1, C3, and C4 in Example P4 of the present invention.

Figure 3 shows the gel electrophoresis images of HBV-positive samples detected by the newly designed Core/pre-core region upstream primer CA, downstream primer CB, upstream primer CC, and downstream primer CD according to the embodiment of the present invention.

Figure 4 shows the gel electrophoresis images of HBsAg-negative children's samples detected by the upstream primer CA, downstream primer CB, upstream primer CC, and downstream primer CD of the Core/pre-core region of the embodiment of the present invention.

Figure 5a and Figure 5b show the comparison of the nucleotide sequences of the HBV Core/pre-core region of 5 children in the embodiment of the present invention.

Detailed ways

Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention.

In the comparison results shown in Figure 2b, Figure 5a and Figure 5b, a dash "-" indicates a blank, and a dot "." indicates the same base as the HBVC gene.

Main experimental reagents: serum virus DNA extraction kit, purchased from Qiagen; 2×Taq PCR MasterMix (product number KT201-02, specification 5ml, containing dye), 100bp marker, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; Agarose was purchased from HraGene Company; GoodViewTM nucleic acid dye was purchased from Beijing Saibaisheng Bioengineering Company.

Experimental samples: 100 serum samples of HBsAg-negative children hospitalized in the Children's Hospital Affiliated to Chongqing Medical University. ) completed 3 hepatitis B vaccinations; (4) no history of blood transfusion; (5) no other hepatitis virus such as HAV, HCV infection; (6) no HIV infection; (7) aged over 8 months. Serum samples were collected from 1 HBV positive child.

Serum extraction of HBV DNA experiment: use the serum virus DNA extraction kit, according to the kit operating instructions, extract the HBV DNA in the serum samples.

Nested PCR experiment: 25 μL nested PCR amplification reaction system is as follows: 12.5 μL 2×Taq PCR Master Mix, 1 μL forward primer, 1 μL reverse primer, 5 μL DNA template (HBV DNA), 5.5 μL ddH ₂ O.

Nested PCR reaction parameters are as follows: pre-denaturation: temperature is 94°C, time is 3min; denaturation: temperature is 94°C, time is 30s, annealing: temperature is different for different reaction primers, time is 30s, extension: temperature is 72°C, The time is 1 min, after 34 cycles of denaturation annealing and extension; the temperature of extension is 72° C., and the time is 5 min.

The annealing temperature of different primers is different, the annealing temperature of P region and S region is 62°C, the annealing temperature of X region is 55°C, the annealing temperature of C region and PS region is 55°C in the first round, and 62°C in the second round; The primer sequences are shown in Table 1 below.

Table 1

Newly designed Core/pre-core region primers: Select the conserved region of the Core/pre-core region gene of HBV DNA to design primers, which are synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

The upstream primer CA is located in the 1797-1815 base region upstream of the Core/pre-core region, and the sequence is: 5'-GTCTGTTCACCAGCACCAT-3' (SEQ ID NO: 1);

The downstream primer CB is located in the 2377-2395 base region of the Core/pre-core region, and the sequence is: 5'-TCTGCGAGGCGAGGGAGTT-3' (SEQ ID NO: 2);

The upstream primer CC is located in the 1877-1896 base region of the Core/pre-core region, and the sequence is: 5'-CTGTGCCTTGGGTGGCTTTG-3' (SEQ ID NO: 3);

The downstream primer CD is located in the 2345-2362 base region of the Core/pre-core region, and the sequence is: 5'-CCTGCCTCGTCGTCTAAC-3' (SEQ ID NO: 4).

The configuration of the first round of PCR amplification reaction reagents is:

The parameters of the first round of PCR amplification reaction are as follows: pre-denaturation: temperature is 94°C, time is 3min; denaturation: temperature is 94°C, time is 30s, annealing: first round of annealing temperature is 55°C, time is 30s, extension: temperature It is 72°C, the time is 1min, after 34 cycles of denaturation annealing and extension; and then extended, the temperature is 72°C, and the time is 5min.

Using the first-round PCR product as a template, the second-round PCR reaction was carried out.

The configuration of the second round of PCR amplification reaction reagents is:

Element Final concentration Mother liquor concentration Dosage (μL) 2×Taq PCR Master Mix 1× 2× 12.5 Upstream primer CC 0.4μM 10μM 1 downstream primer CD 0.4μM 10μM 1 HBV DNA — — 5 ddH ₂ O — — 5.5 total capacity — — 25

The parameters of the second round of PCR amplification reaction are as follows: pre-denaturation: temperature is 94°C, time is 3min; denaturation: temperature is 94°C, time is 30s, annealing: second round of annealing temperature is 55°C, time is 30s, extension: temperature It is 72°C, the time is 1min, after 34 cycles of denaturation annealing and extension; and then extended, the temperature is 72°C, and the time is 5min.

Two rounds of nested PCR products were subjected to agarose gel electrophoresis experiments respectively. GoodViewTM nucleic acid dye was added to the gel preparation, the agarose concentration was 1%, the voltage was 160V, and the electrophoresis time was 20 minutes. The experimental results were observed in the gel imaging system. Expected experimental results: the first-round product has a band at 600bp, and the second-round product has a band at 500bp.

The experimental results are as follows:

1. Nested PCR results of the original primers: Use the primers in Table 1 to detect HBV DNA in the serum of HBV-positive children. The electrophoresis results are: the target bands can be amplified in all regions.

2. The results of detecting HBV DNA in the serum of HBsAg-negative children with the original primers: use the primers in Table 1 to detect HBV DNA in the serum of HBsAg-negative children. Multiple bands other than bands (see Figure 1, Note: 1: ddH ₂ O control; 2-11: 10 HBsAg-negative samples; 12: negative control; 13: HBsAg-positive children’s samples; 14: 100bp marker). In the Core/pre-core area of 100 children’s specimens, only P4 had a weak target band (see Figure 2a, Note: 1: P4; 2: Negative control; 3: HBV positive specimen; 4: 100bp marker), The comparison results of the sequenced gene sequences showed that the P4 gene sequence is shown in Figure 2b, and compared with the gene sequence of the HBV C genotype, it was found that it was the HBV gene sequence. That is, one of the 100 HBsAg-negative specimens amplified the HBV C gene.

SEQ ID NO: 5 shows the HBV C gene sequence used for comparison;

SEQ ID NO: 6 shows the sequence of the P4 gene used for comparison, and the sequence is obtained by sequencing the original primers after nested PCR amplification.

4. Nested PCR results of newly designed Core/pre-core region primers: upstream primer CA and downstream primer CB are the first round of primers, upstream primer CC and downstream primer CD are the second round of primers, two rounds of nested PCR experiments, Observe the electrophoresis results of the HBV-positive samples in the gel imaging system: the first round of PCR products has a band at 600 bp, and the second round of PCR products has a band at 500 bp, which means that the primers have successfully amplified the target band. The electropherogram is shown in Figure 3. Note: 1: ddH ₂ O control; 2: negative control; 3: HBV positive sample; 4: 100bp marker.

5. Newly designed Core/pre-core region primers for detection of HBV DNA in the serum of HBsAg-negative children: use the newly designed Core/pre-core region primers to detect HBV DNA in the serum of HBsAg-negative children, and compare the results of primers C1, C3, and C4 It was found that the electrophoresis results were: only the target band appeared (see Figure 4, Note: 1: ddH ₂ O control; 2-11: 10 HBsAg negative samples; 12: negative control; 13: HBsAg positive children's samples; 14 : 100bp marker), 5 of the 100 children’s specimens had the target bands, the sequence comparison results of the sequenced genes are shown in Figure 5a and Figure 5b, and compared with the gene sequence of the HBV C genotype, it was found that they were all HBV genes The HBV C gene was amplified in 5 specimens out of 100 HBsAg-negative specimens.

The sequencing results of the five samples are as follows:

SEQ ID NO: 7 shows the P4 gene sequence used for comparison, which is obtained by sequencing the primers designed in this example after nested PCR amplification;

SEQ ID NO: 8 shows the P17 gene sequence used for comparison, which is obtained by sequencing the primers designed in this example after nested PCR amplification;

SEQ ID NO: 9 shows the P23 gene sequence used for comparison, which is obtained by sequencing the primers designed in this example after nested PCR amplification;

SEQ ID NO: 10 shows the P29 gene sequence used for comparison, which is obtained by sequencing the primers designed in this example after nested PCR amplification;

SEQ ID NO: 11 shows the sequence of the P72 gene used for comparison, which is obtained by nested PCR amplification of the primers designed in this example and sequenced.

The analysis of the experimental results is as follows:

Occult hepatitis B virus infection has been proven to be closely related to the occurrence and development of liver cirrhosis, primary liver cancer and other diseases, and may also increase the risk of HBV infection in patients with blood transfusion, dialysis, liver transplantation, etc., which has great potential harm. Occult HBV infection can be divided into serologically positive OBI and serologically negative OBI according to whether the hepatitis B core antibody (Hepatits B core Antibody, HBcAb) and hepatitis B surface antibody (Hepatits B surface Antibody, HBsAb) are positive, and serologically positive OBI is hepatitis B core antibody positive, with or without hepatitis B surface antibody positive; seronegative is negative for both.

Currently, detection of HBV DNA is the gold standard for detection of occult HBV infection. The detection of occult hepatitis B virus infection uses specific primers for highly conserved regions of different HBV genes to perform nested PCR or real-time quantitative PCR with high sensitivity. Nested PCR detection generally detects at least 3 conserved regions of the HBV genome, such as S region, X region, and C region. Due to the high sensitivity of nested PCR, in order to reduce false positives during the experiment, a negative control and a blank control must be set up for each experiment. When the results of the negative control and the blank control are negative, the positive results of the sample can be adopted. .

Therefore, the present embodiment collected 100 HBsAg-negative children's serum specimens. By extracting the HBV DNA of the specimens and performing a nested PCR experiment, according to the results of electrophoresis and sequencing results, it was found that when primers C1, C3, and C4 amplify the HBV C gene, Only the target band appeared in P4, and the target band was weak, and its gene sequence was obtained by sequencing, and compared with the gene sequence of HBV C genotype, it was found that the similarity of the two gene sequences was high, that is, the gene sequence of P4 was HBV gene sequence.

Primer design plays an important role in nested PCR experiments. Improper design of PCR primers can easily lead to deviations in experimental results: electrophoresis results show that multiple bands other than the target band can be amplified, and the target band is weak or does not appear. In this example, through the verification of the primers C1, C3, and C4 in the Core/pre-core region, it was found that multiple bands other than the target band appeared in the electrophoresis results. The primers were analyzed and found to have the following problems: (1) more than 3 consecutive bases GGG appear at the 3' end of primer C1, which may lead to an increase in the probability of false priming; (2) the base at the 3' end of primer C4 is A, its amplification efficiency at the mismatched position is significantly higher than that of other bases; (3) The GC content of primer C3 is higher than 60%, and the difference between the GC content of the upstream and downstream primers is too large, which is not conducive to the PCR reaction. In view of the above problems, and according to the distribution of HBV genotypes in my country, this embodiment redesigned the primers for the Core/pre-core region, and found through verification that the primers can be used to amplify HBV DNA.

In this example, by using the newly designed upstream primers CA, CB, CC, and CD to detect 100 serum samples of HBsAg-negative children, it was found that the target bands were successfully amplified in 5 samples. The gene sequences of 5 specimens were obtained by sequencing, and compared with the gene sequences of HBV C genotype, it was found that these gene sequences were highly similar to HBV, and point mutations could be seen. The gene sequences of these 5 specimens were all HBV gene sequences . That is, in the P4 specimen in which the target gene was successfully amplified by primers C1, C3, and C4, the newly designed primers CA, CB, CC, and CD also successfully amplified the target gene; Among the 4 specimens with the target gene, the newly designed CA, CB, CC, CD primers successfully amplified the target gene. The newly designed CA, CB, CC, CD primers greatly improved the detection efficiency.

In summary, according to the genotype of HBV in my country, the present invention designs primers suitable for diagnosing occult HBV infection in the Chinese population by analyzing the conserved sequence of the HBV gene.

The sequences of the Core/pre-core regions of HBV B genotype and C genotype are completely consistent, therefore, the primers of the present invention are mainly suitable for the diagnosis of HBV B genotype and C genotype hepatitis B virus infection.

The above-mentioned embodiments only illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical ideas disclosed in the present invention should still be covered by the claims of the present invention.

references:

1. Lavanchy D.Hepatitis B virus epidemiology,disease burden,treatment,and current and emerging prevention and control measures[J].J Viral Hepat,2004,11(2):97-107.

2.West D J,Waston B,Lichtman J,et al.Persistence of immunological memory for twelve years in children given hepatitis B vaccine in infancy[J].Pediatr Infect Dis J,1994,13(8):745-7.

3. Ministry of Health. Remarkable results in controlling hepatitis B in my country [EB/OL].

http://www.nhfpc.gov.cn/jkj/s3582/201307/518216575e544109b2caca07fca3b430.shtml.

4. Raimondo G, Allain J P, Brunetto M R, et al. Statements from the Taormina expert meeting on occult hepatitis B virus infection [J]. J Hepatol, 2008; 49(4):652-657.

5. Chakvetadze C, Roussin C, Roux J, et a1. Efficacy of hepatitis B sero-vaccination in newborns of African HBsAg positive mothers [J]. Vaccine, 2011, 29(16): 2846—2849.

6. Shahmoradi S, Yahyapour Y, Mahmoodi M, et al. High prevalence of occulthepatitis B virus infection in children born to HBsAg positive mothers despite prophylaxis with hepatitis Bvaccination and HBIG [J]. J Hepatol, 2012; 57:515–521.

7. Sabrina C, Claudio A, Pierluca P, et al.A statistical model based onserological parameters for predicting occult HBV infection: implications for organ/blood donations[J].New Microbiologica.2015,38(1):39-49.

8.Hollinger F B, Habibollahi P, Daneshmand A, et al.Occult hepatitis Binfection in chronic hemodialysis patients: current concepts and strategy[J].Hepatitis Monthly.2010,19(2):175.

9. LledóJ, Fernández C, Gutiérrez M L, et al. Management of occulthepatitis B virus infection: an update for the clinician [J]. World Journal of Gastroenterology. 2011, 17(12): 1563-1568.

10.Torbenson M,Thomas D L.Occult hepatitis B[J].Lancet Infect Dis,2002,2(8):479–486.

11. Raimondo G, Pollicino T, Levrero M, et al. Occult hepatitis B virus infection and hepatocellular carcinoma development in patients with chronicepatitis C[J].Hepatology.2011,54(1):373-4.

12. Zhang Xinyu, Gao Yanning.PCR primer design and software usage skills[J].Bioinformatics,2004,2(4):15-18.

SEQUENCE LISTING

<110> Children's Hospital Affiliated to Chongqing Medical University

<120> Occult hepatitis B virus detection kit and its detection method

<130> PCQYK

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 19

<212>DNA

<213> Artificial

<220>

<223> upstream primer CA

<400> 1

gtctgttcac cagcaccat 19

<210> 2

<211> 19

<212>DNA

<213> Artificial

<220>

<223> downstream primer CB

<400> 2

tctgcgaggc gagggagtt 19

<210> 3

<211> 20

<212>DNA

<213> Artificial

<220>

<223> Upstream Primer CC

<400> 3

ctgtgccttgggtggctttg 20

<210> 4

<211> 18

<212>DNA

<213> Artificial

<220>

<223> downstream primer CD

<400> 4

cctgcctcgt cgtctaac 18

<210> 5

<211> 639

<212>DNA

<213> Artificial

<220>

<223> HBV C

<400> 5

atgcaacttt ttcacctctg cctaatcatc tcatgttcat gtcctactgt tcaagcctcc 60

aagctgtgcc ttgggtggct ttggggcatg gacattgacc cgtataaaga atttggagca 120

tctgtggagt tactctcttt tttgccttct gacttctttc cttctattcg agatctcctc 180

gacaccgcct ctgctctgta tcgggaggcc ttagagtctc cggaacattg ttcacctcac 240

catacagcac tcaggcaagc tattctgtgt tggggtgagt taatgaatct ggccacctgg 300

gtgggaagta atttggaaga cccagcatcc agggaattag tagtcagcta tgtcaatgtt 360

aatatgggcc taaaaatcag acaactattg tggtttcaca tttcctgtct tacttttgga 420

agagaaactg ttcttgagta tttggtgtct tttggagtgt ggattcgcac tcctcccgct 480

tacagaccac caaatgcccc tatcttatca aacacttccgg aaactactgt tgttagacga 540

cgaggcaggt cccctagaag aagaactccc tcgcctcgca gacgaaggtc tcaatcgccg 600

cgtcgcagaa gatctcaatc tcgggaatct caatgttag 639

<210> 6

<211> 460

<212>DNA

<213> Artificial

<220>

<223> P4C

<400> 6

ctcataatgt gccttgggtg gctttggggc atggacattg accccgtataa agaatttgga 60

gcttctgtgg agttactctc ttttttgcct tctgacttct ttccttctat tcgagatctc 120

ctcgacaccg cctctgctct gtatcgggag gccttagagt ctccggaaca ttgttcacct 180

caccatacgg cactcaggca agctatcctg tgttggggtg agttgatgaa tctagccacc 240

tgggtgggaa gtaatttgga agatccagca tccagggaat tagtagtcag ctatgtcaac 300

gttaatatgg gcctaaaaat cagacaacta ttgtggtttc aatttcctg tcttactttt 360

gggagagaaa ctgttcttga atatttggtg tcttttggag tgtggattcg cactcctcct 420

gcatatagac caccaaatgc ccctatctta tcaacacttc 460

<210> 7

<211> 432

<212>DNA

<213> Artificial

<220>

<223> P4CD

<400> 7

agataggggc atttggtggt ctatatgcag gaggagtgcg aatccaacact ccaaaagaca 60

ccaaatattc aagaacagtt tctctcccaa aagtaagaca ggaaatgtga aaccacaata 120

gttgtctgat ttttaggccc atattaacgt tgacatagct gactactaat tccctggatg 180

ctggatcttc caaattactt cccacccagg tggctagatt catcaactca ccccaacaca 240

ggatagcttg cctgagtgcc gtatggtgag gtgaacaatg ttccggagac tctaaggcct 300

cccgatacag agcagaggcg gtgtcgagga gatctcgaat agaaggaaag aagtcagaag 360

gcaaaaaaga gagtaactcc acagaagctc caaattcttt atacgggtca atgtccatgc 420

cccaaagcca ca 432

<210> 8

<211> 437

<212>DNA

<213> Artificial

<220>

<223> P17CD

<400> 8

gataagatag gggcattgg tggtctgtaa gcaggagggag tgcgaatcca cactccaaaa 60

gataccaaat actcaaggac agtttctctt ccaaaagtaa gacaggaaat gtgaaaccac 120

agtagttgtc tgatttttag gcccatgtta acattgacat agctgactac taattccctg 180

gatgctgggt cttccaaatt acttcccacc caggtggcca gattcatcaa ctcaccccaa 240

cacagaatag cttgcctgag tgcagtatgg tgaggtgagc aatgttccgg agactctaag 300

gcctcccgat atagagcaga ggcggtgtcg aggagatctc gaatagaagg aaagaagtca 360

gacggcaaaa aagagagtaa ctccacagaa gctccaaatt ctttatacgg gtcaatgtcc 420

atgccccaaa gccacaa 437

<210> 9

<211> 434

<212>DNA

<213> Artificial

<220>

<223> P23CD

<400> 9

tgatagatag gggcatttgg tggtctgtaa gcaggagggag tgcgaatcca cactccaaaa 60

gataccaaat actcaaggac agtttctctt ccaaaagtaa gacaggaaat gtgaaaccac 120

agtagttgtc tgatttttag gcccatgtta acattgacat agctgactac taattccctg 180

gatgctgggt cttccaaatt acttcccacc caggtggcca gattcatcaa ctcaccccaa 240

cacagaatag cttgcctgag tgcagtatgg tgaggtgagc aatgttccgg agactctaag 300

gcctcccgat atagagcaga ggcggtgtcg aggagatctc gaatagaagg aaagaagtca 360

gacggcaaaa aagagagtaa ctccacagaa gctccaaatt ctttatacgg gtcaatgtcc 420

atgccccaaa gccc 434

<210> 10

<211> 430

<212>DNA

<213> Artificial

<220>

<223> P29CD

<400> 10

agataggggc atttggtggt ctgtaagcag gaggagtgcg aatccacacc ccaaaagata 60

ccaaatactc aagggcagtt tctcttccaa aagtaagaca ggaaatgtga aaccacagta 120

gttgtctgat ttttaggccc atgttaacat tgacatagct gactactaat tccctggatg 180

ctgggtcttc caaattactt cccacccagg tggccagatt catcaactca ccccaacaca 240

gaatagcttg cctgagtgca gtatggtgag gtgagcaatg ttccggagac tctaaggcct 300

cccgatatag agcagaggcg gtgtcgagga gatctcgaat agaaggaaag aagtcagacg 360

gcaaaaaaga gagtaactcc acagaagctc caaattcttt atacgggtca atgtccatgc 420

cccaaagccc 430

<210> 11

<211> 426

<212>DNA

<213> Artificial

<220>

<223> P72CD

<400> 11

ggggcatttg gtggtctgta agcaggagga gtgcgaatcc acactccaaa agataccaaa 60

tactcaagga cagtttctct tccaaaagta agacaggaaa tgtgaaacca cagttttgt 120

ctgattttta ggcccatgtt aacattgaca tagctgacta ctaattccct ggatgctggg 180

tcttccaaat tacttcccac ccaggtggcc agattcatca actcacccca aacacagaata 240

gcttgcctga gtgcagtatg gtgaggtgag caatgttccg gagactctaa ggcctcccga 300

tatagagcag aggcggtgtc gaggagatct cgaatagaag gaaagaagtc agacggcaaa 360

aaagagagta actccacaga agctccaaat tctttatacg ggtcaatgtc catgccccaa 420

agcccc 426

Claims (11)

1. a kind of primer sets for the detection of invisible hepatitis B, which is characterized in that including sense primer CA, downstream primer CB, sense primer CC, downstream primer CD；

The sequence of the sense primer CA such as SEQ ID NO：Shown in 1；

The sequence of the downstream primer CB such as SEQ ID NO：Shown in 2；

The sequence of the sense primer CC such as SEQ ID NO：Shown in 3；

The sequence of the downstream primer CD such as SEQ ID NO：Shown in 4.

2. the kit for being used to detect invisible hepatitis B containing primer sets according to claim 1.

3. kit according to claim 2, it is characterised in that：The sense primer CA, downstream primer CB are the first round Specific primer, the sense primer CC, downstream primer CD are the second wheel specific primer.

4. kit according to claim 2, it is characterised in that：The kit further include PCR premixed liquids, HBV DNA, One or more combinations in water.

5. kit according to claim 4, it is characterised in that：The PCR premixed liquids contain Taq archaeal dna polymerases, One or more combinations in dNTPs, standard Taq enzyme reaction buffer, enzyme stabilizers.

6. kit according to claim 5, it is characterised in that：The PCR premixed liquids are 2 × Taq PCR Master Mix。

7. primer sets according to claim 1 or claim 2-5 any one of them kit are detecting invisible second Application in hepatovirus.

8. application according to claim 7, it is characterised in that：Including at least following steps：

1）Extract the HBV DNA in sample to be tested；

2）Nest-type PRC reacts：Using the sense primer CA, downstream primer CB to step 1）The HBV DNA of acquisition carry out first PCR amplification is taken turns, then using the amplified production of first round PCR as template, second is carried out using the sense primer CC, downstream primer CD Take turns PCR amplification；

3）Judge whether purpose band occur according to the Gel electrophoresis results of pcr amplification product.

9. application according to claim 8, it is characterised in that：Step 1）Described in sample to be tested be selected from serum, blood plasma, complete Blood, pure viral cultures and the Vector factors for carrying this viroid.

10. application according to claim 8, it is characterised in that：Step 2）In, first round pcr amplification reaction condition is as follows： Pre-degeneration：94 DEG C of temperature, time 3min；Denaturation：94 DEG C of temperature, time 30s；Annealing：55 DEG C of temperature, time 30s；Extension：Temperature 72 DEG C of degree, time 1min；After cycle by 34 denaturation annealing extensions, re-extend, 72 DEG C of temperature, time 5min.

11. application according to claim 8, it is characterised in that：Step 2）In, the second wheel pcr amplification reaction condition is as follows： Pre-degeneration：94 DEG C of temperature, time 3min；Denaturation：94 DEG C of temperature, time 30s；Annealing：55 DEG C of temperature, time 30s；Extension：Temperature 72 DEG C of degree, time 1min；After cycle by 34 denaturation annealing extensions, re-extend, 72 DEG C of temperature, time 5min.