HBV integration site chrX:111,009,033 real-time fluorescence quantitative PCR detection kit

Abstract

The present invention belongs to the technical field of virus detection, and in particular, relates to a real-time fluorescence quantitative PCR detection primer and kit for HBV integration site chrX:111,009,033. The present invention develops a kit for quantitatively detecting integration site chrX:111,009,033 by using chimeric primer fluorescence PCR technology, with β-globin as the internal reference gene, which fills the gap in the quantitative detection of HBV integration in the world, and will effectively promote the occurrence of HBV integration, carcinogenic mechanism and other research.

Description

Technical Field

[0001] The invention belongs to the technical field of virus detection, and in particular relates to a real-time fluorescence quantitative PCR detection kit for HBV integration site chrX:111,009,033. Background Art

[0002] There are about 2 billion people infected with hepatitis B virus (HBV) in the world, of which about 240 million are chronic hepatitis B (CHB) patients. About one million people die each year from liver failure, cirrhosis, and hepatocellular carcinoma (HCC) caused by CHB. After HBV infection, the mature virus adheres to the surface of the host liver cells and enters the liver cells. Some viral gene components enter the nucleus of the liver cells. HBV DNA is converted into covalently closed circular DNA (cccDNA) in the form of loose circular structures (rcDNA), and some DNA is integrated into the host genes. The use of traditional methods such as Alu-PCR to detect that HBV randomly integrates into the host genes. In recent years, high-throughput sequencing technology (WGS) has been used to detect HBV-related HCC patients and found that host genes, especially tumor-related cell proliferation genes such as TERT, MLL4, CCNE1, NUP85, AGBL5, etc., have dominant integration sites. The HBV integration sites and frequencies of these genes in tumor tissue hepatocytes are significantly higher than those in the surrounding non-tumor tissues, suggesting that HBV integration may cause HCC. The oncogenic mechanism of HBV integration may include: ① Integration causes host chromosome instability; ② Affects the function of host genes around the integration site, such as inactivation or expression inhibition of tumor suppressor genes, or overexpression activation of oncogenes; ③ Integration forms HBV truncated proteins and virus-host gene fusion proteins with carcinogenic effects. Early detection and timely elimination of HBV integrated hepatocytes in hepatitis B patients are of great clinical significance for preventing HCC and completely curing chronic hepatitis B, and is one of the important strategies for HBV-related HCC gene therapy in the future. However, due to the randomness of HBV integration and the diversity of inserted viral sequences, there are currently no HBV integration detection kits available in the world, and it is impossible to conduct systematic cell research on HBV integration, resulting in no breakthrough progress in the occurrence and carcinogenic mechanism of HBV integration. The applicant has been engaged in the research of HBV integration and carcinogenic mechanism for a long time. In 2018, the HBV integration site chrX:111,009,033 for the insertion of the PAK3 gene was first discovered in HepG2.2.15 cells using inverse nested PCR (InvPCR) in the INSERMU1052 Molecular Biology Laboratory in Lyon, France (Ruan P, Dai XF, Sun J, He CP, Huang C, Zhou R, Chemin I. Integration of hepatitis B virus DNA into p21-activated kinase 3 (PAK3) gene in HepG2.2.15 cells [J]. Virus Genes. 2020, 56 (2): 168-173).

[0003] Therefore, there is an urgent need to develop primers, probes and kits that can quickly and accurately detect the HBV integration site chrX:111,009,033. Summary of the invention

[0004] Aiming at the HBV integration site chrX:111,009,033, the present invention provides a real-time fluorescence quantitative PCR detection kit for the HBV integration site chrX:111,009,033, which can quickly, sensitively and accurately quantitatively detect the integration site chrX:111,009,033.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] In the first aspect of the present invention, a real-time fluorescence quantitative PCR detection kit for HBV integration site chrX:111,009,033 is provided, the kit comprising a chimeric primer having a nucleotide sequence as shown in SEQ ID NO.4, a real-time fluorescence quantitative PCR detection primer pair for HBV integration site chrX:111,009,033 having a nucleotide sequence as shown in SEQ ID NO.1-2, and a probe having a nucleotide sequence as shown in SEQ ID NO.3.

[0007] Furthermore, the kit also includes a primer pair with a nucleotide sequence as shown in SEQ ID NO.5-6 and a probe with a nucleotide sequence as shown in SEQ ID NO.7.

[0008] Furthermore, the kit also includes: a positive standard for quantitative detection, wherein the positive standard is a plasmid containing an amplification sequence of the HBV integration site chrX:111,009,033 in a concentration gradient.

[0009] Furthermore, the kit further comprises: ultrapure water or RNase Free H 2 O, 5×PCR Buffer and Enzyme Mix.

[0010] Furthermore, when the kit is used, the reaction system includes a single-strand extension system and a real-time fluorescence quantitative PCR amplification system;

[0011] The single-strand extension system includes the extracted nucleic acid and a chimeric primer as shown in SEQ ID NO.4 to obtain a single-strand extension product;

[0012] The real-time fluorescence quantitative PCR amplification system includes the single-stranded extension product, the HBV integration site chrX:111,009,033 real-time fluorescence quantitative PCR detection primer pair with a nucleotide sequence as shown in SEQ ID NO.1-2, and a probe with a nucleotide sequence as shown in SEQ ID NO.3, 5×PCR Buffer, Enzyme Mix, and ultrapure water or RNase Free H 2 O.

[0013] Further, the single-strand extension system includes 5±1 μl of the extracted nucleic acid, 1±0.3 μl of the chimeric primer as shown in SEQ ID NO.4, and obtains a single-strand extension product;

[0014] The real-time fluorescence quantitative PCR amplification system includes 5±1 μl of the single-stranded extension product, 0.75±0.3 μl of each of the real-time fluorescence quantitative PCR detection primer pairs of the HBV integration site chrX:111,009,033 with a nucleotide sequence as shown in SEQ ID NO.1-2, 1±0.3 μl of the probe with a nucleotide sequence as shown in SEQ ID NO.3, 4±1 μl of 5×PCR Buffer, 5±1 μl of Enzyme Mix, and ultrapure water or RNase Free H 2 0 to 20 μl.

[0015] Preferably, the positive standard comprises standard 1, with a concentration of 1×10 1 copies / mL; Standard 2, concentration is 1×10 2 copies / mL; standard 3, concentration is 1×10 3 copies / mL; standard 4, concentration is 1×10 4 copies / mL; standard 5, concentration 1×10 5 copies / mL, standard 6, concentration 1×10 6 copies / mL; standard 7, concentration 1×10 7 copies / mL.

[0016] Furthermore, the kit also includes:

[0017] Positive control: HBV integration site chrX:111,009,033 amplified sequence plasmid standard;

[0018] Negative control: RNase Free H 2 O.

[0019] Internal standard primers FP: SEQ ID NO. 5, and RP: SEQ ID NO. 6;

[0020] Fluorescent probe: SEQ ID NO.7.

[0021] Furthermore, when the kit is used, the reaction system includes a single-strand extension system and a real-time fluorescence quantitative PCR amplification system;

[0022] The single-strand extension system includes the extracted nucleic acid and a chimeric primer as shown in SEQ ID NO.4 to obtain a single-strand extension product;

[0023] The real-time fluorescence quantitative PCR amplification system includes the single-stranded extension product, the HBV integration site chrX:111,009,033 real-time fluorescence quantitative PCR detection primer pair with a nucleotide sequence as shown in SEQ ID NO.1-2, and a probe with a nucleotide sequence as shown in SEQ ID NO.3, 5×PCR Buffer, Enzyme Mix, and ultrapure water or RNase Free H 2 O.

[0024] Furthermore, the single-strand extension system includes 5±1 μl of extracted nucleic acid, 1±0.3 μl of chimeric primer as shown in SEQ ID NO.4, 5±1 μl of Mix containing DNA polymerase I, ultrapure water or RNase Free H 2 0 to 20 μl to obtain single-strand extension products;

[0025] The real-time fluorescence quantitative PCR amplification system includes 5±1 μl of the single-stranded extension product, 0.75±0.3 μl of each of the real-time fluorescence quantitative PCR detection primer pairs of the HBV integration site chrX:111,009,033 with a nucleotide sequence as shown in SEQ ID NO.1-2, 1±0.3 μl of the probe with a nucleotide sequence as shown in SEQ ID NO.3, 4±1 μl of 5×PCR Buffer, 5±1 μl of Enzyme Mix, and ultrapure water or RNase Free H 2 0 to 20 μl.

[0026] In the second aspect of the present invention, a quantitative detection standard for HBV integration site chrX:111,009,033 is provided, and the preparation method of the standard is as follows:

[0027] The chimeric primer R with the nucleotide sequence shown in SEQ ID NO.8 and the Alu primer R1 with the nucleotide sequence shown in SEQ ID NO.9 were used to perform PCR amplification on the DNA extracted from the cells to obtain an amplified product, which was confirmed by the improved InvPCR technology to be a specific amplified fragment containing the full-length sequence of the viral insertion at the integration site chrX:111,009,033;

[0028] Insert the specific amplification fragment into the PMD-18T vector to obtain a quantitative detection standard for the HBV integration site chrX:111,009,033.

[0029] In the second aspect of the present invention, a method for using the real-time fluorescence quantitative PCR detection kit for the HBV integration site chrX:111,009,033 is provided, including:

[0030] Using the extracted nucleic acid as a template, perform single-strand extension with a chimeric primer having a nucleotide sequence as shown in SEQ ID NO.4 to obtain a single-strand extension product;

[0031] Using the single-strand extension product as a template, perform real-time fluorescence PCR amplification with the real-time fluorescence quantitative PCR detection primer pair for the HBV integration site chrX:111,009,033 having a nucleotide sequence as shown in SEQ ID NOs.1-2 to obtain an amplification curve, analyze the amplification curve, and make a judgment; the amplification reaction system includes the real-time fluorescence quantitative PCR detection primer pair and probe for the HBV integration site chrX:111,009,033.

[0032] Further, the reaction system for the real-time fluorescence PCR amplification includes: sample template, the real-time fluorescence quantitative PCR detection primers and probe for the HBV integration site chrX:111,009,033, 5×PCR Buffer, and Enzyme Mix.

[0033] The amplification program is: preheat at 95°C for 5 min, denature at 95°C for 10 s, anneal at 54°C for 10 s, and extend at 72°C for 10 s for a total of 40 cycles.

[0034] Further, the principle for analyzing and judging the amplification curve is:

[0035] When Ct ≤ 40 in the FAM fluorescence channel, the sample is judged to be positive for the HBV integration site chrX:111,009,033;

[0036] When 40 < Ct ≤ 45 in the FAM fluorescence channel, repeat the experiment once. If Ct is still within this range or less than 40, the sample is judged to be positive for the HBV integration site chrX:111,009,033, otherwise the sample is judged to be negative for the HBV integration site chrX:111,009,033;

[0037] When there is no amplification curve in the FAM fluorescence channel and Ct ≤ 45 in the HEX fluorescence channel, the sample is judged to be negative for the HBV integration site chrX:111,009,033;

[0038] When there is no amplification curve in the FAM fluorescence channel and there is no amplification curve in the HEX channel, it is judged that the experiment is abnormal and the sample RNA needs to be re-extracted and re-amplified.

[0039] In summary, the advantages and positive effects of the present invention are:

[0040] 1. The present invention provides a real-time fluorescent PCR detection primer pair, probe, kit and detection method for HBV integration site chrX:111,009,033, which can quickly, accurately and sensitively detect HBV integration site chrX:111,009,033, with strong specificity and high sensitivity, up to 5 copies / mL. The quantitative linear range of quantitative analysis is good; the experimental results have good repeatability and high precision.

[0041] 2. After a large number of innovative experiments, the present application has obtained the primer pairs, probes, kits and detection methods of the present application, which solves the problem of detecting the HBV integration site chrX:111,009,033 with a pair of primer pairs and a probe, and making it highly specific and sensitive, which is also the biggest technical difficulty of the present application. Ordinary HBV quantitative detection has no requirements for the detection part, and it can be selected from the full length of 3200bp, and it is easy to find a pair of primers that completely match; however, the HBV integration site chrX:111,009,033 of the present application belongs to cccDNA, and technicians in this field usually design primers through a section of notch to detect cccDNA. However, the method has too few options and it is difficult to design primers with strong specificity. The inventors of the present application creatively designed primers from both sides of a fixed chimeric point, and used in combination with the probe with strong specificity and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is the mechanism of HBV integration;

[0043] Figure 2 It is a strategy of inserting the full-length sequence of the viral fragment at the integration site chrX:111,009,033 using the improved InvPCR technology;

[0044] Figure 3 The PCR results are: A. The integration site chrX:111,009,033 inserts the full-length virus fragment; B. The detection result of the right binding site of the virus-host chimera; C. The detection result of the left binding site of the virus-host chimera; D. The plasmid containing the integration site chrX:111,009,033 inserts the full-length virus sequence;

[0045] Figure 4These are the results of Sanger sequencing: A. The right binding site of the virus-host chimera; B. The left binding site of the virus-host chimera; C. The full-length viral sequence is inserted into the integration site chrX:111,009,033.

[0046] Figure 5 It is a plasmid containing the HBV integration site chrX:111,009,033 with the full-length viral sequence inserted;

[0047] Figure 6 It is the InvPCR technology that detects HBV integration in host genes;

[0048] Figure 7 It is a schematic diagram of the TaqMan PCR reaction;

[0049] Figure 8 . is a schematic diagram of quantitative detection of integration site chrX:111,009,033 by chimeric primer fluorescence PCR. A. Artificially designed base sequence; B. Chimeric sequence containing PAK3 gene and HBX gene.

[0050] Fig. 9 This is the amplification curve of the HBV integration site chrX:111,009,033 standard;

[0051] Fig.10 is the standard concentration standard curve;

[0052] Fig.11 It is the virus real-time fluorescence quantitative PCR amplification curve of 3 HBV integration site chrX:111,009,033 positive specimens and 33 negative specimens;

[0053] Fig.12 This is the amplification curve of the internal reference gene β-globin. DETAILED DESCRIPTION

[0054] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0055] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0056] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0057] The present application will be described in detail below with reference to embodiments, comparative examples and experimental data.

[0058] Example 1 Development of Real-time Fluorescence Quantitative PCR Detection Kit

[0059] 1. HBV integration site chrX:111,009,033 quantitative detection standard (containing the plasmid with the full-length sequence of the virus inserted into the integration site chrX:111,009,033)

[0060] ① Using the known InvPCR technology ( Figure 6 ) Detection of HBV integration in HepG2.2.15 cells [Tu T, Jilbert AR. Detection of hepatocyte clones containing integrated hepatitis B virus DNA using inverse nested PCR [J]. Methods Mol Biol. 2017, 1540: 97-118.];

[0061] ② According to the detected integration site chrX:111,009,033 virus-host chimera right binding site sequence, chimeric primer R and Alu primer R1 and Alu primer F1 were designed (see Table 1). Using DNA extracted from HepG2.2.15 cells as substrate, chimeric primer R and Alu primer R1 and Alu primer F1 were used for PCR reaction: 95℃ preheating for 5min, 95℃ melting for 10s, 56℃ annealing for 10s, and 72℃ extension for 3min for a total of 40 cycles. The results showed that a 1255bp fragment was amplified between chimeric primer R and Alu primer R1;

[0062] Table 1

[0063]

[0064]

[0065] ③ According to the characteristics that the PCR amplification products of chimeric primer R (SEQ ID NO.8) and Alu primer R1 (SEQ ID NO.9) contain Alu restriction sites (AGCT) at both ends, the known InvPCR technology was improved, that is, Alu restriction endonuclease was used instead of NcoI restriction endonuclease. 10U of Alu restriction endonuclease was added to 2μg of PCR amplification products of chimeric primer R (SEQ ID NO.8) and Alu primer R1 (SEQ ID NO.9), incubated at 37℃ for 1h, and inactivated at 80℃ for 20m. 500U of T4 ligase was added, ligated at 25℃ for 2h, and inactivated at 70℃ for 20m. Then 5U of BsiHKAI and 5U of SphI-HF restriction endonuclease were added, incubated at 37℃ for 1h (SphI-HF is suitable for temperature) and then incubated at 65℃ for 1h (BsiHKAI is suitable for temperature), and 1μg of DNA was extracted as the InvPCR reaction template. The first round of PCR used improved InvPCR primers F1 (SEQ ID NO.13) and R1 (SEQ ID NO.14): 95℃ preheating for 5min, 95℃ melting for 15s, 54℃ annealing for 15s, and 72℃ extension for 3min for a total of 40 cycles. The reaction product was diluted 1000 times as a template for the second round of PCR reaction, and the improved InvPCR primers F2 (SEQ ID NO.15) and R2 (SEQ ID NO.16) were used as primer pairs for amplification. The reaction conditions were the same as the first round. Finally, a 900bp fragment was amplified. Sanger sequencing technology showed that the PCR amplification product of the chimeric primer R and Alu primer R1 was a specific amplified fragment containing the full-length sequence of the integration site chrX:111,009,033 virus insertion;

[0066] ④ To detect the remaining undetected sequence of the above-mentioned specific amplified fragment, primers IN F2 (SEQ ID NO.11) and Alu R2 (SEQ ID NO.12) were designed to perform PCR reaction on the above-mentioned specific amplified fragment (i.e., the 1255bp fragment amplified between the chimeric primer R and the Alu primer R1): 95°C preheating for 5min, 95°C melting for 10s, 56°C annealing for 10s, and 72°C extension for 14s for a total of 40 cycles. Finally, the integration site chrX:111,009,033 was detected to insert the full-length sequence of the virus.

[0067] ⑤ The 1255bp specific amplified fragment containing the full-length sequence of the virus inserted at the integration site chrX:111,009,033 obtained in step ② is loaded into the PMD-18T vector, and a plasmid containing the full-length sequence of the virus inserted at the integration site chrX:111,009,033 is produced by molecular cloning technology, and the plasmid is used as a quantitative detection standard.

[0068] 2. Primers and probe combinations for real-time fluorescence quantitative PCR detection of HBV integration site chrX:111,009,033

[0069] (1) In this example, a chimeric primer consisting of 28 bases (as shown in Table 2, 5'- TCGCTTTCGGGTCCCT TTTAATACCCAA-3'), the 5' end underlined part is a 16-base sequence designed artificially and has no homology with the virus-host gene sequence detected, and the 12 bases at the 3' end include 7 PAK3 gene sequences and 5 HBX gene sequences. First, use this chimeric primer to extend the extracted product for 15 cycles of single strand extension, PCR reaction: 95℃ preheating for 5min, 95℃ melting for 10s, 50℃ annealing for 20s, 72℃ extension for 1min for a total of 15 cycles. At the same time, β-globin is used as an internal standard, and the internal standard primers and probes are shown in Table 2.

[0070] Table 2

[0071]

[0072] (2) A real-time fluorescence quantitative PCR detection primer pair for the HBV integration site chrX:111,009,033 whose nucleotide sequence is shown in SEQ ID NO.1-2, and a nucleic acid probe whose nucleotide sequence is shown in SEQ ID NO.3.

[0073] (3) The kit also includes a positive control: HBV integration site chrX:111,009,033 standard; a negative control: RNase Free H 2 O; 5×PCR Buffer and Enzyme Mix. The preparation method of 5×PCR Buffer and Enzyme Mix is ​​as follows:

[0074] Preparation of 5×PCR Buffer:

[0075]

[0076] After mixing, store in a refrigerator at -20℃.

[0077] Enzyme Mix is ​​formulated with:

[0078]

[0079] After mixing, store in a refrigerator at -20℃.

[0080] 3. Real-time fluorescence PCR detection method for HBV integration site chrX:111,009,033

[0081] (1) Extraction of nucleic acids

[0082] ① First, add 25 ml and 30 ml of anhydrous ethanol to buffers 1 and 2 respectively; add 30 μg / ml carrier RNA to the rinse solution;

[0083] ②Put 30μl protease into a 1.5ml centrifuge tube;

[0084] ③ Take 200 μl of the sample and add it to this tube, add 5 μl of internal standard solution, and mix thoroughly;

[0085] ④ Add 200 μl of rinse solution (containing 30 μg / ml carrier RNA) to each tube, mix and oscillate for 30 seconds, and incubate at 70°C for 10 minutes;

[0086] ⑤ Add 250 μl of anhydrous ethanol, mix thoroughly and shake for 30 seconds, and lyse at room temperature for 5 minutes;

[0087] ⑥ Add the above lysate to the centrifuge column, centrifuge at 8000rpm for 1min, and discard the centrifuge in the collection tube; put the filter column back on the collection tube, suck all the remaining mixed solution in step ③ into the filter column, centrifuge and discard the centrifuge;

[0088] ⑦ Add 500 μl buffer 1 to the filter column, centrifuge at 12000 rpm for 1 min, and discard the centrifuge liquid in the collection tube;

[0089] ⑧ Take another clean 2ml collection tube, move the centrifuged filter column to a new collection tube, add 500μl buffer 2 to the filter column, centrifuge at 12000rpm for 1min. Repeat step ⑧ once;

[0090] ⑨ Move the filter column to a clean collection tube, centrifuge at 12000rpm for 3min, and then place the filter column at 37℃ for 15min to dry the filter membrane;

[0091] ⑩Put the filter column on a 1.5ml Eppendorf tube and add 50μl RNase-free H 2 O, let stand at room temperature for 2 min; centrifuge at 12 000 rpm for 2 min, and collect the centrifuge liquid as the extracted nucleic acid;

[0092] (2) Real-time fluorescence quantitative PCR amplification (20 μl system per portion)

[0093] ① Take 5 μl of the extracted nucleic acid as a template, add 1 μl of the chimeric primer shown in Table 2 for 15 cycles of single-strand extension to obtain a single-strand extension product. The PCR reaction procedure is as follows: preheat at 95°C for 5 min, melt at 95°C for 10 s, anneal at 50°C for 20 s, and extend at 72°C for 1 min for a total of 15 cycles.

[0094] ② Using the single-stranded extension product as a substrate, add the real-time fluorescence quantitative PCR detection primer pair for the HBV integration site chrX:111,009,033 with the nucleotide sequences shown in SEQ ID NO.1-2 and the nucleic acid probe with the nucleotide sequence shown in SEQ ID NO.3. At the same time, add 20 μl of PCR reaction solution (the preparation system of the real-time fluorescence quantitative PCR reaction solution is shown in Table 3) to an eight-strip tube for real-time fluorescence quantitative PCR amplification.

[0095] Table 3 Preparation system of the real-time fluorescence quantitative PCR reaction solution

[0096]

[0097] (3) The real-time fluorescence quantitative PCR reaction program is as follows: preheat at 95 °C for 5 min, denature at 95 °C for 10 s, anneal at 54 °C for 10 s, and extend at 72 °C for 10 s for a total of 40 cycles. The amplified fragment length is 140 bp.

[0098] (4) Obtain the real-time fluorescence quantitative PCR amplification result, analyze the amplification curve, and make a judgment. The judgment rules are as follows:

[0099] When Ct ≤ 40 in the FAM fluorescence channel, the sample is judged to be positive for the HBV integration site chrX:111,009,033;

[0100] When 40 < Ct ≤ 45 in the FAM fluorescence channel, repeat the experiment once. If Ct is still within this range or less than 40, the sample is judged to be positive for the HBV integration site chrX:111,009,033, otherwise the sample is judged to be negative for the HBV integration site chrX:111,009,033;

[0101] When there is no amplification curve in the FAM fluorescence channel and Ct ≤ 45 in the HEX fluorescence channel, the sample is judged to be negative for the HBV integration site chrX:111,009,033;

[0102] When there is no amplification curve in the FAM fluorescence channel and no amplification curve in the HEX fluorescence channel either, the current experiment is judged to be abnormal and the specimen RNA needs to be re-extracted and re-amplified.

[0103] 3. Experimental results

[0104] The amplification curve of the HBV integration site chrX:111,009,033 standard product is as Fig. 9 shown. The standard product is a plasmid containing the full-length inserted sequence of the HBV integration site chrX:111,009,033. The concentration of standard product 1 is 1×10 1 copies / mL; the concentration of standard product 2 is 1×10 2 copies / mL; the concentration of standard product 3 is 1×103 copies / mL; the concentration of standard 4 is 1×10 4 copies / mL; the concentration of standard 5 is 1×10 5 copies / mL; the concentration of standard 6 is 1×10 6 copies / mL; the concentration of standard 7 is 1×10 7 copies / mL;

[0105] Fig.10 It is the concentration standard curve of the HBV integration site chrX:111,009,033 standard, and the equation of the standard concentration curve is: y=-6.03x+41.18, wherein x is the log value of the concentration, and y is the Ct value.

[0106] Thirty-six specimens were tested using the method constructed above, and three cases were detected to be positive for the HBV integration site chrX:111,009,033; Fig.11 The real-time fluorescence quantitative PCR amplification curves of the virus of the 3 HBV integration site chrX:111,009,033 positive specimens and 33 negative specimens are shown in Table 4. The virus concentrations of the 3 HBV integration site chrX:111,009,033 positive specimens were automatically analyzed by Roche LightCycler 480 analysis software based on the Ct values ​​of the 3 positive results combined with the standard curve equation. The specific results are shown in Table 4. At the same time, the amplification curves of the internal reference gene β-globin of the 36 specimens were normal, as shown in Table 4. Fig.12 As shown, it shows that the extraction and amplification process of this experiment is normal, and the positive and negative results are accurate.

[0107] Table 4-3 Virus concentrations of samples positive for HBV integration site chrX:111,009,033

[0108]

[0109] Example 2: Performance measurement of real-time fluorescence PCR kit for HBV integration site chrX:111,009,033

[0110] 1. Accuracy verification

[0111] The gold standard for viral nucleic acid detection is genome sequencing. The detection results of this kit are compared with viral genome sequencing to analyze its accuracy. In this example, 4 specimens were selected that were determined to be HBV integration site chrX:111,009,033 by genome sequencing. The results after detection with the kit provided by the present invention are shown in Table 5 below. As can be seen from the results, all 4 positive specimens were detected, indicating that the accuracy of the real-time fluorescence PCR of HBV integration site chrX:111,009,033 provided by the present invention is 100%.

[0112] Table 5 Accuracy analysis of the present invention

[0113]

[0114] 2. Specificity verification

[0115] The specificity of the kit is evaluated by detecting other pathogens. This example selects common pathogen-positive specimens or plasmid simulation-positive specimens, and the results are shown in Table 6. After testing, this invention has no amplification for 22 respiratory and other common pathogen-positive specimens, indicating that the specificity of the real-time fluorescence PCR of the HBV integration site chrX:111,009,033 provided by the present invention is 100%.

[0116] Table 6 Specificity analysis of the present invention

[0117] Positive pathogens Is there an expansion Positive pathogens Is there an expansion Adenovirus No amplification Hepatitis B virus No amplification Influenza A virus No amplification Hepatitis C virus No amplification Influenza B virus No amplification Human immunodeficiency virus No amplification Hepatitis B virus No amplification Staphylococcus aureus No amplification Respiratory syncytial virus No amplification Escherichia coli No amplification Parainfluenza virus No amplification Mycoplasma pneumoniae No amplification Human herpes simplex virus No amplification Chlamydia pneumoniae No amplification Metapneumovirus No amplification Klebsiella pneumoniae No amplification Bocavirus No amplification Group A Streptococcus No amplification Rhinovirus No amplification Pseudomonas aeruginosa No amplification Cytomegalovirus No amplification Haemophilus influenzae No amplification

[0118] 3. Sensitivity detection

[0119] Sensitivity, also known as the minimum detection limit, refers to the probability of detecting the target nucleic acid in the same sample at the lowest dilution gradient after the positive sample is diluted in a gradient, and the probability is statistically >95%. The sample used for sensitivity assessment should be tested no less than 20 times at each concentration level to be evaluated, and at least 19 positive amplification signals are qualified. Here, we dilute the positive standard plasmid of the HBV integration site chrX:111,009,033 according to a certain copy number multiple, and each dilution is divided into 20 samples on average. The method of this invention is used for detection, and the copy number of 19 or more positive copies is the minimum detection limit. The results are shown in Table 7. It has been verified that at a concentration of 5 copies / ml, the detection rate is 95%, and at a concentration lower than this, the detection rate is less than 95%. It can be seen that the sensitivity of the real-time fluorescence PCR of the HBV integration site chrX:111,009,033 provided by the present invention is 5 copies / mL.

[0120] Table 7 Sensitivity analysis of the present invention

[0121]

[0122] 4. Accuracy test

[0123] Precision refers to the consistency of the results of multiple tests on the same positive sample. The coefficient of variation (CV) is less than 5, which is considered to be good precision. In this example, 4 positive samples were tested 3 times at each concentration gradient, and the results are shown in Table 8 below. 8 Within the copy / mL range, the intra-batch CV of this invention is <5%, and the inter-batch CV is <5%, with good precision.

[0124] Table 8 Accuracy analysis of the present invention

[0125]

[0126] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0127] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0128] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A real-time fluorescence quantitative PCR detection kit for HBV integration site chrX:111,009,033, characterized in that: The kit includes a chimeric primer with a nucleotide sequence as shown in SEQ ID NO.4, a real-time fluorescence quantitative PCR detection primer pair for HBV integration site chrX:111,009,033 with a nucleotide sequence as shown in SEQ ID NO.1-2, and a probe with a nucleotide sequence as shown in SEQ ID NO.

3.

2. The HBV integration site chrX:111,009,033 real-time fluorescence quantitative PCR detection kit according to claim 1, characterized in that: The kit also includes a primer pair with a nucleotide sequence as shown in SEQ ID NO.5-6 and a probe with a nucleotide sequence as shown in SEQ ID NO.

7.

3. The real-time fluorescence quantitative PCR detection kit for HBV integration site chrX:111,009,033 according to claim 2, characterized in that: The kit also includes: a positive standard for quantitative detection.

4. The HBV integration site chrX:111,009,033 real-time fluorescence quantitative PCR detection kit according to claim 3, characterized in that: The positive standard is a plasmid containing the HBV integration site chrX:111,009,033 amplified sequence in a concentration gradient.

5. The real-time fluorescence quantitative PCR detection kit for HBV integration site chrX:111,009,033 according to claim 4, characterized in that: The kit also includes ultrapure water or RNase Free H2O, 5×PCR Buffer and EnzymeMix.

6. The real-time fluorescence quantitative PCR detection kit for HBV integration site chrX:111,009,033 according to claim 5, characterized in that: When the kit is used, the reaction system includes a single-strand extension system and a real-time fluorescence quantitative PCR amplification system; The single-strand extension system includes the extracted nucleic acid and the chimeric primer as shown in SEQ ID NO.4, and the single-strand extension product is obtained by using the single-strand extension system; The real-time fluorescence quantitative PCR amplification system includes the single-stranded extension product, a real-time fluorescence quantitative PCR detection primer pair of HBV integration site chrX:111,009,033 with a nucleotide sequence as shown in SEQ ID NO.1-2, a probe with a nucleotide sequence as shown in SEQ ID NO.3, 5×PCR Buffer, Enzyme Mix, and ultrapure water or RNase Free H2O.

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