CN111057791A - Kit for detecting HBV pgRNA in blood - Google Patents
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- C12Q1/6851—Quantitative amplification
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Abstract
The invention discloses a kit for detecting HBV pgRNA in blood, belonging to the technical field of molecular biology, wherein the kit comprises an amplification reagent, the amplification reagent comprises a PCR reaction solution I, and the PCR reaction solution I comprises primers shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 and probes shown in SEQ ID NO. 4 and SEQ ID NO. 5. The kit provided by the invention utilizes the characteristic that RNA single strand needs reverse transcription, adds a section of specific non-HBV sequence at the 5' end of a reverse transcription primer, and simultaneously uses oligonucleotide for synthesizing the section of sequence as a primer for cDNA amplification, thereby achieving the purpose of specifically amplifying pgRNA, being free from DNA interference in the detection process, effectively improving the sensitivity of the kit in detection, and achieving the lower limit of detection of the pgRNA of hepatitis B virus up to 10 copies/ml.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a kit for detecting HBV pgRNA in blood.
Background
Hepatitis B Virus (HBV) infection is one of the most common causes of liver diseases such as cirrhosis and primary liver cancer. More than 20 hundred million people are infected with hepatitis B virus worldwide, about 2.4 hundred million chronic hepatitis B patients exist, about 9300 million chronic hepatitis B virus carriers exist in China, and about 2000-3000 million patients develop chronic hepatitis B.
Hepatitis B virus belongs to hepadnavirus, after the virus invades human body, it is combined with sodium ion-taurocholic acid cotransporter receptor on the liver cell membrane through preS1 area, after removing the envelope, it enters into the liver cytoplasm, releases loose ring DNA (rcDNA) to enter into the liver cell nucleus, the rcDNA is converted into covalent closed circular DNA (cccDNA) in the liver cell nucleus, and then 4 mRNAs are transcribed by taking cccDNA as template, wherein the length of 3.5Kb of pregenomic RNA (pgRNA) contains all the genetic information on HBV DNA, and pgRNA can be reverse transcribed in cytoplasm to form rcDNA and virus protein, finally assembled into complete HBV, released to the outside of liver cell, and a part of rcDNA enters into the cell nucleus, and then converted into cccDNA, forming the process of hepatitis B virus continuous replication.
The cccDNA can exist in a free form in a liver cell nucleus stably for a long time, so the level of the cccDNA has great correlation with hepatitis B relapse, the detection of the cccDNA needs to puncture and sample the liver of a patient, and invasive examination causes certain damage to the patient. The research shows that pgRNA exists in the serum of a chronic hepatitis B patient, the pgRNA is derived from a non-integrated complete HBV genome and enters blood in the form of encapsidated HBV pgRNA virus particles, the pgRNA can reflect the transcription activity state of cccDNA in the liver, and the serum pgRNA is positively correlated with the level of the cccDNA in the liver in an HBeAg positive HBV infected person, so the pgRNA can be used as a sensitive serum marker for HBV virus replication and prognosis, the HBV pgRNA continuous negative and low HBsAg level can be judged as 'quasi-clinical cure' of chronic hepatitis B, and the method has important significance in guiding the safe drug discontinuation of hepatitis B treatment. However, the current market has few technologies and kits for detecting pgRNA, and the detection of pgRNA is susceptible to interference from DNA, and the sensitivity of the existing products cannot meet the actual demand. Although the chinese patent application No. 201611099305.8 discloses a composition, its application and a kit containing the composition, which can quantitatively detect HBV pgRNA in blood with a sensitivity as low as 5 copies, the detection is based on digital PCR technology, but the kit based on fluorescence quantitative PCR detection technology cannot achieve the above sensitivity.
Disclosure of Invention
In order to overcome the defects of the prior art, the technical problems to be solved by the invention are as follows: provides a high-sensitivity HBV pgRNA detection kit by utilizing a fluorescent quantitative PCR detection technology.
In order to solve the technical problems, the invention adopts the technical scheme that: a kit for detecting HBV pgRNA in blood comprises an amplification reagent, wherein the amplification reagent comprises a PCR reaction solution I, and the PCR reaction solution I comprises primers shown by SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 and probes shown by SEQ ID NO. 4 and SEQ ID NO. 5 (see Table 1).
TABLE 1
The invention has the beneficial effects that: the kit provided by the invention utilizes the characteristic that RNA single strand needs reverse transcription, adds a section of specific non-HBV sequence at the 5' end of a reverse transcription primer, and simultaneously uses oligonucleotide for synthesizing the section of sequence as a primer for cDNA amplification to achieve the purpose of specifically amplifying pgRNA, so that the kit is free from DNA interference in the detection process, the sensitivity of the kit in detection is effectively improved, and the lower limit (the lowest detection limit) of the detection on the pgRNA of hepatitis B virus can reach 10 copies/ml.
Drawings
FIG. 1 is a graph showing the results of the sensitivity test of the kit for detecting HBV pgRNA in blood according to the embodiment of the present invention;
FIG. 2 is a diagram showing the results of the specific detection of the kit for detecting HBV pgRNA in blood according to the embodiment of the present invention.
Detailed Description
In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.
The most key concept of the invention is as follows: adding a specific non-HBV sequence at the 5' end of the reverse transcription primer, and using the oligonucleotide for synthesizing the sequence as a primer for cDNA amplification.
Referring to FIGS. 1 to 2, the kit for detecting HBV pgRNA in blood of the present invention comprises an amplification reagent, wherein the amplification reagent comprises a PCR reaction solution I, and the PCR reaction solution I comprises primers shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and probes shown in SEQ ID NO. 4 and SEQ ID NO. 5.
From the above description, the beneficial effects of the present invention are: the kit provided by the invention utilizes the characteristic that RNA single strand needs reverse transcription, adds a section of specific non-HBV sequence at the 5' end of a reverse transcription primer, and simultaneously uses oligonucleotide for synthesizing the section of sequence as a primer for cDNA amplification to achieve the purpose of specifically amplifying pgRNA, so that the kit is free from DNA interference in the detection process, the sensitivity of the kit in detection is effectively improved, and the lower limit (the lowest detection limit) of the detection on the pgRNA of hepatitis B virus can reach 10 copies/ml.
Further, the PCR reaction solution I also comprises a PCR buffer solution, dATP, dUTP, dGTP and dCTP.
Further, the amplification reagent further comprises an internal standard, and the internal standard is a cloning plasmid.
Further, the amplification reagent also comprises a PCR reaction liquid II, and the PCR reaction liquid II comprises Taq enzyme, UDG enzyme, MMLV reverse transcriptase and RNase inhibitor.
Further, the amplification reagent further comprises a positive quality control product, a negative quality control product and a quantitative reference product, wherein the positive quality control product is an HBV positive serum sample, the negative quality control product is an HBV negative serum sample, and the quantitative reference product is a constant value HBV positive serum sample.
Further, the kit further comprises a pgRNA extraction reagent, wherein the pgRNA extraction reagent comprises lysis solution, inhibitor removing solution, washing solution A, washing solution B, eluent and DNA digestion reaction solution.
As can be seen from the above description, by providing the pgRNA extraction reagent, the rapid detection of HBV pgRNA in blood using the same kit is facilitated.
Further, the lysis solution is guanidine thiocyanate, the inhibitor removing solution is proteinase K, the washing solution A is composed of guanidine thiocyanate and absolute ethyl alcohol, the washing solution B is composed of sodium chloride and absolute ethyl alcohol, the eluent is sterilized purified water, and the DNA digestion reaction solution is DNase I.
Example 1:
a kit for detecting HBV pgRNA in blood comprises components shown in Table 2, wherein sequences of a primer and a probe are shown as SEQ ID NO 1-SEQ ID NO 5;
TABLE 2
Wherein dATP, dUTP, dGTP and dCTP in the PCR reaction solution 1 are 0.5-2mM and Mg2+2-4mM, 200-500nM primer, 100-300nM probe.
The gene sequence of the cloned plasmid in the internal standard is as follows: taggaggctgtaggcataaagttcgtagtggtgtcacacgtctgtctcgcacgctagctcctcaggtgtcgtagtgtggacacatacgggctaaa cag are provided.
The quantitative reference 1, the quantitative reference 2, the quantitative reference 3 and the quantitative reference 4 are respectively 1 × 104copies/ml、1×105copies/ml、1×106copies/ml、1×107Positive serum samples of copies/ml.
The positive quality control product 1 and the positive quality control product 2 are respectively 1 multiplied by 103-5×104copies/ml and 1X 106-5×107Positive serum samples of copies/ml.
Example 2:
the kit of example 1 is used for detecting HBV pgRNA in blood, and the specific steps are as follows:
1. viral nucleic acid extraction
1) And (4) preparing related reagents.
2) N centrifugal tubes of 2.0ml were sampled and numbered (n ═ sample number + positive quality control product × 2+ negative quality control product × 1+ quantitative reference product × 4), and 10 μ l inhibitor removing solution and 400 μ l sample were added, and 600 μ l lysis solution + internal standard plasmid mixed solution (lysis solution: internal standard plasmid 600: 1, the mixture is used at present), uniformly mixed by shaking, instantaneously centrifuged, placed in a metal bath at 70 ℃ for 10min, instantaneously centrifuged and cooled to room temperature;
3) respectively adding 500 μ l ethanol, mixing, centrifuging instantly, adding 10 μ l magnetic bead suspension, mixing, standing at room temperature for 10min, mixing again every 1-2min, and keeping the magnetic bead mixing state;
4) placing the centrifugal tube on a magnetic frame, and absorbing and discarding waste liquid after the magnetic beads are completely adsorbed to the tube wall; taking down the centrifuge tube, respectively adding 1ml of washing solution A, and reversing for several times to uniformly disperse the magnetic beads;
6) placing the centrifugal tube on the magnetic frame again, and absorbing and discarding waste liquid after the magnetic beads are completely adsorbed to the tube wall; taking down the centrifuge tube, respectively adding 1ml of washing solution B, and reversing for several times to uniformly disperse the magnetic beads;
7) placing the centrifuge tube on a magnetic frame, completely adsorbing the magnetic beads to the tube wall, completely absorbing the waste liquid, and repeating the steps 6) and 7);
8) opening the cover of the centrifugal tube, placing the centrifugal tube in a metal bath at 50 ℃ for 30 s-2 min, adding 20 mu l of eluent when the surfaces of the magnetic beads have no water traces, flicking the tube wall to disperse the magnetic beads, placing the centrifugal tube in the metal bath at 50 ℃ for 5min again, and flicking the tube wall again to mix uniformly if the magnetic beads settle to the bottom of the tube;
9) placing the centrifugal tube on a magnetic frame, and taking the supernatant to obtain the HBV nucleic acid after the magnetic beads are completely adsorbed to the tube wall.
2. Preparation of PCR reaction system and sample adding
Taking the PCR reaction solution 1 and the PCR reaction solution 2, preparing a PCR reaction system according to the sample example according to the following system:
PCR reaction solution I7.5. mu.l × n; 1.5. mu.l Xn of PCR reaction solution II;
after fully mixing, 10 mul of each tube is subpackaged into a PCR tube, 15 mul of HBV nucleic acid sample is added into each tube, the final system is 25 mul/tube, mixing evenly and carrying out instantaneous centrifugation.
3. PCR amplification detection
The PCR reaction tube was placed in a fluorescent PCR machine and the program settings are shown in Table 3.
TABLE 3
Wherein the fluorescent signal is collected in stage 3: the fluorescence signal collected was probe-labeled fluorescence (FAM/VIC) at 60 ℃ for 40sec (. multidot.label).
4. Analysis of results
The software automatically quantifies by appropriately adjusting the Start and Stop values and the threshold (to flatten the curve of the negative quality control or below the threshold line) of the baseline based on the image automatically analyzed by the instrument.
5. Quality control
1) Internal standard: the Ct value of 33< of the VIC channel is less than or equal to 35, and an amplification curve is S-shaped;
2) negative quality control product: FAM channel has no Ct value;
3) positive quality control 1: the quantitative value is between 1 × 103-5×104copies/ml；
4) Positive quality control 2: the quantitative value is between 1 × 106-5×107copies/ml；
5) And (3) quantitative reference products: all positive, the linear correlation coefficient is between 0.98 and 1;
the above parameters must all be satisfied, otherwise, the test should be retested.
Example 3:
the kit of example 1 was tested for sensitivity and specificity.
1) Sensitivity detection
HBV quantitative national standard substance is diluted to 1E5, 1E4, 1E3, 1E2, 50, 20, 10copies/ml and below by negative serum, HBV pgRNA is quantitatively detected by using the kit, and the detection result is shown in figure 1.
As can be seen, the lowest detection limit of the kit can reach 10copies/ml, and the result of repeated experiments shows that the detection rate of the virus sample with the concentration of 10copies/ml is 95%.
2) Specificity detection
Clinical HBV negative samples are taken and detected by the kit, the negative samples comprise nucleic acid samples of common pathogens such as hepatitis A virus, hepatitis C virus, herpes simplex virus 1/2, herpes zoster virus, cytomegalovirus, yellow fever virus and the like, the detection results are negative, and the coincidence rate is 100%. The results of the detection are shown in FIG. 2.
As can be seen, the kit has good specificity.
In conclusion, the kit provided by the invention utilizes the characteristic that RNA single strand needs reverse transcription, adds a section of specific non-HBV sequence at the 5' end of a reverse transcription primer, and simultaneously uses oligonucleotide for synthesizing the section of sequence as a primer for cDNA amplification to achieve the purpose of specifically amplifying pgRNA, so that the kit is free from DNA interference in the detection process, the sensitivity of the kit in detection is effectively improved, and the lower limit (the lowest detection limit) of the detection on the pgRNA can reach 10 copies/ml.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.
Sequence listing
<110> Ashian (Fuzhou) Gene medicine inspection laboratory Co., Ltd
<120> kit for detecting HBV pgRNA in blood
<160>5
<170>SIPOSequenceListing 1.0
<210>1
<211>19
<212>DNA
<213> Artificial sequence
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taggaggctg taggcataa 19
<210>2
<211>14
<212>DNA
<213> Artificial sequence
<400>2
aagccaccca aggc 14
<210>3
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<212>DNA
<213> Artificial sequence
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ctgtttagcc cgtatgtgtc c 21
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<213> Artificial sequence
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ctttttcacc tctgcctaat ca 22
<210>5
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<212>DNA
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ctgtctcgca cgctagctcc tca 23
Claims (7)
1. A kit for detecting HBV pgRNA in blood is characterized by comprising an amplification reagent, wherein the amplification reagent comprises a PCR reaction solution I, and the PCR reaction solution I comprises primers shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 and probes shown in SEQ ID NO. 4 and SEQ ID NO. 5.
2. The kit for detecting HBV pgRNA in blood according to claim 1, wherein the PCR reaction solution I further comprises PCR buffer, dATP, dUTP, dGTP and dCTP.
3. The kit for detecting HBV pgRNA in blood according to claim 1, wherein the amplification reagent further comprises an internal standard which is a cloning plasmid.
4. The kit for detecting HBV pgRNA in blood according to claim 1, wherein the amplification reagent further comprises PCR reaction solution II, and the PCR reaction solution II comprises Taq enzyme, UDG enzyme, MMLV reverse transcriptase and RNase inhibitor.
5. The kit for detecting HBV pgRNA in blood according to claim 1, wherein the amplification reagent further comprises a positive quality control product, a negative quality control product and a quantitative reference product, the positive quality control product is an HBV positive serum sample, the negative quality control product is an HBV negative serum sample, and the quantitative reference product is a constant value HBV positive serum sample.
6. The kit for detecting HBV pgRNA in blood according to claim 1, further comprising a pgRNA extraction reagent, wherein the pgRNA extraction reagent comprises a lysis solution, an inhibitor removal solution, a washing solution A, a washing solution B, an eluent and a DNA digestion reaction solution.
7. The kit for detecting HBV pgRNA in blood according to claim 6, wherein the lysis solution is guanidine thiocyanate, the inhibitor removing solution is proteinase K, the washing solution A is composed of guanidine thiocyanate and absolute ethyl alcohol, the washing solution B is composed of sodium chloride and absolute ethyl alcohol, the eluent is sterilized purified water, and the DNA digestion reaction solution is DNase I.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113755642A (en) * | 2021-09-10 | 2021-12-07 | 湖南大学 | High-discrimination detection kit and detection method for HBV pgRNA in trace sample |
| CN115097129A (en) * | 2022-08-24 | 2022-09-23 | 山东子峰生物技术有限公司 | Detection reagent composition for placenta growth factor and soluble fms-like tyrosine kinase-1 |
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2019
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113755642A (en) * | 2021-09-10 | 2021-12-07 | 湖南大学 | High-discrimination detection kit and detection method for HBV pgRNA in trace sample |
| CN113755642B (en) * | 2021-09-10 | 2024-03-29 | 湖南大学 | High-discrimination detection kit and detection method for HBV pgRNA in trace sample |
| CN115097129A (en) * | 2022-08-24 | 2022-09-23 | 山东子峰生物技术有限公司 | Detection reagent composition for placenta growth factor and soluble fms-like tyrosine kinase-1 |
| CN115097129B (en) * | 2022-08-24 | 2023-03-10 | 山东子峰生物技术有限公司 | Detection reagent combination for placenta growth factor and soluble fms-like tyrosine kinase-1 |
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