CN114200127B - Hepatitis B virus enrichment fluorescent PCR detection method - Google Patents
Hepatitis B virus enrichment fluorescent PCR detection method Download PDFInfo
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Abstract
The invention relates to a fluorescence PCR detection method for enriching hepatitis B virus, which comprises the following steps: s1, preparation of immunomagnetic beads: coupling the hepatitis B virus antibody to carboxyl modified super-cis nanometer magnetic beads to obtain immune magnetic beads coupled with the hepatitis B virus antibody, and enriching S2 and hepatitis B virus: mixing and incubating hepatitis B virus positive serum or plasma with immune magnetic beads coupled with hepatitis B virus antibodies; s3, separating the immunomagnetic bead-virus complex by using a magnetic tool, and re-suspending the immunomagnetic bead-virus complex in a salt ion buffer solution for heating and cracking; s4, separating the magnetic beads by using a magnetic tool to obtain enriched and concentrated hepatitis B virus, and directly detecting the liquid in the cracked product by using a fluorescent PCR reagent. The invention greatly improves the detection sensitivity due to enrichment and concentration, has strong specificity through two passes of immune recognition and gene recognition, and uses a rapid PCR reagent to realize short fluorescence PCR detection time.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a fluorescence PCR (polymerase chain reaction) detection method for enriching hepatitis B virus.
Background
Hepatitis b virus (Hepatitis B Virus, HBV) is a pathogen of viral hepatitis b (abbreviated as hepatitis b), and is transmitted through blood and body fluid, and causes infectious diseases in a chronic carrying state, mainly causing liver damage. HBV infection is one of the serious public health problems worldwide. HBV infection is a worldwide epidemic, and about 20 hundred million people worldwide are reported by WHO to be infected with HBV, of which 3.5 hundred million people are chronically HBV infected, and about 100 ten thousand people die each year due to liver failure, cirrhosis and primary hepatocellular carcinoma (hepatocellular rcinoma, HCC) caused by HBV infection. About 1 hundred million HBV carriers in China seriously harm the health of people in China and influence the life quality. In recent years, with the development of detection technology, there is also detection of HBV deoxyribonucleic acid (DNA) in addition to serum immunomarkers for hepatitis B virus. HBV-DNA detection is an important index for accurately judging replication of hepatitis B virus at present. The current HBV-DNA detection method is mainly a real-time fluorescence PCR method. The real-time fluorescence PCR detection technology can amplify a large amount of weak virus DNA, and realizes visual and quantifiable detection by detecting fluorescence accumulation through a detector, and has the characteristics of high detection sensitivity, strong specificity, difficult pollution caused by single-tube operation and the like.
Although the detection sensitivity of the fluorescent PCR method is higher than that of other detection means, a few researches indicate that the existing fluorescent PCR method still has the problem of insufficient sensitivity. After a part of samples are extracted with total nucleic acid (or virus nucleic acid) by using a magnetic bead method or a centrifugal column method, the detection result is in an 'gray area' between positive and negative, and when the detection result of the part of samples is negative, the part of samples cannot be used for excluding that the patient is not infected by influenza virus. The cause of such phenomena may be that the patient is in the early stage or recovery stage of infection, the virus content in the sample is too low, or substances inhibiting amplification detection remain in the clinical sample, etc.; for clinical demands requiring high sensitivity, such as blood screen projects, the existing methods are prone to missed detection. Thus, it is necessary to enrich and concentrate hepatitis B virus by using a suitable method.
The existing HBV-DNA fluorescence PCR detection method is used as an enzymatic reaction, and still has the defects of complicated sample treatment, high requirement on nucleic acid purity, long PCR detection time, high automation cost and the like, and in addition, the existing detection method in clinical examination such as blood sieve and the like can not meet the requirement on sensitivity; the above shortcomings result in the current HBV-DNA fluorescence PCR detection method being limited in application in clinical examination, and especially cannot meet the requirements of clinic and emergency.
Disclosure of Invention
The invention aims to solve the technical problems of complex sample processing, high requirement on nucleic acid purity, long PCR detection time, high automation cost and the like in the prior art.
Based on the above purpose, the invention adopts the following technical scheme:
a fluorescence PCR detection method for enriching hepatitis B virus, which comprises the following steps:
s1, preparation of immunomagnetic beads: coupling the hepatitis B virus antibody to carboxyl modified super-cis nanometer magnetic beads to obtain the immune magnetic beads coupled with the hepatitis B virus antibody,
s2, enrichment of hepatitis B virus: mixing and incubating hepatitis B virus positive serum or plasma with immune magnetic beads coupled with hepatitis B virus antibodies;
s3, separating the immunomagnetic bead-virus complex by using a magnetic tool, and re-suspending the immunomagnetic bead-virus complex in TE buffer solution for heating and cracking;
s4, separating the magnetic beads by using a magnetic tool to obtain enriched and concentrated hepatitis B virus, and directly detecting the liquid in the cracked product by using a fluorescent PCR reagent.
The hepatitis B virus antibody generally refers to a hepatitis B virus surface antibody or a core antibody, and can be a monoclonal antibody or a polyclonal antibody.
The fluorescent PCR detection reagent contains a PCR reaction buffer solution resisting inhibition and anti-interference and a DNA polymerase for rapid amplification, and can effectively amplify and fluorescence detect samples containing various interference substances.
Preferably, the hepatitis B virus antibody is a monoclonal hepatitis B virus surface antibody.
Preferably, the specific method for enriching the hepatitis b virus in the step S2 comprises the following steps: mixing 200-600 μl of serum or plasma with 2-5 μl of immunomagnetic beads, and mixing at room temperature (25-37deg.C) for 6-15 min.
Preferably, the thermal cracking in step S3 is: the virus-releasing nucleic acid is cleaved by heating at 85-95℃for 5-15 minutes.
Preferably, the fluorescent PCR reagent of step S4 comprises:
(1) Carrier RNA (nucleotide analogues) with a final concentration of 5-15 μg/mL;
(2) Tris (tris) at a final concentration of 0.10-0.15M.
In general, the Carrier RNA can not be used as a PCR additive, and the purpose of adding the Carrier RNA into a fluorescent PCR reagent is to reduce the influence of static electricity carried by the PCR tube wall on the reaction process;
in the fluorescent PCR reagent, the general concentration of Tris is 0.01-0.05M, and in the invention, 0.10-0.15 and M high concentration Tris is adopted, so as to improve the buffer capacity and reduce the influence of inhibitors in a sample (without nucleic acid purification) on the reaction process.
Preferably, the total reaction volume of the fluorescent PCR reagent is less than or equal to 40 mu L.
Preferably, the diameter of the super-clockwise nano magnetic beads is 200nm-350nm, and carboxyl (-COOH) groups are arranged on the surfaces of the super-clockwise nano magnetic beads.
Preferably, the preparation of the immunomagnetic beads specifically comprises the following steps: the EDC activation method is used to connect the anti-monoclonal hepatitis B virus surface antibody to the surface of the super-cis nanometer magnetic bead with carboxyl (-COOH) on the surface, and BSA is used to seal the magnetic bead coupled with the antibody.
Preferably, the air-drying preservation method of the immunomagnetic beads comprises the following steps: placing the immunomagnetic beads in an oven, adjusting the temperature to 30-37 ℃, opening ventilation, standing for more than 10 hours, taking out, and sealing and preserving at 2-8 ℃.
Compared with the existing HBV nucleic acid fluorescence PCR detection technology, the invention has the following beneficial effects:
1. the sample adding volume is not limited by operation equipment, the air-dried immunomagnetic beads are easy to store and transport, the superparamagnetic beads are easy to operate, and automation is easy to realize;
2. the sample is simple to process, the detection sensitivity is greatly improved due to enrichment and concentration, the specificity is strong through two ways of immune recognition and gene recognition, and the fluorescent PCR detection time is short by using a rapid PCR reagent.
Drawings
FIG. 1 is a schematic diagram of HBV nucleic acid detection flow of the present invention;
FIG. 2 is a graph showing amplification curves of purification-free fluorescent PCR detection (of the present invention) after HBV enrichment of a sample;
FIG. 3 is a graph of fluorescence PCR detection amplification after sample nucleic acid extraction;
FIG. 4 is a sample stock solution direct fluorescence PCR detection amplification graph;
FIG. 5 is a graph showing the fluorescence PCR detection amplification after thermal cleavage of a virus-immune enriched serum sample (supernatant).
Detailed Description
The technical scheme of the invention is further specifically described by the following specific examples. It should be understood that the practice of the invention is not limited to the following examples, but is intended to be within the scope of the invention in any form and/or modification thereof.
In the present invention, unless otherwise specified, all parts and percentages are by weight, and the equipment, materials, etc. used are commercially available or are conventional in the art. The methods in the following examples are conventional in the art unless otherwise specified.
The magnetic nano-microsphere is a magnetic microsphere with a modified surface and capable of being coupled with an antibody, the surface is provided with carboxyl groups, the size of the microsphere is generally nano-scale, the diameter is preferably 200-600 nm, and in the embodiment of the invention, the diameter of the magnetic microsphere is 300nm.
After the enrichment of the hepatitis B virus, the method can be used for quantitative or qualitative detection of virus nucleic acid by methods such as real-time fluorescence PCR, isothermal amplification and the like.
Example 1
A fluorescence PCR detection method for enrichment of hepatitis B virus, the process flow chart of which is shown in figure 1, comprises the following specific steps:
1. preparation of immunomagnetic beads
(1) Activation of
10mg (25 mg/mL) of carboxyl magnetic beads which are sufficiently shaken are placed in a 2 mL centrifuge tube, and 1mL of 15mM MES buffer (pH 6.0) is added to wash the magnetic beads for 3 times; after separation by a magnetic separator, 100. Mu.L of EDC solution (prepared by 15mM MES buffer (pH 6.0)) with the concentration of 10mg/ml was added, and the mixture was stirred uniformly by a vortex mixer, fixed on a mixer, and activated at room temperature for 30min; the beads were separated with a magnetic separator and the supernatant was discarded.
(2) Coupling of
400 μg of hepatitis B virus surface antibody was added, the beads were resuspended, and the tube was placed on a horizontal shaker and mixed at room temperature for 3 hours (2-4 hours). The beads were separated with a magnetic separator, the supernatant was discarded, and 1mL of PBST buffer was added for 3 washes, each wash requiring thorough mixing and washing on a mixer to remove unbound antibody.
(3) Closure
In the centrifuge tube containing the magnetic beads, 1mL of 15mM MES buffer (pH 6.0) containing 1% BSA was added, mixed well by vortex mixer, fixed on a mixer, closed at room temperature by 2 h, and the coupled magnetic beads were separated by magnetic separator.
(4) Air-drying preservation
Placing the immunomagnetic beads in an oven, adjusting the temperature to 30-37 ℃, opening ventilation, standing for more than 10 hours, taking out, and sealing and preserving at 2-8 ℃; the solubilization was performed by adding 500. Mu.L of 1 XTE buffer before use in the immunoenrichment.
2. HBV nucleic acid detection reagent (hands-free fluorescence PCR method)
(1) PCR reaction solutions were prepared according to Table 1:
TABLE 1
| Material | Final concentration | System amount (mu L) of 25 mu L |
| 5 Xq PCRmix (containing enzyme) | 1× | 5 |
| Carrier RNA（1μg/uL） | 10μg/mL | 0.25 |
| Tris（1M） | 0.12M | 3 |
| MgCl 2 （1M） | 2mM | 0.05 |
| HBV upstream primer | 0.2μM | 0.05 |
| HBV downstream primer | 0.2μM | 0.05 |
| HBV fluorescent probe | 0.15μM | 0.375 |
| Internal standard upstream primer | 0.1μM | 0.025 |
| Internal standard downstream primer | 0.1μM | 0.025 |
| Internal standard fluorescent probe | 0.05μM | 0.0125 |
| H 2 O | 11.17 |
(2) Split charging PCR reaction liquid
The PCR reaction solution was dispensed into PCR tubes at 20. Mu.L/reaction.
3. Sample processing
4 clinical samples were taken, and each sample was treated in parallel with the following three groups:
(1) Treatment group-immune enrichment group
Adding 10 mu L of immunomagnetic beads into a centrifuge tube, taking 200 mu L of sample, and blowing the gun head for 3 times;
incubating for 15 minutes at room temperature;
centrifugation, magnetic separation using a magnetic rack, discarding the supernatant, and adding 25. Mu.L TE buffer (10 mM Tris-HCl 1mM EDTA pH=8.0) to the remaining beads;
heated at 90℃for 5 minutes. Magnetic separation is carried out by using a magnetic frame;
adding 5 mu L of supernatant into a PCR tube, and preparing for on-machine detection;
(2) Control group 1-extraction test group
Adding 200 mu L of sample into a nucleic acid extraction or purification reagent, extracting nucleic acid, adding 5 mu L of extracted nucleic acid into a PCR tube, and preparing for on-machine detection;
(3) Control group 2-stock solution detection group
After the samples are uniformly mixed, directly taking 5 mu L of the samples, adding the samples into a PCR tube, and preparing for on-machine detection;
supernatant detection
Taking the supernatant obtained after the magnetic separation of the immune enrichment group (1) as a sample, heating at 90 ℃ for 5 minutes, taking 5 mu L of the supernatant, adding the supernatant into a PCR tube, and preparing for on-line detection.
4. On-machine detection
Placing the sample-added PCR tube into a fluorescent PCR instrument, performing fluorescent PCR amplification, and detecting the sample by using a detection program: the temperature is 52 ℃ for 2min; the temperature is 95 ℃ for 1min; (95 ℃ C. For 5s;58 ℃ C. For 30s and fluorescence collected) 45 cycles. After the equipment detection program is run, observing an amplification curve graph, and reading a Ct value;
the results (fluorescence curve Ct values) of the treatment groups are shown in Table 2 and FIGS. 2 to 5. FIG. 2 is a graph showing amplification curves of purification-free fluorescent PCR detection (of the present invention) after HBV enrichment of a sample; FIG. 3 is a graph of fluorescence PCR detection amplification after sample nucleic acid extraction; FIG. 4 is a sample stock solution direct fluorescence PCR detection amplification graph; FIG. 5 is a graph showing the fluorescence PCR detection amplification after thermal cleavage of a virus-immune enriched serum sample (supernatant).
TABLE 2
| Sample concentration (IU/mL) | Immune enriched group | Extraction detection | Direct amplification | Supernatant fluid |
| About 2E+4 | 27.38 | 27.87 | 29.63 | 33.63 |
| About 2E+3 | 30.04 | 30.11 | 33.36 | 36.47 |
| About 2E+2 | 33.08 | 33.71 | 37.96 | 40.55 |
| About 50 | 34.66 | 35.43 | 39.61 | no Ct |
Analysis of Table 2 and FIGS. 2-5 shows that the detection after immune enrichment treatment (invention) is basically consistent with the detection efficiency (Ct value is smaller and efficiency is higher) of the conventional detection control group after extraction, and the detection result of the invention is better at low concentration; the detection result of the invention is obviously superior to that of a direct amplification control group. And comparison with the supernatant detection result shows that the immunity enrichment has better virus capturing capability.
In this specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, so that the same or similar parts between the embodiments are referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant points refer to the description of the method section.
The invention provides a method for detecting the hepatitis B virus enriched fluorescence PCR in detail. The principles and embodiments of the present invention have been described herein with reference to specific examples, the description of which is intended only to facilitate an understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims.
Claims (6)
1. A hepatitis b virus enriched fluorescent PCR detection reagent, characterized in that the reagent comprises:
an immunomagnetic bead coupled with antibody of hepatitis B virus,
TE buffer
A fluorescent PCR reagent comprising: (1) Carrier RNA at a final concentration of 5-15 μg/mL; (2) tris at a final concentration of 0.10-0.15M; the total reaction volume of the fluorescent PCR reagent is less than or equal to 40 mu L;
the preparation of the immunomagnetic beads coupled with the hepatitis B virus antibody comprises the following steps: coupling the hepatitis B virus antibody to carboxyl modified super-cis nanometer magnetic beads to obtain immune magnetic beads coupled with the hepatitis B virus antibody;
the detection method of the reagent comprises the following steps:
s1, enrichment of hepatitis B virus: mixing and incubating hepatitis B virus positive serum or plasma with immune magnetic beads coupled with hepatitis B virus antibodies;
the specific enrichment method of the hepatitis B virus comprises the following steps: mixing 200-600 μl of serum or plasma with 2-5 μl of immunomagnetic beads, and mixing at 25-37deg.C for 6-15 min;
s2, separating the immunomagnetic bead-virus complex by using a magnetic tool, and re-suspending the immunomagnetic bead-virus complex in TE buffer solution for heating and cracking; the thermal cracking is as follows: heating at 85-95deg.C for 5-15 min to lyse virus-releasing nucleic acid;
s3, separating the magnetic beads by using a magnetic tool to obtain enriched and concentrated hepatitis B virus, and directly detecting the liquid in the cracked product by using a fluorescent PCR reagent.
2. The hepatitis b virus enriched fluorescent PCR detection reagent of claim 1, wherein the hepatitis b virus antibody is a monoclonal hepatitis b virus surface antibody.
3. The hepatitis b virus enriched fluorescent PCR detection reagent as claimed in claim 1, wherein the diameter of the super-clockwise nano magnetic beads is 200nm-350nm, and the surface of the super-clockwise nano magnetic beads has carboxyl (-COOH) groups.
4. The hepatitis b virus enriched fluorescent PCR detection reagent of claim 1, wherein the immunomagnetic bead preparation is specifically: the EDC activation method is used to connect the anti-monoclonal hepatitis B virus surface antibody to the surface of the super-cis nanometer magnetic bead with carboxyl (-COOH) on the surface, and BSA is used to seal the magnetic bead coupled with the antibody.
5. The hepatitis b virus enriched fluorescent PCR detection reagent as claimed in claim 1, wherein the air-drying preservation method of the immunomagnetic beads is as follows: placing the immunomagnetic beads in an oven, adjusting the temperature to 30-37 ℃, opening ventilation, standing for more than 10 hours, taking out, and sealing and preserving at 2-8 ℃.
6. The hepatitis b virus enriched fluorescent PCR detection reagent of claim 1, wherein the fluorescent PCR reagent further comprises: 1 Xq PCRmix,2mM MgCl 2 And 0.15. Mu.M HBV fluorescent probe, 0.2. Mu.M HBV upstream and downstream primer each, 0.05. Mu.M internal standard fluorescent probe, 0.1. Mu.M internal standard upstream and downstream primer each, and deionized water.
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| WO2017181339A1 (en) * | 2016-04-19 | 2017-10-26 | 廖世奇 | Method and kit for simultaneous detection of protein ligand and gene |
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