CN114200127A - Hepatitis B virus enrichment fluorescence PCR detection method - Google Patents

Hepatitis B virus enrichment fluorescence PCR detection method Download PDF

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CN114200127A
CN114200127A CN202111349937.6A CN202111349937A CN114200127A CN 114200127 A CN114200127 A CN 114200127A CN 202111349937 A CN202111349937 A CN 202111349937A CN 114200127 A CN114200127 A CN 114200127A
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CN114200127B (en
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陈文�
孙艳
刘松林
胡丹枫
朱小娟
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Hangzhou Danwei Biotechnology Co ltd
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Abstract

The invention relates to a hepatitis B virus enrichment fluorescence PCR detection method, which comprises the following steps: s1, preparation of immunomagnetic beads: coupling the hepatitis B virus antibody to the carboxyl modified superparamagnetic nano-bead to obtain the immunomagnetic bead coupled with the hepatitis B virus antibody, and S2, enriching the hepatitis B virus: mixing and incubating hepatitis B virus positive serum or plasma with immunomagnetic beads coupled with hepatitis B virus antibodies; s3, separating the immunomagnetic bead-virus compound by using a magnetic tool, and resuspending the immunomagnetic bead-virus compound in a salt ion buffer solution for heating and cracking; s4, separating the magnetic beads by a magnetic tool to obtain enriched and concentrated hepatitis B virus, and directly detecting the liquid in the cleavage product by a fluorescent PCR reagent. The invention greatly improves the detection sensitivity due to enrichment and concentration, has strong specificity through two measures of immune recognition and gene recognition, and uses a rapid PCR reagent to shorten the fluorescence PCR detection time.

Description

Hepatitis B virus enrichment fluorescence PCR detection method
Technical Field
The invention relates to the field of molecular biology, in particular to a hepatitis B virus enrichment fluorescence PCR detection method.
Background
Hepatitis B Virus (HBV) is a pathogen of Hepatitis B, and is transmitted through blood and body fluid, and has an infectious disease in a chronic carrier state, mainly causing liver damage. Infection with HBV is one of the serious public health problems worldwide. HBV infection is worldwide prevalent, and it is reported by WHO that about 20 million people worldwide are infected with HBV, of which about 3.5 million people with chronic HBV infection die of liver failure, cirrhosis and primary hepatocellular carcinoma (HCC) due to HBV infection every year by about 100 million people. About 1 hundred million HBV carriers exist in China, which seriously harm the health of Chinese people and influence the quality of life. In recent years, with the development of detection technology, detection of hepatitis B virus is accompanied by detection of HBV deoxyribonucleic acid (DNA) in addition to serum immune marker of hepatitis B. The detection of HBV-DNA is an important index for accurately judging the 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, realizes visual and quantifiable detection by detecting fluorescence accumulation through a detector, and has the characteristics of high detection sensitivity, strong specificity, difficult pollution of single-tube operation and the like.
Although the detection sensitivity of the fluorescence PCR method is higher than that of other detection means, a lot of researches indicate that the current fluorescence PCR method still has the problem of insufficient sensitivity. After a part of samples are extracted by a magnetic bead method or a centrifugal column method to obtain total nucleic acid (or virus nucleic acid) of the samples, the detection result is in a gray area between positive and negative, and when the detection result of the part of samples is negative, the part of samples cannot exclude that a patient is not infected with influenza virus. The reasons for such phenomena may be that the patient is in the initial stage of infection or recovery stage, resulting in low virus content in the sample, or residual substances inhibiting amplification detection in clinical samples, etc.; for clinical requirements requiring high sensitivity, such as blood screening projects and the like, the existing method is easy to cause missed detection. Therefore, the hepatitis B virus needs to be enriched and concentrated by using a proper method.
The existing HBV-DNA fluorescent PCR detection method, as an enzymatic reaction, 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 the existing detection method can not meet the requirement on sensitivity in clinical tests such as blood screening and the like; the above disadvantages cause the application of the current HBV-DNA fluorescent PCR detection method in clinical examination to be limited, and especially cannot meet the requirements of outpatient and emergency treatment.
Disclosure of Invention
The invention aims to solve the technical problems of overcoming the defects in the prior art, providing a hepatitis B virus DNA detection method, and solving the defects of complex sample treatment, high requirement on nucleic acid purity, long PCR detection time, high automation cost and the like in the prior detection technology.
Based on the purpose, the invention adopts the following technical scheme:
an enrichment fluorescence PCR detection method for hepatitis B virus, which comprises the following steps:
s1, preparation of immunomagnetic beads: coupling the hepatitis B virus antibody to the carboxyl modified superparamagnetic nano-bead to obtain the immunomagnetic bead coupled with the hepatitis B virus antibody,
s2, enrichment of hepatitis B virus: mixing and incubating hepatitis B virus positive serum or plasma with immunomagnetic beads coupled with hepatitis B virus antibodies;
s3, separating the immunomagnetic bead-virus complex by using a magnetic tool, and resuspending the immunomagnetic bead-virus complex in TE buffer solution for heating and cracking;
s4, separating the magnetic beads by a magnetic tool to obtain enriched and concentrated hepatitis B virus, and directly detecting the liquid in the cleavage product by a fluorescent PCR reagent.
The hepatitis B virus antibody generally refers to hepatitis B virus surface antibody or core antibody, and can be monoclonal antibody or polyclonal antibody.
The fluorescent PCR detection reagent contains a PCR reaction buffer solution with inhibition resistance and interference resistance and DNA polymerase for rapid amplification, and can effectively amplify and perform fluorescent detection on 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 step S2 is: mixing 200 and 600 mu L of serum or plasma with 2-5 mu L of immunomagnetic beads, and uniformly mixing for 6-15 minutes at room temperature (25-37 ℃).
Preferably, the thermal cracking in step S3 is: heating at 85-95 deg.C for 5-15 min to crack virus and release nucleic acid.
Preferably, the fluorescent PCR reagent of step S4 includes:
(1) carrier RNA (nucleotide analogs) at a final concentration of 5-15. mu.g/mL;
(2) tris (tris) at a final concentration of 0.10-0.15M.
In general, 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 carried by the PCR tube wall on the reaction process;
in the fluorescent PCR reagent, the concentration of Tris is generally 0.01-0.05M, and in the invention, 0.10-0.15M high concentration of Tris is adopted, so as to improve the buffer capacity and reduce the influence of an inhibitor (not subjected to nucleic acid purification) in a sample 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 superparamagnetic nano bead is 200nm to 350nm, and the surface of the superparamagnetic nano bead is provided with carboxyl (-COOH) groups.
Preferably, the preparation of the immunomagnetic beads specifically comprises the following steps: connecting the anti-monoclonal hepatitis B virus surface antibody to the surface of a superparamagnetic nano magnetic bead with carboxyl (-COOH) on the surface by using an EDC activation method, and sealing the magnetic bead coupled with the antibody by using BSA.
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 ℃, starting ventilation, standing for more than 10 hours, taking out, and sealing and storing at 2-8 ℃.
Compared with the existing HBV nucleic acid fluorescence PCR detection technology, the invention has the beneficial effects that:
1. the sample adding volume is not limited by operation equipment, the air-dried immunomagnetic beads are easy to store and transport, the use of superparamagnetic beads is easy to operate, and automation is easy to realize;
2. the sample is easy to process, the detection sensitivity is greatly improved due to enrichment and concentration, the specificity is strong through two measures of immune recognition and gene recognition, and the fluorescence PCR detection time is short due to the use of a rapid PCR reagent.
Drawings
FIG. 1 is a schematic diagram of the HBV nucleic acid detection process of the present invention;
FIG. 2 is a graph showing the amplification curve of a sample after HBV enrichment in a fluorescence PCR assay without purification according to the present invention;
FIG. 3 is a graph showing the fluorescence PCR detection amplification curve after the extraction of the sample nucleic acid;
FIG. 4 is a graph of a direct fluorescence PCR detection amplification curve of a sample stock solution;
FIG. 5 is a fluorescent PCR detection amplification curve diagram of a serum sample (supernatant) after virus immune enrichment.
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
In the present invention, all parts and percentages are by weight, unless otherwise specified, and the equipment and materials used are commercially available or commonly used 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 which is modified on the surface and can be coupled with an antibody, the surface of the magnetic nano-microsphere is provided with carboxyl groups, the size of the magnetic nano-microsphere is generally nano-scale, the preferred diameter is 200 nm-600 nm, and in the embodiment of the invention, the diameter of the magnetic nano-microsphere is 300 nm.
After enriching 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 hepatitis B virus enrichment fluorescence PCR detection method is shown in figure 1, and comprises the following specific steps:
1. preparation of immunomagnetic beads
(1) Activation of
10mg (25 mg/mL) of fully shaken carboxyl magnetic beads are placed in a 2 mL centrifuge tube, and 1mL of 15mM MES buffer solution (pH6.0) is added to wash the magnetic beads for 3 times; separating with magnetic separator, adding 100 μ L EDC solution (prepared with 15mM MES buffer solution (pH6.0)) with concentration of 10mg/ml, mixing well with vortex mixer, fixing on mixer, and activating at room temperature for 30 min; the magnetic beads were separated by a magnetic separator and the supernatant was discarded.
(2) Coupling of
Add 400. mu.g of hepatitis B virus surface antibody, mix the resuspended beads, place the tube on a horizontal shaker, mix well for 3 hours (2-4 hours) at room temperature. Separating magnetic beads with magnetic separator, discarding supernatant, adding 1mL PBST buffer solution to wash for 3 times, and mixing and washing thoroughly on mixer to remove unbound antibody.
(3) Sealing of
1mL of 15mM MES buffer (pH6.0) containing 1% BSA was added to the above centrifuge tube containing magnetic beads, mixed by a vortex mixer, fixed on a mixer, blocked at room temperature for 2 hours, and coupled magnetic beads were separated by a magnetic separator.
(4) Air-drying and storing
Placing the immunomagnetic beads in an oven, adjusting the temperature to 30-37 ℃, starting ventilation, standing for more than 10 hours, taking out, and sealing and storing at 2-8 ℃; 500. mu.L of 1 XTE buffer was added for reconstitution before use for immuno-enrichment.
2. Preparing HBV nucleic acid detecting reagent (extraction-free fluorescent PCR method)
(1) PCR reaction solutions were prepared as in table 1:
TABLE 1
Material(s) Final concentration 25 μ L System dosage (μ L)
5 Xq PCRmix (containing enzyme) 1× 5
Carrier RNA（1μg/uL） 10μg/mL 0.25
Tris（1M） 0.12M 3
MgCl2（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
H2O 11.17
(2) Subpackaging PCR reaction solution
The PCR reaction solution was dispensed into a PCR tube at a volume of 20. mu.L/reaction.
3. Sample processing
4 clinical samples were taken, each sample being treated in parallel with three groups of treatments:
(1) treatment group-Immunocontraction group
Adding 10 mu L of immunomagnetic beads into a centrifuge tube, taking 200 mu L of sample, and blowing and beating the sample by a gun head for 3 times;
incubation for 15 minutes at room temperature;
centrifuging, performing magnetic separation with a magnetic frame, discarding the supernatant, and adding 25 μ L of TE buffer (10 mM Tris-HCl 1mM EDTA pH = 8.0) to the remaining beads;
heating at 90 deg.C for 5 min. Performing magnetic separation by using a magnetic frame;
taking 5 mu L of supernatant, adding the 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 for nucleic acid extraction, adding 5 mu L of extracted nucleic acid into a PCR tube, and preparing for detection on a computer;
(3) control group 2 stock solution test group
After the samples are mixed evenly, directly sampling 5 mu L of the sample, adding the sample 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 in the step (1) as a sample, heating the sample at 90 ℃ for 5 minutes, taking 5 mu L of the supernatant, adding the supernatant into a PCR tube, and preparing for detection on a computer.
4. Detection on machine
And (3) putting the PCR tube with the sample added into a fluorescence PCR instrument for fluorescence PCR amplification, wherein the detection procedure comprises the following steps: maintaining at 52 deg.C for 2 min; maintaining at 95 deg.C for 1 min; (95 ℃ for 5 s; 58 ℃ for 30s and fluorescence collected) 45 cycles. After the operation of the equipment detection program is finished, observing an amplification curve graph, and reading a Ct value;
the detection 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 the amplification curve of a sample after HBV enrichment in a fluorescence PCR assay without purification according to the present invention; FIG. 3 is a graph showing the fluorescence PCR detection amplification curve after the extraction of the sample nucleic acid; FIG. 4 is a graph of a direct fluorescence PCR detection amplification curve of a sample stock solution; FIG. 5 is a fluorescent PCR detection amplification curve diagram of a serum sample (supernatant) after virus immune enrichment.
TABLE 2
Sample concentration (IU/mL) Immunity enrichment group Extraction assay 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
Table 2 and fig. 2-5 were analyzed, and it can be seen that the detection after the immuno-enrichment treatment (the present invention) was substantially identical to the detection efficiency of the detection control group after the conventional extraction (the efficiency is higher for small Ct value), and the detection result of the present invention was more excellent at low concentration; the detection result of the invention is obviously superior to that of a direct amplification control group. And the comparison with the detection result of the supernatant liquid shows that the immune enrichment of the invention has better virus capture capacity.
The embodiments are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same or similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
The hepatitis B virus enrichment fluorescence PCR detection method provided by the invention is described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims (9)

1. A hepatitis B virus enrichment fluorescence PCR detection method is characterized by comprising the following steps:
s1, preparation of immunomagnetic beads: coupling the hepatitis B virus antibody to the carboxyl modified superparamagnetic nano-bead to obtain the immunomagnetic bead coupled with the hepatitis B virus antibody,
s2, enrichment of hepatitis B virus: mixing and incubating hepatitis B virus positive serum or plasma with immunomagnetic beads coupled with hepatitis B virus antibodies;
s3, separating the immunomagnetic bead-virus complex by using a magnetic tool, and resuspending the immunomagnetic bead-virus complex in TE buffer solution for heating and cracking;
s4, separating the magnetic beads by a magnetic tool to obtain enriched and concentrated hepatitis B virus, and directly detecting the liquid in the cleavage product by a fluorescent PCR reagent.
2. The hepatitis b virus-enriched fluorescent PCR detection method according to claim 1, wherein the hepatitis b virus antibody is a monoclonal hepatitis b virus surface antibody.
3. The fluorescence PCR detection method for hepatitis B virus enrichment according to claim 1, wherein the specific method for enriching the hepatitis B virus in step S2 is as follows: mixing 200 and 600 mu L of serum or plasma with 2-5 mu L of immunomagnetic beads, and uniformly mixing for 6-15 minutes at room temperature (25-37 ℃).
4. The fluorescence-enriched PCR detection method for hepatitis B virus of claim 1, wherein the thermal cleavage in step S3 is: heating at 85-95 deg.C for 5-15 min to crack virus and release nucleic acid.
5. The hepatitis b virus-enriched fluorescent PCR detection method according to claim 1, wherein the fluorescent PCR reagents of step S4 comprise:
(1) carrier RNA with a final concentration of 5-15 mug/mL;
(2) tris at a final concentration of 0.10-0.15M.
6. The hepatitis B virus-enriched fluorescent PCR detection method according to claim 1, wherein the total reaction volume of the fluorescent PCR reagents is less than or equal to 40 μ L.
7. The hepatitis B virus-enriched fluorescence PCR detection method of claim 1, wherein the diameter of the superparamagnetic beads is 200nm to 350nm, and the surface of the superparamagnetic beads has carboxyl (-COOH) groups.
8. The hepatitis b virus-enriched fluorescent PCR detection method according to claim 1, wherein the preparation of the immunomagnetic beads specifically comprises: connecting the anti-monoclonal hepatitis B virus surface antibody to the surface of a superparamagnetic nano magnetic bead with carboxyl (-COOH) on the surface by using an EDC activation method, and sealing the magnetic bead coupled with the antibody by using BSA.
9. The fluorescence-enriched PCR detection method for hepatitis B virus according to claim 1, wherein the air-dried preservation method for immunomagnetic beads comprises: placing the immunomagnetic beads in an oven, adjusting the temperature to 30-37 ℃, starting ventilation, standing for more than 10 hours, taking out, and sealing and storing at 2-8 ℃.
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Cited By (1)

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CN115326683A (en) * 2022-08-12 2022-11-11 四川成电医联科技咨询有限公司 Magnetic bead calibration white blood cell sub-group counting method based on impedance pulse time delay method

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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
CN113322302A (en) * 2021-06-02 2021-08-31 重庆医科大学 Immunocapture molecular detection method for HBV complete virus particles

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Publication number Priority date Publication date Assignee Title
US20130078612A1 (en) * 2011-09-26 2013-03-28 Asiagen Corporation Method for detecting microorganisms and a kit thereof
CN103805574A (en) * 2014-01-26 2014-05-21 江西农业大学 Method for enriching water body hepatitis A viruses based on immunomagnetic beads
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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