CN117230258A - EB virus detection method of culture amplification combined PCR for improving sensitivity - Google Patents
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Abstract
The invention provides a method for detecting EB virus by combining culture amplification with PCR, which improves sensitivity. The method of the invention is to take the lysate to inoculate human B lymphocyte by lysing the cells possibly infected by EB virus; inoculating B lymphocyte, continuously culturing, adding chemical agent TPA for promoting EB virus gene synthesis in the culturing process, and screening to obtain optimal TPA use concentration to fully amplify potential EB virus; and detecting the cultured B cells by using a PCR method to determine the presence or absence of the infectious EB virus. The excellent effect of the EB virus PCR detection method is that the invention provides a method for effectively detecting infectious EB virus for cell lines which cannot be directly detected by PCR method due to EB virus sequence expression in cell nucleus, thereby avoiding false positive and improving the whole sensitivity of EB virus detection.
Description
Technical Field
The invention relates to the field of biological detection, in particular to virus detection, especially EB virus detection.
Background
EB virus (Epstein-Barr virus, EB virus) was found and isolated in 1964 in biopsies of Burkitt's Lymphoma (BL). EB virus belongs to the gamma herpes virus subfamily and is one of 9 viruses which have been identified to infect humans only. EB virus is an icosahedral, dodecameric portal structure (viral genome translocation machinery) and capsid-related epidermal complex structure. The EB virus genome is approximately 172kb and consists of a linear double stranded DNA molecule encoding more than 85 genes. The coding genes are currently known to have 6 nuclear antigens (EB virus nucleic antigen, EBNA), 3 latent membrane proteins (latent membrane protein, LMP), small non-polyadenylation RNAs, EB virus encoded small RNAs (EBER RNA, EBER) 1 and 2, microRNAs and several early lytic genes [ tingting, xu Sihong, zhou Haiwei. EB virus laboratory detection technology research progress [ J ]. J. Molecular diagnostics and therapeutics, 2020, 12 (11): 1587-1590.
EB virus mainly proliferates in oropharynx epithelial cells, a large amount of virus exists in oropharynx secretion, the toxin expelling time can reach several months, and some virus carriers can even intermittently or continuously expel toxin for several years. EB virus can infect T lymphocyte, B lymphocyte, nasopharyngeal and oropharyngeal squamous cell, salivary gland and gastric gland, thyroid gland epithelial cell, smooth muscle and follicular dendritic cell. The detection method of EB virus mainly comprises serological and molecular biological methods. The heteroagglutination antibody assay (heterophile antibody test, HAb) is one of the usual serological methods and is a simple but non-specific assay. HAb depends on the ability of patient serum or plasma to agglutinate horse, goat or sheep erythrocytes [ Nagpal B, sreeshla H, kishale a, et al Evaluation of infectious mononucleosis status among a cohort of dental students [ J ]. IntJ Adv Med,2016,3 (1): 116-119 ]. HAb is commonly used in the diagnosis and screening of primary and recurrent infections and is a sensitive diagnostic test for infectious mononucleosis (infectious mononucleosis, IM).
The serological antibody assay for EB virus conventionally includes immunofluorescence (immunofluorescence assays, IFAs), enzyme-linked immunosorbent assay (enzyme immunoassays, EIAs) and western blot assay in combination with chemiluminescent immunoassay (CLIA). After EB virus infects cells, capsid antigen (viralcapsid antigen, VCA), early Antigen (EA), membrane Antigen (MA), EBNA, etc. can be expressed, and each antigen expression stimulates the production of the corresponding antibody. There are different types of specific assays for EB virus antibodies (VCA-IgG, VCA-IgM, EBNA-IgG, EBNA-IgM and EA-IgG) [ manting, xu Sihong, zhou Haiwei. EB Virus laboratory detection techniques development [ J ]. Journal of molecular diagnostics and treatment 2020, 12 (11): 1587-1590 @)
Quantitative polymerase chain reaction methods are the most common molecular biology methods that rely on the amplification of conserved nucleic acid sequences and use fluorescent probes or intercalating dyes to quantify nucleic acids of interest. The risk of amplicon contamination is lower because the reaction mixture is contained in a sterile, closed container. The standard method for judging whether tissue is infected by EB virus is in situ hybridization of EB virus coded small RNA (EBER). The principle of the method is that a digoxin-marked EBER probe, a horseradish peroxidase-marked anti-digoxin antibody and DAB chromogenic liquid are sequentially dripped on a treated tumor tissue paraffin section and then hematoxylin staining is carried out, and finally a doctor reads the section, and the result is that a brown yellow nuclear staining chromogenic area of a tumor cell is more than or equal to 5% positive, or negative [ see patent document CN115287336A, publication date 2022-11-04 ].
For the PCR detection of EB virus in the field, the prior art discloses a quantitative detection method of EB virus DNA, which adopts a nucleic acid releasing agent to rapidly crack and release EB virus DNA in a sample, adopts different extraction modes for a plasma sample, a peripheral blood sample, a throat swab sample and a negative positive control, is matched with components such as PCR reaction liquid, and the like, and adopts a real-time fluorescent quantitative PCR detection technology on a fluorescent quantitative PCR instrument to realize quantitative detection of the EB virus DNA through the change of fluorescent signals (see patent document CN108754024A, publication date 2018.11.06). The prior art also discloses an MNP marking site, a primer composition, a kit and application thereof for EB virus detection, wherein the MNP marking site refers to a genome region which is screened on an EB virus genome and is separated from other species and has a plurality of nucleotide polymorphisms in the species, and comprises marking sites of MNP-1-MNP-15. The MNP marker locus can specifically identify the EB virus and accurately detect variation; the primers do not interfere with each other, and the multiplex amplification and sequencing technology is integrated, so that sequence analysis can be performed on all the marker loci of multiple samples at one time [ see patent document CN113718057A, publication date 2021.11.30 ].
However, the current methods for detecting EB virus by PCR method have defects that the method directly takes sample nucleic acid for PCR detection, and the main defects include: (1) If the virus content in the sample is low, false negative results can be caused; (2) It is impossible to determine whether the detected EB virus has also infectious activity.
In addition, in the prior art, NK cells form immortalized cells through EB virus infection, but the EB virus gene is continuously existed in the immortalized NK cell genes, and EB virus with infection activity can be generated, and related genes of EB virus replication in NK cells can be knocked out through a gene editing technology, but the possibility of generating bioactive viruses still exists. And the NK cells infected by the EB virus are mostly in a latent infection state, and the phenomenon of cell splitting can not occur. It is difficult to determine whether NK cells release bioactive EB virus by the existing detection technology.
Therefore, there is a need in the art to provide a PCR detection method that has high sensitivity and can determine whether or not EB virus in a sample is infectious.
Disclosure of Invention
In order to solve the above-mentioned defects of the existing EB virus detection technology, the invention provides a detection method of EB virus culture amplification combined PCR, which can improve the sensitivity of EB virus detection and can also determine whether the EB virus in the sample has infectivity. The invention solves the problem of detecting the EB virus with biological activity by the following improved and optimized technical scheme.
The invention provides a method for detecting EB virus by combining culture amplification and PCR for non-diagnosis purpose, which is characterized by comprising the following steps:
(1) Lysing cells possibly infected by EB virus, filtering the lysed supernatant, and inoculating human B lymphocytes;
(2) Continuously culturing inoculated human B lymphocytes, continuously culturing and observing for 3-14 days, continuously adding chemical agent TPA (Phorbol 12-tetradecanoate 13-acetate, phorbol 12-myristate 13-acetate, CAS: 16561-29-8) for promoting EB virus gene synthesis in the culturing process, keeping the use concentration of TPA at 0.01-5 ng/mL, and fully amplifying potential EB virus;
(3) Culturing for 3-14 days, collecting human B lymphocytes, and washing the cells for 2-5 times;
(4) And (3) performing cell lysis, nucleic acid extraction and PCR detection on the human B lymphocyte sample obtained in the step (3) to judge whether the infectious EB virus exists or not.
Further, the optimal use concentration of TPA in step (2) is 0.25 ng/mL.
Further, the cells possibly infected with the EB virus in the step (1) are NK cells, optionally NK cells knocked out genes of the EB virus integrated on the chromosome; the continuous culture and observation period in the step (2) is 7 days; washing the cells in step (3) using PBS or DPBS; the step (3) of cleaning the cells ensures that EV virus and residual EB virus genes which do not have infectious activity in the culture supernatant are removed; and (3) culturing the cell line on the 7 th day in the step (3), and collecting B lymphocytes.
Further, if the PCR detection result of the sample is positive, the step (4) proves that the infectious EB virus exists in the sample.
Further, the PCR detection in the step (4) is real-time quantitative PCR.
Furthermore, the PCR detection in the step (4) adopts a specific primer and a probe, the forward primer sequence is shown as SEQ ID No.1, the reverse primer sequence is shown as SEQ ID No.2, and the probe sequence is shown as SEQ ID No. 3.
Further, the PCR detection in the step (4) is set as follows: s1:95 ℃ for 15min; s2: the fluorescence acquisition temperature was 72℃for 45 cycles at 95℃for 15S,55℃for 30S, and 72℃for 30S.
Another aspect of the invention provides the use of TPA at a concentration of 0.01-5 ng/mL in an EB virus PCR detection method for non-diagnostic purposes.
Further, the TPA was used at a concentration of 0.25 ng/mL.
The EB virus detection method combining culture amplification and PCR provided by the invention has the following beneficial technical effects:
1. and adding a chemical agent TPA for promoting the gene synthesis of the EB virus in the culture process, screening to obtain the optimal TPA use concentration of 0.25ng/mL, fully amplifying the potential EB virus, and screening to obtain the optimal TPA use concentration of 0.25ng/mL so as to ensure that the EB virus amplification efficiency is highest, thereby improving the sensitivity of the subsequent PCR detection.
2. Human B lymphocyte with clear origin is used as an indicator cell, EB virus amplification is carried out, the detection sensitivity is obviously improved, and the minimum detection limit can reach 0.05 TCID 50 。
3. The method comprises the steps of detecting the infectious EB virus, inoculating B cells after lysing sample cells and filtering, removing cell fragments and virus genes integrated on host cell chromosomes, and eliminating interference of EB virus genes in the sample cells on detection results.
4. The EV virus and residual EB virus gene interference which do not have infectious activity in the culture supernatant are eliminated by cleaning the cultured B cells, and if the B cells have positive PCR detection results, the presence of the infectious EB virus in the sample is indicated.
Drawings
FIG. 1 shows the flow of detection of infectious EB virus in NK cells of the present invention.
FIG. 2 shows the PCR results of the negative control, positive control and interference group samples according to the present invention.
Detailed Description
The media, cells, reagents or consumables, and instruments used in the present invention are commercially available in the art.
Example 1 determination of optimal working concentration of TPA in EB Virus PCR detection method
The day before the start of the test, cells were prepared according to the following table 1.
TABLE 1 parameters such as cell concentration
Remarks: all cells in the assay were tested at 36℃and 5% CO, unless otherwise specified 2 Culturing in a wet environment.
The cell state prepared according to table 2 was observed to ensure that the cell morphology was normal. The cells were pipetted and mixed well and the cell suspension was aliquoted into centrifuge tubes, 1 ml/tube. 1000 Centrifuge at room temperature for 5 min. After centrifugation, the supernatant was discarded, 5.0-6.0 mL of RPMI-1640 complete medium containing TPA at various concentrations (0. 0 ng/ml, 0.01 ng/ml, 0.05ng/ml, 0.25ng/ml, 1.25 ng/ml, 5 ng/ml) was added and the well mixed cells were pipetted. Transfer the cell suspension to T-25cm in its entirety 2 In a flask. The cells were returned to the incubator for culturing.
Cell passage: passaging was performed every 3 to 4 days for 4 times in total. 0.5-2.0 ml cell suspensions (depending on growth rate and passaging schedule) were dispensed into centrifuge tubes and centrifuged at room temperature for 5 min. Collecting supernatant, and storing at a temperature of less than or equal to-20deg.C. To the corresponding centrifuge tube, 5.0-6.0. 6.0 ml of RPMI-1640 complete medium containing TPA at various concentrations (0 ng/ml, 0.01 ng/ml, 0.05ng/ml, 0. 0.25ng/ml, 1.25 ng/ml, 5 ng/ml) was added and the mixed cells were pipetted. Transfer of the cell suspension to T-25cm in its entirety 2 In a flask. The cells were returned to the incubator for culturing.
After passage 4, culture was continued for 3-4 days, all cell suspensions were transferred to centrifuge tubes and centrifuged at room temperature for 5 min. Collecting supernatant, and storing at a temperature of less than or equal to-20deg.C. And (3) carrying out real-time quantitative PCR detection on all the collected cell supernatants, carrying out data analysis on detection results, and selecting the TPA concentration which generates the most EB virus to be 0.25ng/mL, wherein the optimal infection time is 7 days. The original results are shown in Table 2.
TABLE 2 PCR detection of corresponding EB Virus CT values obtained for TPA concentration and days of culture
Example 2 treatment of EB Virus in NK cells
NK cells possibly containing active EB virus are lysed (the NK cells knock out EB virus genes integrated on chromosomes; the EB virus gene knock out can be carried out by the method disclosed in patent document (publication No. CN111926016B, publication day 20221209)), and the lysate is filtered and inoculated with human B lymphocytes. The flow of detecting whether NK cells are infected with active EB virus is shown in the following figure 1, and the flow is briefly described as follows: after the sample, interference, negative and positive control groups were seeded with human B lymphocytes, 0.25ng/mL TPA was added, wherein the interference and positive control groups were added with EV virus. The cells were observed in culture and harvested on day 7, washed three times and subjected to real-time quantitative PCR detection. The day before the start of the assay, B cells for assay were prepared using a Petri dish or T25 flask, each flask was inoculated with 1.0X10 6 Individual cells. Remarks: all cells in the assay procedure were incubated at 36.+ -. 1 ℃, 5.+ -. 1% CO, unless otherwise specified 2 Culturing in a wet environment.
On the day of detection, each flask of B cells was treated as follows:
negative Control (NC), medium with final concentration of 0.25ng/mL TPA was added;
positive control group (PC), medium containing TPA at final concentration of 0.25ng/mL and EB virus 5TCID were added 50 ；
Test sample group (Sa), 1.0X10 th is added 7 Cell lysate 3 mL, while meeting the TPA concentration of 0.25ng/mL working concentration;
interference control group (IC), add 1.0X10 7 Cell lysate 3 mL and EB virus 5TCID 50 And TPA was added at a final concentration of 0.25 ng/mL.
And after the treatment, the culture flask is put back into the incubator.
B cell culture and observation:
the cell status was observed every day, all cells collected on day 7 of culture were subjected to the subsequent PCR test of example 3, and the test results were subjected to data analysis to determine whether or not the sample cells contained infectious EB virus.
Example 3 PCR detection of EB Virus
1. The target sequence plasmid was subjected to gradient dilution containing 1X 10 per 1. Mu.L 7 、1×10 6 、1×10 5 、1×10 4 、1×10 3 、 1×10 2 1X 10 1 Standard for each copy of target sequence (used as positive control).
2. Specific primer and probe designs and screens table 3 below:
TABLE 3 EB Virus-specific primers and probes
3. Nucleic acid extraction step: nucleic acid extraction reference commercial kit: JD-SOP-QC-2-155 viral genome extraction procedure
4. Preparing a reaction system: the reaction system comprises a premix, a forward primer, a reverse primer, a probe and a template, wherein the premix is QIAGEN 2X QuantiTect Probe PCR Master Mix;
TABLE 4 preparation of premix
5. Sample loading, wherein samples shown in the following table are respectively added into sample holes, and the specific details are shown in table 5:
TABLE 5 sample and final sample volume
6. Performing fluorescent quantitative PCR reaction, and setting a reaction program by adopting a verified PCR instrument;
S1：95℃ 15min。
s2: the fluorescence acquisition temperature was 72℃for 45 cycles at 95℃for 15S,55℃for 30S,72℃for 30S, and S2.
7. Viral nucleic acid extraction: viral nucleic acid extraction procedure reference commercial kit: QIAamp DNA Mini Kit (250), QIAGEN 51306.
Example 4 results criteria and related experimental results
Judgment standard:
the result of the negative control PCR detection of EB virus is negative.
The result of positive control PCR detection of EB virus is positive.
Interference group samples do not interfere with EB virus detection positive results.
The PCR results for the negative control, positive control, and interference group samples of example 3 are shown in fig. 2, where the threshold line is 1.15. The specific results are as follows: the negative control result is negative, the positive control and the interference group result are positive, and the result shows that the components in the sample, such as cell lysate and the like, do not influence the detection result.
Because the cell washing is performed before the PCR detection, viruses and nucleic acid fragments possibly existing in the supernatant are washed away, namely EB virus nucleic acid which is not infected into B cells or EB virus particles are washed away, and the infected EB virus is infected into the B cells to replicate and amplify in the cells. Thus, if the PCR in the test sample set is positive, it indicates the presence of infectious EB virus in the sample.
Example 5 EB Virus detection of samples of different Virus titres
For different EB virus samples, including EB virus of inoculation experiment group 1 (5 TCID 50 ) EB virus of experimental group 2 (0.5 TCID) 50 ) EB virus of experimental group 3 (0.05 TCID) 50 ) Measles of the negative control group and Measles virus group (5 TCID) 50 ) The experimental results obtained by using the TPA concentration of 0.25ng/mL, which was the most abundant for EB virus production, determined in example 1, and the optimal infection time of 7 days, and the EB virus treatments and PCR assays of examples 2 and 3 are shown in Table 6 below. Furthermore, sample 1 NK Cell was provided, and it was clearly known that there was no infectious EB virus in this sample 1 before detection, which was detected by the method of this example, and the detection results are shown in Table 6 as negative results. However, the sample 1 NK Cell was directly subjected to PCR detection, i.e., the PCR detection of the virus of example 3 EB, without using the detection method of the culture amplification combined PCR of the present invention, and the junction was detectedThe result is positive. As can be seen, the conventional PCR detection method in the prior art can lead to false positive results, and the method for combining the culture amplification with the PCR detection can avoid false positive.
TABLE 6 experimental data for EB Virus detection of different samples
Experimental results show that the minimum detection limit of the method is 0.05 TCID 50 The method improves the PCR detection sensitivity of the EB virus by culturing and amplifying the virus and using TPA treatment with optimized concentration. In addition, the conventional PCR detection method in the prior art can lead to false positive results, and the method for combining the culture amplification and the PCR detection can avoid false positive.
The above examples of the present disclosure are merely examples for clearly illustrating the present disclosure and are not limiting of the embodiments of the present disclosure. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modifications, equivalent substitutions, improvements, etc. that fall within the spirit and principles of the present disclosure are intended to be included within the scope of the claims of the present disclosure.
Claims (9)
1. A method for detecting an epstein barr virus by combining culture amplification and PCR for non-diagnostic purposes, comprising the steps of:
(1) Lysing cells possibly infected by EB virus, filtering the lysed supernatant, and inoculating human B lymphocytes;
(2) Continuously culturing the inoculated human B lymphocytes, continuously culturing and observing for 3-14 days, continuously adding a chemical agent TPA for promoting the gene synthesis of the EB virus in the culturing process, keeping the use concentration of the TPA to be 0.01-5 ng/mL, and fully amplifying the potential EB virus;
(3) Culturing for 3-14 days, collecting human B lymphocytes, and washing the cells for 2-5 times;
(4) And (3) performing cell lysis, nucleic acid extraction and PCR amplification detection on the human B lymphocyte sample obtained in the step (3), and judging whether the infectious EB virus exists or not.
2. The method of claim 1, wherein the TPA in step (2) is optimally used at a concentration of 0.25 ng/mL.
3. The method according to claim 1 or 2, wherein the cells potentially infected with epstein barr virus in step (1) are NK cells, optionally NK cells knocked out epstein barr virus genes integrated on the chromosome; the continuous culture and observation in the step (2) are 7 days; washing the cells in step (3) using PBS or DPBS; the number of times of cell washing in the step (3) is 3; and (3) culturing the cell line on the 7 th day in the step (3), and collecting B lymphocytes.
4. The method according to claim 1 or 2, wherein in step (4), if the sample PCR test result is positive, the presence of infectious epstein barr virus in the sample is confirmed.
5. The method according to claim 1 or 2, wherein the PCR detection in step (4) is real-time quantitative PCR.
6. The method according to claim 5, wherein the PCR detection in step (4) uses specific primers and probes, the forward primer sequence of which is shown in SEQ ID No.1, the reverse primer sequence of which is shown in SEQ ID No.2, and the probe sequence of which is shown in SEQ ID No. 3.
7. The method according to claim 1 or 2, wherein the PCR detection in step (4) is performed by the following procedure: s1:95 ℃ for 15min; s2: the fluorescence acquisition temperature was 72℃for 45 cycles at 95℃for 15S,55℃for 30S,72℃for 30S, and S2.
8. The application of TPA with the concentration of 0.01-5 ng/mL in the EB virus PCR detection method for non-diagnosis purpose.
9. The method of claim 8, wherein the TPA is used at a concentration of 0.25 ng/mL.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5911982A (en) * | 1995-10-06 | 1999-06-15 | National Science Council | Hz-1 virus persistence-associated-gene 1 (PAG1) promoter uses therefor, and compositions containing same or products therefrom |
| US20040180331A1 (en) * | 1998-03-04 | 2004-09-16 | Marcel Bartolina Hendrikus Johannes Vervoort M.B.H.J. | Oligonucleotides for the amplification and detection of epstein barr virus (EBV) nucleic acid |
| US20090270268A1 (en) * | 2005-12-16 | 2009-10-29 | Ribovax Biotechnologies Sa | Methods for Obtaining Immortalized Antibody Secreting Cells |
| WO2013000982A1 (en) * | 2011-06-27 | 2013-01-03 | Vivalis | Method for screening cells |
| CN106947835A (en) * | 2017-04-24 | 2017-07-14 | 首都医科大学附属北京友谊医院 | The authentication method of ebv infection lymphocyte subgroup and its application |
| CN116144835A (en) * | 2022-09-09 | 2023-05-23 | 安徽医科大学第二附属医院 | Method for improving sensitivity of identifying EBV-infected lymphocyte subpopulation and application thereof |
-
2023
- 2023-11-10 CN CN202311491010.5A patent/CN117230258B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5911982A (en) * | 1995-10-06 | 1999-06-15 | National Science Council | Hz-1 virus persistence-associated-gene 1 (PAG1) promoter uses therefor, and compositions containing same or products therefrom |
| US20040180331A1 (en) * | 1998-03-04 | 2004-09-16 | Marcel Bartolina Hendrikus Johannes Vervoort M.B.H.J. | Oligonucleotides for the amplification and detection of epstein barr virus (EBV) nucleic acid |
| US20090270268A1 (en) * | 2005-12-16 | 2009-10-29 | Ribovax Biotechnologies Sa | Methods for Obtaining Immortalized Antibody Secreting Cells |
| WO2013000982A1 (en) * | 2011-06-27 | 2013-01-03 | Vivalis | Method for screening cells |
| CN106947835A (en) * | 2017-04-24 | 2017-07-14 | 首都医科大学附属北京友谊医院 | The authentication method of ebv infection lymphocyte subgroup and its application |
| CN116144835A (en) * | 2022-09-09 | 2023-05-23 | 安徽医科大学第二附属医院 | Method for improving sensitivity of identifying EBV-infected lymphocyte subpopulation and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 张梅!710061, 司履生, 金莉, 王一理, 耿宜苹: "重组人单链白细胞介素12融合基因的构建、真核表达及生物学活性测定", 中华血液学杂志, no. 12, pages 1 - 5 * |
| 陈莉;朱远源;: "肿瘤研究的新热点――EB病毒", 肿瘤防治研究, no. 10, pages 750 - 754 * |
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