CN110607400B

CN110607400B - Tissue in-situ hybridization diagnosis and detection system for BKV and application thereof

Info

Publication number: CN110607400B
Application number: CN201910950375.7A
Authority: CN
Inventors: 朱同玉; 吴楠楠; 胡春兰; 张小楠; 石业静
Original assignee: SHANGHAI PUBLIC HEALTH CLINICAL CENTER
Current assignee: SHANGHAI PUBLIC HEALTH CLINICAL CENTER
Priority date: 2019-10-08
Filing date: 2019-10-08
Publication date: 2022-12-27
Anticipated expiration: 2039-10-08
Also published as: CN110607400A

Abstract

The invention relates to the technical field of gene diagnosis, and particularly discloses an in-situ nucleic acid diagnosis and detection system for BKV, which comprises a probe for detecting BKV, wherein the sequences of the probe are respectively the sequences shown as SEQ ID Nos. 1-40 or complementary chains thereof. The invention also discloses the application of the system in preparing a diagnosis or detection reagent/kit of the BKV. The tissue in-situ hybridization diagnosis and detection system of the BKV has high sensitivity and good specificity, can be used for accurately diagnosing the activation of the BKV so as to guide clinical symptomatic medication, and improves the sensitivity and specificity of BKV tissue diagnosis. The application of the BKV in-situ hybridization technology can greatly improve the sensitivity and specificity of BKV tissue diagnosis, reduce the harm caused by BKV related diseases, and have wide market application prospect.

Description

Tissue in-situ hybridization diagnosis and detection system for BKV and application thereof

Technical Field

The invention relates to the technical field of gene diagnosis, in particular to an in-situ nucleic acid diagnosis and detection system for BKV and application thereof.

Background

In-Situ Hybridization (ISH) is a technique for quantifying and localizing specific nucleic acid (DNA and RNA) targets In fixed tissues and cells using nucleic acid molecular probes, and is one of the tissue diagnosis methods commonly used In clinic, which can obtain temporal and spatial information related to gene expression and genetic loci. Immunohistochemistry (IHC) is the most common method in tissue diagnosis, and is a technology for qualitative, localized and quantitative determination of corresponding antigens (i.e. proteins) by using the principle of antigen-antibody specific binding and performing a color reaction through antibody labeling. BK polyomavirus (BKV) is an opportunistic infectious virus which easily causes serious urinary system diseases in people with low immunity. Currently, no BKV-related immunohistochemistry or in situ hybridization patents have been detected from patent retrieval systems. There are a number of diagnostic methods for BKV immunohistochemistry in scientific literature, but there are few reports on BKV in situ hybridization.

BKV belongs to the polyomaviridae family. Currently, the clinical tissue diagnosis of BKV adopts an immunohistochemical method, and the antibody is cross-recognized by an antibody against the large T antigen of BKV, which is a monkey polyoma virus SV40 large T antigen. Therefore, this detection technique has the following disadvantages: 1) The specificity is poor, and misjudgment is easily caused. That is, other polyomaviruses such as BKV, SV40, or BKV's close JC virus (JCV) can be recognized by the antibody, and the detection result cannot be determined as which polyomavirus is. The pathogenicity and treatment regimens of different polyomaviruses vary, and therefore tissue diagnosis specificity is required for symptomatic administration. 2) The sensitivity is poor, and the judgment is easy to miss. The lack of sensitivity is a common problem in immunohistochemical detection. BKV mainly affects organ transplant recipients such as kidney transplantation, and the specificity of tissue diagnosis is that the symptoms of tissue inflammation caused by BKV activation are very similar to those caused by acute organ rejection, but the immunosuppressive schemes for them are completely opposite. Therefore, once the BKV-related nephritis is judged to be the BKV false negative and is judged to be acute rejection, the immunosuppression intensity needs to be increased, which may lead to severe consequences such as worsening of the symptoms of the BKV-related nephritis and loss of function of transplanted kidney. At present, sporadic reports about BKV in situ hybridization diagnosis exist in the literature, but the target site is not limited within the gene coding region, or only the BKV conserved region is considered and the specific region is not considered, so that the problem that the sensitivity of the target site only aiming at DNA (with small quantity) is poor is caused; or the probe targets the BKV, the JCV and the SV40 at the same time and cannot be distinguished, and the like.

Disclosure of Invention

The BKV is a latent infection opportunistic virus and is widely distributed in people, and the adult IgG antibody positive rate reaches over 90 percent. Once the immunity of the body is reduced, such as taking immunosuppressant, infecting HIV, getting pregnant and suffering from cancer, the BKV is easily activated to cause serious urinary system diseases, including: 1) Causes BKV-related nephropathy (BKVAN) to develop in-6% of kidney transplant recipients, which can lead to 16-40% of transplanted kidneys losing work even under treatment; 2) Causing hemorrhagic cystitis in about-10% of hematopoietic stem cell transplant recipients; 3) The activation of BKV increases the risk of development and metastasis of some tumors, such as bladder cancer. Due to the lack of effective antiviral drugs, lowering the immunosuppressive strength is the only effective anti-BKV regimen for organ transplant recipients, which in turn carries the risk of organ rejection. At present, although the blood and urine fluorescence quantitative PCR diagnosis result can also be used as a reference basis for judging the activation of the BKV, the tissue diagnosis is the gold standard for judging the BKVAN, and the tissue immunohistochemical large T antigen positive is generally used as a standard at present.

BKVAN and organ rejection both cause inflammatory responses and are very similar in tissue symptoms and difficult to distinguish. The treatment regimen for the inflammatory response is to increase the immunosuppressive strength, the regimen for BKVAN is the exact opposite, and the immunosuppressive strength needs to be decreased to restore some immunity against the virus. Therefore, the tissue diagnosis of BKV requires high precision and sensitivity. On the other hand, as the currently clinically generally adopted immunohistochemical technology cannot distinguish BKV and JCV or other polyomaviruses, and the pathogenicity and treatment schemes of different polyomaviruses are different, for example, JCV mainly causes diseases of the brain, SV40 has been distributed in the european and american population to a certain extent since it has historically contaminated polio vaccine, but there is no report of causing diseases. Therefore, it is clinically necessary to know exactly whether the disease is caused by BKV, and to decide what treatment scheme to use. In summary, aiming at the defects in the prior art, the invention provides a tissue in-situ hybridization diagnosis and detection system for the BKV, which is used for accurately diagnosing the activation of the BKV so as to guide clinical symptomatic medication and improve the sensitivity and specificity of BKV tissue diagnosis.

Based on the above, the first aspect of the present invention provides a tissue in situ hybridization diagnosis and detection system for BKV, which comprises 40 probes for detecting BKV, wherein the sequences of the probes are respectively the sequences shown in SEQ ID nos. 1 to 40 or their complementary strands. Further, the probes are labeled with a chemical dye and a fluorescent luminescent group, respectively.

The second aspect of the invention provides a kit for tissue in situ hybridization diagnosis and detection of the BKV, which comprises a probe for detecting the BKV, wherein the sequence of the probe is a sequence shown as SEQ ID No: 1-40 or a complementary chain thereof.

In a third aspect of the invention, the nucleotide sequence pair for tissue in situ hybridization diagnosis and detection of the BKV is provided, and the nucleotide sequence pair is a sequence shown as SEQ ID No. 1-40 or a complementary strand thereof.

In a fourth aspect of the invention, the probe set for tissue in situ hybridization diagnosis and detection of BKV is provided, and the probe set consists of 40 probes, and the sequences of the probes are respectively the sequences shown as SEQ ID Nos. 1-40 or complementary strands thereof.

In a fifth aspect of the invention, there is provided the use of a pair or set of nucleotide sequences as described above in the preparation of a diagnostic or detection reagent/kit for BKV.

Further, the diagnosis or detection is specifically: BKV, SV40 and JCV are distinguished.

The invention has the advantages that:

1. the conservation is high. The diagnosis and detection system of the invention compares all 315 BKV sequences on NCBI with 12 clinical strain sequences sequenced by us (not uploaded to NCBI temporarily), and covers broad BKV strains. The designed probe sequence can cover all the BKV strains known at present.

2. The sensitivity is high. For the tissue diagnosis of the BKV, the sensitivity is the key and the central importance for judging the BKV related nephropathy and the adjustment of immunosuppressant. The diagnostic and detection system of the present invention improves diagnostic sensitivity by three aspects: the method has the advantages that the large T gene targeted by a detection target is massively expressed in the early and late stages of the BKV lytic infection and the BKV latent infection; secondly, the designed probe simultaneously targets DNA and mRNA, particularly the mRNA expression quantity of a large T gene is very high, and each DNA can transcribe a large amount of mRNA, so that the background concentration of the detection target is higher, and the sensitivity is improved; and the system comprises 40 conservative and specific probe combinations, and each probe can cascade and amplify signals, so that the diagnostic sensitivity can be obviously improved.

3. The BKV in-situ hybridization technology can greatly improve the sensitivity and specificity of BKV tissue diagnosis, reduce the harm brought by BKV related diseases, and has wide market application prospect.

Drawings

FIG. 1 is a schematic diagram of the genome structure of BKV.

FIG. 2. Example of conservative, specific probe selection.

FIG. 3 shows a comparison of the diagnostic sensitivity of immunohistochemistry and in situ hybridization of tissue samples from kidney transplant recipients. The sensitivity of the in-situ hybridization diagnosis and detection system is higher than that of immunohistochemistry.

FIG. 4. The immunofluorescence assay for the BKV large T antigen is not specific.

FIG. 5 shows that the fluorescence in situ hybridization technique of the present invention can specifically detect large T nucleic acid of BKV.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications can be made to the present invention by those skilled in the art after reading the present specification, and these equivalents also fall within the scope of the invention defined by the appended claims.

The following detailed description of the present invention is provided in connection with the accompanying drawings.

(1) Analyzing virological characteristics of the BKV and selecting a detection target.

The BKV is a DNA double-stranded virus, has a genome with the total length of about 5300bp, and comprises early encoding genes (encoding regulatory proteins large T and small T), late encoding genes (encoding Agno protein and virus structural proteins VP1, VP2 and VP 3) and a non-encoding regulatory region NCCR. The invention designs a BKV specific probe in a large T region based on the following reasons:

1) In the tissues of the bladder cancer, kidney cancer and other BKV continuously infected tissues, the BKV genome is often integrated into a host genome or copied in a double-stranded DNA loop form, and the existing research shows that only the expression of large T antigen can be detected at the moment, but the expression of VP1 cannot be detected;

2) In the tissue of lytic infection such as renal tubule, the early and late expression protein can be detected, but generally, the expression amount of the large T is higher than that of VP1, and the former is expressed in the early and late stages, and the period is longer;

3) The large T gene occupies about half of the full length of the BKV, and is suitable for screening probes for designing a conserved region and a specific region of the BKV. And the long fragment gene has the advantages that the corresponding mRNA fragment is also long, the probe which can simultaneously aim at the plus-strand DNA/mRNA is suitable for being designed, compared with the limited BKV DNA level in the cell, the mRNA level is far higher than the DNA level in the large T expression period, and the diagnostic sensitivity can be obviously improved.

FIG. 1 is a schematic diagram of the genome structure of BKV. The dark red color in the figure is the early coding region, containing the small T gene (sT) and the large T gene (LT, containing introns), and the black color is the late coding region, containing the Agno, VP1, VP2, and VP3 genes, with all VP1, VP2, and VP3 genes overlapping. Green is a non-coding regulatory region (NCCR) containing the viral origin of replication (indicated by circles) and the bidirectional promoter sequence.

(2) And designing a probe combination with high BKV coverage and good JCV specificity from the target area.

The biggest interference of the BKV diagnosis is JCV, the genome matching degree of the JCV and the genome matching degree of the JCV is as high as 75%, the matching degree of the JCV and the genome matching degree of the conservative region is more than 90%, and the JCV and the genome matching degree of the conservative region overlap at the position of the human body, for example, the JCV and the genome matching degree of the conservative region are hidden in urinary epithelium to carry out latent infection, and the JCV and the genome matching degree of the conservative region are easy to activate in urine and blood. While BKV mainly causes urinary system diseases and JCV mainly causes brain diseases. Therefore, the diagnostic system of BKV needs to exclude the interference of JCV. The MEGA7 software is used for carrying out sequence array arrangement on 327 existing BKV large T sequences (including 12 BKV sequences uploaded on NCBI and newly sequenced by people) and selecting a conserved region on a large T gene. Finally, a conservative region of about 1680bp (counted by the first base of an Agno gene initiation codon as 1, the position of the conservative region is 2365-3765bp,4235-4515bp large T antigen mRNA coding positive chain sequence) is selected from the large T gene region of the BKV, and the conservative region is compared with JCV and SV40 to find that the specificity is good, and is selected to design a specific probe. To ensure probe specificity, we first select a representative strain of BKV (SHA-30) to label conserved regions by sequence array alignment, with base sites that are completely conserved represented by lower case letters and sites that are not conserved represented by upper case letters. The same method as the arrangement of the BKV sequences is utilized to arrange 604 JCV sequences on NCBI into an array, then SHA-30 is brought into the JCV array and is manually compared and arranged, and a fragment with low matching degree with JCV is selected from a conserved region of a selected BKV large T gene to be selected as a probe target point.

As shown in figure 2, the BKV strain SHA-30 is marked by upper and lower case letters, the lower case represents a conserved site, then 604 JCV sequence arrays are introduced, the positions of the corresponding sites are adjusted, and a fragment with low matching degree with JCV is selected from the conserved region of the BKV large T gene to be selected as a probe target. Yellow highlights represent a selected probe target sequence, the upper asterisk indicates that all JCV strains and SHA-30 strains are singly conserved at the base at the site, and blank indicates that at least 1 strain has different bases at the site. Thus, the more spaces above the conserved segment corresponding to the lower case bases of SHA-30 represent greater specificity compared to JCV.

Finally, based on these principles and analysis results, we selected 40 probes from the conserved regions, and the probe sequences are shown in the following table. And the synthetic 2-type probes were designed for chemochromic tissue in situ hybridization (probe labeled with 3' digoxigenin) and fluorochromic in situ hybridization (FISH, probe labeled with Alexa Fluor 647), respectively.

TABLE 1 Probe sequences

(3) The sensitivity of the in-situ hybridization diagnosis and detection system is higher than that of immunohistochemistry.

To verify the advantage of our in situ hybridization system diagnosis in sensitivity over traditional immunohistochemical diagnosis. We select kidney biopsy samples of kidney transplant recipients who have been subjected to BKV immunohistochemical diagnosis, and perform in situ hybridization detection using the in situ hybridization diagnosis and detection system (40 probe sets) of the present invention and compare the results with immunohistochemical detection results. As shown in FIG. 3, the results show that the in situ hybridization diagnosis and detection system of the present invention can well detect BKV in renal tissue samples, and in small sample quantity comparison, all in situ hybridization results of samples with positive immunohistochemical diagnosis are positive, but samples with negative immunohistochemical detection and positive in situ hybridization system detection are also found. The BKV in-situ hybridization system designed by the invention is proved to have higher sensitivity and good feasibility.

FIG. 3 shows partial results of immunohistochemistry and in situ hybridization diagnostic comparisons of tissue samples from kidney transplant recipients, negative and positive 2 groups of samples for known immunohistochemistry results. The left 2 samples of the kit have negative results of CH-K and HJW-K immunohistochemistry, but have positive results of HJW-K in situ hybridization. The right 2 immunohistochemistry and in situ hybridization results were both positive. This suggests that our in situ hybridization system may be more sensitive than clinically used immunohistochemical assays. Note: blue arrows point to typical SV 40T antigen positive nuclei.

(4) The in-situ hybridization diagnosis and detection system has good specificity.

In many documents concerning BKV and JCV, antibodies against SV40 large T antigen were used for detection of large T antigen, suggesting that this diagnostic method is not specific to polyoma virus interiors. Immunofluorescence is the same principle as immunohistochemistry but is more sensitive than the latter and can be detected by fluorescence. We therefore used cell experiments to select HEK293T cells stably expressing large T antigen, a JCV large T gene transfected bladder

cancer cell line

5637, and 5637 infected with BKV and another bladder cancer cell line TCCSUP representing large T targets of SV40, JCV and BKV, respectively. The commercial large T antigen monoclonal antibody (PAb 416, abcam) is used for immunofluorescence detection, the system is used for fluorescence in situ hybridization detection, and the diagnosis specificity of the two methods is compared.

HEK293T (human kidney germ cells containing stably expressed SV40 large T gene), TCCSUP (human bladder cancer fourth stage cells) and 5637 (human bladder cancer second stage cells) were passaged into chamber slides, TCCSUP and 5637 were infected with BKV virus (MOI = 0.3), and immunofluorescence was performed using SV40 large T antigen antibody 5 days later; 5637 cells were transfected with JCV large T plasmid (constructed into pCDNA3.1 expression plasmid) by Fugene transfection reagent (Promega) and immunofluorescence assay using SV40 large T antigen antibody 1 day later, with uninfected or untransfected cells as controls. As shown in fig. 4, red fluorescence is large T protein signal of BKV and blue is DAPI stained nuclei.

The results show that the large T targets of SV40, JCV and BKV in the three groups are positive through immunofluorescence detection, while the fluorescence in situ hybridization result shows that only the BKV infected cells can detect fluorescence signals, and no signals are detected in JCV transfected or HEK293T cells containing SV40 large T genes. The good specificity of the in situ hybridization system is proved, and the defect of the large T protein diagnosis on the specificity is further verified.

The fluorescence in situ hybridization technology can specifically detect the large T nucleic acid of the BKV. As shown in FIG. 5, the experimental grouping, pre-infection and transfection treatments were the same as those in FIG. 4, and were consistent with those in immunofluorescence assay, and the cells were fixed at the late stage and then detected by fluorescence in situ hybridization detection procedure, where green fluorescence is the large T nucleic acid (DNA/mRNA) signal of BKV and blue is DAPI-stained nuclei.

In conclusion, the tissue in-situ hybridization diagnosis and detection system for the BKV has the following advantages:

1) The conservation property is high.

The system compares all 315 BKV sequences on NCBI with 12 sequenced clinical strain sequences (not uploaded to NCBI temporarily), and covers broad BKV strains. The designed probe sequence can cover all the BKV strains known at present.

2) The specificity is strong.

The sequence similarity of the BKV, the JCV and the SV40 is very high, and the expressed large T protein can be cross-recognized by an SV40 large T antibody, so that the immunohistochemical diagnosis taking the large T protein as a target is determined to be not specific (figure 4). The target BKV DNA and mRNA can select specific target spots to design probes according to nucleic acid sequences, can well solve the problem, and has good early-stage pre-experiment effect (figure 5).

3) The sensitivity is high.

For the tissue diagnosis of BKV, sensitivity is the key to judging BKV-related nephropathy and adjustment of immunosuppressive agents, which is important. The present system thus improves the sensitivity of the diagnosis by three aspects: the large T gene is selected as a detection target, and is expressed in large quantity in the early and late stages of the BKV lytic infection and the BKV latent infection; secondly, the designed probe simultaneously targets DNA and mRNA, particularly the mRNA expression quantity of a large T gene is very high, and each DNA can transcribe a large amount of mRNA, so that the background concentration of the detection target is higher, and the sensitivity is improved; and the system comprises 40 conservative and specific probe combinations, and each probe can cascade and amplify signals, so that the diagnostic sensitivity can be obviously improved.

The invention has social and economic benefits.

1) Social benefits.

More than 90% of adults are carriers of the BKV, and once the immunity of the organism is reduced, the adults can suffer from the BKV threat, and serious urinary system diseases such as BKV-related nephropathy, hemorrhagic cystitis, bladder cancer and the like are caused. Due to the lack of effective antiviral drugs, organ transplant recipients need to reduce the immunosuppressive strength (bear the risk of organ rejection) to combat BKV. This places high demands on the sensitivity and specificity of BKV diagnostics. The currently generally adopted immunohistochemical method has poor sensitivity and no specificity, and easily causes missed diagnosis and misdiagnosis, thereby causing serious consequences. Therefore, the development of the BKV in-situ hybridization method can greatly improve the sensitivity and specificity of the BKV tissue diagnosis and reduce the harm brought by the BKV related diseases.

2) And (4) economic benefits.

The BKV mainly threatens organ transplantation patients, immunosuppressor users of autoimmune diseases, nephropathy and the like, people infected by Human Immunodeficiency Virus (HIV), and people with low immunity such as bladder cancer, renal cell carcinoma and the like. With organ transplant recipients (especially kidney transplants and hematopoietic stem cell transplants), HIV infected individuals, and patients with bladder cancer being more threatened. Because of the large number of these people, BKV is detected as a hard requirement. Therefore, the diagnostic system has a wide market. See table below for details.

The data source is as follows:

[1]http://www.sohu.com/a/298390365_120044167；

[2]https://item.btime.com/04mvqcaks7huvnvoapkdrfhnfsp

[3]Xu LP,Wu DP,Han MZ,Huang H,Liu QF,Liu DH,Sun ZM,Xia LH,Chen J,Wang HX,Wang C,Li CF,Lai YR,Wang JM,Zhou DB,Chen H,Song YP,Liu T,Liu KY,Huang XJ.A review of hematopoietic cell transplantation in China:data and trends during 2008-2016.Bone Marrow Transplant.2017Nov；52(11):1512-1518.

[4]https://www.thepaper.cn/newsDetail_forward_2663539

[5] heyutong, li Daojuan, lidong, liqu, zhengrongshan, zhang thinking, zenghongmei, chenwanqing, hergi 2014, china bladder cancer pathogenesis and death analysis, chinese tumor journal, 2018.40 (9), 647-652.

[6] Korean Soviet force, zhangwise, chenwanqing, li Chang Ling, chinese bladder cancer status and prevalence trend analysis cancer progression, 2013,11 (1).

The above description is only a preferred embodiment of the present invention, and it should be noted that, for a person skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be considered as the protection scope of the present invention.

SEQUENCE LISTING

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Claims

1. A tissue in-situ hybridization diagnosis and detection system for BK polyomaviruses (BKVs) is characterized by comprising 40 probes for detecting the BKVs, wherein the sequences of the probes are respectively the sequences shown in SEQ ID Nos. 1 to 40 or complementary chains thereof.

2. The tissue in situ hybridization diagnostic and detection system for BK polyomavirus (BKV) according to claim 1, wherein the probes are labeled with a chemical dye and a fluorescent emitting group, respectively.

3. A kit for tissue in-situ hybridization diagnosis and detection of BK polyomavirus (BKV) comprises a probe for detecting BKV, and is characterized in that the sequence of the probe is as shown in SEQ ID Nos. 1 to 40 or a complementary chain thereof.

4. A probe set for tissue in situ hybridization diagnosis and detection of BK polyomavirus (BKV), which is characterized by consisting of 40 probes, wherein the sequences of the probes are respectively shown as SEQ ID Nos. 1 to 40 or complementary strands thereof.

5. Use of a panel of probes according to claim 4 for the preparation of diagnostic or detection reagents for BK polyomavirus (BKV).

6. Use according to claim 5, wherein said diagnosis or detection is in particular: BKV, SV40 and JCV are distinguished.