CN110607400A

CN110607400A - Tissue in-situ hybridization diagnosis and detection system for BKV and application thereof

Info

Publication number: CN110607400A
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: 2019-12-24
Anticipated expiration: 2039-10-08
Also published as: CN110607400B

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 a corresponding antigen (i.e., protein) by using the principle of antigen-antibody specific binding and performing a color reaction through antibody labeling. BK polyomavirus (BKV), a type of opportunistic infectious virus that is prone to cause severe urinary system disease in immunocompromised people. Currently, no BKV-related immunohistochemical or in situ hybridization patents were detected from patent retrieval systems. There are a number of diagnostic methods in scientific literature relating to BKV immunohistochemistry, 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 antibodies against the large T antigen of BKV are cross-recognized by antibodies against the large T antigen of simian polyoma virus SV 40. 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 closely related JC virus (JCV) can be recognized by the antibody, and the result of the detection cannot be determined as to 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 for 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, JCV and SV40 at the same time and cannot be distinguished, and the like.

Disclosure of Invention

The BKV is a latent infection opportunistic virus which 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, etc., 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 causes inflammatory responses with organ rejection and is 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 from 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 due to the contamination of polio vaccine historically, 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 invention provides a tissue in situ hybridization diagnosis and detection system for BKV, which comprises 40 probes for detecting BKV, and the sequences of the probes are respectively the sequences shown in SEQ ID Nos. 1-40 or complementary strands thereof. Further, the probes are labeled with a chemical dye and a fluorescent luminescent group, respectively.

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.

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

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

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

Further, the diagnosing or detecting is specifically: a distinction is made between BKV, SV40 and JCV.

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 currently known BKV strains.

2. 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 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 targets DNA and mRNA at the same time, particularly the mRNA expression quantity of the 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 of the invention 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 the results of comparing 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 shows that the immunofluorescence detection of the BKV large T antigen has no specificity.

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. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

The following detailed description of the present invention will be made with reference to the accompanying drawings.

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

BKV is a DNA double-stranded virus with a genome of about 5300bp in length, and comprises early coding genes (encoding regulatory proteins large T and small T), late coding genes (encoding Agno protein and virus structural proteins VP1, VP2, VP3), and non-coding 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 bladder cancer, kidney cancer and other BKV persistent infection, the BKV genome is often integrated into the host genome or copied in the form of a double-stranded DNA loop, 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 in the large T expression period is far higher than the DNA level, 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 region.

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. 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 the 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 specificity is also good after the conservative region is compared with JCV and SV40, so that a specific probe is selected. To ensure the specificity of the probe, we first select a representative strain of BKV (SHA-30) and align the labeled conserved regions by sequence array, and represent the completely conserved base sites in lower case letters and the non-conserved sites in upper case letters. The same method as that of the arrangement of the BKV sequences is utilized to arrange 604 JCV sequences on NCBI into an order array, then SHA-30 is brought into the JCV sequence array to carry out manual comparison arrangement, and a segment 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 conserved sites, 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 open spaces above the conserved segment corresponding to the lower case bases of SHA-30 represent greater specificity compared to JCV.

Finally, we selected 40 probes from the conserved regions according to these principles and analysis results, and the probe sequences are shown in the following table. And the synthetic 2-class 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 undergone 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 were negative in CH-K and HJW-K immunohistochemistry results, but positive in HJW-K in situ hybridization results. The right 2 immunohistochemistry and in situ hybridization results were positive. This suggests that our in situ hybridization system may be more sensitive than clinically used immunohistochemical assays. Note: blue arrows point to the 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 the detection of large T antigen, suggesting that this diagnostic method is not specific to the interior of polyoma virus. 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 (PAb416, 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 embryo cells containing stably expressed SV40 large T gene), TCCSUP (human bladder cancer fourth-stage cells) and 5637 (human bladder cancer second-stage cells) are passed to a chamber glass slide, the TCCSUP and 5637 are infected with the BKV virus (MOI is 0.3), and after 5 days, immunofluorescence detection is carried out by using SV40 large T antigen antibody; 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.

As a result, the large T targets of SV40, JCV and BKV in the three groups are positive by 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 transfection 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 experiments, and the cells were detected by fluorescence in situ hybridization detection after the late stage of cell fixation, where green fluorescence is the large T nucleic acid (DNA/mRNA) signal of BKV and blue is DAPI-stained cell nuclei.

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

1) the conservation is high.

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

2) The specificity is strong.

The sequence similarity of the BKV, JCV and SV40 is 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 nonspecific (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 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 targets DNA and mRNA at the same time, particularly the mRNA expression quantity of the 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 can easily cause missed diagnosis and misdiagnosis to cause 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.Areview of hematopoietic cell transplantation in China:data and trends during2008-2016.Bone Marrow Transplant.2017Nov；52(11):1512-1518.

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

[5] haoyongtong, Li Daozuan, Lidong, Lidi, Zheng Rong shou, Zhang Xin Mei, Chenwangqing, He Jie, 2014, China bladder cancer morbidity and mortality analysis, China tumor journal, 2018.40(9),647, 652.

[6] Korean Sujun, Zhang thinking, 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 those 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 regarded 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 BKV is characterized by comprising 40 probes for detecting BKV, wherein the sequences of the probes are respectively the sequences shown as SEQ ID Nos. 1-40 or complementary chains thereof.

2. The system for BKV in-situ tissue hybridization diagnosis and detection according to claim 1, wherein the probes are labeled with a chemical dye and a fluorescent fluorophore, respectively.

3. A kit for tissue in-situ hybridization diagnosis and detection of the BKV comprises a probe for detecting the BKV, and is characterized in that the sequence of the probe is a sequence shown as SEQ ID No: 1-40 or a complementary chain thereof.

4. A nucleotide sequence pair for tissue in-situ hybridization diagnosis and detection of BKV is characterized in that the nucleotide sequence pair is a sequence shown as SEQ ID No. 1-40 or a complementary chain thereof.

5. Use of the pair of nucleotide sequences of claim 4 in the preparation of a diagnostic or detection reagent for BKV.

6. Use according to claim 5, wherein said diagnosis or detection is in particular: a distinction is made between BKV, SV40 and JCV.

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

8. Use of a set of probes according to claim 7 in the preparation of a diagnostic or detection reagent for BKV.

9. Use according to claim 8, wherein said diagnosis or detection is in particular: a distinction is made between BKV, SV40 and JCV.