Method for detecting HPV E6 / E7 mRNA expression in cells and detection kit

Abstract

The invention discloses a method for detecting HPV E6 / E7 mRNA expression in cells and a detection kit. A lock-type probe is combined with HPV E6 / E7 mRNA in the cells in a targeted mode, signal amplification is achieved through rolling circle amplification, a rolling circle amplification product is detected through an HRP enzyme labeled detection probe, and HRP enzyme is catalyzed and developed under the action of a chromogenic substrate. According to the invention, accurate shunting of low-level and atypical cytopathy can be realized, the operation is simple, and more tumor patients needing cervical cancer detection can be covered.

Description

Technical Field

[0001] The present invention belongs to the technical field of RNA expression detection, and particularly relates to a method for detecting the expression of HPV E6 / E7 mRNA in cells. Background Art

[0002] In 2021, cervical cancer has become the most common gynecological reproductive tract malignant tumor globally. In recent years, the phenomenon of low-age onset has increased significantly. Its occurrence can be effectively controlled through the examination and treatment of precancerous lesions. The incidence of cervical cancer can be reduced by 70%-90% in the population with close screening. Persistent infection with high-risk human papillomavirus (HR-HPV) is a necessary factor for the occurrence of cervical cancer and precancerous lesions. That is, during the process of cervical carcinogenesis, HPV infection is the most critical link. In a woman's lifetime, the probability of being infected with high-risk HPV is more than 70%, but only less than 10% of women develop cervical cancer or cervical intraepithelial neoplasia (CIN). The main reason is that 80% of women's HPV infections are transient.

[0003] HPV E6 and E7 viral oncoproteins play a key role in promoting cell carcinogenesis. HPV infection begins in the basal layer of stratified squamous epithelium. Through continuous infection of basal cells, the epithelial upper basal layer is formed, and the viral genome replication switches to a high-copy number mode. Then, virus particles are released during epithelial desquamation, resulting in the infection of adjacent cells. The HPV genome can integrate with the host genome or remain in an episomal form. 83% of HPV-positive cervical cancer cases show evidence of HPV genome integration into host cells. The E6 protein is generally present in the cell membrane and nucleus, and the E6 protein is closely related to the transforming activity and cell proliferation activity of the virus. The protein E7 is mainly distributed in the nucleus and plays an important role in cell malignant transformation. In addition, an important function of the E7 oncoprotein is to directly inactivate the key factors in the G1-S phase transition, and these key factors tightly regulate cell proliferation. E6 and E7 encode two oncoproteins. When the viral DNA integrates with the host cell genome, it leads to the overexpression of the E6 and E7 genes, which is the main cause of HPV carcinogenesis.

[0004] Currently, the main detection target of common HPV detection methods is HPV DNA. However, a positive HPV DNA test only indicates the presence of the virus in cells, but does not show whether it has integrated into the host genome and caused cell lesions. Moreover, most HPV infections are transient and will eventually be cleared by the human immune system. Therefore, over-reliance on HPV DNA test results has problems of high false positives and low specificity, and misdiagnosis can cause great psychological and financial burdens on patients. Currently, the tissue biopsy, which is the gold standard for diagnosis, is cumbersome, and the p16 immunohistochemistry, which is used as an auxiliary basis, depends on the experience of pathologists. The interpretation of p16 is subjective, and there is no unified standard to determine the positive and negative immunohistochemical cut-off points. Detecting HPV E6 / E7 mRNA is considered the "gold standard" for tumor pathological molecular detection. Using RNA in-situ detection technology, the infection status of HPV virus in tumor cells can be observed more intuitively. However, the current HPV RNA detection technology has the disadvantages of high cost and complex operation, and has not been popularized yet. Summary of the Invention

[0005] One object of the present invention is to provide a composition for detecting the expression of HPV E6 / E7 mRNA in cells, which is characterized by comprising SEQ ID NO: 1 - SEQ ID NO: 15.

[0006] Another object of the present invention is to provide a kit for detecting the expression of HPV E6 / E7 mRNA in cells, which comprises the above composition.

[0007] A third object of the present invention is to provide a method for detecting the expression of HPV E6 / E7 mRNA in cells, which is simple to operate, can cover more tumor patients in need of cervical cancer detection, and provides valuable reference information for the clinical diagnosis and treatment of HPV patients.

[0008] The technical solution of the present invention is as follows:

[0009] A method for detecting the expression of HPV E6 / E7 mRNA in cells, comprising the following steps:

[0010] (1) Obtain the HPV gene sequence, screen the highly specific target recognition sequence, perform reverse complementation on it, and then insert an irrelevant sequence in the middle of the two sequences to obtain a complete padlock probe; design its corresponding rolling circle amplification primer and detection probe according to the padlock probe;

[0011] (2) Drop the cells to be tested into the staining groove fixed on the glass slide, add a fixing agent to fix the RNA molecules in the cells to be tested;

[0012] (3) After the above-mentioned padlock probe enters the sample to be tested and specifically binds to the target sequence, the 3' end and 5' end of the padlock probe are adjacent to each other head and tail, and are ligated by DNA ligase to form a circular template. Then, a rolling circle amplification primer that specifically binds to the irrelevant sequence in the middle is added, and rolling circle amplification is carried out under the action of DNA polymerase to obtain a rolling circle amplification product;

[0013] (4) After hybridizing the rolling circle amplification product with the detection probe, in situ detection of HPV E6 / E7 mRNA is carried out;

[0014] (5) The developed cells are immersed in hematoxylin staining solution and EA / OG staining solution for staining and mounting, and observed under a microscope.

[0015] In a preferred embodiment of the present invention, the padlock probe used in the method is the sequence shown in SEQ ID NO: 1-NO: 13, the rolling circle amplification primer used in the method is the sequence shown in SEQ ID NO: 14, and the detection probe used in the method is the sequence shown in SEQ ID NO: 15.

[0016] In a preferred embodiment of the present invention, the detection probe is labeled with HRP to detect the rolling circle amplification product. Under the action of DAB, the HRP enzyme catalyzes the development.

[0017] In a preferred embodiment of the present invention, the cells to be tested include artificially cultured cells, liquid-based thin-layer cells, and cells dissociated from tissues.

[0018] The beneficial effects of the present invention are as follows: The present invention provides a set of padlock probes for detecting the expression of HPV E6 / E7 mRNA, and the detection probe labeled with HRP more conveniently realizes the visualization of genes, highly integrates in situ detection technology, rolling circle amplification technology, and cytology technology, and realizes the detection and analysis of HPV E6 / E7 mRNA in cells. Brief Description of the Drawings

[0019] The present invention will be further described below with reference to the drawings and embodiments.

[0020] Figure 1 It is the experimental principle diagram of the present invention.

[0021] Figure 2 It is the experimental result diagram of Example 1 of the present invention.

[0022] Figure 3 It is one of the experimental result diagrams of Example 2 of the present invention.

[0023] Figure 4 It is the second experimental result diagram of Example 2 of the present invention. Detailed Embodiments

[0024] The technical solutions of the present invention will be further described and illustrated through specific embodiments below.

[0025] Example 1 Detection of HPV E6 / E7 mRNA on HeLa cell slides

[0026] 1. Cell culture and fixation

[0027] After culturing HeLa cells in RPMI 1640 (containing 10% FBS) for 48 h, they were treated with trypsin to form a cell suspension, seeded on sterile slides with polylysine on the surface, and re-cultured for 24 h. They were rinsed 3 times with DEPC-PBS for 3 min each time; fixed with 4% PFA prepared with DEPC-PBS at room temperature for 30 min; rinsed twice again with DEPC-PBS, and then dehydrated with gradient ethanol: 70%, 85%, 100% for 5 min each, and air-dried.

[0028] 2. Pretreatment of cell samples

[0029] The cells were permeabilized by adding 0.1 M HCl and incubated at room temperature for 5 min. After washing twice with DEPC-PBS containing 0.1% (v / v) Tween 20, they were washed twice with DEPC-PBST. Subsequently, they were incubated with an endogenous peroxidase blocking solution (3% H 2 O 2 solution) at room temperature for 5 min to reduce the activity of endogenous peroxidase in the samples, and washed three times with DEPC-PBST for 5 min each.

[0030] 3. In-situ nucleic acid detection, specifically including the following steps:

[0031] (1) Recognition hybridization of probe and target gene

[0032] 50 μL of a hybridization reaction mixture containing a final concentration of 10% formamide, 6×SSC, and 0.1 μM probe was added to the sample and incubated at 37 °C for 2 h to allow the probe to hybridize directly and complementarily with the mRNA molecules. The padlock probes hybridizing with the target gene have a recognition sequence specifically complementary to the target sequence at the 5' and 3' ends respectively, and the probe sequences are shown in Table 1.

[0033] Table 1

[0034]

[0035]

[0036] (2) Probe ligation into a loop

[0037] After the padlock probes hybridize with the target gene sequences, they are washed three times with DEPC-PBST. Then, 50 μL of a ligation reaction mixture containing glycerol at a final concentration of 50%, BSA at 0.2 μg / μL, 1× SplintR buffer (NEB), SplintR ligase at 0.1 U / μL (NEB), and RiboLock RNase inhibitor at 1 U / μL (Thermo) is added to the sample and incubated at 37 °C for 1 h.

[0038] (3) Rolling circle amplification primer hybridization

[0039] The sample is washed three times with 2× SSC and 20% formamide, 5 min each time, and then washed three times with DEPC-PBST. Next, 50 μL of a hybridization reaction mixture containing formamide at a final concentration of 10%, 6× SSC, and 0.1 μM primer is added to the sample and incubated at 37 °C for 0.5 h to allow the primer to hybridize with the padlock probes bound to the target sequences.

[0040] (4) Rolling circle amplification

[0041] After washing three times with DEPC-PBST, 50 μL of a hybridization mixture containing glycerol at a final concentration of 5%, BSA at 0.2 μg / μL, 1× Phi29 polymerase buffer (Thermo), 1 mM dNTPs, and Phi29 DNA polymerase at 1 U / μL (Thermo) is added to the sample and reacted at 37 °C for 5 h.

[0042] (5) HRP enzyme-labeled probe detection

[0043] After washing three times with DEPC-PBST, 50 μL of a hybridization mixture containing glycerol at a final concentration of 20%, BSA at 0.4 μg / μL, 1× Phi29 polymerase buffer (Thermo), 0.05% Tween 20, 2× SSC, and 0.1 μM HRP probe is added to the sample and incubated at room temperature for 0.5 h.

[0044] (6) HRP-DAB development

[0045] After washing three times with DEPC-PBST, 50 μL of DAB reaction solution (DAB buffer: DAB Substrate (20×): DAB Chromogen (20×) = 18:1:1) is added to the sample and incubated for 1 - 3 min.

[0046] (7) Hematoxylin staining

[0047] 1) The sample is immersed and washed three times with DEPC-PBST, 3 min each time;

[0048] 2) After immersing the glass slide in hematoxylin staining solution twice, let it stand for 5 min for staining;

[0049] 3) Rinse the glass slide with running water for 30 s to wash away the residual hematoxylin staining solution;

[0050] 4) Immerse the glass slide in 0.5% hydrochloric acid alcohol differentiation solution for 4 - 5 s for differentiation;

[0051] 5) Rinse the glass slide with running water for 30 s to wash away the residual 0.5% hydrochloric acid alcohol differentiation solution;

[0052] 6) After immersing the glass slide in 1×TBST buffer twice, let it stand for 1 min for bluing;

[0053] 7) Rinse the glass slide with running water for 30 s to wash away the residual 1×TBST buffer;

[0054] 8) Immerse in 95% ethanol I and II for 20 s each, and 100% ethanol I and II for 20 s each;

[0055] 9) Dry in an oven, seal with neutral resin, and observe under a microscope.

[0056] The results are shown in Figure 1 、 2 ( Figure 1 Schematic diagram of HPV E6 / E7 mRNA detection. After the padlock probe hybridizes to the target sequence, it undergoes rolling circle amplification to amplify the signal, binds to the detection probe that can make the substrate develop color, and catalyzes the development of the image. Figure 2 The blue - purple ones are Hela cell nuclei, and the brown signal dots around the cell nuclei are the detection results of HPV E6 / E7 mRNA).

[0057] Example 2 Detection of HPV mRNA E6 / E7 on liquid - based thin - layer cell samples (TCT)

[0058] 1. Preparation of liquid - based thin - layer cell samples:

[0059] (1) After shaking the high - grade lesion cervical exfoliated cell sample on an oscillator, take 2 mL of the sample and add it to 6 mL of cell dilution solution, and shake again for 30 s;

[0060] (2) Add 4 mL of cell extraction solution to a centrifuge tube. Filter the diluted 8 mL cell sample and add it to the tube. Centrifuge at 1000 rpm for 2 min; aspirate and discard the upper 8 mL of liquid, centrifuge at 2000 rpm for 10 min, and discard the supernatant;

[0061] (3) Add 500 μL of buffer to the centrifuge tube and shake for 25 s;

[0062] (4) Fix the staining groove on the glass slide, transfer the cell suspension mixed in the centrifuge tube, let it stand for natural precipitation for 10 min, discard the excess liquid, add the rinsing solution (absolute ethanol: isopropanol = 1:1), and discard it after 30 s.

[0063] (5) Rinse with DEPC-PBS, 3×3 min; fix with 4% PFA prepared with DEPC-PBS at room temperature for 30 min; after rinsing twice with DEPC-PBS again, dehydrate with gradient ethanol: 70%, 85%, 100% for 5 min each, and air dry.

[0064] 2. Pretreatment of cell samples

[0065] Add 0.1 M HCl to permeabilize the cells and incubate at room temperature for 5 min. After washing twice with DEPC-PBS containing 0.1% (v / v) Tween 20, wash twice with DEPC-PBST. Subsequently, incubate with the endogenous peroxidase blocking solution (3% H 2 O 2 solution) at room temperature for 5 min to reduce the activity of endogenous peroxidase in the sample, and wash three times with DEPC-PBST, 5 min each time.

[0066] 3. In-situ nucleic acid detection, specifically including the following steps:

[0067] (1) Recognition hybridization of the probe and the target gene

[0068] Add 50 μL of the hybridization reaction mixture containing 10% formamide, 6×SSC and 0.1 μM probe at the final concentration to the sample, and incubate at 37 °C for 2 h to enable the probe to specifically hybridize and complement with the mRNA molecules in situ. The padlock probe hybridized with the target gene has a recognition sequence specifically complementary to the target sequence at the 5' and 3' ends respectively. The probe sequence is shown in Table 1.

[0069] (2) Ligation of the probe into a loop

[0070] After the padlock probe hybridizes with the target gene target sequence, wash three times with DEPC-PBST, and continue to add 50 μL of the ligation reaction mixture containing 50% glycerol, 0.2 μg / μL BSA, 1×SplintR buffer (NEB), 0.1 U / μL SplintR ligase (NEB) and 1 U / μL RiboLock RNase inhibitor (Thermo) to the sample, and incubate at 37 °C for 1 h.

[0071] (3) Hybridization of the rolling circle amplification primer

[0072] Wash three times with 2×SSC and 20% formamide for 5 min each time, and then wash three times with DEPC-PBST. Continue to add 50 μL of hybridization reaction mixture containing 10% formamide, 6×SSC, and 0.1 μM primer to the sample, and incubate at 37 °C for 0.5 h to hybridize the primer with the padlock probe bound to the target sequence.

[0073] (4) Rolling circle amplification

[0074] After washing three times with DEPC-PBST, add 50 μL of hybridization mixture containing 5% glycerol, 0.2 μg / μL BSA, 1×Phi29 polymerase buffer (Thermo), 1 mM dNTPs, and 1 U / μL Phi29 DNA polymerase (Thermo) to the sample, and react at 37 °C for 5 h.

[0075] (5) Detection with HRP-labeled probe

[0076] After washing three times with DEPC-PBST, add 50 μL of hybridization mixture containing 20% glycerol, 0.4 μg / μL BSA, 1×Phi29 polymerase buffer (Thermo), 0.05% Tween 20, 2×SSC, and 0.1 μM HRP probe to the sample, and incubate at room temperature for 0.5 h.

[0077] (6) HRP-DAB development

[0078] After washing three times with DEPC-PBST, add 50 μL of DAB reaction solution (DAB buffer: DAB Substrate (20х): DAB Chromogen (20х) = 18:1:1) to the sample and incubate for 1 - 3 min.

[0079] (7) Papanicolaou staining

[0080] 1) Immerse and wash three times with DEPC-PBST for 3 min each time;

[0081] 2) Add 500 μL of hematoxylin staining solution to the staining tank and stain for 2 min;

[0082] 3) Add 500 μL of buffer and wash twice to remove the residual hematoxylin staining solution;

[0083] 4) Add 500 μL of rinsing solution and rinse once;

[0084] 5) Add 500 μL of EA / OG staining solution and stain for 1 min;

[0085] 6) Add 500 μL of rinsing solution and rinse twice;

[0086] 7) Rinse the glass slide with running water for 30 s to wash away the residual 1×TBST buffer;

[0087] 8) Immerse in 95% ethanol I and II for 20 s each, and immerse and shake in 100% ethanol I and II for 20 s each;

[0088] 9) Dry in an oven, seal the slide with neutral resin, and observe under a microscope.

[0089] The results are shown in Figure 3 and 4 . ( Figure 3 For the HPV E6 / E7 test results on the liquid-based thin-layer cell samples of low-grade lesions, there are few signal points. Figure 4 For the HPV E6 / E7 test results on the liquid-based thin-layer cell samples of high-grade lesions, there are many signal points).

Claims

1. A composition for detecting the expression of HPV E6 / E7 mRNA in cells, characterized in that, it comprises SEQ ID NO: 1 - SEQ ID NO:

15.

2. A kit for detecting the expression of HPV E6 / E7 mRNA in cells, characterized in that, the kit comprises the composition according to claim 1.

3. A method for detecting the expression of HPV E6 / E7 mRNA in cells, characterized in that, it comprises the following steps: (1) Obtain the HPV gene sequence, screen out a highly specific target recognition sequence, perform reverse complementation on it, and then insert an irrelevant sequence in the middle of the two sequences to obtain a complete padlock probe; design its corresponding rolling circle amplification primer and detection probe according to the padlock probe; (2) Drop the cells to be tested into a staining trough fixed on a glass slide, add a fixative to fix the RNA molecules in the cells to be tested; (3) After the padlock probe in step (1) enters the sample to be tested and specifically binds to the target sequence, the 3' end and 5' end of the padlock probe are adjacent to each other head and tail, and it is ligated by DNA ligase to form a circular template, then add a rolling circle amplification primer that specifically binds to the middle irrelevant sequence, and perform rolling circle amplification under the action of DNA polymerase to obtain a rolling circle amplification product; (4) Hybridize the rolling circle amplification product with the detection probe, and then perform in-situ detection of HPV E6 / E7 mRNA; (5) Immerse the developed cells in hematoxylin staining solution and EA / OG staining solution for staining and mounting, and observe under a microscope.

4. The method for detecting the expression of HPV E6 / E7 mRNA in cells according to claim 3, characterized in that, in step (1), the padlock probe used is the sequence shown in SEQ ID NO: 1 - NO: 13, the rolling circle amplification primer used is the sequence shown in SEQ ID NO: 14, and the detection probe used is the sequence shown in SEQ ID NO:

15.

5. The method for detecting the expression of HPV E6 / E7 mRNA in cells according to claim 4, characterized in that, after the padlock probe hybridizes with the target gene target sequence, wash it three times with DEPC-PBST, add 50 μL of a ligation reaction mixture containing 50% glycerol, 0.2 μg / μL BSA, 1×SplintR buffer (NEB), 0.1 U / μL SplintR ligase (NEB) and 1 U / μL RiboLock RNase inhibitor (Thermo) to the sample, and incubate at 37°C for 1 h.

6. The method for detecting the expression of HPV E6 / E7 mRNA in cells according to claim 4, characterized in that, The conditions for rolling circle amplification are as follows: after washing three times with DEPC-PBST, add 50 μL of hybridization mixture containing glycerol with a final concentration of 5%, BSA at 0.2 μg / μL, 1×Phi29 polymerase buffer (Thermo), 1 mM dNTPs, and 1 U / μL Phi29 DNA polymerase (Thermo) to the sample, and react at 37 °C for 5 h.

7. The method for detecting the expression of HPV E6 / E7 mRNA in cells according to claim 3, characterized in that the detection probe is labeled with HRP to detect the rolling circle amplification product, and under the action of DAB, the HRP enzyme catalyzes the development.

8. The method for detecting the expression of HPV E6 / E7 mRNA in cells according to claim 3, characterized in that The detection conditions for the HRP enzyme-labeled probe are as follows: after washing three times with DEPC-PBST, add 50 μL of hybridization mixture containing glycerol with a final concentration of 20%, BSA at 0.4 μg / μL, 1×Phi29 polymerase buffer (Thermo), 0.05% Tween 20, 2×SSC, and 0.1 μM HRP probe to the sample, and incubate at room temperature for 0.5 h.

9. The method for detecting the expression of HPV E6 / E7 mRNA in cells according to claim 3, characterized in that in step (2), the cells to be tested include at least one of artificially cultured cells, liquid-based thin-layer cells, and cells dissociated from tissues.

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