Human papilloma virus mRNA detection kit for auxiliary diagnosis of cervical cancer patients

Abstract

The invention discloses a human papilloma virus mRNA (messenger ribonucleic acid) detection kit for auxiliary diagnosis of cervical cancer patients. The human papilloma virus mRNA detection kit comprises a nucleic acid probe group and a series of nucleic acid probes AMP1-AMP5, wherein the nucleic acid probe group is used for being combined with a human papilloma virus mRNA target, and the nucleic acid probes AMP1-AMP5 are used for signal amplification. The human papilloma virus mRNA detection kit provided by the invention can assist pathologists in identifying and diagnosing cervical cancer patients, and provides a basis for clinicians to formulate a therapeutic schedule.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pathological detection, and particularly relates to a human papillomavirus mRNA detection kit for auxiliary diagnosis of cervical cancer patients. BACKGROUND

[0002] Cervical cancer is one of the common gynecological malignant tumors, and its incidence rate ranks the second in female malignant tumors in China, following breast cancer. However, it is worth noting that the average age of cervical cancer in recent years is gradually decreasing, and there is a trend of youth. Therefore, it is necessary to standardize the diagnosis and treatment of cervical cancer in the country. On the other hand, the occurrence of cervical cancer can be effectively controlled through the examination and treatment of precancerous lesions. The experience of western countries shows that the incidence of cervical cancer in closely screened populations has been reduced by 70%-90%.

[0003] It has been confirmed that persistent infection of high-risk human papillomavirus (HPV) is a necessary factor for the occurrence of cervical cancer and precancerous lesions, that is, HPV infection is the most critical link in the process of cervical cancer. In a woman's life, the probability of infection with high-risk HPV is more than 70%, but less than 10% of women develop cervical cancer or cervical intraepithelial neoplasia (CIN), the main reason is that 80% of women's HPV infection is transient. In addition to the role of persistent high-risk HPV infection, other endogenous and exogenous factors also need to participate and act together to cause the occurrence of cervical cancer. Therefore, the risk factors that trigger cervical cancer can be divided into two categories: one is biological factors, that is, persistent infection of high-risk HPV; the other is exogenous behavioral risk factors.

[0004] The HPV detection method has a high false negative rate (6%-8%), so it is essential to accurately detect high-risk HPV in cervical lesions. Current HPV detection methods include hybrid capture II (HC-II), HPV DNA in situ hybridization, polymerase chain reaction (PCR), and HPV mRNA in situ hybridization (RISH), which have different sensitivities and specificities. Because the expression level of HPV mRNA is low, the traditional RNA in situ hybridization detection technology cannot detect obvious positive signals, which leads to false negative detection results. In view of this, it is necessary to study a human papillomavirus mRNA detection kit for auxiliary diagnosis of cervical cancer patients. SUMMARY

[0005] In order to solve the problems of the prior art, the purpose of the present application is to provide a human papillomavirus mRNA detection kit for auxiliary diagnosis of cervical cancer patients, which can assist pathologists in differential diagnosis of cervical cancer patients and provide a basis for clinical doctors to develop treatment plans through in situ hybridization detection technology.

[0006] To achieve the above object, the present application adopts the technical solutions as follows:

[0007] The human papillomavirus mRNA detection kit for auxiliary diagnosis of cervical cancer patients comprises:

[0008] The nucleic acid probe set comprises a plurality of nucleic acid probes, the nucleic acid probe comprises a complementary pairing region combined with a human papillomavirus mRNA target and a signal amplification binding region combined with a signal amplification probe set, the complementary pairing region and the signal amplification binding region are connected through C6 Spacer spatial modification, the sequence of the complementary pairing region of the plurality of nucleic acid probes is shown in SEQ ID NO: 1-20, and the sequence of the signal amplification binding region is shown in SEQ ID NO: 21.

[0009] The signal amplification probe set comprises nucleic acid probe AMP1, nucleic acid probe AMP2, nucleic acid probe AMP3, nucleic acid probe AMP4 and nucleic acid probe AMP5.

[0010] Preferably, the structures of the nucleic acid probe AMP1, the nucleic acid probe AMP2, the nucleic acid probe AMP3 and the nucleic acid probe AMP4 are all "L" type, all have the signal amplification connection region b at the bottom, and all have the signal amplification connection region a at the 5' end.

[0011] Preferably, the sequence of the nucleic acid probe AMP1 is shown in SEQ ID NO: 22, and the signal amplification connection region b is combined with the signal amplification binding region with the sequence shown in SEQ ID NO: 21.

[0012] Preferably, the sequence of the nucleic acid probe AMP2 is shown in SEQ ID NO: 23, and the signal amplification connection region b is combined with the signal amplification connection region a of the nucleic acid probe AMP1.

[0013] Preferably, the sequence of the nucleic acid probe AMP3 is shown in SEQ ID NO: 24, and the signal amplification connection region b is combined with the signal amplification connection region a of the nucleic acid probe AMP2.

[0014] Preferably, the sequence of the nucleic acid probe AMP4 is shown in SEQ ID NO: 25, and the signal amplification connection region b is combined with the signal amplification connection region a of the nucleic acid probe AMP3.

[0015] Preferably, the sequence of the nucleic acid probe AMP5 is shown in SEQ ID NO: 26, and the 5' and 3' ends are both modified with a digoxin molecule (D) combined with the signal amplification connection region a of the nucleic acid probe AMP4.

[0016] Preferably, the signal amplification binding region with the sequence shown in SEQ ID NO: 21 is located at the 3' end of the nucleic acid probe, and has a length of 12-16 bp.

[0017] Preferably, the structural formula of the C6 Spacer is:

[0018]

[0019] The advantages of this invention are:

[0020] (1) The detection kit of the present invention provides 10 sets of in situ hybridization probes targeting different regions of human papillomavirus mRNA, which can avoid false negative results caused by RNA degradation, assist pathologists in differentiating and diagnosing patients with cervical cancer caused by human papillomavirus, and provide a basis for clinicians to formulate treatment plans.

[0021] (2) The detection kit of the present invention can amplify the mRNA signal by hundreds of times through a cascade amplification process, which greatly increases the sensitivity of the in situ hybridization detection technology. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the signal amplification principle of the detection kit in this invention;

[0023] Figure 2 The staining results of the kits in the experimental group and control groups 1-3 are shown in the figure (A, experimental group, B, control group 1, C, control group 2, D, control group 3).

[0024] Figure 3 Comparison of incubation temperatures for human papillomavirus mRNA probes in tissue samples ((A) 40℃ incubation; (B) 37℃ incubation).

[0025] Figure 4 For comparison of different repair methods ((A) incubation at 100℃ for 5 min; (B) incubation at 100℃ for 10 min; (C) incubation at 100℃ for 15 min). Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0027] Example 1: Design of a human papillomavirus mRNA in situ hybridization detection probe

[0028] like Figure 1 As shown, the human papillomavirus (HPV) mRNA in situ hybridization detection probe comprises two parts: a complementary pairing region and a signal amplification binding region. The complementary pairing region can pair with the base sequence of HPV mRNA to achieve probe binding; the signal amplification binding region can bind to the signal amplification linker b of AMP1 to achieve cascaded signal amplification. Similarly, the AMP2-AMP4 probes also contain two signal amplification linker regions, with the same function as the HPV mRNA in situ hybridization detection probe.

[0029] The complementary pairing region and the signal amplification binding region play the role of linker to increase the spatial distance through the special C6 Spacer spatial sub-modification structure, preventing the complementary pairing region and the signal amplification binding region from affecting the base complementary pairing of each other due to the reason of spatial steric hindrance. Moreover, this sequence will not be complementary to the base sequence of nucleic acid, which can increase the specificity of in situ hybridization probe. The sequences of the human papillomavirus mRNA in situ hybridization detection probe and the signal amplification probe are shown in Table 1.

[0030] Table 1 Sequences of human papillomavirus mRNA in situ hybridization detection probe and signal amplification probe

[0031]

[0032]

[0033] Among them, the sequences of the 20 nucleic acid probes in the nucleic acid probe group are as follows: SEQ ID NO: 1-C6 Spacer-SEQ ID NO: 21; SEQ ID NO: 2-C6 Spacer-SEQ ID NO: 21; SEQ ID NO: 3-C6 Spacer-SEQ ID NO: 21; SEQ ID NO: 4-C6 Spacer-SEQ ID NO: 21; SEQ ID NO: 5-C6 Spacer-SEQ ID NO: 21; SEQ ID NO: 6-C6 Spacer-SEQ ID NO: 21; SEQ ID NO: 7-C6 Spacer-SEQ ID NO: 21; SEQ ID NO: 8-C6 Spacer-SEQ ID NO: 21;

[0034] SEQ ID NO: 9-C6 Spacer-SEQ ID NO: 21;

[0035] SEQ ID NO: 10-C6 Spacer-SEQ ID NO: 21;

[0036] SEQ ID NO: 11-C6 Spacer-SEQ ID NO: 21;

[0037] SEQ ID NO: 12-C6 Spacer-SEQ ID NO: 21;

[0038] SEQ ID NO: 13-C6 Spacer-SEQ ID NO: 21;

[0039] SEQ ID NO: 14 - C6 Spacer - SEQ ID NO: 21;

[0040] SEQ ID NO: 15 - C6 Spacer - SEQ ID NO: 21;

[0041] SEQ ID NO: 16 - C6 Spacer - SEQ ID NO: 21;

[0042] SEQ ID NO: 17 - C6 Spacer - SEQ ID NO: 21;

[0043] SEQ ID NO: 18 - C6 Spacer - SEQ ID NO: 21;

[0044] SEQ ID NO: 19 - C6 Spacer - SEQ ID NO: 21;

[0045] SEQ ID NO: 20 - C6 Spacer - SEQ ID NO: 21.

[0046] The length of the signal amplification binding region of the nucleic acid probe in the nucleic acid probe set is a range value (12-16 bp in length) because it is not a single continuous sequence. If the length is too long, there is a greater probability of binding to the nucleic acid sequence of the non-target region, resulting in reduced specificity. If the length is too short, it will not be firmly combined with the signal amplification sequence, resulting in reduced sensitivity.

[0047] Example 2, the staining method of the human papilloma virus mRNA detection kit, comprising the following specific steps:

[0048] 1. Tissue section pretreatment

[0049] 1.1 65-70℃ baking sheet fixation for 1-2h;

[0050] 1.2 Detergent for 5min, repeat 3 times;

[0051] 1.3 Anhydrous ethanol, 95% ethanol, 75% ethanol gradient washing 3min, TBS buffer flushing once;

[0052] 1.4 Dry moisture, draw a circle around the tissue with a water-resistant pen 2-3mm, drop the peroxidase blocking agent to completely cover the tissue sample, and place it at room temperature for 10min, then wash it with TBS buffer for 2 times;

[0053] 2. Repair

[0054] 2.1 Put the tissue section into the repair solution, take it out after boiling water bath for 15min;

[0055] 2.2 Dry the water, add 100 μL pepsin to the slice to completely cover the slice, incubate at 37°C for 2-5 min, then rinse twice with TBS buffer;

[0056] 3. Probe hybridization

[0057] 3.1 Add 100 μL HPV probe to the slice to completely cover the slice;

[0058] 3.2 Incubate the slice at 40°C for 2 h, then rinse twice with TBS buffer;

[0059] 4. Probe signal amplification

[0060] 4.1 Wash twice with TBS buffer to remove excess HPV probe, then add 100 μL nucleic acid probe AMP1, incubate at 40°C for 30 min;

[0061] 4.2 Wash twice with TBS buffer to remove excess nucleic acid probe AMP1, then add 100 μL nucleic acid probe AMP2, incubate at 40°C for 30 min;

[0062] 4.3 Wash twice with TBS buffer to remove excess nucleic acid probe AMP2, then add 100 μL nucleic acid probe AMP3, incubate at 40°C for 30 min;

[0063] 4.4 Wash twice with TBS buffer to remove excess nucleic acid probe AMP3, then add 100 μL nucleic acid probe AMP4, incubate at 40°C for 30 min;

[0064] 4.5 Wash twice with TBS buffer to remove excess nucleic acid probe AMP4, then add 100 μL nucleic acid probe AMP5, incubate at 40°C for 30 min;

[0065] 5. Antibody incubation: Dry the water, add 100 μL digoxin antibody to completely cover the slice, incubate at 37°C for 30 min, then rinse twice with TBS buffer;

[0066] 6. Color re-staining

[0067] 6.1 DAB color development: Add 100 μL of freshly prepared DAB color developing solution, incubate at room temperature for 10 min, then rinse three times with TBS buffer;

[0068] 6.2 Hematoxylin color development: Add 100 μL of hematoxylin staining solution, incubate at room temperature for 3 min, then rinse three times with TBS buffer,

[0069] 7. Dehydration and mounting

[0070] 7.1 75% ethanol, 95% ethanol, anhydrous ethanol gradient dehydration for 3 min each;

[0071] 7.2 Xylene room temperature washing for 3 min, repeated 3 times, and mounted with neutral resin;

[0072] 7.3 Reading: positive localization is mainly in the cytoplasm.

[0073] Example 3, staining effect of human papillomavirus mRNA detection kit

[0074] In order to increase the sensitivity of human papillomavirus mRNA in situ hybridization detection, the probes corresponding to SEQ ID NO: 1-20 are mixed, the signal cascade amplification and staining are carried out by using the staining method in Example 2, and experimental groups and control groups 1-3 are set. Experimental group: adding AMP1, AMP2, AMP3, AMP4, AMP5; control group 1: adding AMP1, AMP2, AMP3, AMP5; control group 2: adding AMP1, AMP2, AMP5; control group 3: adding AMP1, AMP5. The staining effects of the detection kits of the experimental group and control groups 1-3 are tested respectively, and the results are shown in Figure 2 .

[0075] As shown in Figure 2 , with the increase of the number of signal amplification, the staining effect is enhanced, and the staining intensity of the experimental group with 5-stage signal amplification Figure 3 (A) is the highest, without any non-specific coloring, and the contrast is strong.

[0076] Example 4, comparison of HPV probe incubation temperature in tissue samples

[0077] The operation process of the detection kit of this example is the same as that of Example 2, and the specific difference is that the HPV probe incubation temperature is set to 37℃.

[0078] As shown in Figure 3 , by comparison, it can be known that the HPV probe in the tissue sample has the best effect when incubated at 40℃.

[0079] Example 5, comparison of different denaturation conditions of human papillomavirus mRNA in tissue samples

[0080] The human papillomavirus mRNA in the tissue sample can form a secondary structure, and the present application can eliminate the secondary structure of the human papillomavirus mRNA by high-temperature boiling repair, i.e. incubating at 100℃ for 5, 10 and 15 min and then quickly cooling in cold water, so that the probe can be combined smoothly. The operation process of the detection kit of this example is the same as that of Example 2.

[0081] As shown in Figure 4As shown, the staining effect of human papilloma virus mRNA in the tissue sample can be best achieved by incubation at 100℃ for 15 min in the case of high-temperature boiling repair.

[0082] The basic principles, main features and advantages of the present application are shown and described above. Those skilled in the art should understand that the above examples do not limit the present application in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the protection scope of the present application.

Claims

1. A human papillomavirus mRNA detection kit for the auxiliary diagnosis of cervical cancer patients, characterized by, Comprise: A nucleic acid probe set comprising a plurality of nucleic acid probes, the nucleic acid probes comprising a complementary pairing region that binds to a human papillomavirus mRNA target and a signal amplification binding region that binds to a signal amplification probe set, the complementary pairing region and the signal amplification binding region being connected by a C6 Spacer steric modification, the sequence of the complementary pairing region of the plurality of nucleic acid probes being as set forth in SEQ ID NOs: 1-20, the sequence of the signal amplification binding region being as set forth in SEQ ID NO: 21; The signal amplification probe set comprises nucleic acid probe AMP1, nucleic acid probe AMP2, nucleic acid probe AMP3, nucleic acid probe AMP4 and nucleic acid probe AMP5.

2. The human papillomavirus mRNA detection kit for the auxiliary diagnosis of cervical cancer patients according to claim 1, characterized by, The structures of nucleic acid probe AMP1, nucleic acid probe AMP2, nucleic acid probe AMP3 and nucleic acid probe AMP4 are all "L" type, all have signal amplification connection region b at the bottom, and all have signal amplification connection region a at the 5' end.

3. The human papillomavirus mRNA detection kit for the auxiliary diagnosis of cervical cancer patients according to claim 2, characterized by, The sequence of nucleic acid probe AMP1 is as set forth in SEQ ID NO: 22, and the signal amplification connection region b thereof is combined with the signal amplification binding region having the sequence as set forth in SEQ ID NO:

21.

4. The human papillomavirus mRNA detection kit for the auxiliary diagnosis of cervical cancer patients according to claim 3, characterized by, The sequence of nucleic acid probe AMP2 is as set forth in SEQ ID NO: 23, and the signal amplification connection region b thereof is combined with the signal amplification connection region a of nucleic acid probe AMP1.

5. The human papillomavirus mRNA detection kit for the auxiliary diagnosis of cervical cancer patients according to claim 4, characterized by, The sequence of nucleic acid probe AMP3 is as set forth in SEQ ID NO: 24, and the signal amplification connection region b thereof is combined with the signal amplification connection region a of nucleic acid probe AMP2.

6. The human papillomavirus mRNA detection kit for the auxiliary diagnosis of cervical cancer patients according to claim 5, characterized by, The sequence of nucleic acid probe AMP4 is as set forth in SEQ ID NO: 25, and the signal amplification connection region b thereof is combined with the signal amplification connection region a of nucleic acid probe AMP3.

7. The human papillomavirus mRNA detection kit for the auxiliary diagnosis of cervical cancer patients according to claim 6, characterized by, The sequence of nucleic acid probe AMP5 is as set forth in SEQ ID NO: 26, and is combined with the signal amplification connection region a of nucleic acid probe AMP4, and both the 5' and 3' ends thereof are modified with a digoxin molecule.

8. The human papillomavirus mRNA detection kit for the auxiliary diagnosis of cervical cancer patients according to claim 1, characterized by, The signal amplification binding region having the sequence as set forth in SEQ ID NO: 21 is located at the 3' end of the nucleic acid probe, and has a length of 12-16 bp.

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