Kit for detecting mRNA (messenger Ribonucleic Acid) in E6 / E7 regions of 14 high-risk human papilloma viruses

Abstract

The invention discloses a kit for detecting mRNA (messenger Ribonucleic Acid) in E6 / E7 regions of 14 high-risk human papilloma viruses, and relates to the technical field of pathogen detection, and the technical key points are as follows: the kit is used for detecting E6 / E7 mRNA of at least one high-risk HPV, the high-risk HPV comprises HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 types, and the HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 types can be used for detecting the E6 / E7 mRNA of at least one high-risk HPV. The kit comprises a primer probe combination for detecting the 14 high-risk HPVs and a primer probe combination for detecting the reference gene ACTB. By detecting HPV E6 / E7mRNA, the kit can provide higher specificity than traditional DNA detection and reduce false negative results, so that the accuracy of cervical cancer screening is improved, the kit can early recognize high-risk HPV infection, facilitates timely discovery of cervical cancer precancerous lesions and provides possibility for early intervention, and the kit can distinguish at least 14 high-risk HPVs and has good application prospects. Accurate virus genotype information is provided for clinic, and personalized treatment and prognosis evaluation are facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogen detection, and in particular to a kit for detecting 14 high-risk human papillomavirus E6 / E7 region mRNAs. Background Art

[0002] Human papillomavirus (HPV) is the main pathogenic factor of cervical cancer, among which persistent infection with high-risk HPV is the key to the occurrence of cervical cancer and its precancerous lesions. Cervical cancer, as the second most common disease in gynecological tumors, poses a serious threat to women's health worldwide. In 2020, 604,000 women were diagnosed with cervical cancer worldwide, and about 342,000 women died from the disease. Cervical intraepithelial neoplasia (CIN) is an early lesion of cervical cancer, and it takes about ten years for CIN to develop into cervical cancer. The World Health Organization (WHO) recommends HPV-DNA testing as the preferred method for cervical cancer screening, and updated the guidelines in December 2021 to use HPVmRNA testing as a primary screening test for cervical cancer prevention. HPV's E6 and E7 proteins play a key role in the occurrence of cervical cancer, and they promote carcinogenesis by interfering with cell cycle regulation. Therefore, detecting HPV E6 / E7 mRNA has higher specificity than detecting HPV DNA and can more accurately predict cervical precancerous lesions.

[0003] Although domestic HPV DNA detection kits have developed to a mature stage of complete typing, mRNA detection technology is still in its infancy in China. Most mRNA kits can only perform untyping or partial typing tests, such as AptimaHPV, and cannot fully type high-risk HPV. This limitation restricts the accurate prediction and risk assessment of cervical precancerous lesions. Therefore, the development of a kit that can fully type the mRNA of the E6 / E7 region of 14 high-risk human papillomaviruses is of great significance for improving the accuracy and efficiency of cervical cancer screening. This kit can provide more accurate assessment of the risk of canceration and help doctors develop more effective prevention and treatment strategies. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems and provide a kit for detecting 14 high-risk human papillomavirus E6 / E7 region mRNAs.

[0005] In order to achieve the above object, the technical scheme of the present invention is as follows: a kit for detecting 14 types of high-risk human papillomavirus E6 / E7 region mRNA, the kit is used for amplifying and detecting HPV mRNA across the E6 and E7 segments, the kit is used for detecting the E6 / E7 mRNA of at least one high-risk HPV, and the kit comprises the following components:

[0006] (1) Specific primers and probes for detecting the internal reference gene ACTB, wherein the primer sequences are shown in SEQ ID NOs. 1 to 2, and the probe sequence is shown in SEQ ID NO. 3;

[0007] (2) Specific primers and probes for detecting HPV 16 E6 / E7 region mRNA, the primer sequences are shown in SEQ ID NOs. 4 to 5, and the probe sequence is shown in SEQ ID NO. 6;

[0008] (3) Specific primers and probes for detecting HPV 18 E6 / E7 region mRNA, the primer sequences are shown in SEQ ID NOs. 7 to 8, and the probe sequence is shown in SEQ ID NO. 9;

[0009] (4) Specific primers and probes for detecting HPV 31E6 / E7 region mRNA, the primer sequences of which are shown in SEQ ID NOs. 10 to 11, and the probe sequence of which is shown in SEQ ID NO. 12;

[0010] (5) Specific primers and probes for detecting HPV 33E6 / E7 region mRNA, the primer sequences of which are shown in SEQ ID NOs. 13 to 14, and the probe sequence of which is shown in SEQ ID NO. 15;

[0011] (6) Specific primers and probes for detecting HPV 35E6 / E7 region mRNA, the primer sequences of which are shown in SEQ ID NOs. 16 to 17, and the probe sequence of which is shown in SEQ ID NO. 18;

[0012] (7) Specific primers and probes for detecting HPV 39E6 / E7 region mRNA, the primer sequences are shown in SEQ ID NOs. 19 to 20, and the probe sequence is shown in SEQ ID NO. 21;

[0013] (8) Specific primers and probes for detecting HPV 45E6 / E7 region mRNA, the primer sequences are shown in SEQ ID NOs. 22 to 23, and the probe sequence is shown in SEQ ID NO. 24;

[0014] (9) Specific primers and probes for detecting HPV 51E6 / E7 region mRNA, the primer sequences are shown in SEQ ID NO. 25-26, and the probe sequence is shown in SEQ ID NO. 27;

[0015] (10) Specific primers and probes for detecting HPV 52 E6 / E7 region mRNA, the primer sequences are shown in SEQ ID NOs. 28 to 29, and the probe sequence is shown in SEQ ID NO. 30;

[0016] (11) Specific primers and probes for detecting HPV 56 E6 / E7 region mRNA, the primer sequences are shown in SEQ ID NOs. 31 to 32, and the probe sequence is shown in SEQ ID NO. 33;

[0017] (12) Specific primers and probes for detecting HPV 58 E6 / E7 region mRNA, the primer sequences are shown in SEQ ID NO.34-35, and the probe sequence is shown in SEQ ID NO.36;

[0018] (13) Specific primers and probes for detecting HPV 59E6 / E7 region mRNA, the primer sequences are shown in SEQ ID NOs. 37 to 38, and the probe sequence is shown in SEQ ID NO. 39;

[0019] (14) Specific primers and probes for detecting HPV 66 E6 / E7 region mRNA, the primer sequences are shown in SEQ ID NOs. 40 to 41, and the probe sequence is shown in SEQ ID NO. 42;

[0020] (15) Specific primers and probes for detecting HPV 68 E6 / E7 region mRNA, wherein the primer sequences are shown in SEQ ID NOs. 43 to 44, and the probe sequence is shown in SEQ ID NO. 45.

[0021] The present invention is further configured as follows: the kit also includes components for nucleic acid extraction and purification, including lysate, proteinase K, DNase I, and buffer for washing and elution.

[0022] The present invention is further configured as follows: the kit also includes PrimeScript Buffer, PrimeScript RT Enzyme Mix I, Oligo dT Primer, Random 6mers and RNase FreedH2O for reverse transcription reaction.

[0023] The present invention is further configured as follows: the kit also includes TaqProbe2X qPCR-Multiplex Mastermix for multiplex fluorescence quantitative PCR reaction and a fluorescent probe for detection.

[0024] Another object of the present invention is to provide a method for using the above-mentioned human papillomavirus E6 / E7 mRNA typing detection kit, the method comprising the following steps:

[0025] S1: Extract and purify RNA from the sample to be tested;

[0026] S2: prepare cDNA;

[0027] S3: cDNA is detected by multiplex fluorescence quantitative PCR to determine the type of high-risk HPV present in the sample.

[0028] The present invention is further configured as follows: the method further comprises analyzing the PCR product to determine the genotype of HPV.

[0029] Another object of the present invention is to provide the use of the above-mentioned kit in cervical exfoliated cell samples.

[0030] Compared with the prior art, the present solution has the following beneficial effects: the present invention can more directly evaluate the activity and carcinogenic potential of HPV by detecting the E6 / E7 mRNA of HPV. The presence of E6 / E7 mRNA indicates that the virus is actively interfering with the cycle regulation of host cells, which is a key step in the development of cervical cancer. Therefore, this detection method can provide more accurate prognostic information and help doctors better evaluate the disease status and treatment needs of patients; the specific primers and probes in the kit provided by the present invention are designed to cover the E6 / E7 region mRNA of 14 high-risk HPVs, ensuring the high specificity of the detection, which reduces the false positive results caused by the cross reaction of non-target sequences, and also reduces the false negative results caused by the detection limit not being low enough;

[0031] The kit of the present invention integrates a full set of reagents and steps from nucleic acid extraction to reverse transcription and then to multiplex fluorescent quantitative PCR, greatly simplifying the experimental operation. This one-stop solution not only reduces the time and complexity of laboratory preparation, but also reduces errors caused by improper operation and improves the efficiency of the entire detection process. Through precise typing, the kit of the present invention can help doctors determine the specific HPV genotype, which is crucial for formulating personalized treatment plans. Understanding the specific HPV genotype can help doctors choose the most appropriate treatment method, improve treatment effects, and reduce unnecessary treatments and side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1-15 The primer-probe combination standard curves of 1-ACTB, 2-HPV16, 3-HPV18, 4-HPV31, 5-HPV33, 6-HPV35, 7-HPV39, -HPV45, 9-HPV51, 10-HPV52, 11-HPV56, 12-HPV58, 13-HPV59, 14-HPV66, and 15-HPV68 in the embodiments of the present invention are shown in order;

[0033] Figure 16-30They are, respectively, the standard curves of 2-HPV16, 3-HPV18, 4-HPV31, 5-HPV33, 6-HPV35, 7-HPV39, 8-HPV45, 9-HPV51, 10-HPV52, 11-HPV56, 12-HPV58, 13-HPV59, 14-HPV66, 15-HPV68, and 1-ACTB type plasmid sensitivity experiments in the embodiments of the present invention. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0035] Example 1: A kit for detecting 14 high-risk human papillomavirus E6 / E7 region mRNA, the kit comprises: specific primers and probes: specific primers and probes designed for the E6 / E7 mRNA region of 14 high-risk HPVs and primers and probes for the internal reference gene ACTB, the kit is used for amplification and detection of HPV mRNA across the E6 and E7 segments. The primer and probe sequences are as follows:

[0036] Table 1 Primer probe sequences

[0037]

[0038]

[0039] Nucleic acid extraction and purification components: including lysis buffer, proteinase K, DNase I, and buffer for washing and elution. Reverse transcription reaction components: including PrimeScript Buffer, PrimeScript RT Enzyme Mix I, Oligo dTPrimer, Random 6mers, and RNase Free dH2O. Multiplex fluorescence quantitative PCR reaction components: including TaqProbe2X qPCR-Multiplex Mastermix and fluorescent probes for detection.

[0040] Example 2: Application of the kit of Example 1 in cervical exfoliated cell samples

[0041] 1. Extraction and purification of nucleic acids

[0042] Reagent preparation: 1. Prepare anhydrous ethanol and 85% ethanol. 2. Buffer preparation: When the boxed kit is opened for the first time, add anhydrous ethanol to Buffer TBB, Buffer PBR2, and Buffer PBR4 as indicated on the bottle label and mix well. 3. DNase I preparation: Use the DNase dissolve Buffer provided in the kit to dissolve the DNase I dry powder.

[0043] Sample pretreatment: Take an appropriate amount of cell sample, centrifuge at 12000rpm for 3min, discard the supernatant, add 400μL lysis buffer (Buffer TLB) and 10μL proteinase K (Proteinase K), vortex to mix, incubate at 56℃ for 15min, and centrifuge at room temperature ≥12000rpm for 5min.

[0044] On the machine: 1. Take a 96-well plate and add samples and reagents to the wells according to the table below. Note: The total volume of each well cannot exceed 900μl, otherwise it may overflow. 2. Start the instrument, place the magnetic rod cover in the instrument, and put the 96-well plate with samples and reagents into the instrument. The 96-well plate surface settings and reagent amounts are shown in Table 2 below, corresponding to the magnetic rod cover. Note: The magnetic rod cover must be placed in the wells where the reagents are added, otherwise it will corrode the magnetic rod and affect subsequent experiments.

[0045] Table 2 96-well plate layout and reagent dosage

[0046]

[0047]

[0048] 3. Execute the program "Tissue RNA-1". The program settings of "Tissue RNA-1" are shown in Table 3 below.

[0049] 4. After the program "Tissue RNA-1" is completed, add 700μl Buffer PBR4 to the 3rd column and the 9th column, and execute the program "Tissue RNA-2". The program settings of "Tissue RNA-2" are shown in Table 4. Note: Replacing the magnetic rod cover at this time can increase the product 260 / 230 ratio.

[0050] 5. After the program is completed, the eluted products are in columns 6 and 12. It is recommended to dispense the eluted products into clean centrifuge tubes and store them at -80°C to avoid repeated freezing and thawing.

[0051] Table 3 Tissue RNA-1 program settings

[0052]

[0053] Table 4 Tissue RNA-2 program settings

[0054]

[0055] 2. Reverse Transcription Reaction

[0056] First, prepare the RT reaction solution (10μl system) according to the following components: 5X PrimeScript Buffer (for RealTime): 2μL; PrimeScript RT Enzyme Mix I: 0.5μL; Oligo dT Primer (50μM): 0.5μL; Random 6mers (100μM): 2μL; Total RNA: 12μL; RNase Free dH2O: add to 10μL; Reverse transcription program: reverse transcription reaction at 37°C for 15 minutes, followed by reverse transcriptase inactivation reaction at 85°C for 5 seconds.

[0057] 3. Multiplex Fluorescence Quantitative PCR

[0058] The probe reaction groups are grouped as shown in Table 5 below:

[0059] Table 5 Probe reaction group group

[0060] Serial number 1 group 2 groups 3 groups Fluorescence modification 1 1-H-ACTB 5-HPV-33 4-HPV-31 FAM-MGB 2 2-HPV-16 6-HPV-35 7-HPV-39 VIC-MGB 3 3-HPV-18 8-HPV-45 12-HPV-58 TAMRA-MGB 4 9-HPV-51 11-HPV-56 14-HPV-66 ROX-MGB 5 10-HPV-52 15-HPV-68 13-HPV-59 CY5-MGB

[0061] Primer probe combination ratio: Prepare each set of primer probes in a ratio of 0.4:0.4:0.4, and then mix them in a ratio of 1:1:1. Prepare the multiplex fluorescence quantitative PCR reaction system (20μL) according to the following ratios: TaqProbe 2X qPCR-Multiplex Mastermix: 8μL; combination probe primer: 6μL; cDNA template: 3μL; dH2O up to 20μL;

[0062] Reaction procedure: 10 minutes of pre-denaturation at 25°C, 2 minutes of denaturation at 95°C, and then 50 cycles of 15 seconds of denaturation at 95°C and 1 minute of annealing and extension at 60°C. The corresponding fluorescence signal was collected at 60°C to determine the type of high-risk HPV present in the sample.

[0063] Result analysis: Figure 1-15 As shown, the PCR product is analyzed to determine the HPV genotype. By reading the fluorescence amplification curve and Ct value, the HPV type present in the sample can be identified.

[0064] 4. Sensitivity Experiment

[0065] The present invention constructs a plasmid with 14 high-risk HPV cDNA as template sequences, namely HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, and performs a sensitivity test of the kit. The plasmid is sequentially diluted 10 times in a gradient, and 8 low-concentration gradients are selected for experiments. 6 μL of each concentration gradient is taken as a template for amplification, and the copy numbers of the 8 concentration gradients are respectively: 1×108 copies / ul to 1×101 copies / ul, and multiple RT-PCR amplification and detection are performed.

[0066] When conducting sensitivity tests, we conducted three independent experiments on each concentration gradient of plasmid template to ensure the reliability and repeatability of the results. Experimental data showed that all probes could stably detect the target sequence at a concentration of 1×106 copies / μL, showing good sensitivity. As the plasmid concentration decreased, some probes were still able to detect signals at concentrations of 1×104 copies / μL and 1×103 copies / μL, but the signal intensity was weakened. At concentrations of 1×102 copies / μL and 1×10 copies / μL, the detection ability of some probes began to decline, but more than half of the probes were still able to provide detectable signals.

[0067] Through statistical analysis, we calculated the Ct value (threshold cycle number) of each probe at different concentrations and drew standard curves. These curves showed a linear relationship between the Ct value and the plasmid concentration, further verifying the sensitivity and quantitative ability of the kit. In the range of 1×106 copies / μL to 1×103 copies / μL, the slope and R^2 values ​​of the standard curves of most probes were within the acceptable range, indicating that the kit has a good linear relationship and quantitative accuracy.

[0068] The experimental results are as attached Figure 16-30 As shown, the experimental results show that the kit has a high detection sensitivity for 14 high-risk HPV types, and most probes can detect targets as low as 10 copies / μL. This proves that the kit has high sensitivity in practical applications and can detect low-abundance HPV mRNA, which is of great significance for early identification of high-risk HPV infection and timely detection of cervical precancerous lesions.

[0069] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A kit for detecting 14 types of high-risk human papillomavirus E6 / E7 region mRNA, the kit is used to detect at least one high-risk HPV E6 / E7 mRNA, characterized in that: The kit is used for amplification and detection of HPV mRNA across E6 and E7 segments, and the kit comprises the following components: (1) Specific primers and probes for detecting the internal reference gene ACTB, wherein the primer sequences are shown in SEQ ID NOs. 1 to 2, and the probe sequence is shown in SEQ ID NO. 3; (2) Specific primers and probes for detecting HPV 16 E6 / E7 region mRNA, the primer sequences of which are shown in SEQ ID NOs. 4 to 5, and the probe sequence of which is shown in SEQ ID NO. 6; (3) Specific primers and probes for detecting HPV 18 E6 / E7 region mRNA, the primer sequences of which are shown in SEQ ID NOs. 7 to 8, and the probe sequence of which is shown in SEQ ID NO. 9; (4) Specific primers and probes for detecting HPV 31E6 / E7 region mRNA, the primer sequences of which are shown in SEQ ID NOs. 10 to 11, and the probe sequence of which is shown in SEQ ID NO. 12; (5) Specific primers and probes for detecting HPV 33E6 / E7 region mRNA, the primer sequences of which are shown in SEQ ID NOs. 13 to 14, and the probe sequence of which is shown in SEQ ID NO. 15; (6) Specific primers and probes for detecting HPV 35E6 / E7 region mRNA, the primer sequences of which are shown in SEQ ID NOs. 16 to 17, and the probe sequence of which is shown in SEQ ID NO. 18; (7) Specific primers and probes for detecting HPV 39E6 / E7 region mRNA, the primer sequences are shown in SEQ ID NOs. 19 to 20, and the probe sequence is shown in SEQ ID NO. 21; (8) Specific primers and probes for detecting HPV 45E6 / E7 region mRNA, the primer sequences of which are shown in SEQ ID NOs. 22 to 23, and the probe sequence of which is shown in SEQ ID NO. 24; (9) Specific primers and probes for detecting HPV 51E6 / E7 region mRNA, the primer sequences are shown in SEQ ID NOs. 25 to 26, and the probe sequence is shown in SEQ ID NO. 27; (10) Specific primers and probes for detecting HPV 52 E6 / E7 region mRNA, the primer sequences of which are shown in SEQ ID NOs. 28 to 29, and the probe sequence of which is shown in SEQ ID NO. 30; (11) Specific primers and probes for detecting HPV 56 E6 / E7 region mRNA, the primer sequences are shown in SEQ ID NOs. 31 to 32, and the probe sequence is shown in SEQ ID NO. 33; (12) Specific primers and probes for detecting HPV 58 E6 / E7 region mRNA, the primer sequences are shown in SEQ ID NOs. 34 to 35, and the probe sequence is shown in SEQ ID NO. 36; (13) Specific primers and probes for detecting HPV 59E6 / E7 region mRNA, the primer sequences are shown in SEQ ID NOs. 37 to 38, and the probe sequence is shown in SEQ ID NO. 39; (14) Specific primers and probes for detecting HPV 66 E6 / E7 region mRNA, the primer sequences are shown in SEQ ID NOs. 40 to 41, and the probe sequence is shown in SEQ ID NO. 42; (15) Specific primers and probes for detecting HPV 68 E6 / E7 region mRNA, wherein the primer sequences are shown in SEQ ID NOs. 43 to 44, and the probe sequence is shown in SEQ ID NO.

45.

2. The kit according to claim 1, wherein: The kit also includes components for nucleic acid extraction and purification, including lysis solution, proteinase K, DNase I, and buffer for washing and elution.

3. The kit according to claim 1, wherein: The kit also includes PrimeScript Buffer, PrimeScript RT Enzyme Mix I, Oligo dT Primer, Random6mers and RNase Free dH2O for reverse transcription reaction.

4. The kit according to claim 1, wherein: The kit also includes TaqProbe 2X qPCR-Multiplex Mastermix for multiplex fluorescence quantitative PCR reaction and fluorescent probes for detection.

5. A method for using the human papillomavirus E6 / E7 mRNA typing detection kit according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: S1: Extract and purify RNA from the sample to be tested; S2: prepare cDNA; S3: cDNA is detected by multiplex fluorescence quantitative PCR to determine the type of high-risk HPV present in the sample.

6. The method according to claim 5, characterized in that: The method further comprises analyzing the PCR product to determine the HPV genotype.

7. Use of the kit as claimed in claim 1 in cervical exfoliated cell samples.

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