A primer combination and its application in preparing HPV typing detection products

Abstract

The present invention discloses a primer combination and its application in preparing an HPV typing detection product. The present invention is based on multiplex PCR and combined with time-of-flight mass spectrometry, and provides a primer combination that can perform typing detection on the 20 types of HPV in the same reaction system for the genomes of 20 types of HPV including HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82, and also provides an HPV typing detection kit containing the primer combination. The detection specificity, sensitivity and precision of the 20 types of HPV typing detection using the kit of the present invention are good, and the detection results are accurate, which is conducive to the early screening of cervical cancer and realizes early detection and early treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and more specifically, relates to a primer combination and its application in preparing HPV typing detection products. Background Art

[0002] Human papillomavirus (HPV) is a type of tiny covalent double-stranded circular DNA virus that is tropistic for human epithelium and mucosa and can cause a variety of diseases. Among them, persistent high-risk HPV infection is the main cause of cervical intraepithelial neoplasia (cervical precancerous lesions) and cervical cancer. The nucleic acid detection of high-risk HPV is used for cervical cancer screening, which can effectively increase the detection rate of cervical precancerous lesions and achieve early detection and early treatment.

[0003] High-risk HPV includes HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82. Although there are reports of high-risk HPV typing detection reagents, they still have shortcomings such as inability to detect multiple infections, incomplete detection of high-risk HPV types, and inability to detect all high-risk HPV types at once.

[0004] Multiplex PCR combined with time-of-flight mass spectrometry (MALDI-TOF-MS) is an efficient molecular diagnostic technology with advantages such as high throughput and rapid detection. Using it for HPV typing detection can overcome the shortcomings of being unable to detect multiple infections. However, the HPV typing detection products based on multiplex PCR combined with time-of-flight mass spectrometry that have been reported still have shortcomings such as incomplete detection of high-risk HPV types. If you want to cover more HPV types, the design of primers and other materials will be more difficult, making it difficult to develop reagents that can perform typing detection on HPV including 18 high-risk HPV types. Summary of the invention

[0005] In view of the problems existing in the above-mentioned prior art, the present invention provides a primer combination, which can be used to realize the typing detection of HPV including 18 high-risk HPV types.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] The present invention targets the genomes of 18 high-risk HPV types (HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82) and low-risk HPV types (HPV 6 and 11) associated with the occurrence of genital warts (condyloma acuminatum), and obtains a primer combination that can be used for typing and detection of the above 20 types of HPV by multiplex PCR combined with time-of-flight mass spectrometry.

[0008] Specifically, the primer combination includes a multiplex PCR amplification primer set and a single base extension primer set; wherein the multiplex PCR amplification primer set includes PCR amplification primer pairs of HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82, and the nucleotide sequences of the primer pairs are shown in SEQ ID NOs. 1 to 40, respectively; the single base extension primer set includes single base extension primers of HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82, and the nucleotide sequences of the extension primers are shown in SEQ ID NOs. 43 to 62, respectively.

[0009] As an optional embodiment, the present invention designs corresponding PCR amplification primer pairs and single-base extension primers using the gene GAPDH as an internal reference.

[0010] Specifically, the nucleotide sequences of the PCR amplification primer pair for the gene GAPDH designed by the present invention that can be used in the same reaction system with the above-mentioned multiple PCR amplification primer set are shown as SEQ ID NO.41 and SEQ ID NO.42, and the nucleotide sequence of the single base extension primer for the gene GAPDH that can be used in the same reaction system with the above-mentioned single base extension primer set is shown as SEQ ID NO.63.

[0011] The primer combination of the present invention is a primer combination with the best detection effect obtained through continuous adjustment, testing and optimization. The primer combination combined with time-of-flight mass spectrometry can realize the typing detection of 20 types of HPV including HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82 at the same time, with good detection specificity, high sensitivity, precision and accuracy. Therefore, the present invention also requests protection for the use of the primer combination in the preparation of HPV typing detection products.

[0012] Optionally, the product is a kit.

[0013] Specifically, the HPV includes HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and / or 82.

[0014] The invention also provides a kit containing the primer combination.

[0015] Specifically, the kit is an HPV typing detection kit, and the HPV includes HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and / or 82.

[0016] Specifically, the kit also contains reagents required for PCR reaction, SAP reaction and single base extension reaction.

[0017] Specifically, the reagents required for the PCR reaction include PCR enzyme, dNTP mix, MgCl2 and PCR Buffer.

[0018] Specifically, the reagents required for the SAP reaction include SAP enzyme and SAP Buffer.

[0019] Specifically, the reagents required for the single-base extension reaction are Termination Mix, iPLEXPro enzyme and its Buffer (Buffer plus).

[0020] Specifically, the kit also contains a positive quality control product and a blank control.

[0021] Optionally, the positive control is a recombinant plasmid or cell containing genomic DNA of HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and / or 82.

[0022] In a specific embodiment of the present invention, the positive control product is cells containing HPV18 genomic DNA, and the blank control is ultrapure water.

[0023] Specifically, the positive quality control product is obtained by inserting the full genome sequence of HPV 18 into a pcDNA5FRT vector and then introducing it into a human cell line.

[0024] Optionally, the kit also contains reagents required for extracting sample DNA.

[0025] The present invention also provides a method for using the kit, comprising the following steps:

[0026] S1. Extract sample DNA;

[0027] S2. Performing PCR amplification reaction using the extracted sample DNA as a template to obtain PCR amplification product 1;

[0028] S3. Performing SAP reaction with PCR amplification product 1 as template to obtain PCR amplification product 2;

[0029] S4. Performing a single base extension reaction using PCR amplification product 2 as a template to obtain PCR amplification product 3;

[0030] S5. Add ultrapure water to the PCR amplification product 3, desalt the sample using DP-TOF, spot the sample onto the chip, and use time-of-flight mass spectrometry to obtain the test sample data, and use the data with no problems in the quality control results to judge the results.

[0031] Optionally, the reagent used to extract sample DNA is a nucleic acid extraction or purification reagent (magnetic bead method DR-9600-KZ type) produced by Chaozhou Kaipu Biochemical Co., Ltd.

[0032] Specifically, the PCR amplification reaction system in S2 was: 10×PCR Buffer 0.5 μL, 25 mM MgCl2 0.4 μL, 25 mM dNTP mix 0.1 μL, 5 U / μL PCR enzyme 0.2 μL, PCR primer mixture 1 μL, DNA template 2 μL and water 0.8 μL; the PCR amplification reaction program was: 95°C 2 min; 45 cycles: 95°C 30 sec, 56°C 30 sec, 72°C 1 min; 72°C 5 min; 4°C insulation.

[0033] Specifically, in S3, a SAP reaction mixture is directly added to the PCR amplification product 1 obtained in S2 to perform a SAP reaction; the SAP reaction mixture contains 0.17 μL of 10×SAP Buffer, 0.3 μL of 1.7 U / μL SAP enzyme and 1.53 μL of water; the SAP reaction procedure is: 37°C 40 min; 85°C 5 min; and insulation at 4°C.

[0034] Specifically, in S4, an extension reaction mixture is directly added to the PCR amplification product 2 obtained in S3 to perform a single base extension reaction; the extension reaction mixture contains 0.2 μL of 10×Buffer Plus, 0.2 μL of 10 mM Termination Mix, 0.04 μL of 32 U / μL iPLEXPro, 0.94 μL of extension primer mixture and 0.62 μL of water; the extension reaction procedure is as follows:

[0035]

[0036] The present invention has the following beneficial effects:

[0037] The present invention is based on multiplex PCR and combined with time-of-flight mass spectrometry, and provides a primer combination that can perform typing detection on the 20 types of HPV in the same reaction system for the genomes of 20 types of HPV including HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82, and also provides an HPV typing detection kit containing the primer combination. The kit has good specificity, high sensitivity and precision for typing detection of the 20 types of HPV, and accurate detection results, which is conducive to early screening of cervical cancer and early detection and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the quality control flow chart of the time-of-flight mass spectrometry platform.

[0039] Figure 2 This is the time-of-flight mass spectrum of the GAPDH gene.

[0040] Figure 3 This is the time-of-flight mass spectrum of the HPV18 positive quality control product.

[0041] Figure 4 This is the time-of-flight mass spectrum of the HPV16 reference.

[0042] Figure 5 This is the time-of-flight mass spectrum of the HPV18 reference. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0044] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0045] Example 1 Obtaining a primer combination

[0046] The present invention is directed to the genomes of 18 high-risk HPVs (HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82) and low-risk HPVs (HPV 6 and 11) associated with the occurrence of genital warts (condyloma acuminatum), and first designs multiple sets of PCR amplification primer pairs and single base extension primers. At the same time, corresponding PCR amplification primer pairs and single base extension primers are designed with human GAPDH gene as an internal reference. On the basis of the designed primers, the typing detection effect is tested using clinical samples, primers with poor detection effect are eliminated, and the primer sequence and primer combination are continuously adjusted in combination with the test results (such as redesigning the primer sequence, or replacing individual bases in the primer with hypoxanthine, etc.). Through a process of repeated testing and adjustment, a primer combination was obtained that can be used to perform typing detection of the 20 types of HPV in the same reaction system based on PCR combined with time-of-flight mass spectrometry.

[0047] The primer combination obtained by the present invention includes a multiplex PCR amplification primer set and a single base extension primer set, wherein the sequences of the PCR amplification primer pairs and the single base extension primers contained therein are shown in Table 1 and Table 2, respectively. To avoid redundancy, only the final primer combination is listed in the specification.

[0048] Table 1 Primer sets for multiplex PCR amplification

[0049]

[0050] Table 2 Single base extension primer sets

[0051]

[0052] Table 2

[0053]

[0054] Note: "I" in the primer sequences shown in Tables 1 and 2 is hypoxanthine. According to the editing rules of WIPO Sequence software, only the symbols listed in "WIPO ST.26 Annex I Part 1" can appear in the nucleotide sequence. Therefore, "n" (meaning other) is used in the sequence listing to represent the "I" appearing in the table.

[0055] Example 2 20 HPV typing test kit

[0056] Based on the primer combination described in Example 1, the present invention also provides a kit containing the primer combination and a method for using the kit, which can be used to perform typing detection on 20 types of HPV including HPV6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82.

[0057] In addition to the primer combination, the kit also contains the following components:

[0058] (1) Reagents required for PCR amplification: including PCR enzyme, dNTP mix, MgCl2, and 10×PCR Buffer;

[0059] (2) Reagents required for SAP reaction: including SAP enzyme and SAP Buffer;

[0060] (3) Reagents required for single-base extension reaction: including iPLEXPro enzyme, Termination Mix and BufferPlus;

[0061] (4) Positive quality control and blank control: The positive quality control has a concentration of 1×10 6 The blank control substance is ultrapure water; the positive quality control substance is obtained by inserting the full genome sequence of HPV 18 into the pcDNA5FRT vector and then introducing it into the human cell line.

[0062] The method for typing and detecting the 20 types of HPV using the kit (how to use the kit) is as follows:

[0063] Before use, the synthesized multiple PCR amplification primer set was dissolved in ultrapure water to prepare a PCR amplification primer mixture containing each PCR amplification primer pair, and the concentration of each amplification primer was 0.5 μM / L; at the same time, the synthesized single-base extension primer set was dissolved in ultrapure water to prepare an extension primer mixture containing each single-base extension primer, and the concentration of each extension primer was 9 μmol / L.

[0064] The specific steps are as follows:

[0065] S1. Extract sample DNA;

[0066] In the present invention, the reagent used to extract sample DNA is a nucleic acid extraction or purification reagent (magnetic bead method DR-9600-KZ type) produced by Chaozhou Kaipu Biochemical Co., Ltd.;

[0067] S2. Perform PCR amplification reaction using the extracted sample DNA as a template; add the prepared PCR amplification reaction system to a 384-well plate, seal it with a plate sealing film, centrifuge it instantly, throw all the liquid on the tube wall to the bottom of the tube, eliminate bubbles, and place the 384-well plate into a PCR instrument for reaction to obtain a 384-well plate containing PCR amplification product 1;

[0068] The PCR amplification reaction system was as follows: 10×PCR Buffer 0.5 μL, 25 mM MgCl2 0.4 μL, 25 mM dNTPmix 0.1 μL, 5 U / μL PCR enzyme 0.2 μL, PCR primer mixture 1 μL, DNA template 2 μL and water 0.8 μL; the PCR amplification reaction program was as follows: 95°C 2 min; 45 cycles: 95°C 30 sec, 56°C 30 sec, 72°C 1 min; 72°C 5 min; 4°C insulation;

[0069] S3. Perform SAP reaction with PCR amplification product 1 as template; dispense the prepared SAP reaction mixture into the 384-well plate containing PCR amplification product 1, adhere it with a sealing film, centrifuge it instantly, throw all the liquid on the tube wall to the bottom of the tube, put the 384-well plate into the PCR instrument for reaction, and obtain a 384-well plate containing PCR amplification product 2;

[0070] The SAP reaction mixture contained 0.17 μL of 10×SAP Buffer, 0.3 μL of 1.7 U / μL SAP enzyme, and 1.53 μL of water. The SAP reaction procedure was as follows: 37°C for 40 min; 85°C for 5 min; and 4°C insulation.

[0071] S4. Perform a single base extension reaction using PCR amplification product 2 as a template; dispense the prepared extension reaction mixture into a 384-well plate containing PCR amplification product 2, adhere it with a sealing film, centrifuge it instantly, throw all the liquid on the tube wall to the bottom of the tube, eliminate bubbles, and place the 384-well plate into a PCR instrument for reaction to obtain a 384-well plate containing PCR amplification product 3;

[0072] The extension reaction mixture contained 0.2 μL of 10× Buffer Plus, 0.2 μL of 10 mM Termination Mix, 0.04 μL of 32 U / μL iPLEXPro, 0.94 μL of extension primer mixture, and 0.62 μL of water. The extension reaction procedure was as follows:

[0073]

[0074] S5. Add ultrapure water (16 μL / well) to the 384-well plate containing the PCR amplification product 3 obtained in S4 and centrifuge instantly. Desalt the sample using DP-TOF. After spotting the sample onto the chip, use time-of-flight mass spectrometry to obtain the test sample data. Use the data with no problems in the quality control results to judge the results.

[0075] The instrument model used for time-of-flight mass spectrometry analysis in the present invention is DP-TOF, and the mass spectrometer parameters and check settings are shown in Table 3.

[0076] Table 3 Mass spectrometer instrument parameters and checkbox settings

[0077]

[0078] Note: ③ In the chip spotting module, add resin to the plate position according to the test requirements; when testing one chip at a time, check Plate 1 or Plate 2 according to the plate position used; when testing two chips at a time, check Plate 1 and Plate 2. After completing the above settings, click the start button "Start Chip Preparation Module" icon to start the automated detection process;

[0079] Once the automated testing process begins, the testing system completes the entire process through the following steps:

[0080] a. Product purification: desalting resin and extension product are mixed to remove excess salt ions in the extension product; related parameter "resin volume";

[0081] b. Sample transfer: transfer the purified extension product to the corresponding matrix point on the chip for co-crystallization; related parameters are "start spotting status";

[0082] c. Excitation and detection: Instantaneous laser irradiation is performed on the co-crystallized product to ionize the substance to be tested; the ionized product passes through the vacuum flight channel and reaches the detector. The detector completes the collection of the pulse signal generated by the ion, and finally the analysis is completed through the software; the relevant parameters are "maximum acquisition times, minimum number of high-quality spectra, and maximum number of high-quality spectra". After completion, the report data is exported.

[0083] According to the quality control flow chart of the time-of-flight mass spectrometry platform (such as Figure 1 The quality control results were analyzed as shown in Table 4, and the results were judged by referring to Table 5.

[0084] Table 4

[0085]

[0086] Table 5

[0087]

[0088] Example 3 Accuracy test

[0089] The present invention uses positive references of HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82 (the positive references are identified clinical samples) as accuracy references, and tests the accuracy of HPV typing detection by the kit described in Example 2. The detection method is the same as that in Example 2, and the accuracy of the detection results is verified by sequencing.

[0090] The number, name, properties and concentration of the accuracy reference materials are shown in Table 6.

[0091] Table 6 Accuracy reference

[0092]

[0093] Table 6

[0094]

[0095] By comparing the test results of the kit and the sequencing results, it can be seen that the test results of the accuracy reference shown in Table 6 using the kit of the present invention are completely consistent with the sequencing results, and both are positive, indicating that the kit of the present invention can be used to type HPV6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82, and the test results are accurate.

[0096] Because there are many samples tested, only the detection patterns of GAPDH gene in HPV18 positive quality control, HPV18 positive quality control, and HPV16 and HPV18 reference products are given as examples, respectively. Figures 2 to 5 shown.

[0097] Example 4 Specificity Detection

[0098] The present invention uses positive references of HPV 42, 43 and 81, Ureaplasma urealyticum, Chlamydia trachomatis, Neisseria gonorrhoeae and herpes simplex virus type II (the positive references are identified clinical samples) as specific references to detect the detection specificity of the kit described in Example 2.

[0099] The number, name, property and concentration of the specific reference substances are shown in Table 7.

[0100] Table 7 Specific reference substances

[0101]

[0102] The detection results of the kit of the present invention for the specific reference substances shown in Table 7 were all negative, indicating that the detection specificity of the kit was good and non-specific amplification would not occur.

[0103] Example 5 Detection limit detection

[0104] The concentrations of the accuracy references shown in Table 6 were all adjusted to 1.00E+05 copies / mL, and the detection limits of the kit described in Example 2 were tested as detection limit references. During the test, the detection limit references were gradient diluted, and the detection limit test results are shown in Table 8. As can be seen from Table 8, when the sample concentration in a single reaction system is greater than or equal to 200 copies, all types of HPV can be detected, indicating that the detection sensitivity of the kit described in the present invention is good. See Table 8 for the specific detection limits of each type.

[0105] Table 8 Detection limits of various HPV types

[0106]

[0107] Example 6 Precision Testing

[0108] The present invention uses the precision reference materials shown in Table 9 to test the detection precision of the kit described in Example 2, and repeats the test 10 times. The negative samples described in Table 9 are cervical exfoliated cell samples that are tested to be free of the HPV types detected by the present invention, and the rest are clinical samples that have been identified.

[0109] Table 9 Precision reference products

[0110]

[0111] After testing, all test results of the negative samples were negative, and all test results of the other reference samples were positive. The repeated test results of the negative and positive samples were consistent, indicating that the detection precision of the kit of the present invention is high.

[0112] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A primer combination, characterized in that: Including multiplex PCR amplification primer set and single base extension primer set; The multiplex PCR amplification primer set includes PCR amplification primer pairs of HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82, and the nucleotide sequences of the primer pairs are shown in SEQ ID NOs. 1 to 40, respectively; The single base extension primer set includes single base extension primers of HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82, and the nucleotide sequences of the extension primers are shown in SEQ ID NOs. 43 to 62 respectively; The n involved in the relevant primer sequences is hypoxanthine.

2. The primer combination according to claim 1, characterized in that The multiplex PCR amplification primer set also includes a PCR amplification primer pair for the GAPDH gene, and the nucleotide sequence of the primer pair is shown in SEQ ID NO.41 and SEQ ID NO.42; the single base extension primer set also includes a single base extension primer for the GAPDH gene, and the nucleotide sequence of the extension primer is shown in SEQ ID NO.

63.

3. Use of the primer combination according to claim 1 or 2 in preparing an HPV typing detection product, characterized in that: The HPVs include HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82.

4. A kit comprising the primer combination according to claim 1 or 2.

5. The kit according to claim 4, characterized in that The kit is an HPV typing detection kit, and the HPV includes HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and / or 82.

6. The kit according to claim 5, characterized in that The kit also contains reagents required for PCR reaction, SAP reaction and single base extension reaction.

7. The kit according to claim 6, characterized in that The reagents required for the PCR reaction include PCR enzyme, dNTPmix, MgCl2 and PCR Buffer.

8. The kit according to claim 6, characterized in that The reagents required for the SAP reaction include SAP enzyme and SAP Buffer.

9. The kit according to claim 6, characterized in that The reagents required for the single base extension reaction include Termination Mix, iPLEXPro enzyme and its buffer.

10. The kit according to claim 6, characterized in that: The kit also contains a positive quality control product and a blank control.

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