Composition and kit for detecting and typing HPV (human papillomavirus) and application

Abstract

The invention belongs to the field of molecular biology detection, particularly relates to HPV detection, and more particularly relates to a composition for detecting 26 HPVs and typing the 26 HPVs based on an asymmetric PCR (Polymerase Chain Reaction) single tube. According to the combined detection composition provided by the invention, an asymmetric multiplex PCR analysis method is mainly utilized, and different targets are detected by detecting targets on different pathogens, so that the 26 HPVs are detected and distinguished in a single-tube reaction system at the same time, and a targeted strategy is provided for subsequent treatment. According to the composition, the time required by a traditional detection method is shortened, the whole detection process only needs 40-50 minutes, the timeliness of multiple PCR is improved, and clinical doctors can provide strategies for prevention and treatment of infection caused by the pathogens in a targeted mode.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection, and specifically, relates to the detection of HPV, and more specifically, to a composition for detecting and typing 26 types of HPV based on asymmetric PCR in a single tube. Background Art

[0002] Human papillomavirus (HPV) belongs to the family Papillomaviridae. It is a small, non-enveloped circular double-stranded DNA virus with a genome length of about 8,000 base pairs (bp) and is divided into three functional regions, namely the early transcription region (E region), the late transcription region (L region) and the non-transcription region (long control region, LCR). Human papillomavirus infects humans through direct or indirect contact with contaminated objects or sexual transmission. The virus is not only host-specific, but also tissue-specific. It can only infect human skin and mucosal epithelial cells, causing a variety of papilloma or warts on human skin and proliferative lesions of the genital epithelium.

[0003] According to the pathogenicity or carcinogenicity of each genotype, it can be divided into two categories: low-risk and high-risk. Genital HPV infection is common in women with a history of sexual activity. According to statistics, 70% to 80% of women will have at least one HPV infection in their lifetime, but most infections are self-limiting. More than 90% of infected women will have an effective immune response and can clear the infection within 6 to 24 months without any long-term health intervention. Persistent high-risk HPV infection is the main cause of cervical intraepithelial neoplasia and cervical cancer.

[0004] The current HPV genotyping detection method is the flow fluorescence hybridization method: this technology hybridizes the PCR amplification product with the cross-linked fluorescent marker probe on the microsphere, and finally detects the fluorescent signal on the multifunctional flow array instrument. The disadvantages are that the operation is complicated and cumbersome, the price is expensive, and professional technicians are required to operate. The PCR-Taqman MGB probe typing method is currently a commonly used detection method because of its simple operation, accurate typing, and closed-tube operation throughout the process. However, due to the limitations of the instrument's signal acquisition channel, each reaction can only detect a limited number of HPV types, and the throughput is low. When multi-tube detection is adopted, the economic cost and time cost of the detection will be greatly increased. Melting curve method: Add probes labeled with fluorescent groups and quenching groups to the PCR system, amplify single-stranded oligonucleotide sequences complementary to the probe sequence during the PCR process, and add a melting curve analysis process after amplification to obtain a melting curve and obtain the melting point (Tm value) of each type. However, the disadvantage is that the melting point ranges of the melting peaks between the various targets are close, and adjacent melting peaks are prone to interfere with each other, resulting in fusion peaks.

[0005] There is a need in this field for a multiplex PCR identification method from single-tube amplification to hybridization, which can realize the rapid detection of multiple pathogens at the same time, greatly shorten the time required for traditional detection methods, improve the timeliness of multiplex PCR, and at the same time can quickly detect whether 26 high-risk human papillomaviruses are present in the sample and type them, providing a new technical option for the prevention and treatment of HPV. Summary of the invention

[0006] In view of this, in a first aspect, the present invention provides a composition for detecting and typing HPV, comprising a primer and probe set for detecting at least 8 of the following 26 targets:

[0007] Upstream primers, downstream primers and probes for detecting HPV6 as shown in SEQ ID NOs: 1 to 3;

[0008] Upstream primers, downstream primers and probes for detecting HPV11 as shown in SEQ ID NOs: 4 to 6;

[0009] Upstream primers, downstream primers and probes for detecting common HPV16 as shown in SEQ ID NOs: 7 to 9;

[0010] Upstream primers, downstream primers and probes for detecting HPV18 as shown in SEQ ID NOs: 10 to 12;

[0011] Upstream primers, downstream primers and probes for detecting HPV31 as shown in SEQ ID NOs: 13 to 15;

[0012] Upstream primers, downstream primers and probes for detecting HPV33 as shown in SEQ ID NOs: 16 to 18;

[0013] Upstream primers, downstream primers and probes for detecting HPV35 as shown in SEQ ID NOs: 19 to 21;

[0014] Upstream primers, downstream primers and probes for detecting HPV39 as shown in SEQ ID NOs: 22 to 24;

[0015] Upstream primers, downstream primers and probes for detecting HPV40 as shown in SEQ ID NOs: 25 to 27;

[0016] Upstream primers, downstream primers and probes for detecting HPV42 as shown in SEQ ID NOs: 28 to 30;

[0017] Upstream primers, downstream primers and probes for detecting HPV43 as shown in SEQ ID NOs: 31 to 33;

[0018] Upstream primers, downstream primers and probes for detecting HPV44 as shown in SEQ ID NOs: 34 to 36;

[0019] Upstream primers, downstream primers and probes for detecting HPV45 as shown in SEQ ID NOs: 37 to 39;

[0020] Upstream primers, downstream primers and probes for detecting HPV51 as shown in SEQ ID NOs: 40 to 42;

[0021] Upstream primers, downstream primers and probes for detecting HPV52 as shown in SEQ ID NOs: 43 to 45;

[0022] Upstream primers, downstream primers and probes for detecting HPV53 as shown in SEQ ID NOs: 46 to 48;

[0023] Upstream primers, downstream primers and probes for detecting HPV54 as shown in SEQ ID NOs: 49 to 51;

[0024] Upstream primers, downstream primers and probes for detecting HPV55 as shown in SEQ ID NOs: 52 to 54;

[0025] Upstream primers, downstream primers and probes for detecting HPV56 as shown in SEQ ID NOs: 55 to 57;

[0026] Upstream primers, downstream primers and probes for detecting HPV57 as shown in SEQ ID NOs: 58 to 60;

[0027] Upstream primers, downstream primers and probes for detecting HPV58 as shown in SEQ ID NOs: 61 to 63;

[0028] Upstream primers, downstream primers and probes for detecting HPV59 as shown in SEQ ID NOs: 64 to 66;

[0029] Upstream primers, downstream primers and probes for detecting HPV66 as shown in SEQ ID NOs: 67 to 69;

[0030] Upstream primers, downstream primers and probes for detecting HPV67 as shown in SEQ ID NOs: 70 to 72;

[0031] The upstream primers, downstream primers and probe for detecting HPV68 as shown in SEQ ID NOs: 73 to 75; or

[0032] The upstream primers, downstream primers and probe for detecting HPV73 are shown in SEQ ID NOs: 76 to 78.

[0033] Furthermore, the composition includes primers and probe sets for detecting at least 8, 10, 12, 15, 18, 20, 22, 24, 25, and 26 of the above 26 targets.

[0034] The combined detection composition provided by the present invention mainly utilizes the analysis method of asymmetric multiplex PCR to detect different targets by detecting targets on different pathogens, thereby simultaneously realizing the detection and differentiation of 26 types of HPV in a single-tube reaction system, so as to provide a targeted strategy for subsequent treatment. The composition of the present invention greatly shortens the time required for traditional detection methods, and the entire detection process only takes 40-50 minutes, which improves the timeliness of multiplex PCR, allowing clinicians to provide targeted strategies for the prevention and treatment of infections caused by the above pathogens.

[0035] In a specific embodiment, the present invention provides a composition for detecting and typing HPV, comprising:

[0036] Upstream primers, downstream primers and probes for detecting HPV6 as shown in SEQ ID NOs: 1 to 3;

[0037] Upstream primers, downstream primers and probes for detecting HPV11 as shown in SEQ ID NOs: 4 to 6;

[0038] Upstream primers, downstream primers and probes for detecting common HPV16 as shown in SEQ ID NOs: 7 to 9;

[0039] Upstream primers, downstream primers and probes for detecting HPV18 as shown in SEQ ID NOs: 10 to 12;

[0040] Upstream primers, downstream primers and probes for detecting HPV31 as shown in SEQ ID NOs: 13 to 15;

[0041] Upstream primers, downstream primers and probes for detecting HPV33 as shown in SEQ ID NOs: 16 to 18;

[0042] Upstream primers, downstream primers and probes for detecting HPV35 as shown in SEQ ID NOs: 19 to 21;

[0043] Upstream primers, downstream primers and probes for detecting HPV39 as shown in SEQ ID NOs: 22 to 24;

[0044] Upstream primers, downstream primers and probes for detecting HPV40 as shown in SEQ ID NOs: 25 to 27;

[0045] Upstream primers, downstream primers and probes for detecting HPV42 as shown in SEQ ID NOs: 28 to 30;

[0046] Upstream primers, downstream primers and probes for detecting HPV43 as shown in SEQ ID NOs: 31 to 33;

[0047] Upstream primers, downstream primers and probes for detecting HPV44 as shown in SEQ ID NOs: 34 to 36;

[0048] Upstream primers, downstream primers and probes for detecting HPV45 as shown in SEQ ID NOs: 37 to 39;

[0049] Upstream primers, downstream primers and probes for detecting HPV51 as shown in SEQ ID NOs: 40 to 42;

[0050] Upstream primers, downstream primers and probes for detecting HPV52 as shown in SEQ ID NOs: 43 to 45;

[0051] Upstream primers, downstream primers and probes for detecting HPV53 as shown in SEQ ID NOs: 46 to 48;

[0052] Upstream primers, downstream primers and probes for detecting HPV54 as shown in SEQ ID NOs: 49 to 51;

[0053] Upstream primers, downstream primers and probes for detecting HPV55 as shown in SEQ ID NOs: 52 to 54;

[0054] Upstream primers, downstream primers and probes for detecting HPV56 as shown in SEQ ID NOs: 55 to 57;

[0055] Upstream primers, downstream primers and probes for detecting HPV57 as shown in SEQ ID NOs: 58 to 60;

[0056] Upstream primers, downstream primers and probes for detecting HPV58 as shown in SEQ ID NOs: 61 to 63;

[0057] Upstream primers, downstream primers and probes for detecting HPV59 as shown in SEQ ID NOs: 64 to 66;

[0058] Upstream primers, downstream primers and probes for detecting HPV66 as shown in SEQ ID NOs: 67 to 69;

[0059] Upstream primers, downstream primers and probes for detecting HPV67 as shown in SEQ ID NOs: 70 to 72;

[0060] Upstream primers, downstream primers and probes for detecting HPV68 as shown in SEQ ID NOs: 73 to 75; and

[0061] The upstream primers, downstream primers and probe for detecting HPV73 are shown in SEQ ID NOs: 76 to 78.

[0062] Further, in some embodiments, the composition of the present invention may include one or more of the above primer and probe pairs. In the present invention, "set" refers to upstream and downstream primers and probes that match each other for detecting a target.

[0063] The composition of the present invention can be combined into any combination of detection corresponding to 26 targets. Those skilled in the art can combine as needed to detect which targets, that is, to combine the primers and probe pairs corresponding to the targets. These combinations are all included in the present invention, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, ... 20, 21, 22, 23, 24, 25, 26 targets.

[0064] For example, it may include any 23 of the above 26 sets of primers and probes, it may include any 21 of the above 26 sets of primers and probes, it may include any 16 of the above 26 sets of primers and probes, it may include any 14 of the above 26 sets of primers and probes, it may include any 13 of the above 26 sets of primers and probes, and it may include any 11 of the above 26 sets of primers and probes.

[0065] In some specific embodiments, the compositions of the invention are used for asymmetric PCR.

[0066] In some specific embodiments, the downstream primer of the composition of the present invention has a fluorescent group, and the fluorescent groups may be the same or different. Further, the 5' end of the downstream primer of the composition of the present invention has a fluorescent group.

[0067] In a specific embodiment, the fluorescent group is CY5.

[0068] Furthermore, in the composition, the upstream and downstream primers and the probe are separated.

[0069] Furthermore, the composition also includes a carrier. Still further, the carrier includes the probe in the composition of the present invention.

[0070] In some specific embodiments, the carrier is a chip.

[0071] Furthermore, one of the upstream and downstream primers in the composition is a limiting primer, and the other is a non-limiting primer.

[0072] Furthermore, the concentration ratio of the limiting primer to the non-limiting primer is 1:1 to 1:50. Preferably, the concentration ratio of the limiting primer to the non-limiting primer is 1:5 to 1:50, and more preferably, 1:5 to 1:20.

[0073] Using the above ratio further increases the yield of single-stranded products, thereby increasing the probability of product-probe binding, making the amplification and hybridization reaction processes more efficient, while reducing nonspecific binding between products and between probes.

[0074] In a second aspect, the present invention provides use of the above-mentioned composition of the present invention in preparing a kit for detecting and typing HPV.

[0075] In a third aspect, the present invention provides a kit for detecting HPV, wherein the kit comprises the composition of the present invention as described above.

[0076] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0077] Furthermore, the kit also includes dNTP(U)s, PCR buffer and Mg 2+ At least one of .

[0078] Furthermore, the kit also includes at least one of DNA polymerase and uracil glycosylase (UNG).

[0079] Furthermore, the kit also includes at least one of a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0080] Furthermore, the kit also includes a nucleic acid release reagent, a nucleic acid extraction reagent, dNTP, dUTP, uracil glycosylase (UNG), DNA polymerase, UNG enzyme, PCR buffer and Mg 2+ At least one of .

[0081] Furthermore, the concentration of the DNA polymerase is 2U / μL to 5U / μL, for example, the DNA polymerase may be Taq enzyme.

[0082] In a specific embodiment, the kit of the present invention comprises Taq enzyme, UDG enzyme, Mg 2+ , dNTP(U)s, primers, probes and PCR buffer.

[0083] In some specific embodiments, the total volume of the reactants in a single PCR reaction tube is 25 μL to 50 μL.

[0084] In a fourth aspect, a method for detecting and typing HPV is provided, the method comprising the following steps:

[0085] 1) Extracting nucleic acid from the sample to be tested;

[0086] 2) using the composition of the present invention or the primers in the kit of the present invention as described above to perform asymmetric PCR detection on the nucleic acid obtained in step 1) to obtain an amplified product;

[0087] 3) using the composition of the present invention or the probe in the kit of the present invention as described above to hybridize with the amplification product obtained in step 2);

[0088] 4) Detect and analyze the results.

[0089] Provided is a use of a composition for preparing a reagent for detecting and typing HPV, wherein the detection comprises the following steps:

[0090] 1) Extracting nucleic acid from the sample to be tested;

[0091] 2) using the composition of the present invention or the primers in the kit of the present invention as described above to perform asymmetric PCR detection on the nucleic acid obtained in step 1) to obtain an amplified product;

[0092] 3) using the composition of the present invention or the probe in the kit of the present invention as described above to hybridize with the amplification product obtained in step 2); and

[0093] 4) Detect and analyze the results.

[0094] In some specific embodiments, steps 2) and 3) are performed in a microfluidic chip. Furthermore, step 2) is performed in an amplification reaction chamber in a microfluidic chip; and step 3) is performed in a hybridization reaction chamber in a microfluidic chip.

[0095] Furthermore, the vector of the present invention is in a hybridization reaction chamber.

[0096] In the present invention, the sample used for detection may be cervical exfoliated cells, etc., but is not limited thereto.

[0097] Furthermore, the reaction conditions of the asymmetric PCR are:

[0098] 95°C, 1 min; 95°C, 15 s, 60°C, 30 s, 40 cycles.

[0099] Furthermore, the reaction conditions of the hybridization are:

[0100] Incubate at 60°C for 5 min.

[0101] Furthermore, the method of detecting and typing HPV is for non-diagnostic purposes.

[0102] In this article, the term "non-diagnostic purpose" means that it is not intended to obtain information on whether an individual is infected with the above pathogens and suffers from the infection. For example, the method can detect whether the above pathogens are present in the environment and culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 a is a photograph of a single-tube amplification 26-plex microarray for detecting 1000 copies / mL of the composition of the present invention; Figure 1 b is the NTC result;

[0104] Figure 2 a is a comparison of different asymmetric ratios of the composition of the present invention - microarray photograph; Figure 2 b is the NTC result. DETAILED DESCRIPTION

[0105] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0106] Example 1: Primers and probes used in the present invention

[0107] The primers and probes used in the present invention are shown in Table 1 below:

[0108] Table 1

[0109]

[0110]

[0111]

[0112] Example 2: Method for detecting and typing HPV

[0113] The samples to be tested are tested and typed according to the following method:

[0114] (1) A certain concentration of DNA is obtained from the sample to be tested by conventional nucleic acid extraction methods in the art to serve as a template;

[0115] (2) Configure the asymmetric PCR reaction system according to the following template:

[0116] PCR buffer system 8 μL, DNA polymerase 50 U / μL 2 μL, restriction primer F 0.1 μL, non-restriction primer R 1 μL, 0.1% DEPC water 1.4 μL, template DNA 10 μL.

[0117] (3) Perform asymmetric PCR according to the following amplification procedure to obtain a single-stranded amplification product:

[0118] The multiplex amplification program was: 95°C, 1 minute; 95°C, 15 s, 60°C, 30 s, 40 cycles;

[0119] (4) Microarray hybridization: The amplified product of step (3) is transported to the hybridization reaction chamber by microfluidics to react with the microarray chip, incubated at 60° C. for 5 min, rinsed with hybridization cleaning solution for 1-2 min, and the device performs emission spectrum reading.

[0120] Example 3: Test results of the test samples of the composition of the present invention

[0121] The primers and probes shown in Example 1 were used to detect 26 HPV genotypes according to the method of Example 2. The specific results are shown in Figure 2. Figure 1 As shown in Table 2. As can be seen from the table, 26 HPV genotypes are 3 ~10 6 (E3~E6) can be detected. Figure 1 a is a photograph of a single-tube amplification 26-plex microarray for detecting 1000 copies / mL of the composition of the present invention; Figure 1 b is the NTC result.

[0122] Table 2

[0123]

[0124]

[0125] Note: The interpretation criteria of the analysis results of QArray Analyser software: a value > 1000 is positive, a value < 1000 is invalid / negative, and an NTC value > 1000 indicates non-specific binding of the amplified product to the probe.

[0126] Example 4: Detection results of primers in different ratios of the composition of the present invention

[0127] The present invention takes HPV16 as an example, and verifies the yield of amplified products and the hybridization efficiency with the probe under different ratios by setting the restrictive and non-restrictive primers in the following ratios. The ratios of restrictive and non-restrictive primers used in the present invention are: 1:1, 1:5, 1:10, 1:20, 1:50, 1:100. The test results are shown in Table 3 and Figure 2 As shown, Figure 2 a is a comparison of different asymmetric ratios of the composition of the present invention - microarray photograph; Figure 2 b is the NTC result. It can be seen from the table and the figure that the ratios of 1:10 and 1:20 increase the yield of single-stranded products, thereby increasing the binding efficiency of the products and the probes, making the amplification and hybridization reaction processes more efficient.

[0128] Table 3

[0129]

[0130] Note: The interpretation criteria of the analysis results of QArray Analyser software: a value > 1000 is positive, a value < 1000 is invalid / negative, and an NTC value > 1000 indicates non-specific binding of the amplified product to the probe.

Claims

1. A composition for detecting and typing HPV, comprising a primer and probe set for detecting at least 8 of the following 26 targets: Upstream primers, downstream primers and probes for detecting HPV6 as shown in SEQ ID NOs: 1 to 3; Upstream primers, downstream primers and probes for detecting HPV11 as shown in SEQ ID NOs: 4 to 6; Upstream primers, downstream primers and probes for detecting common HPV16 as shown in SEQ ID NOs: 7 to 9; Upstream primers, downstream primers and probes for detecting HPV18 as shown in SEQ ID NOs: 10 to 12; Upstream primers, downstream primers and probes for detecting HPV31 as shown in SEQ ID NOs: 13 to 15; Upstream primers, downstream primers and probes for detecting HPV33 as shown in SEQ ID NOs: 16 to 18; Upstream primers, downstream primers and probes for detecting HPV35 as shown in SEQ ID NOs: 19 to 21; Upstream primers, downstream primers and probes for detecting HPV39 as shown in SEQ ID NOs: 22 to 24; Upstream primers, downstream primers and probes for detecting HPV40 as shown in SEQ ID NOs: 25 to 27; Upstream primers, downstream primers and probes for detecting HPV42 as shown in SEQ ID NOs: 28 to 30; Upstream primers, downstream primers and probes for detecting HPV43 as shown in SEQ ID NOs: 31 to 33; Upstream primers, downstream primers and probes for detecting HPV44 as shown in SEQ ID NOs: 34 to 36; Upstream primers, downstream primers and probes for detecting HPV45 as shown in SEQ ID NOs: 37 to 39; Upstream primers, downstream primers and probes for detecting HPV51 as shown in SEQ ID NOs: 40 to 42; Upstream primers, downstream primers and probes for detecting HPV52 as shown in SEQ ID NOs: 43 to 45; Upstream primers, downstream primers and probes for detecting HPV53 as shown in SEQ ID NOs: 46 to 48; Upstream primers, downstream primers and probes for detecting HPV54 as shown in SEQ ID NOs: 49 to 51; Upstream primers, downstream primers and probes for detecting HPV55 as shown in SEQ ID NOs: 52 to 54; Upstream primers, downstream primers and probes for detecting HPV56 as shown in SEQ ID NOs: 55 to 57; Upstream primers, downstream primers and probes for detecting HPV57 as shown in SEQ ID NOs: 58 to 60; Upstream primers, downstream primers and probes for detecting HPV58 as shown in SEQ ID NOs: 61 to 63; Upstream primers, downstream primers and probes for detecting HPV59 as shown in SEQ ID NOs: 64 to 66; Upstream primers, downstream primers and probes for detecting HPV66 as shown in SEQ ID NOs: 67 to 69; Upstream primers, downstream primers and probes for detecting HPV67 as shown in SEQ ID NOs: 70 to 72; The upstream primers, downstream primers and probe for detecting HPV68 as shown in SEQ ID NOs: 73 to 75; or The upstream primers, downstream primers and probe for detecting HPV73 are shown in SEQ ID NOs: 76 to 78.

2. The composition according to claim 1, characterized in that The composition includes primers and probe sets for detecting at least 20 of the above 26 targets.

3. The composition according to claim 2, characterized in that The composition includes primers and probe sets for detecting the above 26 targets.

4. The composition according to claim 1, characterized in that One of the upstream and downstream primers in the composition is a limiting primer, and the other is a non-limiting primer; the concentration ratio of the limiting primer to the non-limiting primer is 1:1 to 1:

50.

5. The composition according to any one of claims 1 to 4, characterized in that The composition also includes a carrier.

6. The composition according to claim 5, characterized in that The carrier is a chip.

7. Use of the composition according to any one of claims 1 to 6 in the preparation of a kit for detecting HPV.

8. A kit for detecting HPV, comprising the composition according to any one of claims 1 to 6.

9. Use of a composition for preparing a reagent for detecting and typing HPV, the detection comprising the following steps: 1) Extracting nucleic acid from the sample to be tested; 2) using the composition according to any one of claims 1 to 6 or the primers in the kit according to claim 8 to perform asymmetric PCR detection on the nucleic acid obtained in step 1) to obtain an amplified product; 3) using the composition according to any one of claims 1 to 6 or the probe in the kit according to claim 8 to hybridize with the amplification product obtained in step 2); and 4) Detect and analyze the results.

10. The use according to claim 9, characterized in that The steps 2) and 3) are performed in a microfluidic chip.

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