Nucleic acid composition for human papilloma virus nucleic acid detection, kit and application thereof

Abstract

The invention provides a nucleic acid composition for human papilloma virus nucleic acid detection, a kit and application thereof, and belongs to the field of gene detection. The kit provided by the invention comprises probes corresponding to 18 genotypes of HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV82, as well as 17 forward primers and 15 reverse primers corresponding to the 18 genotypes, and further comprises an amplification reaction solution, a negative control, a positive control and a quality control internal reference. The kit provided by the invention can be used for simultaneously detecting 18 high-risk human papilloma viruses in a single reaction, can be used for typing common HPV16 and HPV18, and is simple, rapid, high in specificity and high in sensitivity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a gene detection kit, and in particular to a nucleic acid composition, a kit and an application thereof for detecting human papillomavirus nucleic acid. Background Art

[0002] Human papilloma virus (HPV) is a type of double-stranded circular DNA virus that mainly infects the skin and mucosal tissues, and can cause warts, benign tumors and cancer. The diameter of HPV virus particles is about 55nm, the nucleocapsid is icosahedral symmetrical, and there are 72 capsomers. HPV is a small DNA virus, without an envelope, and has a double-stranded closed circular DNA genome of 7.2-8kb. The genome is divided into three functional regions: ① Early region (E): about 4.5kb, containing 6 subregions including E1, E2, E4-E7: ② Late region (L), about 2.5kb, containing two subregions L1L2: Long regulatory region (LCR), located between the end of L region and the beginning of E region, generally 800-900bp. E1 is involved in viral replication, and its encoded protein has ATP-dependent helicase activity: E2 is involved in transcriptional regulation, and the protein encoded by E3 is the main viral transcription factor; E4 encodes late cytoplasmic proteins, which are related to viral maturation: E5 has weak transformation activity: E6 and E7 are mainly related to cell transformation and tumorigenicity: The L region encodes two proteins, L1 is the main species-specific antigen, and L2 is the type-specific antigen. L1 or L1 / L2 can assemble into virus-like particles in eukaryotic cells: LCR, also known as URR, contains DNA replication and expression regulatory elements. The expression of viral genes is regulated by viral and host cell transcription factors, including E2, LCR and intracellular trans-regulatory factors. Their changes can cause overexpression of transformation proteins and cause tumors. According to the latest statistics from the International Human Papillomavirus Reference Center, more than 200 HPV genotypes have been discovered. According to the carcinogenic potential of each subtype, HPV is usually divided into two categories: high-risk and low-risk. High-risk HPV infection can cause male and female genital cancer, cervical cancer, etc. Almost all cervical squamous cell carcinomas and more than 85% of cervical adenocarcinomas are caused by HPV infection. A large number of studies have confirmed that 18 subtypes, including HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV82, are the most common high-risk HPV. Among them, HPV16 and HPV18 are the two most harmful genotypes, causing 55% and 15% of cervical cancer cases respectively. The diagnostic technology that can type HPV16 and 18 is of great reference significance for patients with HPV positive test results.

[0003] At present, the detection of HPV is mainly based on molecular biology methods at the DNA level, which is the most sensitive detection method and provides an effective means for detecting low-level viral infections. The subtype of infected HPV, the duration of infection and the content of HPV affect the process of carcinogenesis. The methods based on the DNA level mainly include nucleic acid hybridization detection and polymerase chain reaction. Based on PCR technology, many other experimental methods for HPV detection have also been derived, such as universal primer PCR, type-specific primer PCR, restriction fragment length polymorphism analysis, reverse hybridization, etc. For type-specific primer PCR, this is a detection method that selects type-specific primers to amplify sample DNA. When using this method for detection, multiple PCR reactions must be performed simultaneously. It is a time-consuming and labor-intensive detection method with very large limitations. Universal primer PCR is a method that designs primers for the conserved sequences of various HPV subtypes, and generally amplifies the L1 region. Traditional HPV detection is mainly a method that combines morphological methods with immunological methods, but the specificity and sensitivity of traditional detection methods are not ideal. Pap smear has been a basic screening method widely used for half a century, but in recent years, the problems of Pap smear in the diagnosis of cervical cancer and precancerous lesions have become increasingly obvious. Although thin-layer liquid-based technology has been introduced to improve the Pap smear method, which has greatly improved experimental operations and reduced false positives, the high operating costs have prevented this technology from becoming a mainstream operating technology. Gene chip technology is a technology that relies on solid phase carriers. Although this technology has many advantages, this method is mainly used to study the relationship between cervical diseases and HPV subtypes, and cannot be used and promoted on a large scale in clinical testing.

[0004] The real-time fluorescence quantitative PCR method based on polymerase chain reaction technology is currently the most widely used technology for HPV-DNA detection. This technology refers to the addition of a specific fluorescent probe at the same time as a pair of primers during PCR amplification. The probe is an oligonucleotide with a reporter fluorescent group and a quencher fluorescent group labeled at both ends. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; during PCR amplification, the 5'-3' exonuclease activity of the Taq enzyme will enzymatically degrade the probe, separating the reporter fluorescent group and the quencher fluorescent group, so that the fluorescence monitoring system can receive the fluorescent signal, that is, every time a DNA chain is amplified, a fluorescent molecule is formed, achieving complete synchronization between the accumulation of the fluorescent signal and the formation of the PCR product.

[0005] Compared with ordinary PCR, the experimental results of real-time fluorescence PCR are more intuitive and more real-time. Real-time fluorescence PCR is detected by adding fluorescent groups to the PCR system and relying on the accumulation of fluorescent signals. The experimental results are absolutely quantitative with standard curves and Ct values. A study compared the application of the second-generation hybrid capture method and fluorescent PCR in the clinical detection of human papillomavirus. According to the experimental results, the two different detection methods have a good correlation with high-risk cervical cancer lesions, but fluorescent PCR has more advantages in sensitivity and specificity. In addition, the total positive detection rate of real-time fluorescent PCR is also higher in the study, which suggests that fluorescent PCR can be used as an effective detection method for clinical detection, providing a basis for the effective detection of human papillomavirus. Multiple fluorescent PCR can simultaneously amplify two or more pairs of target genes due to the differences between fluorescent groups. This method organically combines the high sensitivity of ordinary PCR, the high specificity of DNA hybridization and the high accuracy of spectral technology, overcoming the shortcomings and limitations of traditional PCR in many aspects. The closed-tube operation of real-time PCR can effectively eliminate the cross-contamination caused by aerosols in nucleic acid amplification, and the accuracy and specificity of qualitative or quantitative DNA templates are greatly improved. Multiplex fluorescence PCR has been widely used in many fields such as gene testing, sexually transmitted disease testing, and genetic disease testing due to abnormal gene expression.

[0006] Prior art CN104195247B uses multiplex fluorescence PCR technology to detect 18 HPV genotypes in the same tube amplification system. At the same time, the three-color high-risk HPV fluorescence detection kit can detect 15 common high-risk HPV genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 82) and three rare high-risk HPV genotypes (26, 53, 73). The four-color high-risk HPV fluorescence detection kit can detect 13 common high-risk HPV genotypes (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 82) and three rare high-risk HPV genotypes (26, 53, 73) at the same time, and can also specifically type high-risk HPV genotypes 16 and 18. The invention also adds UNG enzyme to prevent contamination, making the sensitivity of the kit of the present invention higher. This technology has a small number of probes and poor anti-interference ability.

[0007] Prior art CN110904278B discloses a 13-type high-risk HPV multiplex fluorescence detection kit, including a hot-start Taq enzyme system, a dUTP UNG anti-contamination system, tailing primers, universal primers, fluorescent probes, negative controls and positive controls, and the fluorescent probes are HPV-specific probes; the kit of the invention is suitable for qualitatively detecting 13 high-risk HPV genotypes that can cause cervical cancer in cervical exfoliated cells and genital urinary tract secretion samples, and typing 16 and 18 genotypes; the kit uses a homologous tailing system to reduce interference between multiple primers, and realizes the detection of 13 HPV genotypes in the same tube fluorescent PCR amplification system. The invention detects a small number of HPV genotypes at the same time, and the design of primers and probes is relatively cumbersome.

[0008] Fluorescence quantitative PCR technology has been widely used in the detection of HPV, but the current common method of this type of method for detecting high-risk HPV often designs a probe for each HPV subtype. The detection of multiple high-risk HPV subtypes requires multiple probes. The cost of probe synthesis is high, and different probes are prone to interference, resulting in poor detection specificity. Summary of the invention

[0009] In view of the problems existing in the prior art, the present invention provides a nucleic acid composition, a kit and applications thereof for detecting human papillomavirus nucleic acid. The provided kit has a small number of probes, strong detection specificity and high sensitivity.

[0010] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0011] In one aspect, the present invention provides a nucleic acid composition, comprising probes for HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV82 and corresponding primers;

[0012] Preferably, the probe comprises:

[0013] A probe for detecting HPV16, as shown in SEQ ID NO.1;

[0014] A probe for detecting HPV18, as shown in SEQ ID NO.2;

[0015] Probes for detecting HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV82 are shown in SEQ ID NO.3-5.

[0016] Preferably, the primers include 17 forward primers and 15 reverse primers for HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV82.

[0017] Preferably, the primers include:

[0018] The forward and reverse primers for detecting HPV16 are shown in SEQ ID NO.6 and SEQ ID NO.23, respectively;

[0019] The forward and reverse primers for detecting HPV18 are shown in SEQ ID NO.7 and SEQ ID NO.24 respectively;

[0020] Forward and reverse primers for detecting HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV82, wherein the forward primer is shown in SEQ ID NO.8-22 and the reverse primer is shown in SEQ ID NO.25-37.

[0021] The nucleic acid composition consists of 5 probes, 17 forward primers, and 15 reverse primers, which can be applied to 18 types of HPV detection, of which 3 probes cover 16 subtypes. Through this merging strategy, the nucleic acid composition provided by the present invention can reduce the mutual interference caused by too many primers and probes compared to the one-to-one amplification combination, while reducing costs.

[0022] Preferably, the 5' end of the probe is labeled with a fluorescent group, and the fluorescent group includes any one of Cyan500, FAM, VIC, Cy3, JOE, TAMRA, Texas Red, ROX, Cy5, Cy5.5, TET, HEX, and NED.

[0023] Preferably, the 3' end of the probe is labeled with a quencher group, and the quencher group includes any one of MGB, TAMRA, BHQ1, BHQ2, BHQ3, NFQ, DABCYL or Eclipse.

[0024] Preferably, the probes corresponding to SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3-5 have different fluorescent groups. By setting different fluorescent groups, probes corresponding to different fluorescence can be separated for distinguishing different genes.

[0025] Specifically, the 5' end of the probe corresponding to SEQ ID NO.1 is a CY5 fluorescent group, and the 3' end is a BHQ2 quenching group; the 5' end of the probe corresponding to SEQ ID NO.2 is a ROX fluorescent group, and the 3' end is a BHQ2 quenching group; the 5' end of the probe corresponding to SEQ ID NO.3-5 is a FAM fluorescent group, and the 3' end is a MGB quenching group.

[0026] On the other hand, the present invention provides a kit for detecting human papillomavirus nucleic acid, the kit comprising the above-mentioned nucleic acid composition, an amplification reaction solution, a negative control, a positive control and a quality control internal reference.

[0027] Preferably, the amplification reaction solution comprises a reaction buffer, a hot-start Taq enzyme, dNTPs, dUTP and UNG enzyme.

[0028] The reaction buffer is an important component of the PCR reaction. It provides a stable chemical environment and contains a variety of components that help the reaction proceed. The PCR reaction buffer usually uses a 10mmol / L Tris-HCl (pH 8.3-9.0, room temperature) buffer system. This system has a certain pH buffering capacity and provides a stable chemical environment for the reaction. The buffer contains divalent cations that can activate the active center of DNA polymerase, generally Mg 2+ (Generally provided in the form of MgCl2). 2+ The concentration of can significantly affect the specificity of PCR amplification products. The buffer may also contain other ingredients, such as 50mmol / L K + , which is conducive to the annealing of primers and templates. Some buffers also contain gelatin or serum albumin and detergents to stabilize Taq DNA polymerase.

[0029] UNG enzyme, or uracil-DNA glycosylase, is an important enzyme in the human body, mainly used for anti-contamination in PCR reactions. UNG enzyme can effectively catalyze the hydrolysis of the N-glycosidic bond between uracil bases and sugar-phosphate backbones in single-stranded or double-stranded DNA, but has no activity against RNA; when used in combination with dUTP, a PCR anti-contamination system can be established to ensure the accuracy of PCR amplification results. Before the PCR reaction, UNG enzyme can degrade PCR products containing uracil and eliminate residual PCR contamination. Natural, unmodified DNA is not affected by UNG enzyme and can continue to be used as a template for PCR amplification.

[0030] Preferably, the negative control comprises sterile deionized water, TE buffer or the above-mentioned reaction buffer.

[0031] Specifically, the negative control is TE buffer.

[0032] Preferably, the positive control comprises a plasmid or DNA fragment containing 18 HPV subtype genes within the detection range of the kit.

[0033] Preferably, the quality control internal reference includes a specific probe complementary to an internal reference gene such as β-actin, tubulin, glyceraldehyde-3-phosphate dehydrogenase or ribonuclease P30, and a corresponding forward primer and reverse primer.

[0034] Internal reference genes, also known as housekeeping genes, are internal references. Their expression in various tissues and cells is relatively constant. They are often used as references when detecting changes in gene expression levels. Their function is to correct the amount of sample loading and the experimental errors in the loading process to ensure the accuracy of the experimental results. By detecting the amount of internal reference in each sample, it can be used to correct the loading error, so that the semi-quantitative results are more reliable. Generally, a gene that does not change its expression under the condition of the treatment factor should be selected as an internal reference. Commonly used internal reference genes include GAPDH, β-actin, 18sRNA, 28sRNA, B2M, ACTB, SDHA, HPRT1, ARBP, etc.

[0035] Preferably, the 5' end of the specific probe is labeled with a fluorescent group, and the fluorescent group includes any one of Cyan500, FAM, VIC, Cy3, JOE, TAMRA, Texas Red, ROX, Cy5, Cy5.5, TET, HEX or NED; the 3' end of the specific probe is labeled with a quencher group, and the quencher group includes any one of MGB, TAMRA, BHQ1, BHQ2, BHQ3, NFQ, DABCYL or Eclipse.

[0036] Preferably, the 5'-end fluorescent group of the specific probe is different from the 5'-end fluorescent group of the probe in the above nucleic acid composition, so that the HPV gene can be distinguished from the internal reference gene and the possibility of interference is reduced.

[0037] Specifically, the quality control internal reference comprises a specific probe whose sequence is complementary to the ribonuclease P30 (RPP30) gene as shown in SEQ ID NO.38, a forward primer whose sequence is shown in SEQ ID NO.39, and a reverse primer whose sequence is shown in SEQ ID NO.40.

[0038] Specifically, the 5' end of the specific probe is a VIC fluorescent group, and the 3' end is a BHQ1 quenching group.

[0039] On the other hand, the present invention provides the use of the above-mentioned nucleic acid composition in detecting high-risk human papillomavirus, wherein the application is a non-disease diagnosis or treatment application.

[0040] Preferably, the application includes preparing the above-mentioned nucleic acid composition into a high-risk human papillomavirus nucleic acid detection kit.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] 1. The present invention provides a set of primer-probe combinations for detecting high-risk human papillomaviruses HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV82, and realizes typing detection of HPV16 and HPV18 with strong specificity and high sensitivity.

[0043] 2. The primer-probe combination provided by the present invention is obtained through reasonable design and optimization, has a small quantity and low cost, and has good specificity when used for PCR amplification.

[0044] 3. The kit provided by the present invention utilizes multiplex fluorescence PCR technology to detect 18 HPV genotypes in the same reaction system, with high detection throughput, greatly reducing screening costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Negative control amplification plot.

[0046] Figure 2 Amplification plot of positive control. DETAILED DESCRIPTION

[0047] It is worth noting that the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0048] Example 1: Primer and probe design

[0049] This example is a multiplex PCR primer and probe design, which is as follows:

[0050] The sequences of 18 high-risk HPVs were obtained from the NCBI database. According to the principle of complementary base pairing, primer pairs and probes complementary to the DNA sequences of the 18 HPVs were designed. When designing primers, it is also necessary to avoid the formation of dimers between primers and avoid nonspecific amplification. The probes should avoid base G at the 5' end. Base merger design should be performed for one base in the probe for different HPV types to reduce the number of probes. At the same time, the ribonuclease RPP30 gene was used as the internal reference gene, and the conservative segment of the ribonuclease RPP30 gene was preferred to design a pair of primers and probes. The specific nucleotide sequences of the primers and probes and their modifications (synthesized by Beijing Qingke Biotechnology Co., Ltd.) are shown in Table 1.

[0051] Table 1 Primer probe sequences and modifications

[0052]

[0053]

[0054]

[0055] In Table 1, the fluorescent group used by the HPV16 probe is CY5, and the quenching group is BHQ2, the fluorescent group used by the HPV18 probe is ROX, and the quenching group is BHQ2, the fluorescent group used by the other 16 HPV types is FAM, and the quenching group is MGB, R in the sequence represents A or G, and the primers have no special modifications; the fluorescent group used by the RPP30 probe is VIC, and the quenching group is BHQ1, and the primers have no special modifications.

[0056] Example 2: Kit components and methods of use

[0057] This embodiment provides components of a 16+2 high-risk human papillomavirus nucleic acid detection kit, wherein the kit includes 34 primers and 6 probes for 18 high-risk HPVs and internal reference genes provided in Example 1, which are prepared into a primer-probe mixture according to the proportions in the table below.

[0058] Table 2 Primer-probe mixture

[0059]

[0060]

[0061] The kit also includes a 2× amplification reaction solution, which is composed of a PCR buffer, dNTPs, dUTPs, Taq DNA polymerase, UNG enzyme, and the like.

[0062] The kit also includes a positive control, which is an engineered bacterial plasmid containing HPV16, HPV18, HPV58 and RPP30 gene fragments within the detection range of the kit and has been confirmed by sequencing, and the negative control is TE buffer.

[0063] The method of using the above kit is as follows:

[0064] (1) obtaining genomic DNA of the sample to be tested;

[0065] (2) using the DNA of the sample to be tested as a template and using the above kit to perform fluorescent quantitative PCR amplification to determine whether the sample to be tested contains high-risk human papillomavirus;

[0066] First, prepare the PCR reaction system, take the same volume (10 μL) of the sample DNA, positive control and negative control, and add them to the PCR reaction system. The details are shown in Table 3:

[0067] Table 3 Multiplex PCR system

[0068] Reagent name Volume per reaction (μL) Primer probe mixture 5 2× Amplification Reaction Solution 15 template 10 Total volume 30

[0069] Then PCR amplification was performed, and the amplification reaction procedure is shown in Table 4.

[0070] Table 4 Multiplex PCR program

[0071]

[0072] The determination method for each fluorescent channel detection is as follows:

[0073] If the Ct value is greater than 38 or there is no Ct value, the result is negative; if the Ct value exists and is ≤38, the result is positive.

[0074] Example 3: Kit Performance

[0075] In order to verify the performance of the kit provided in Example 2, the negative control and positive control of the kit were tested on a fluorescent quantitative PCR instrument (Hangzhou Bioray Technology, model: FQD-96A). Figure 1 and Figure 2 As shown, the negative control had no amplification curve, and the positive controls HPV16, HPV18, HPV58 and RPP30 amplified normally.

[0076] 18 plasmids containing high-risk human papillomavirus DNA fragments that have been confirmed by sequencing were used in different combinations to prepare templates, and detection was carried out on a fluorescent quantitative PCR instrument. The high-risk HPV types detected by the kit of the present invention were consistent with the template combination preparation, and HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV82 were all detected normally. HPV16 and HPV18 had no crossover with each other or with the other 16 high-risk HPVs. The detection results are shown in Table 5.

[0077] Table 5 Detection of different template combinations

[0078]

[0079]

[0080]

[0081] Note: HPV16 corresponds to the CY5 channel, HPV18 corresponds to the ROX channel, the other 16 high-risk HPV types correspond to the FAM channel, and the internal standard RPP30 corresponds to the VIC channel.

[0082] Example 4: Applicability of the kit for clinical samples

[0083] In order to verify the applicability of the kit provided in Example 2 to clinical samples, the present invention selected 50 clinical samples, tested them on a fluorescent quantitative PCR instrument, and compared them with the hospital diagnosis results. The results of the comparative experiment are shown in Table 6. One hospital-diagnosed HPV13, HPV16, HPV18, HPV57, and HPV59 positive samples were detected by this kit, and the FAM channel, CY5 channel, and ROX channel were all positive; 3 HPV16 positive samples were diagnosed by this kit, and the FAM channel and ROX, CY5 channel were negative, and the CY5 channel was positive; 1 hospital-diagnosed HPV16, HPV42, and HPV82 positive samples were detected by this kit, and the FAM channel and CY5 channel were positive, and the ROX channel was negative; 1 hospital-diagnosed HPV18 positive sample, and the FAM channel and CY5 channel were negative, and the ROX channel was positive; 1 hospital-diagnosed HPV18 and HPV53 positive samples were detected by this kit, and the FAM channel and ROX channel were positive, and the CY5 channel was negative; 3 hospitals-diagnosed HPV31, HPV33, and 2 other positive samples were detected by this kit. HPV35 positive samples, 2 HPV39 positive samples diagnosed in hospitals, 1 HPV42 and HPV53 positive samples diagnosed in hospitals, 1 HPV45 positive sample diagnosed in hospitals, 2 HPV51 positive samples diagnosed in hospitals, 1 HPV51 and HPV68 positive samples diagnosed in hospitals, 3 HPV52 positive samples diagnosed in hospitals, 1 HPV52 and HPV58 positive samples diagnosed in hospitals, 2 HPV53 positive samples diagnosed in hospitals, 3 HPV56 positive samples diagnosed in hospitals, 3 HPV58 positive samples diagnosed in hospitals, 1 HPV59 and HPV81 positive samples diagnosed in hospitals, 4 HPV66 positive samples diagnosed in hospitals, 3 HPV68 positive samples diagnosed in hospitals, this kit detected positive FAM channel, negative ROX and CY5 channels; 3 HPV negative samples diagnosed in hospitals, this kit detected negative FAM, ROX and CY5 channels. In summary, the test results of this kit are consistent with the hospital diagnosis results, and the test results are shown in Table 6.

[0084] Table 6 Clinical sample testing

[0085]

[0086]

[0087] Note: HPV16 corresponds to the CY5 channel, HPV18 corresponds to the ROX channel, the other 16 high-risk HPV types correspond to the FAM channel, and the internal standard RPP30 corresponds to the VIC channel; “-” indicates that the test result is negative.

[0088] Experimental Example 5: Sensitivity Test

[0089] In order to verify the sensitivity of the kit provided in Example 2, the copy number calculation formula was used: copies / mL (copy number) = plasmid concentration (μg / ml) × Avogadro constant / plasmid molecular weight to calculate the copy number of the positive plasmid, the positive plasmid was diluted, and the diluted low-concentration positive plasmid was detected on a fluorescent quantitative PCR instrument. The test results are shown in Table 7. When the concentration was diluted ≥0.5 copies / μl, the FAM channel, CY5 channel, and ROX channel were all positive; when the concentration was diluted to 0.1 copies / μl, there was no amplification in the FAM channel, CY5 channel, and ROX channel, and the sensitivity of the kit could reach 0.5 copies / μl (i.e., 5 copies / well).

[0090] Table 7 Sensitivity test

[0091]

[0092] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A nucleic acid composition, characterized in that The nucleic acid composition includes probes corresponding to 18 genotypes including HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV82, and forward primers and reverse primers corresponding to the 18 genotypes. The sequence of the probe is the sequence shown in SEQ ID NO.1-5.

2. The nucleic acid composition according to claim 1, characterized in that The sequence of the forward primer is the sequence shown in SEQ ID NO.6-22.

3. The nucleic acid composition according to claim 1, characterized in that The sequence of the reverse primer is the sequence shown in SEQ ID NO.23-37.

4. The nucleic acid composition according to claim 1, characterized in that The 5' end of the probe is labeled with a fluorescent group, and the fluorescent group is any one of Cyan500, FAM, VIC, Cy3, JOE, TAMRA, Texas Red, ROX, Cy5, Cy5.5, TET, HEX or NED.

5. The nucleic acid composition according to claim 1, characterized in that The 3' end of the probe is labeled with a quencher group, and the quencher group is any one of MGB, TAMRA, BHQ1, BHQ2, BHQ3, NFQ, DABCYL or Eclipse.

6. The nucleic acid composition according to any one of claims 1, 4 or 5, characterized in that The probes corresponding to SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3-5 have different fluorescent groups.

7. The nucleic acid composition according to claim 6, characterized in that The 5' end of the probe corresponding to SEQ ID NO.1 is a CY5 fluorescent group, and the 3' end is a BHQ2 quenching group; the 5' end of the probe corresponding to SEQ ID NO.2 is a ROX fluorescent group, and the 3' end is a BHQ2 quenching group; the 5' end of the probe corresponding to SEQ ID NO.3-5 is a FAM fluorescent group, and the 3' end is a MGB quenching group.

8. A kit for detecting human papillomavirus nucleic acid, characterized in that: The kit comprises the nucleic acid composition according to any one of claims 1 to 7.

9. The kit according to claim 8, characterized in that The kit also includes an amplification reaction solution, a negative control, a positive control and a quality control internal reference.

10. The kit according to claim 9, characterized in that The amplification reaction solution includes a reaction buffer, a hot start Taq enzyme, dNTPs, dUTP and UNG enzyme.

11. The kit according to claim 9, characterized in that The negative control comprises sterile deionized water, TE buffer or the reaction buffer described in claim 10.

12. The kit according to claim 9, characterized in that The positive control includes a plasmid or DNA fragment containing 18 HPV subtype genes within the detection range of the kit.

13. The kit according to claim 9, characterized in that The quality control internal reference includes a specific probe complementary to the β-actin gene, the tubulin gene, the glyceraldehyde-3-phosphate dehydrogenase gene or the ribonuclease P30 gene and a corresponding forward primer and reverse primer.

14. The kit according to claim 13, characterized in that The 5' end of the specific probe is labeled with a fluorescent group, and the fluorescent group is any one of Cyan500, FAM, VIC, Cy3, JOE, TAMRA, Texas Red, ROX, Cy5, Cy5.5, TET, HEX or NED; the 3' end of the specific probe is labeled with a quenching group, and the quenching group is any one of MGB, TAMRA, BHQ1, BHQ2, BHQ3, NFQ, DABCYL or Eclipse.

15. The kit according to any one of claims 13 to 14, characterized in that The 5'-end fluorescent group of the specific probe is different from the 5'-end fluorescent group of the probe according to claim 1 .

16. The kit according to claim 13, characterized in that The quality control internal reference comprises a specific probe whose sequence is complementary to the ribonuclease P30 gene as shown in SEQ ID NO.38, a forward primer whose sequence is shown in SEQ ID NO.39, and a reverse primer whose sequence is shown in SEQ ID NO.

40.

17. The kit according to claim 15, characterized in that The 5' end of the specific probe is a VIC fluorescent group, and the 3' end is a BHQ1 quenching group.

18. Use of the nucleic acid composition according to any one of claims 1 to 7 in detecting high-risk human papillomavirus, characterized in that: The application is non-disease diagnosis or treatment application.

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