CN111455108A - Kit and detection method for detecting 14 high-risk HPV (human papilloma Virus) types - Google Patents
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a kit and a detection method for detecting 14 high-risk HPV types. The kit for detecting 14 high-risk HPV types comprises HPV reaction liquid, HPV positive control and HPV negative control, wherein the HPV reaction liquid consists of primers and PCR buffer solution, the HPV positive control is plasmid DNA, and the HPV negative control is wild-type human genome DNA. The invention has the advantages of high detection speed, the whole detection process including the extraction step can be completed only in 150min, and the invention has the characteristics of good specificity, low price, high accuracy and simple operation, and can completely meet the actual requirements of clinical rapid detection.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit and a detection method for detecting 14 high-risk HPV types.
Background
HPV virus is the abbreviation of human papilloma virus, a genus of papilloma vacuolato virus A of the papovaviridae family, a sexually transmitted disease caused by infection with spherical DNA virus. Cervical cancer is second only to breast cancer in prevalence in women. About 13 million cases of new cervical cancer in China each year and about 3 million deaths, almost all cervical cancers are caused by high-risk HPV infection, and in the female population infected with high-level lesion in China, the HPV types with the top ten infection rates are as follows: 16. 52, 58, 18, 33, 31, 51, 53, 68, 59. It is necessary to distinguish between types in HPV detection. Different HPV types cause different clinical manifestations, and can be divided into skin low-risk type, skin high-risk type, mucosa low-risk type and mucosa high-risk type according to different invading tissue parts; among the high-risk types of the mucous membrane, HPV-16, 18, 30, 31, 33, 35 and 39 are related to cervical cancer, rectal cancer, oral cancer, tonsil cancer and the like, especially HPV-16 and 18 can cause cervical cancer and cervical intraepithelial advanced lesion.
The currently common gene mutation detection methods mainly include a sequencing method and a fluorescent quantitative PCR method. These methods all have certain drawbacks. Sequencing methods have low sensitivity (only 20%), resulting in high false negatives, and also have many steps and complicated procedures. The fluorescence quantitative PCR method is the mainstream detection method at present, has higher sensitivity and simple operation, but also has the defects of large demand on a detection sample, lower detection flux and low accuracy. Therefore, how to find a gene detection method which is low in sample detection demand, high in accuracy and high in detection speed is very important.
Disclosure of Invention
The invention aims to provide a kit for detecting 14 high-risk HPV types, which is low in price and high in accuracy; the invention also provides a detection method adopting the kit for detecting 14 high-risk HPV types, which has the advantages of simple operation, high detection speed, low demand on detection samples, high detection flux and high accuracy.
The kit for detecting 14 high-risk HPV types comprises HPV reaction liquid, HPV positive control and HPV negative control, wherein the HPV reaction liquid consists of primers and PCR buffer solution, the HPV positive control is plasmid DNA, and the HPV negative control is wild-type human genome DNA;
the primer comprises PH-16-M-F, PH-16-M-R, PH-18-M-F, PH-18-M-R, PH-31-M-F, PH-33-M-R, PH-35-M-F, PH-39-M-F, PH-39-M-R, PH-45-M-F, PH-51-M-F, PH-51-M-R, PH-M-F, PH-52-M-R, PH-56-M-F, PH-58-M-F, PH-59-M-F, PH-59-M-R, PH-66-M-F, PH-66-M-R and PH-68-M-F;
PH-16-M-F is PH-16-M-F1 or PH-16-M-F2,
PH-16-M-R is PH-16-M-R1 or PH-16-M-R2,
PH-18-M-F is PH-18-M-F1 or PH-18-M-F2,
PH-18-M-R is PH-18-M-R1 or PH-18-M-R2,
PH-31-M-F is PH-31-M-F1 or PH-31-M-F2,
PH-33-M-R is PH-33-M-R1 or PH-33-M-R2,
PH-35-M-F is PH-35-M-F1 or PH-35-M-F2,
PH-39-M-F is PH-39-M-F1 or PH-39-M-F2,
PH-39-M-R is PH-39-M-R1 or PH-39-M-R2,
PH-45-M-F is PH-45-M-F1 or PH-45-M-F2,
PH-51-M-F is PH-51-M-F1 or PH-51-M-F2,
PH-51-M-R is PH-51-M-R1 or PH-51-M-R2,
PH-52-M-F is PH-52-M-F1 or PH-52-M-F2,
PH-52-M-R is PH-52-M-R1 or PH-52-M-R2,
PH-56-M-F is PH-56-M-F1 or PH-56-M-F2,
PH-58-M-F is PH-58-M-F1 or PH-58-M-F2,
PH-59-M-F is PH-59-M-F1 or PH-59-M-F2,
PH-59-M-R is PH-59-M-R1 or PH-59-M-R2,
the PH-66-M-F is PH-66-M-F1 or PH-66-M-F2,
PH-66-M-R is PH-66-M-R1 or PH-66-M-R2,
PH-68-M-F is PH-68-M-F1 or PH-68-M-F2;
PH-16-M-F1, PH-16-M-F2, PH-16-M-R1, PH-16-M-R2, PH-18-M-F1, PH-18-M-F2, PH-18-M-R1, PH-18-M-R2, PH-31-M-F1, PH-31-M-F2, PH-33-M-R1, PH-33-M-R2, PH-35-M-F1, PH-35-M-F2, PH-39-M-F1, PH-39-M-F2, PH-39-M-R1, PH-39-M-R2, PH-45-M-F1, PH-M-F3936, and PH-31-M-F2, PH-45-M-F2, PH-51-M-F1, PH-51-M-F2, PH-51-M-R1, PH-51-M-R2, PH-52-M-F1, PH-52-M-F2, PH-52-M-R1, PH-52-M-R2, PH-56-M-F1, PH-56-M-F2, PH-58-M-F1, PH-58-M-F2, PH-59-M-F1, PH-59-M-F2, PH-59-M-R1, PH-59-M-R2, PH-66-M-F1, PH-66-M-F2, PH-51-M-F2, PH-52-M-F3524, PH-M-F3527, PH-M-F3683, PH-59-M-R1, PH-M, The sequence of PH-66-M-R1, PH-66-M-R2, PH-68-M-F1 and PH-68-M-F2 is shown as SEQ ID NO. 01-42.
The primer is preferably selected from PH-16-M-F1, PH-16-M-R1, PH-18-M-F1, PH-18-M-R1, PH-31-M-F1, PH-33-M-R1, PH-35-M-F1, PH-39-M-F1, PH-39-M-R1, PH-45-M-F1, PH-51-M-F1, PH-51-M-R1, PH-52-M-F1, PH-52-M-R1, PH-56-M-F1, PH-58-M-F1, PH-59-M-F1, PH-59-M-R1, PH-16-M-R1, PH-39-M-F1, PH-M-R1, PH-45-M-F1, PH-51-M-F1, PH-, PH-66-M-F1, PH-66-M-R1 and PH-68-M-F1.
The primer is preferably selected from PH-16-M-F2, PH-16-M-R2, PH-18-M-F2, PH-18-M-R2, PH-31-M-F2, PH-33-M-R2, PH-35-M-F2, PH-39-M-F2, PH-39-M-R2, PH-45-M-F2, PH-51-M-F2, PH-51-M-R2, PH-52-M-F2, PH-52-M-R2, PH-56-M-F2, PH-58-M-F2, PH-59-M-F2, PH-59-M-R2, PH-16-M-R2, PH-39-M-F2, PH-M-R2, PH-45-M-F2, PH-51-M-F2, PH-, PH-66-M-F2, PH-66-M-R2 and PH-68-M-F2.
According to the human HPV gene sequence, the invention designs fluorescent primers aiming at 14 types of HPV.
The primer sequences of SEQ ID NO. 01-42 are shown in Table 1.
TABLE 1 SEQ ID NO. 01-42 primer sequences
The PCR buffer solution comprises the following components:
the kit for detecting 14 high-risk HPV types takes cell sample DNA as a detection sample.
The detection method adopting the kit for detecting the 14 high-risk HPV types comprises the following steps:
(1) extracting human genome DNA of a cell sample;
(2) adopting HPV reaction liquid in a kit for detecting 14 high-risk HPV types to construct a PCR amplification reaction system for carrying out PCR reaction;
(3) performing capillary electrophoresis on the PCR product;
(4) and judging the detection result according to the fragment size, the fluorescence labeling color and the peak value height of the amplified product displayed by the capillary electrophoresis peak image.
The PCR amplification reaction system in the step (2) is as follows:
the PCR reaction conditions in step (2): pre-denaturation at 95 ℃ for 3 min for 1 cycle; denaturation at 94 ℃ for 5 seconds, annealing at 60 ℃ for 10 seconds, extension at 72 ℃ for 30 seconds, and 40 cycles; extension at 72 ℃ for 10 min, 1 cycle.
The 14 HPV types detected by the kit are 16 types, 18 types, 31 types, 33 types, 35 types, 39 types, 45 types, 51 types, 52 types, 56 types, 58 types, 59 types, 66 types and 68 types.
The invention adopts a method of combining high multiplex PCR (high multiplex PCR) and high-precision capillary electrophoresis to complete the high-precision detection of a plurality of gene mutation sites simultaneously. In the multiplex PCR, a plurality of primers are added to a reaction system to simultaneously amplify a plurality of mutation detection sites, generally 2 to 6 sites. More primers are added into the high multiplex PCR reaction system to detect more mutation sites (such as 40-100 mutations), so that the demand of initial detection samples is greatly reduced, the detection flux is improved, and the cost is saved. The primers used in the high multiplex PCR are fluorescently labeled, and the PCR product can be subjected to fragment analysis by using a sequencing-grade fluorescence capillary electrophoresis apparatus (such as an ABI3500 sequencer). The capillary electrophoresis has high resolution, can distinguish the difference of one base, and greatly improves the accuracy and the sensitivity of mutation detection.
Compared with the current mainstream PCR method, the method combining the high multiplex PCR and the high-precision capillary electrophoresis has greatly improved sensitivity and specificity. The sensitivity is greatly improved from the common PCR product to the fluorescence labeling PCR product through ultraviolet light excitation detection and then to the fluorescence labeling PCR product through ultraviolet light excitation detection and single-frequency laser excitation detection. Such methods are also used in some advanced companies, but are still less. In addition to the need for precise detection of multiple mutations simultaneously, another important aspect of mutation detection is the need for highly specific detection methods that distinguish mutations from wild-type and other similar mutations. Any sample has a large amount of wild-type DNA, and many mutations occur in the same region or even at the same site, so that non-specific signals appear due to the competition between primers and mutant/wild-type templates. The method of the invention mainly controls the generation of non-specific amplification through the following aspects, thereby increasing the specificity:
1. designing a primer: the invention designs the primers as a single primer and a universal primer. The sequence of the single primer is completely matched with a certain subtype, so that the amplification specificity can be ensured, and the universal primer is selected from conserved regions among the subtypes, and can amplify upstream primers and downstream primers of two or more subtypes at different design distances, so that different subtypes are amplified into PCR products with different lengths, and the subtypes are distinguished.
2. PCR buffer and Taq enzyme: reagent components beneficial to sensitivity and amplification specificity are added into the PCR buffer solution formula used in the invention, and the components are optimized. The Taq enzyme is lack of 3 '-5' exo function, so that amplification is not generated even if the primer is mismatched to a great extent, and specificity is further improved.
3.PCR cycling program: according to the special design of the primers, the PCR circulation adopts proper renaturation temperature, and the specificity of primer combination is ensured aiming at the central area of the short primer, and simultaneously the primer is ensured to be combined with mutation templates as many as possible so as to increase the sensitivity.
The invention develops a kit for detecting 14 HPV subtypes with high sensitivity based on high multiplex PCR (high multiplex PCR) and a high-precision capillary electrophoresis method. The kit takes cell sample DNA as a detection sample, and can determine the infection of HPV virus by detecting HPV subtype.
The invention has the following beneficial effects:
the invention adopts specific fluorescent primers, and can realize rapid detection of 14 subtypes of HPV in a cell sample.
(1) The high multiplex PCR (high multiplex PCR) and the high-precision capillary electrophoresis technology are applied to detect the FFPE clinical samples, 14 subtypes of HPV are detected simultaneously by a single tube, and the result is obvious;
(2) by using the PCR buffer solution, the amplification efficiency, stability and specificity are greatly improved;
(3) the method has the advantages of high detection speed, capability of completing the whole detection process including the extraction step in only 150min, good specificity, low price, high accuracy and simplicity in operation, and can completely meet the actual requirements of clinical rapid detection.
Drawings
FIG. 1 is a capillary electrophoresis chart of the positive control result of example 1.
FIG. 2 is a capillary electrophoresis chart of the negative control result of example 1.
FIG. 3 is a capillary electrophoresis chart of the blank control result of example 1.
FIG. 4 is a capillary electrophoresis chart showing the type 16 positive results of the clinical specimen in example 1.
FIG. 5 is a capillary electrophoresis chart showing type 18 positive results of the clinical specimen in example 1.
FIG. 6 is a capillary electrophoresis chart showing type 52 positive results of the clinical specimen in example 1.
FIG. 7 is a capillary electrophoresis chart showing the type 33 positive results of the clinical specimen in example 1.
FIG. 8 is a capillary electrophoresis chart showing the type 58 positive results of the clinical specimen in example 1.
FIG. 9 is a capillary electrophoresis chart of type 66 positive results of the clinical specimen of example 1.
FIG. 10 is a capillary electrophoresis chart showing the type 68 positive results of the clinical specimen in example 1.
FIG. 11 is a capillary electrophoresis chart of the positive control result of example 2.
FIG. 12 is a capillary electrophoresis chart of the negative control result of example 2.
FIG. 13 is a capillary electrophoresis image of the blank control result of example 2.
FIG. 14 is a capillary electrophoresis chart showing the type 16 positive results of the clinical specimen in example 2.
FIG. 15 is a capillary electrophoresis chart showing type 18 positive results of the clinical specimen in example 2.
FIG. 16 is a capillary electrophoresis chart showing type 52 positive results of the clinical specimen in example 2.
FIG. 17 is a capillary electrophoresis chart showing the type 33 positive results of the clinical specimen in example 2.
FIG. 18 is a capillary electrophoresis chart showing the type 58 positive results of the clinical specimen in example 2.
FIG. 19 is a capillary electrophoresis chart of type 66 positive results of the clinical specimen of example 2.
FIG. 20 is a capillary electrophoresis chart of type 68 positive results of the clinical specimen of example 2.
Detailed Description
The present invention is further described below with reference to examples.
The composition of the kit for detecting 14 HPV subtypes according to the invention is shown in Table 2.
TABLE 2 kit Components for detection of HPV subtypes
Example 1
In this example, the plasmid constructed by genetic engineering was used as a positive plasmid, and wild-type human genomic DNA was used as a control. 14 subtypes of HPV and 70 cases of HPV clinical samples are detected by using the high multiplex PCR (high multiplex PCR) and high-precision capillary electrophoresis technology of the invention, and the detection is compared with the fluorescent quantitative PCR detection. The method comprises the following specific steps:
1. and (3) detection sample treatment and DNA extraction:
(1) and (2) plasmid treatment and extraction, wherein each plasmid is extracted by adopting a plasmid extraction kit, the specific extraction operation steps are operated according to the kit instruction, the extracted particles are dissolved in Tris-EDTA (10 mmol/L, pH 8.0), the quality of extraction is detected by Nanodrop, the concentration of the particles is determined, then Tris-EDTA (10 mmol/L, pH 8.0) is used for adjusting the plasmid concentration to different copy numbers and is used as a PCR template, and 5 mu L is taken for PCR amplification.
(2) The method for extracting the cell DNA comprises the steps of placing a collected cervical swab sample in a centrifugal tube, extracting the DNA by adopting a cervical swab DNA extraction kit, wherein the specific extraction operation steps are operated according to the kit specification, dissolving the extracted DNA in Tris-EDTA (10 mmol/L, pH 8.0), detecting the extraction quality through Nanodrop, determining the concentration of the extracted DNA, adjusting the DNA concentration to different concentrations by using Tris-EDTA (10 mmol/L, pH 8.0) to serve as a PCR template, and taking 5 mu L for PCR reaction amplification.
2. The PCR amplification reaction system was constructed as shown in Table 3.
TABLE 3 example 1PCR amplification reaction System
PCR reaction conditions: pre-denaturation at 95 ℃ for 3 min for 1 cycle; denaturation at 94 ℃ for 5 seconds, annealing at 60 ℃ for 10 seconds, extension at 72 ℃ for 30 seconds, and 40 cycles; extension at 72 ℃ for 10 min, 1 cycle.
4. Capillary electrophoresis detection, which adopts mixed PCR products, denaturants and fragment size markers for detection, and the detection system is shown in Table 4.
Table 4 example 1 capillary electrophoresis detection system
| Name (R) | Volume of |
| HI-DI | 7-9μL |
| Rox350 | 0.5-2μL |
| PCR product | 0.5-2μL |
| Total | 10μL |
5. Capillary electrophoresis was performed using the ABI3500 by fragment analysis, and the detection results were judged according to the size of the fragment of the amplified product, the color of the fluorescence label, and the height of the peak, which are shown in FIG. 1 as the peak of the capillary electrophoresis.
6. Among 70 clinical samples tested, 10 HPV types 16, 6 HPV types 18, 6 HPV types 52, 5 HPV types 33, 3 HPV types 58, 2 HPV types 66, 6 HPV types 68 and 32 negative samples were tested, and blank controls were simultaneously set for each group, and the results are shown in FIGS. 2, 3, 4, 5, 6, 7, 8, 9 and 10.
Example 2
In this example, the plasmid constructed by genetic engineering was used as a positive plasmid, and wild-type human genomic DNA was used as a control. The high multiplex PCR (high multiplex PCR) and high-precision capillary electrophoresis technology are used for detecting 14 types and 70 cases of HPV clinical samples. The method comprises the following specific steps:
1. and (3) detection sample treatment and DNA extraction:
(1) and (2) plasmid treatment and extraction, wherein each plasmid is extracted by adopting a plasmid extraction kit, the specific extraction operation steps are operated according to the kit instruction, the extracted particles are dissolved in Tris-EDTA (10 mmol/L, pH 8.0), the quality of extraction is detected by Nanodrop, the concentration of the particles is determined, then Tris-EDTA (10 mmol/L, pH 8.0) is used for adjusting the plasmid concentration to different copy numbers and is used as a PCR template, and 5 mu L is taken for PCR amplification.
(2) The method for extracting the cell DNA comprises the steps of placing a collected cervical swab sample in a centrifugal tube, extracting the DNA by adopting a cervical swab DNA extraction kit, wherein the specific extraction operation steps are operated according to the kit specification, dissolving the extracted DNA in Tris-EDTA (10 mmol/L, pH 8.0), detecting the extraction quality through Nanodrop, determining the concentration of the extracted DNA, adjusting the DNA concentration to different concentrations by using Tris-EDTA (10 mmol/L, pH 8.0) to serve as a PCR template, and taking 5 mu L for PCR reaction amplification.
2. The PCR amplification reaction system was constructed as shown in Table 5.
TABLE 5 example 2PCR amplification reaction System
PCR reaction conditions: pre-denaturation at 95 ℃ for 3 min for 1 cycle; denaturation at 94 ℃ for 5 seconds, annealing at 60 ℃ for 10 seconds, extension at 72 ℃ for 30 seconds, and 40 cycles; extension at 72 ℃ for 10 min, 1 cycle.
4. The mixed PCR product, denaturant and fragment size marker were used for detection, and the detection system is shown in Table 6.
TABLE 6 EXAMPLE 2 capillary electrophoresis detection System
| Name (R) | Volume of |
| HI-DI | 7-9μL |
| Rox350 | 0.5-2μL |
| PCR product | 0.5-2μL |
| Total | 10μL |
5. Capillary electrophoresis was performed by using the ABI3500 for fragment analysis, and the detection result was judged from the size of the fragment, the color of the fluorescence label, and the height of the peak of the amplified product, which are shown in FIG. 11 as the peak of the capillary electrophoresis.
6. Among 70 clinical samples tested, 10 HPV types 16, 6 HPV types 18, 6 HPV types 52, 5 HPV types 33, 3 HPV types 58, 2 HPV types 66, 6 HPV types 68 and 32 negative samples were tested, and blank controls were simultaneously set for each group, and the results are shown in FIGS. 12, 13, 14, 15, 16, 17, 18, 19 and 20.
The results of the examples 1-2 show that the coincidence rate of the system of the invention and the fluorescent quantitative PCR method reaches 100%, see Table 7, and further prove the accuracy, rapidity and low cost of the detection of the system of the invention. Therefore, compared with a fluorescence quantitative PCR method, the method for detecting the HPV in the FFPE sample has the same accuracy, but has higher speed and lower cost, and can meet the requirement of the rapid detection of the HPV.
TABLE 7 comparison of capillary electrophoresis with fluorescent quantitative PCR
Note: the detection time in table 7 is the time of capillary detection.
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<400>28
gatgttctgc ttgtccatc 19
<210>29
<211>21
<212>DNA
<213>Artificial
<400>29
cgggtcatgt ttggggtgct g 21
<210>30
<211>21
<212>DNA
<213>Artificial
<400>30
gccgtcatgt ttggggtgct g 21
<210>31
<211>21
<212>DNA
<213>Artificial
<400>31
agggcgctgt gcagtgtgtt g 21
<210>32
<211>20
<212>DNA
<213>Artificial
<400>32
tcgcgctgtg cagtgtgttg 20
<210>33
<211>21
<212>DNA
<213>Artificial
<400>33
aggacagtgt cgtgggtgtc g 21
<210>34
<211>21
<212>DNA
<213>Artificial
<400>34
acaacagtgt cgtgggtgtc g 21
<210>35
<211>19
<212>DNA
<213>Artificial
<400>35
gtggttcagc tcgtctagc 19
<210>36
<211>19
<212>DNA
<213>Artificial
<400>36
acggttcagc tcgtctagc 19
<210>37
<211>21
<212>DNA
<213>Artificial
<400>37
cgggtcatgt ttgcagtgtt g 21
<210>38
<211>21
<212>DNA
<213>Artificial
<400>38
gcggtcatgt ttgcagtgtt g 21
<210>39
<211>19
<212>DNA
<213>Artificial
<400>39
gttgttcagc ttgtctagc 19
<210>40
<211>19
<212>DNA
<213>Artificial
<400>40
cgagttcagc ttgtctagc 19
<210>41
<211>21
<212>DNA
<213>Artificial
<400>41
aggacagtgt cggcactgct g 21
<210>42
<211>21
<212>DNA
<213>Artificial
<400>42
ctgacagtgt cggcactgct g 21
Claims (7)
1. A kit for detecting 14 high-risk HPV types is characterized by comprising HPV reaction liquid, HPV positive control and HPV negative control, wherein the HPV reaction liquid consists of primers and PCR buffer solution, the HPV positive control is plasmid DNA, and the HPV negative control is wild-type human genome DNA;
the primer comprises PH-16-M-F, PH-16-M-R, PH-18-M-F, PH-18-M-R, PH-31-M-F, PH-33-M-R, PH-35-M-F, PH-39-M-F, PH-39-M-R, PH-45-M-F, PH-51-M-F, PH-51-M-R, PH-M-F, PH-52-M-R, PH-56-M-F, PH-58-M-F, PH-59-M-F, PH-59-M-R, PH-66-M-F, PH-66-M-R and PH-68-M-F;
PH-16-M-F is PH-16-M-F1 or PH-16-M-F2,
PH-16-M-R is PH-16-M-R1 or PH-16-M-R2,
PH-18-M-F is PH-18-M-F1 or PH-18-M-F2,
PH-18-M-R is PH-18-M-R1 or PH-18-M-R2,
PH-31-M-F is PH-31-M-F1 or PH-31-M-F2,
PH-33-M-R is PH-33-M-R1 or PH-33-M-R2,
PH-35-M-F is PH-35-M-F1 or PH-35-M-F2,
PH-39-M-F is PH-39-M-F1 or PH-39-M-F2,
PH-39-M-R is PH-39-M-R1 or PH-39-M-R2,
PH-45-M-F is PH-45-M-F1 or PH-45-M-F2,
PH-51-M-F is PH-51-M-F1 or PH-51-M-F2,
PH-51-M-R is PH-51-M-R1 or PH-51-M-R2,
PH-52-M-F is PH-52-M-F1 or PH-52-M-F2,
PH-52-M-R is PH-52-M-R1 or PH-52-M-R2,
PH-56-M-F is PH-56-M-F1 or PH-56-M-F2,
PH-58-M-F is PH-58-M-F1 or PH-58-M-F2,
PH-59-M-F is PH-59-M-F1 or PH-59-M-F2,
PH-59-M-R is PH-59-M-R1 or PH-59-M-R2,
the PH-66-M-F is PH-66-M-F1 or PH-66-M-F2,
PH-66-M-R is PH-66-M-R1 or PH-66-M-R2,
PH-68-M-F is PH-68-M-F1 or PH-68-M-F2;
PH-16-M-F1, PH-16-M-F2, PH-16-M-R1, PH-16-M-R2, PH-18-M-F1, PH-18-M-F2, PH-18-M-R1, PH-18-M-R2, PH-31-M-F1, PH-31-M-F2, PH-33-M-R1, PH-33-M-R2, PH-35-M-F1, PH-35-M-F2, PH-39-M-F1, PH-39-M-F2, PH-39-M-R1, PH-39-M-R2, PH-45-M-F1, PH-M-F3936, and PH-31-M-F2, PH-45-M-F2, PH-51-M-F1, PH-51-M-F2, PH-51-M-R1, PH-51-M-R2, PH-52-M-F1, PH-52-M-F2, PH-52-M-R1, PH-52-M-R2, PH-56-M-F1, PH-56-M-F2, PH-58-M-F1, PH-58-M-F2, PH-59-M-F1, PH-59-M-F2, PH-59-M-R1, PH-59-M-R2, PH-66-M-F1, PH-66-M-F2, PH-51-M-F2, PH-52-M-F3524, PH-M-F3527, PH-M-F3683, PH-59-M-R1, PH-M, The sequence of PH-66-M-R1, PH-66-M-R2, PH-68-M-F1 and PH-68-M-F2 is shown as SEQ ID NO. 01-42.
2. The kit for detecting typing of 14 high-risk HPVs according to claim 1, wherein the primers consist of PH-16-M-F1, PH-16-M-R1, PH-18-M-F1, PH-18-M-R1, PH-31-M-F1, PH-33-M-R1, PH-35-M-F1, PH-39-M-F1, PH-39-M-R1, PH-45-M-F1, PH-51-M-F1, PH-51-M-R1, PH-52-M-F1, PH-52-M-R1, PH-56-M-F1, PH-58-M-F1, PH-16-M-F1, or PH-M-F1, PH-59-M-F1, PH-59-M-R1, PH-66-M-F1, PH-66-M-R1 and PH-68-M-F1.
3. The kit for detecting typing of 14 high-risk HPVs according to claim 1, wherein the primers consist of PH-16-M-F2, PH-16-M-R2, PH-18-M-F2, PH-18-M-R2, PH-31-M-F2, PH-33-M-R2, PH-35-M-F2, PH-39-M-F2, PH-39-M-R2, PH-45-M-F2, PH-51-M-F2, PH-51-M-R2, PH-52-M-F2, PH-52-M-R2, PH-56-M-F2, PH-58-M-F2, PH-16-M-F2, or PH-M-F2, PH-59-M-F2, PH-59-M-R2, PH-66-M-F2, PH-66-M-R2 and PH-68-M-F2.
4. The kit for detecting typing of 14 high risk HPVs according to claim 1, wherein the PCR buffer consists of:
5. a detection method using the kit for detecting typing of 14 high-risk HPVs according to any one of claims 1 to 4, characterized by comprising the steps of:
(1) extracting human genome DNA of a cell sample;
(2) adopting HPV reaction liquid in a kit for detecting 14 high-risk HPV types to construct a PCR amplification reaction system for carrying out PCR reaction;
(3) performing capillary electrophoresis on the PCR product;
(4) and judging the detection result according to the fragment size, the fluorescence labeling color and the peak value height of the amplified product displayed by the capillary electrophoresis peak image.
6. The detection method according to claim 5, wherein the PCR amplification reaction system in step (2) is as follows:
7. the detection method according to claim 5, wherein the PCR reaction conditions in step (2): pre-denaturation at 95 ℃ for 3 min for 1 cycle; denaturation at 94 ℃ for 5 seconds, annealing at 60 ℃ for 10 seconds, extension at 72 ℃ for 30 seconds, and 40 cycles; extension at 72 ℃ for 10 min, 1 cycle.
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