CN113481322A - Composite amplification system and kit for typing detection of 16-valent HPV (human papillomavirus) - Google Patents
Composite amplification system and kit for typing detection of 16-valent HPV (human papillomavirus) Download PDFInfo
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Abstract
The invention belongs to the technical field of biological detection, and discloses a composite amplification system for typing detection of 16-valent HPV (human papillomavirus) and a kit thereof, wherein the amplification system comprises 26 pairs of detection primers, and the nucleotide sequences of the detection primers are shown as SEQ ID No. 1-38. The amplification system and the method can detect 16 types of HPV types, and the selected types are corresponding types of 16-valent vaccines, so that the method has high degree of feasibility and practical detection significance. Wherein, the commonly used 9-valent detectable typing has two detection sites which are respectively set in an L1 region and an E region of an HPV genome, and can ensure that the HPV typing detected by the kit is more efficient, accurate and sensitive.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a composite amplification system and a kit for typing detection of a 16-valent HPV virus.
Background
Human Papilloma Virus (HPV) is a Papilloma vacuolatum Virus A genus of papovaviridae, and is a sexually transmitted disease caused by infection of spherical DNA Virus, and the main infected areas are Human epidermis and mucosal squamous epithelium, and HPV is known to cause benign tumor and wart of Human. The virus has high specificity, and 130 viruses are separated so far, including types of HPV6, 11, 42, 43, 44 and the like, and cause benign lesions such as external genital condyloma and the like, including cervical intraepithelial hypopathy (CINI). This is a low risk category.
High risk types of HPV including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, etc. are associated with the occurrence of cervical cancer and cervical intraepithelial height lesions (CIN ii/iii), especially HPV16 and 18. Long-term infection may be associated with cervical cancer in women. The long time is needed from HPV infection to cervical cancer occurrence, so that whether the HPV infection is caused or not can be detected as early as possible, and the cervical cancer occurrence can be prevented. Recent guidelines for cervical cancer screening also suggest high-risk HPV typing detection as a first-line cervical cancer screening method.
The methods currently used clinically for detecting HPV include cytology methods, immunohistochemistry, in situ hybridization, dot hybridization, nucleic acid blotting, PCR, and the like, and among them, the method with the highest sensitivity by PCR is the most widely used method at present. The detection method based on the PCR technology also comprises a membrane hybridization method, a gene chip method, a reverse dot hybridization method, a flow fluorescence hybridization method, a fluorescence probe method and the like after the specific primer PCR and the universal primer PCR. Different methods have respective advantages and disadvantages, cannot give consideration to the characteristics of simple operation, high sensitivity, good specificity, low price and strong objectivity, and can only select a proper and effective detection method according to different requirements to meet the detection requirements.
The HPV viral genome is small, containing only about 7900 base pairs. The viral genome is divided into 3 parts: early gene region (E), late gene region (L), and long regulatory region (LCR) that distinguishes early from late. The regulatory regions primarily regulate viral transcription, controlling the production of viral proteins and infectious particles. The early regions encode the proteins E6, E7, E1, E2, E4, E3 and E5 and are mainly involved in viral DNA replication and transcription. The late regions encode the L1, L2 proteins, the major and minor coat proteins of the virus, respectively. According to the functional characteristics and sequence conservation characteristics of different gene regions, most of the selected targets for detecting human papillomavirus DNA by using a fluorescence labeling PCR method are an E6E7 region or an L1 region. The sequence of the L1 region is well conserved and is the main basis for distinguishing different subtypes, but there is a certain probability that the HPV L1 gene is deleted in the process of HPV viral DNA integration into the human genome.
Disclosure of Invention
Aiming at the problems, the invention provides a composite amplification system and a kit for 16-valent HPV virus typing detection, which are simple to operate, high in sensitivity, good in specificity and low in price.
The technical purpose is achieved, the technical effect is achieved, and the invention is realized through the following technical scheme:
the multiplex amplification system for typing and detecting the 16-valent HPV in the invention is 26 pairs of detection primers designed on the basis of 16 selected HPV typing viruses, and the nucleotide sequences of the multiplex amplification system are shown in the following table.
Specifically, the primers include 18 pairs of detection primers corresponding to the L region and the E region detection sites in 9 HPV typing viruses, and another 7 pairs of E region detection sites for detecting other 7 high-risk HPV viruses, and 1 pair of detection primers for beta-globin of human genomic DNA.
Further, more than 0.1 to 0.4. mu.M of the detection primers are included in the amplification system.
Further, the kit comprises two different fluorescent markers, and the same fluorescent marker corresponds to the detection sites of the same L region or E region of the HPV typing virus respectively. That is, the fluorescent labels used for the detection sites of the same L region of all HPV typing viruses are the same, the fluorescent labels used for the detection sites of the same E region of all HPV typing viruses are the same, and the fluorescent labels of the detection sites of the L region and the detection sites of the E region are different
Further, TAMRA is specifically used for marking the detection site of the L region, and ROX is used for marking the detection site of the E region.
Further, the ROX marker is also used for marking a beta-globin detection site corresponding to human genomic DNA.
Further, in addition to the fluorescent label disposed at the detection site, an internal standard fluorescence disposed on each pair of detection primers is also included, and the internal standard fluorescence is ORG.
The invention also researches that the reaction conditions of the amplification system during diffusion are as follows: step 1: denaturation at 95 ℃ for 5 min, step 2: denaturation at 94 ℃ for 30 seconds, annealing at 59 ℃ for 30 seconds in step 3, and annealing at step 4: extension at 72 ℃ for 1 minute, repetition of 2 to 4 steps at least 30 times, and final extension at 60 ℃ for 30-60 minutes.
Based on the above amplification system, the present invention aims to prepare a kit for typing and detecting a 16-valent HPV virus by using the above amplification system.
The invention also provides application of the composite amplification system or the kit in HPV virus typing detection.
The invention has the beneficial effects that:
1. and the typing E region of 16 HPV types and the L1 region of 9 high-risk HPV types are simultaneously detected as a control, so that the typing accuracy is high, and the occurrence of false positive and false negative results can be reduced.
2, a three-color fluorescence labeling technology is newly adopted, and the detection result is clear and easy to read. The kit is provided with an internal standard and an allele typing standard substance, can automatically calibrate a detection result, and has simpler and more convenient data analysis and more accurate analysis result.
3. The system has strong specificity and good stability, and no non-specific amplification product is generated after repeated verification, and the signal intensity is stable.
4. The sensitivity is high, and 16 mixed typing plasmid standards are used for detection, so that more than 10copies can be ensured to be free from missing any typing.
5. Positive judgment value is clear, the system can stably detect HPV positive typing with more than 10copies through complicated interference experiments, and when two or more HPV viruses are simultaneously provided, the HPV positive typing is simulated at the ratio of 1: at a ratio of 100, a stable signal intensity can be obtained, thereby establishing a stable positive judgment value.
Drawings
FIG. 1 is a graph showing the amplification effect of random cervical smear samples;
FIG. 2 is a diagram showing the amplification effect of HPV6 and HPV11 typing (national references);
FIG. 3 is a diagram showing the amplification effect of HPV16 and HPV18 typing (national references);
FIG. 4 is a diagram showing the amplification effect of HPV31 and HPV33 typing (national references);
FIG. 5 is a diagram showing the amplification effect of HPV45 and HPV52 typing (national references);
FIG. 6 is a graph showing the amplification effects of HPV58 typing (national reference) and human genomic DNAF 312;
FIG. 7 is a diagram showing the amplification effect of HPV67 and HPV81 (national references);
FIG. 8 is a graph showing the amplification effect of a mixed sample of 16-stage HPV typing (national reference);
FIG. 9 is a schematic of an allelic typing standard;
FIGS. 10 to 17 show the results of the repeatability tests of the kit;
FIGS. 18 to 31 show the results of the minimum detection limit test of the kit.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The following detailed description of the principles of the invention is provided in connection with the accompanying drawings.
1. Primer design
The amplification system of the invention designs 26 pairs of primers, wherein 25 pairs of primers are designed aiming at 16 HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, 25 sites can be detected, and the primers comprise: HPV6-L1, HPV6-E, HPV11-L1, HPV11-E, HPV16-L1, HPV16-E, HPV18-L1, HPV18-E, HPV31-L1, HPV31-E, HPV33-L1, HPV33-E, HPV35-E, HPV39-E, HPV45-L1, HPV45-E, HPV51-E, HPV52-L1, HPV52-E, HPV56-E, HPV58-L1 and HPV58-E, HPV59-E, HPV66-E, HPV 68-E. In addition, a pair of detection sites beta-globin for detecting human genome DNA is also included. The 25 pairs of detection primers and concentrations are shown in table 1.
TABLE 1 nucleotide sequence and concentration of 19 pairs of detection primers
2. Three-color fluorescent marking system
The amplified sites in the amplification system respectively consist of fluorescent markers with two colors, and the fluorescent markers are positioned at the 5' end of one primer in the specific primer pair. The primers specific to the L1 regions of 9 types of types are divided into the same group, and comprise HPV6-L1, HPV11-L1, HPV16-L1, HPV18-L1, HPV31-L1, HPV33-L1, HPV45-L1, HPV52-L1 and HPV58-L1, and are marked by TAMRA. Specific primers of 16 types of typed E regions and a contrast reference beta-globin locus are used as primers which are divided into a second group, HPV6-E, HPV11-E, HPV16-E, HPV18-E, HPV31-E, HPV33-E, HPV35-E, HPV39-E, HPV45-E, HPV51-E, HPV52-E, HPV56-E, HPV58-E, HPV59-E, HPV66-E, HPV68-E and beta-globin, and ROX is adopted for marking. An orange internal standard QD550 and two sets of allele typing calibrators for panel were introduced simultaneously to calibrate HPV typing of the test samples.
Wherein the internal standard QD550 is a collection of PCR products with fluorescent labeling of AF633 of known size 70, 80, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550.
3. Establishment of amplification System Components
The amplification system also comprises a PCR buffer solution, template DNA, Taq polymerase and allele typing standards of each type.
The composition and amount of the PCR Buffer (5 XBuffer) are shown in Table 2.
Table 2: other formulation components of amplification System
| Composition (I) | Unit of | Dosage of |
| KCL | M | 0.01 |
| MgCL2 | M | 0.002 |
| Tris-HCL | M | 0.05 |
| BSA | mg/mL | 0.07 |
| dNTP | mM | 0.2 |
| (NH4)2SO4 | mM | 0.01 |
| DMSO | ％ | 5.5 |
| Ethylene glycol | ％ | 7 |
| Na4P2O7 | mM | 1 |
| Taq enzyme | U | 2 |
The parameters of the various components and components of the amplification system are shown in Table 3.
Table 3: amplification System Components and dosage parameters
| Components | Dosage of |
| 5X Buffer | 2μL |
| Detection primer | 2μL |
| Template DNA | 1-5μL |
| Taq enzyme (5U/. mu.L) | 0.4μL |
| ddH2O | Make up to 10. mu.L |
4. Determination of amplification reaction conditions
The reaction conditions for amplification using the amplification system of the present invention are shown in Table 4.
Table 4: conditions for amplification reaction
5. Determination of Positive judgment value
Respectively mixing the national reference substance of one of the 16 types with other 15 types according to the mixing ratio of 1: 1. 1: 10. 1: 50 and 1: 100, the experiment was performed.
For example, the national reference substance for HPV16 typing, with a concentration of 10copies/uL, was mixed with the other 15 types of 0copies, 10copies, 100copies, 500copies, and 1000copies for detection. The PCR amplification experiment and the capillary electrophoresis experiment are repeated 100 times for each combination, the signal intensity value is established, and the positive judgment value is established.
6. Verification experiment
Detection of national Standard
Human papillomavirus whole genome type national reference (standard contains beta-globin detection site sequence), from the Chinese food and drug testing institute, lot number: 360003-201602. In addition to 16 HPV types contained in the detection kit, HPV67, HPV81 and human genome DNAF312 are randomly selected as templates for amplification detection. The detection results are shown in fig. 2, fig. 3, fig. 4, fig. 5, fig. 6 and fig. 7, and it can be seen from the figure that the detection results of the 9 HPV standards contained in the kit are correct and have no non-specific appearance, and the result of distinguishing the type by the region L1 is consistent with that by the region E. And the detection results of HPV67, HPV81 standard substance and F312 only detect beta-globin, and no other typing and non-specific peaks appear.
Typing standard substance
Mixing 16 HPV typing standard substances contained in the kit in equal proportion, and diluting according to the copy number provided by the standard substances until the copy number of each HPV typing in the mixed solution is below 10 copies/. mu.L. According to the kit amplification system and the program provided by the invention, the amount of the template is 1 mu L, and the detection result after capillary detection is shown in figure 8. It can be seen from the figure that the L1 region and the E region were detected for nine HPV types.
Repeatability of
Repeat detection 102The national reference dilution of the HPV6 and HPV11 of copies/ul is diluted 10 times, the detection results of 10 times are consistent and positive corresponding to the HPV type, and the repetition rate is 100%. The result shows that the technology has better detection repeatability. The results are shown in FIGS. 10 to 17.
Minimum limit of detection
The results show that the national reference dilution of 14 HPV types with the concentrations of 100copies/ul, 50copies/ul and 10copies/ul has positive corresponding HPV types, the detection rate is 100 percent, and only 8 national reference dilutions of 14 HPV types with 1 copy/ul detect 6 undetected HPV types. Therefore, the minimum detection limit of the technology is determined to be 10 copies/reaction, and the technology meets the requirements of the industry standard. The results are shown in FIGS. 18 to 31.
The results of the clinical specimen testing and typing are shown in the following table:
the positive coincidence rate is calculated to be 96.25 percent (95 percent CI: 89.43 percent to 99.22 percent)
The negative coincidence rate is 82.93% (95% CI: 73.02% -90.34%)
The total coincidence rate is 89.5 percent (95 percent CI: 83.73 percent to 93.77 percent)
The Kappa coefficient is 79%
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
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<213> Artificial Sequence (Artificial Sequence)
<400> 16
tgtctaacaa tgatttgctc acgtc 25
<210> 17
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ctatgcactg aagtaactaa ggaag 25
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
actggcagac ggaggaggtg 20
<210> 19
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gaagggtcgc tgcctacact gctg 24
<210> 20
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
tctgctgtcc gtccacttcg tcc 23
<210> 21
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gcttacacac ctggacaaca tgc 23
<210> 22
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
cacavacaag ctttagtgtc agat 24
<210> 23
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
aagattccaa gatataaggg gtcgg 25
<210> 24
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
taacaggtct accaaagtac gaatg 25
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
cgagcacgac aggaacgact 20
<210> 26
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
tctgagtcgg ataattgctc gtg 23
<210> 27
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
taggaacatg gtggacagga cg 22
<210> 28
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
accgtaactg acaacaaaag gtaacg 26
<210> 29
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
cgtagcgacg tagagcaaac tgc 23
<210> 30
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
cgagtatagg tcagttggtt cagga 25
<210> 31
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
acaaacgaag atttcacagc atagc 25
<210> 32
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
gctaacacaa caggtcaaca ggat 24
<210> 33
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
tacctgtgac ccaagtgtaa cg 22
<210> 34
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gtgcatctgt atcctcctca tctga 25
<210> 35
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
tgactttgta tttgcagatt taaga 25
<210> 36
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
tgtaaatcta aaatatattc tct 23
<210> 37
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
ttagcttgtg ctttcaataa gttgat 26
<210> 38
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
actcaggagg ctgaggcagg agaatca 27
Claims (10)
1. A composite amplification system for 16-valent HPV genotyping detection is characterized by comprising 26 pairs of detection primers, wherein the primers are shown as follows:
2. the multiplex amplification system for typing detection of HPV16 valent HPV according to claim 1, wherein the concentrations of the primers are respectively:
3. the multiplex amplification system for the detection of HPV viral typing according to claim 1, characterized in that: the primers include 18 pairs of detection primers corresponding to the detection sites of the L region and the E region in 9 HPV typing viruses, and 7 pairs of primers corresponding to the detection sites of the other 7E regions, and a pair of detection primers for beta-globin of human genomic DNA.
4. The multiplex amplification system for the detection of 16-valent HPV virus typing according to claim 3 further comprising two different fluorescent labels corresponding to the detection sites of the L region or the E region, respectively.
5. The multiplex amplification system for typing detection of HPV16 valent HPV according to claim 4, wherein the detection site in the L region is labeled with TAMRA and the detection site in the E region is labeled with ROX.
6. The multiplex amplification system for typing detection of HPV virus with 16 valency according to claim 5, wherein the ROX marker is also used for marking a detection site for β -globin corresponding to human genomic DNA.
7. The multiplex amplification system for typing detection of HPV virus with 16 valency according to claim 4 or 5, further comprising an internal standard fluorescence (ORG) provided on each pair of detection primers.
8. The multiplex amplification system for typing detection of HPV16 valent HPV virus according to claim 1, wherein the reaction conditions for the diffusion of the amplification system are as follows: step 1: denaturation at 95 ℃ for 5 min, step 2: denaturation at 94 ℃ for 30 seconds, annealing at 59 ℃ for 30 seconds in step 3, and annealing at step 4: extension at 72 ℃ for 1 minute, repetition of 2 to 4 steps at least 30 times, and final extension at 60 ℃ for 30-60 minutes.
9. A kit, characterized in that: comprising an amplification system according to any one of claims 1 to 8.
10. Use of the multiplex amplification system of any one of claims 1 to 8 or the kit of claim 9 in an HPV genotyping assay.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114480728A (en) * | 2021-11-25 | 2022-05-13 | 四川大学华西第二医院 | LAMP primer group and kit for bedside rapid detection of HPV (human papilloma Virus) and using method thereof |
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| CN105087827A (en) * | 2015-08-21 | 2015-11-25 | 北京鑫诺美迪基因检测技术有限公司 | Primer, probe and kit for detecting type-16 HPV (human papillomavirus) |
| CN107841576A (en) * | 2017-12-07 | 2018-03-27 | 广州凯普医药科技有限公司 | 14 kinds of Combining high-risk human papillomavirus E6/E7 areas mRNA detection kits |
| CN111088398A (en) * | 2019-12-18 | 2020-05-01 | 苏州阅微基因技术有限公司 | Complex amplification system for nine-valent HPV typing detection and kit thereof |
| CN111593140A (en) * | 2020-05-21 | 2020-08-28 | 杭州海基生物技术有限公司 | Detection and typing kit for high-risk human papilloma virus |
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| CN105087827A (en) * | 2015-08-21 | 2015-11-25 | 北京鑫诺美迪基因检测技术有限公司 | Primer, probe and kit for detecting type-16 HPV (human papillomavirus) |
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| CN114480728A (en) * | 2021-11-25 | 2022-05-13 | 四川大学华西第二医院 | LAMP primer group and kit for bedside rapid detection of HPV (human papilloma Virus) and using method thereof |
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