CN107130058B - A kind of novel 9 kinds of humam papillomavirus genotype parting kits and method - Google Patents
A kind of novel 9 kinds of humam papillomavirus genotype parting kits and method Download PDFInfo
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Abstract
The present invention relates to a kind of can be combined by ordered series of numbers to be combined with Nano-Au probe method detection method, realize that the detection reaction tube number with less distinguishes the kit and method of more HPV hypotypes.It is combined by a kind of specific ordered series of numbers, realizes and distinguish 9 kinds of typing human papillomavirus hypotypes with 4 detection pipes.This mathematics permutation and combination algorithm is combined by the present invention with molecular diagnostic techniques, in the case where not increasing detection reaction flux, is realized and is distinguished more humam papillomavirus genotypes with minimum reaction tube number, to reduce detection operation difficulty and testing cost.
Description
Technical field
It is specifically a kind of to be used for 9 kinds of human papilloma virus Asias the invention belongs to molecular biotechnology and genetic test field
The kit and method of type parting.
Background technology
Human papilloma virus (Human Papilloma virus, HPV) belongs to Papillomaviridae, is a kind of small molecule
, without envelope coated circular double stranded DNA virus, genome is about 8000 base-pairs (bp), is divided into 3 functional areas, i.e. early stage
Transcriptional domain (areas E), late transcription area (areas L) and nontranscribed domain (long control area, LCR).Human papilloma virus by directly or
Meet contact stain article or the Sex transmitted pathogen mankind.The virus not only has host specificity, and has tissue specificity, only
The skin and mucomembranous epithelial cell that people can be infected cause a variety of papillomas or wart and genital tract epithelial proliferation of human skin
Damage.
For infecting the human papilloma virus of genital tract and anus, according to the other pathogenicity size of each genotype or carcinogenic danger
Property size can be divided into low risk and high-risk-type two major classes.The genital tract human papillomavirus infection tool in the women of sexual intercourse history
There is generality, 70%~80% women has a human papilloma virus infection at least once at it in life according to statistics, but big
Majority infection is self limiting, will appear a kind of effective immune response more than the women of 90% infection, no any long-term
Health intervention when infection can be removed between 6 to 24 months.And the high-risk human mammilla papillomavirus infection of duration is then
The main reason for leading to Cervical intraepitheliaI neoplasia and cervical carcinoma.The result of study of global range is shown, in 99.7% cervical carcinoma
Patient's body detects the presence of high-risk human mammilla papillomavirus DNA, wherein HPV 16,18 types, 45 types and 31
Type infection accounts for 80%.Low risk human papilloma virus is generally related to condyloma acuminatum or low squamous intraepithelial lesion, Ji Shaoyin
Play infiltrating carcinoma.National CFDA has also promulgated that human papilloma virus (HPV) detection of nucleic acids and Genotyping reagent technique examine
Guideline illustrates that human papilloma virus detection has highly important clinical meaning.
According to the homology of HPV, it has been found that more than 120 kinds of type.Currently, having existed some in the market to human papilloma virus
The kit that poison is detected, but the mode that these kits hybridize mostly by way of fluorescence or film is completed to detect.
The step that Nano-Au probe method distinguishes the virus of different subtype mainly has following three phases, and these three stages are equal
It is completed under the conditions of same pipe stopped pipe.First stage：Template amplification is realized by the participation of primer pair, amplification enzyme to target
The highly sensitive amplification of template；Second stage：By the participation of probe and restriction endonuclease, realize to target template to signaling molecule
High efficiency converts；Phase III：It is realized by Nano-Au probe and the high-resolution of signaling molecule is identified.But it is a variety of due to being related to
The differentiation of type, it is complicated when detection, it takes time and effort.
Meanwhile the people for not in the market still being combined the algorithm of mathematics permutation and combination with Nano-Au probe method detection method
Papillomavirus classifying method, it is only necessary to which common PCR cycle instrument can be with the less more human papillomas of pipe number parting
Virus subtype number such as detects the typing of human papillomavirus that 9 kinds of hypotypes are realized in reaction with 5 pipes.
Invention content
Based on this, it is an object of the invention to provide a kind of parting kits and classifying method of 9 kinds of HPV hypotypes, for that will count
Learn permutation and combination algorithm be combined with Nano-Au probe method detection method, without increasing flux, realization with compared with
Few detection reaction tube number (5 pipe) distinguishes 9 kinds of HPV hypotypes.Realize that the technical solution of object above is as follows.
A kind of parting kit of 9 kinds of HPV hypotypes, including following at least four detection pipe, wherein
First pipe includes corresponding detection primer and the probe for 4 kinds of models；
Second pipe includes corresponding detection primer and the detection probe for 4 kinds of models, targeted probe in the second pipe
Model only have it is a kind of identical as model in the first pipe；
Third pipe includes corresponding detection primer and the probe for 3 kinds of models, in third pipe targeted detection visit
The model of needle only has a kind of identical as model in the first pipe and only a kind of identical as model in the second pipe；
4th pipe includes corresponding detection primer and the detection probe for 3 kinds of models, targeted inspection in the 4th pipe
Survey primer and probe model only have it is a kind of identical as model in the first pipe, it is only a kind of identical as model in the second pipe,
And it is identical without the model in a kind of pipe with third.
A kind of classifying method of 9 kinds of HPV hypotypes, includes the following steps：
Following at least four detection pipes of setting, wherein
First pipe includes corresponding detection primer and the probe for 4 kinds of models；
Second pipe includes corresponding detection primer and the detection probe for 4 kinds of models, targeted probe in the second pipe
Model only have it is a kind of identical as model in the first pipe；
Third pipe includes corresponding detection primer and the probe for 3 kinds of models, in third pipe targeted detection visit
The model of needle only has a kind of identical as model in the first pipe and only a kind of identical as model in the second pipe；
4th pipe includes corresponding detection primer and the detection probe for 3 kinds of models, targeted inspection in the 4th pipe
Survey primer and probe model only have it is a kind of identical as model in the first pipe, it is only a kind of identical as model in the second pipe,
And it is identical without the model in a kind of pipe with third；
Often the probe for having detection primer and respective model and gold label general probe is added in pipe, carries out amplification reaction.
Further include having the 5th pipe in one of the embodiments, the 5th pipe is comprising corresponding detection primer and is directed to
The detection probe of positive control.
9 kinds of HPV hypotypes are 6,11,16,18,31,33,35,39,42 in one of the embodiments,.
First pipe includes 11,16,31,42 detection probe in one of the embodiments,；Second pipe comprising 6,
16,33,39 detection probe；Third pipe includes 18,31,33 detection needle；4th pipe includes 35,39,42 detection probe；The
Five pipes include positive control, are the detection primer and probe of β-actin；Or first pipe includes 6,11,16 and 18 detection
Probe；Second pipe includes 6,33,35 and 42 detection probe；Third pipe includes 18,33 and 39 detection needle；4th pipe includes
16,31,42 detection probe；5th pipe includes positive control, is the detection primer and probe of β-actin；Or first pipe
Include 6,11,16 and 18 detection probe；Second pipe includes 6,33,35 and 42 detection probe；Third pipe include 18,33,
With 39 detection needle；4th pipe includes 16,31,42 detection probe；5th pipe includes positive control, is the detection of β-actin
Primer and probe.
A kind of, result interpretation letter low to facility condition requirement is utilized in parting kit of the present invention and detection method
Single, low-cost novel in vitro diagnostic method (nanogold visible detection method), by the mathematical model of mathematics permutation and combination
It is combined with in-vitro diagnosis method, realizes the typing of human papillomavirus detection of highly sensitive, inexpensive, stopped pipe and simplicity.
The invention has the advantages that：
The intersection that different subjects are realized using human papilloma method in the present invention, by the mathematical model of mathematics permutation and combination
It is combined with in-vitro diagnosis method and realizes highly sensitive, inexpensive and easy typing of human papillomavirus detection.Not only entire inspection
It surveys without using fluorescence probe, reagent storage is without being protected from light, it is not necessary to the problem of worrying fluorescence decay, therefore storage condition is more steady
It is fixed.
The mathematics permutation and combination theory that the present invention uses can provide a variety of realizations for 9 kinds of humam papillomavirus genotype partings
The mode of detection, therefore provide a variety of standby detection schemes for typing of human papillomavirus detection.
It can be realized with less inspection in the case where not increasing detection reaction flux using the detection method in the present invention
9 kinds of humam papillomavirus genotypes of reaction tube number point are surveyed, the complexity and testing cost of operation are reduced.
Description of the drawings
Following drawings is not used in for illustrating specific embodiments of the present invention and defines this hair that claim is defined
Bright range.
Fig. 1 be 9 kinds of HPV hypotypes in embodiment 1 each pipe of parting kit in subtype distribution schematic diagram.
Fig. 2 is the detection specific outcome that 9 kinds of humam papillomavirus genotypes are distinguished in 5 pipes detection reaction in embodiment 2.
Fig. 3 is the detection sensitivity result that 9 kinds of humam papillomavirus genotypes are distinguished in 5 pipes detection reaction in embodiment 3.
Fig. 4 is the testing result that 9 kinds of humam papillomavirus genotypes are distinguished in 5 pipes detection reaction in embodiment 4.
9 kinds of humam papillomavirus genotype combinations are distinguished in 5 pipes detection reaction in Fig. 5 embodiments 5.
Fig. 6 is the detection sample results that 9 kinds of humam papillomavirus genotypes are distinguished in 5 pipes detection reaction in embodiment 5.
Specific implementation mode
Unless otherwise defined, all technical and scientific terms used in the present invention and the technical field for belonging to the present invention
The normally understood meaning of technical staff it is identical.The term used in the description of the present invention is intended merely to describe specific reality
The purpose for applying example is not used in the limitation present invention.Term "and/or" used in the present invention includes one or more relevant listed
Any and all combinations of project.
To facilitate the understanding of the present invention, below with reference to relevant drawings to invention is more fully described.In attached drawing
Give presently preferred embodiments of the present invention.But the present invention can realize in many different forms, however it is not limited to this paper institutes
The embodiment of description.Keep the understanding to the disclosure more thorough on the contrary, purpose of providing these embodiments is
Comprehensively.
A kind of novel humam papillomavirus genotype classifying method of the present invention is combined by a kind of specific ordered series of numbers,
It realizes with minimum pipe number typing human papillomavirus hypotype as much as possible.It is examined by means of a kind of qualitative Nano-Au probe method
This mathematics permutation and combination algorithm is combined by survey method with molecular diagnostic techniques, the case where not increasing detection reaction flux
Under, realize and more humam papillomavirus genotypes distinguished with minimum reaction tube number, with reduce detection operation difficulty and detection at
This.This mode is played the role of more notable when parting quantity.
By taking the human papilloma virus of 5 areas under control point, 9 hypotypes as an example, the testing principle of the present invention is described.For to be checked
Survey 21 hypotype human papilloma virus, wherein humam papillomavirus genotype can be following hypotype 6,11,16,18,26,31,
33、35、39、45、51、52、53、56、58、59、66、68、82、40、42、43、54、61、70、72、81、55、62、64、67、
69, arbitrary 9 kinds in 71,73,83,84, the present embodiment selection wherein 6,11,16,18,31,33,35,39,42 etc. 9 kind of hypotype.
The parting kit of 19 kinds of HPV hypotypes of embodiment
A kind of parting kit of 9 kinds of HPV hypotypes, includes following four detection pipe and a control tube, wherein
First pipe includes the detection primer and probe of 4 kinds of models, specially includes 11,16,31 and 42 detection probe；
Second pipe includes the detection primer and probe of 4 kinds of models, in the second pipe the model of targeted detection probe only have it is a kind of with
Model in first pipe is identical, and the specially second pipe includes 6,16,33,39 detection probe；
Third pipe includes the detection primer and probe of 3 kinds of models, and the model of targeted detection probe is only in third pipe
Have a kind of identical as model in the first pipe and only a kind of identical as model in the second pipe, specifically, third pipe includes
18,31,33 detection probe；
4th pipe includes the detection primer and probe of 3 kinds of models, and the model of targeted detection probe is only in the 4th pipe
Have it is a kind of identical as model in the first pipe and only a kind of identical as model in the second pipe, and without a kind of with third pipe
In model it is identical, the specially the 4th pipe include 35,39,42 detection probe；
5th pipe includes positive control, is the detection primer and probe of β-actin.Referring specifically to Fig. 1.
Further include having Hairpinprobe and gold label general probe when above-mentioned often pipe detection.
The detection probe of 9 kinds of hypotypes such as the five pipes detection primer and 6,11,16,18,31,33,35,39,42 is as follows：
Human papilloma parting is in charge of the influence that detection mode is also interfered with each other by the primed probe of each parting itself, detection
It needs to interfere in view of the reagent in the detection of practical parting in the process, through the invention designed probe and primer, Ke Yishi
9 kinds of humam papillomavirus genotype partings of existing 4 pipe detection pipe pair.
The parting kit of 9 kinds of HPV hypotypes described in 2 embodiment 1 of embodiment distinguishes the detection of 9 kinds of humam papillomavirus genotypes
Specificity
The primer and probe being respectively adopted in the present embodiment in embodiment 1 prepares reaction solution, and total volume is 20 μ L, per sample
Detection is divided into the progress of 5 pipes, and often the detected components of pipe are respectively：1 μM of primer, 1 μM of probe and Hairpin probe, 1 μ LAuNP-1
With AuNP-2,0.5U Taq archaeal dna polymerases, corresponding human milk head is added in 2U restriction endonucleases A, 0.2mM dNTP into the system respectively
The target molecules to be measured of tumor virus hypotype, a concentration of 104Copy, the β-actin fragment templates that last pipe is added, reactant
System is configured to response procedures and is：94 DEG C, 2min；94 DEG C, 5s, 72 DEG C, 40s, 35 cycles；72 DEG C, 2min；63 DEG C, 10min；55
DEG C, 30min；10 DEG C, 2min.
The method of the present invention, which detects the step of human papilloma virus of different subtype, mainly a following three phases, and these three
Stage completes under the conditions of same pipe stopped pipe.First stage：Template amplification is realized by the participation of primer pair, amplification enzyme
Highly sensitive amplification to target template；Second stage：By the participation of probe and restriction endonuclease, realizes and target template is divided to signal
The high efficiency conversion of son；Phase III：It is realized by Nano-Au probe and the high-resolution of signaling molecule is identified.Pass through above three
The reaction in a stage can realize under the conditions of stopped pipe, be utilized it is a kind of it is low to facility condition requirement, result interpretation is simple, at
This cheap novel in vitro diagnostic method (nanogold visible detection method), by the mathematical model of mathematics permutation and combination and in vitro
Diagnostic method is combined, and realizes the typing of human papillomavirus detection of highly sensitive, inexpensive, stopped pipe and simplicity.
It takes pictures after reaction, it is as a result shown in Figure 2.Fig. 2's the results show that the first pipe includes 11,16,31,42 inspection
Probing needle, when detection, are separately added into the target molecules to be checked of 6,11,16,18,31,33,35,39,42 etc. 9 kinds of hypotypes, as a result only
There are 11,16,31,42 hypotypes to present positive；Second pipe includes 6,16,33,39 detection probe, when detection is separately added into 6,11,
16, the target molecules to be checked of 18,31,33,35,39,42 etc. 9 kinds of hypotypes, as a result only 6,16,33,39 hypotypes present it is positive；The
Three pipes include 18,31,33 detection needle, and when detection is separately added into waiting for for 6,11,16,18,31,33,35,39,42 etc. 9 kinds of hypotypes
Examine target molecules, as a result only 18,31,33 hypotypes present it is positive；4th pipe includes 35,39,42 detection probe, detects the time-division
Not Jia Ru 6,11,16,18,31,33,35,39,42 etc. 9 kinds of hypotypes target molecules to be checked, as a result only 35,39,42 hypotypes be in
The existing positive；5th pipe includes positive control, is the detection primer and probe of β-actin, which detects β-actin genes simultaneously
Target molecules, be as a result positive.5 pipe of kit detection reaction result of the present invention is consistent with expection, is able to special
Property detect corresponding humam papillomavirus genotype, show that detection specificity is good.
The detection spirit of 9 kinds of humam papillomavirus genotypes is distinguished in the parting kit detection reaction of 39 kinds of HPV hypotypes of embodiment
Sensitivity
It primer and probe in embodiment 1 is respectively adopted in the present embodiment prepares reaction solution, total volume is 20 μ L, often pipe
Detected components are respectively：1 μM of primer, 1 μM of probe and Hairpin probe, 1 μ LAuNP-1 and AuNP-2,0.5U Taq DNA
Polymerase, 2U restriction endonucleases A, 0.2mM dNTP, 10 are separately added into the system5、104、103、102, 10,1 and 0 copy not
With the target molecule to be measured of hypotype, pipe 1 is separately added into the template molecule of 11,16,31,42 hypotype various concentrations；Pipe 2 is separately added into 6,
16, the template molecule of 33,39 hypotype various concentrations；Pipe 3 is separately added into the template molecule of 18,31,33 hypotype various concentrations；Pipe 4
It is separately added into the template molecule of 35,39,42 hypotype various concentrations.Reaction system is configured to response procedures：94 DEG C, 2min；94
DEG C, 5s, 72 DEG C, 40s, 35 cycles；72 DEG C, 2min；63 DEG C, 10min；55 DEG C, 30min；10 DEG C, 2min.
It takes pictures after reaction, it is as a result shown in Figure 3.The result of Fig. 3 shows that 5 pipe of kit detection reaction of the present invention is distinguished
The detection sensitivity of 9 kinds of humam papillomavirus genotypes, detection sensitivity can reach 100 copies and often react.
The detection clinical sample result of 9 kinds of humam papillomavirus genotypes is distinguished in the detection reaction of 45 pipe of embodiment
The primer and probe being respectively adopted in the present embodiment in embodiment 1 prepares reaction solution, and total volume is 20 μ L, per sample
Detection is divided into the progress of 5 pipes, and often the detected components of pipe are respectively：1 μM of primer, 1 μM of probe and Hairpin probe, 1 μ LAuNP-1
With AuNP-2,0.5U Taq archaeal dna polymerases, 2U restriction endonucleases A, 0.2mM dNTP is separately added into different subtype into the system
Sample to be tested, a concentration of 104Copy, reaction system are configured to response procedures and are：94 DEG C, 2min；94 DEG C, 5s, 72 DEG C, 40s,
35 cycles；72 DEG C, 2min；63 DEG C, 10min；55 DEG C, 30min；10 DEG C, 2min.
It takes pictures after reaction, it is as a result shown in Figure 4.Fig. 4's the result shows that kit of the present invention can be detected by 5 pipes
9 kinds of humam papillomavirus genotypes in clinical sample are distinguished in reaction.
The detection reaction of 55 pipe of embodiment distinguish 9 kinds of humam papillomavirus genotypes the combination for being different from embodiment 1 and
Detect sample results
Another combination is schematically illustrated in the present embodiment, as shown in Figure 5.
First pipe includes 6,11,16 and 18 detection probe；Second pipe is visited comprising 6,33,35 and 42 detection
Needle；Third pipe includes 18,33 and 39 detection needle；4th pipe includes 16,31,42 detection probe；5th pipe is comprising positive right
According to for the detection primer and probe of β-actin.It is specific as shown in Figure 5.
The detection probe of 9 kinds of hypotypes such as described five pipes detection primer and 6,11,16,18,31,33,35,39,42 and implementation
Example 1 is identical.
The primer and probe being respectively adopted in the present embodiment in embodiment 1 prepares reaction solution, and total volume is 20 μ L, according to figure
5 combination is divided into the progress of 5 pipes per pattern detection, and often the detected components of pipe are respectively：1 μM of primer, 1 μM of probe and
Hairpin probe, 1 μ LAuNP-1 and AuNP-2,0.5U Taq archaeal dna polymerases, 2U restriction endonucleases A, 0.2mM dNTP, to this
It is separately added into the sample to be tested of different subtype in system, a concentration of 104Copy, reaction system are configured to response procedures and are：94
DEG C, 2min；94 DEG C, 5s, 72 DEG C, 40s, 35 cycles；72 DEG C, 2min；63 DEG C, 10min；55 DEG C, 30min；10 DEG C, 2min.
It takes pictures after reaction, it is as a result shown in Figure 6.Fig. 6's the result shows that kit of the present invention can be detected by 5 pipes
9 kinds of humam papillomavirus genotypes in clinical sample are distinguished in reaction.
Each technical characteristic of embodiment described above can be combined arbitrarily, to keep description succinct, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, it is all considered to be the range of this specification record.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
SEQUENCE LISTING
<110>Guangzhou Bao Chuan Bioisystech Co., Ltd
<120>A kind of novel 9 kinds of humam papillomavirus genotype parting kits and method
<160> 23
<170> PatentIn version 3.3
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Claims (10)
1. a kind of parting kit of 9 kinds of HPV hypotypes using Nano-Au probe method, spy is being, including following at least four
Detection pipe, wherein
First pipe includes the detection probe and corresponding detection primer for 4 kinds of models；
Second pipe includes the detection probe and corresponding detection primer for 4 kinds of models, in the second pipe targeted detection visit
The model of needle only has a kind of identical as model in the first pipe；
Third pipe includes the detection probe and corresponding detection primer for 3 kinds of models, targeted detection in third pipe
The model of probe only has a kind of identical as model in the first pipe and only a kind of identical as model in the second pipe；
4th pipe includes the detection probe and corresponding detection primer for 3 kinds of models, targeted detection in the 4th pipe
The model of primer and detection probe only has a kind of identical as model in the first pipe, only a kind of model phase with the second pipe
Together, and it is identical without the model in a kind of pipe with third.
2. the parting kit of 9 kinds of HPV hypotypes according to claim 1, spy is being,
Further include having the 5th pipe, the 5th pipe includes the detection probe and corresponding detection primer for being directed to positive control.
3. the parting kit of 9 kinds of HPV hypotypes according to claim 2, spy is being,
9 kinds of HPV hypotypes are 6,11,16,18,31,33,35,39 and 42.
4. the parting kit of 9 kinds of HPV hypotypes according to claim 3, spy is being,
First pipe includes 11,16,31,42 detection probe；Second pipe includes 6,16,33,39 detection probe；Third pipe
Include 18,31,33 detection probe；4th pipe includes 35,39,42 detection probe；5th pipe includes positive control, is β-
The detection primer and probe of actin；Or
First pipe includes 6,11,16 and 18 detection probe；Second pipe includes 6,33,35 and 42 detection probe；The
Three pipes include 18,33 and 39 detection probe；4th pipe includes 16,31,42 detection probe；5th pipe includes positive control,
For the detection primer and probe of β-actin.
5. the parting kit of 9 kinds of HPV hypotypes according to claim 4, spy is being,
Detection probe for HPV hypotypes is：
Detection probe for 6 types is SEQ ID NO.11；
Detection probe for 11 types is SEQ ID NO.12；
Detection probe for 16 types is SEQ ID NO.13；
Detection probe for 18 types is SEQ ID NO.14；
Detection probe for 31 types is SEQ ID NO.15；
Detection probe for 33 types is SEQ ID NO.16；
Detection probe for 35 types is SEQ ID NO.17；
Detection probe for 39 types is SEQ ID NO.18；
Detection probe for 42 types is SEQ ID NO.19.
6. according to the parting kit of claim 3-5 9 kinds of HPV hypotypes of any one of them, spy is being,
The detection primer of first pipe is SEQ ID NO.1 and SEQ ID NO.2；
The detection primer of second pipe is SEQ ID NO.3 and SEQ ID NO.4；
The detection primer of the third pipe is SEQ ID NO.5 and SEQ ID NO.6；
The detection primer of 4th pipe is SEQ ID NO.7 and SEQ ID NO.8.
7. a kind of classifying method of 9 kinds of HPV hypotypes using Nano-Au probe method of non-disease diagnostic purpose, spy is being,
Include the following steps：
Following at least four detection pipes of setting, wherein
First pipe includes the detection probe and corresponding detection primer for 4 kinds of models；
Second pipe includes the detection probe and corresponding detection primer for 4 kinds of models, targeted detection in the second pipe
The model of probe only has a kind of identical as model in the first pipe；
Third pipe includes the detection probe and corresponding detection primer for 3 kinds of models, targeted detection in third pipe
The model of probe only has a kind of identical as model in the first pipe and only a kind of identical as model in the second pipe；
4th pipe includes the detection probe and corresponding detection primer for 3 kinds of models, targeted detection in the 4th pipe
The model of primer and detection probe only has a kind of identical as model in the first pipe, only a kind of model phase with the second pipe
Together, and it is identical without the model in a kind of pipe with third；
Often the detection probe for having detection primer and respective model and gold label general probe is added in pipe, carries out amplification reaction.
8. the classifying method of 9 kinds of HPV hypotypes according to claim 7, spy is being,
Further include having the 5th pipe, the 5th pipe includes the detection primer and probe of positive control.
9. the classifying method of 9 kinds of HPV hypotypes according to claim 8, spy is being,
9 kinds of HPV hypotypes are 6,11,16,18,31,33,35,39,42；First pipe includes 11,16,31,42 detection
Probe；Second pipe includes 6,16,33,39 detection probe；Third pipe includes 18,31,33 detection probe；4th pipe includes
35,39,42 detection probe；5th pipe includes positive control, is the detection primer and probe of β-actin；Or
First pipe includes 6,11,16 and 18 detection probe；Second pipe includes 6,33,35 and 42 detection probe；The
Three pipes include 18,33 and 39 detection probe；4th pipe includes 16,31,42 detection probe；5th pipe includes positive control,
For the detection primer and probe of β-actin.
10. the classifying method of 9 kinds of HPV hypotypes according to claim 9, spy is being,
Detection probe for HPV hypotypes is：
Detection probe for 6 types is SEQ ID NO.11；
Detection probe for 11 types is SEQ ID NO.12；
Detection probe for 16 types is SEQ ID NO.13；
Detection probe for 18 types is SEQ ID NO.14；
Detection probe for 31 types is SEQ ID NO.15；
Detection probe for 33 types is SEQ ID NO.16；
Detection probe for 35 types is SEQ ID NO.17；
Detection probe for 39 types is SEQ ID NO.18；
Detection probe for 42 types is SEQ ID NO.19；
The detection primer of first pipe is SEQ ID NO.1 and SEQ ID NO.2；
The detection primer of second pipe is SEQ ID NO.3 and SEQ ID NO.4；
The detection primer of the third pipe is SEQ ID NO.5 and SEQ ID NO.6；
The detection primer of 4th pipe is SEQ ID NO.7 and SEQ ID NO.8.
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