CN109055611A

CN109055611A - For detecting the kit of HPV

Info

Publication number: CN109055611A
Application number: CN201810971577.5A
Authority: CN
Inventors: 黄雅淑; 翁义杰
Original assignee: Shenzhen Xinsi Microbiological Technology Co Ltd
Current assignee: Shenzhen Xinsi Microbiological Technology Co Ltd
Priority date: 2018-08-24
Filing date: 2018-08-24
Publication date: 2018-12-21
Also published as: WO2020037865A1

Abstract

The invention belongs to bioengineering fields, and in particular to a kind of for detecting the kit of HPV.The kit includes the primer and Uvs X, Uvs Y, SSB and archaeal dna polymerase for expanding HPV.In kit provided by the present invention for detecting HPV, the T4 serial enzymes i.e. Uvs X of combination and Uvs Y has been selected to replace existing recombinase, the enzyme system not only can be further improved the sensitivity and specificity of HPV detection, and it can more diversification to the primer selection of amplification HPV, 100-200bp product can be expanded under the conditions of compared with short primer, and then shortens the reaction time, reduce the interference of background signal, it is final to improve detection efficiency, significantly reduce testing cost.

Description

For detecting the kit of HPV

Technical field

The invention belongs to bioengineering fields, and in particular to a kind of for detecting the kit of HPV.

Background technique

Human papillomavirus (Human Papillomavirus, HPV) are a kind of microorganisms also smaller than bacterium, are filtrations One kind of venereal disease poison.In more than 200 kinds of hypotype HPV being currently known, about 40 kinds of hypotypes can invade female genital tract, at least 15 kinds of hypotypes are related with aggressive cervix cancer, referred to as carcinogenicity or high risk type, wherein the 16th type is most commonly seen, the 18th type Take second place, the two has accounted for the 70% of cervix cancer altogether.Human papillomavirus have the host specificity characteristic of height, specially The skin and mucous membrane of the mankind can be infected.Infect the 16th type and 18 type human papillomavirus and cervix cancer, carcinoma of penis, carcinoma of vulva and The formation of cancer of anus etc. is related.

At present on the market there are many kinds of the detection methods of HPV, including hybrid capture, fluorescence in situ hybridization, chip skill Art hybrid method, real-time fluorescence PCR method, spot marking method and constant-temperature amplification method etc., sensibility, the specificity of every kind of detection method are equal It is different.No matter which kind of detection method, cannot all detect all types, but also need be not excessively the problem of worry is failed to pinpoint a disease in diagnosis, It is only related with high-risk HPV because of the generation of cervix cancer, the infection of as noted above 70% cervical cancer patient be 16 types and 18 two, type hypotypes, other high-risk HPVs can be found substantially in existing detection method.Therefore, if high-risk HPV A possibility that testing result is feminine gender, then can exclude cervix cancer substantially.

An important technology that represents expands in constant-temperature amplification (Isothermal Amplification) as recombination enzymatic polymerization enzyme Increase (Recombinase Polymerase Amplification, RPA).This technology is opened by UK corporation Twist Dx Inc One kind of hair can substitute the novel nucleic acids detection technique of normal PCR.Using the technology, can be carried out under room temperature in 30 minutes Monomolecular nucleic acid detection, requirement of the technology to hardware device is lower, particularly suitable for in-vitro diagnosis, animal doctor, food peace Entirely, the fields such as bio-safety, agricultural.But there is also defects for existing RPA amplification technique, such as~500bp- > 500bp The target product selection of collocation (enzyme) be easy to cause poor sensitivity and reaction time long (see document: TwistAmp Manuals.Available from:www.twistdx.co.uk/resources/instruction_manuals/ Twistamp_product_documentatio n/), and expand after product have powerful connections signal interference the problem of (see text It offers: O.Piepenburg, C.H.Williams, D.L.Stemple and N.A.Armes, PLoS Biol., 2006,4, e204,1115–1121.).Therefore, which is further improved.

Summary of the invention

It is an object of the invention to overcome the above-mentioned deficiency of the prior art, provide a kind of for detecting the kit of HPV, purport When solving existing RPA method detection HPV, there are specificities and the not high technical problem of sensitivity.

For achieving the above object, The technical solution adopted by the invention is as follows:

The present invention provides a kind of for detecting the kit of HPV, and the kit includes the primer for expanding HPV, with And Uvs X, Uvs Y, SSB and archaeal dna polymerase.

The combination collocation of enzyme is very crucial in constant-temperature amplification method, and RPA technology depends on three kinds of zymoproteins: can be in conjunction with single Recombinase (Recombinase), single strand binding protein (SSB) and the strand displacement archaeal dna polymerase of chain nucleic acid (Oligonucleolide primers) (Polynerase), and provided by the present invention for detect HPV kit be a kind of kit based on constant-temperature amplification, at this In kit, the T4 serial enzymes i.e. Uvs X of combination and Uvs Y is selected to replace existing recombinase, the relatively existing skill of the enzyme system Art not only can be further improved the sensitivity and specificity of HPV detection, but also can be more more to the primer selection of amplification HPV Memberization can expand 100-200bp product under the conditions of compared with short primer, and then shorten the reaction time, reduce the dry of background signal It disturbs, finally improves detection efficiency, significantly reduce testing cost.

Detailed description of the invention

Fig. 1 is the electrophoretogram of the HPV testing result in the embodiment of the present invention 3；Wherein, 1 is DNA Marker, and 2 be the positive Control, 3 be HPV16 infection DNA sample, 4 be HPV18 infect DNA sample, 5 be negative control, 6 be blank control.

Specific embodiment

In order to which technical problems, technical solutions and advantageous effects to be solved by the present invention are more clearly understood, below in conjunction with Embodiment, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used to explain The present invention is not intended to limit the present invention.

The embodiment of the invention provides a kind of for detecting the kit of HPV, and the kit includes for expanding HPV's Primer and Uvs X, Uvs Y, SSB and archaeal dna polymerase.

The combination collocation of enzyme is very crucial in constant-temperature amplification method, and RPA technology depends on three kinds of zymoproteins: can be in conjunction with single Recombinase, single strand binding protein and the strand displacement archaeal dna polymerase of chain nucleic acid (Oligonucleolide primers), and the embodiment of the present invention provides For to detect the kit of HPV to be a kind of kit based on constant-temperature amplification, in the kit, selected T4 serial enzymes group Closing is that Uvs X and Uvs Y replace existing recombinase, the enzyme system not only can be further improved HPV detection sensitivity and Specificity, and to amplification HPV primer selection can more diversification, 100-200bp can be expanded under the conditions of compared with short primer Product, and then shorten the reaction time, the interference of background signal is reduced, detection efficiency is finally improved, significantly reduces testing cost.

The principle of RPA amplification at present are as follows: the protein-dna that recombinase (Recombinase) is formed in conjunction with primer is compound Object can find homologous sequence in double-stranded DNA, once primer located homologous sequence, Exchange reaction of chain will occur and formed simultaneously Start DNA synthesis, exponential amplification is carried out to the target area in template.The DNA chain and single strand binding protein (SSB) being replaced In conjunction with preventing from further replacing.In this system, open a synthetic reactions by two opposite primers, whole process into Capable very fastly, can generally obtain within 30 minutes can detect horizontal amplified production.During being somebody's turn to do, recombinase, single-stranded knot The mixture of these three enzymes of hop protein and strand displacement archaeal dna polymerase is also active at normal temperature, and optimal reaction temperature is on 37 DEG C of left sides The right side, but these three enzymes combination at present is long there are poor sensitivity and reaction time, the product after amplification has powerful connections signal interference is asked Topic.And in the embodiment of the present invention, on the basis of existing RPA, propose a kind of high quick amplification (Recombinant of recombination Sensitize Amplification, RSA), this method is a kind of improved novel constant-temperature amplification method, applied to being directed to The kit of HPV detection screens optimal enzyme combination (i.e. Uvs X, Uvs Y, SSB and DNA polymerization for product item in the kit Enzyme) existing enzyme is substituted, by simulating original DNA reproduction process, removes normal PCR temperature change to reach expanding effect, arrange in pairs or groups The exclusive primer designed for amplification HPV can shorten entire duplication time-histories, improve the accuracy and efficiency of duplication template, drop The interference of other low DNA/ background signals significantly reduces testing cost to improve detection time.

Further, described for expand the primer of HPV to include for expanding in kit provided in an embodiment of the present invention Increase the first primer pair of HPV16, and the nucleotide sequence of the first primer pair such as SEQ ID NO.1 and SEQ ID NO.2 institute Show.And the primer for expanding HPV includes the second primer pair for expanding HPV18, and second primer pair Nucleotide sequence is as shown in SEQ ID NO.3 and SEQ ID NO.4.Pass through the first primer pair and/or the second primer pair, in conjunction with Distinctive enzyme combined system in kit of the embodiment of the present invention detects high-risk HPV: HPV16 type and HPV18 type.

Specifically, it is searched by NCBI (National Center for Biotechnology Information) The HPV16 (NCBI Reference Sequence:NC_001526.4) and HPV18 (NCBI of GenBank (gene pool) Reference Sequence:NC_001357.1) complete genome sequence, according to the complete genome sequence design primer (primer Length~30bp, G/C content: 30-70%).The first primer designed to and the first primer pair and the present embodiment kit in it is special Some enzyme combined systems combine, can have to HPV16 the and HPV18DNA sequence that ncbi database is found high specificity, about The DNA cloning product of 130~200bp.

Specific primer sequence is as follows:

HPV16Primer Forward (SEQ ID NO.1):

5'-gtttcaggacccacaggagcgacccagaaa-3'；

HPV16Primer Reverse (SEQ ID NO.2):

5'-aacataacgacaagattacaacaaggtagt-3'；

HPV18Primer Forward (SEQ ID NO.3):

5'-atccaacacggcgaccctacaagctacctg-3'；

HPV18Primer Reverse (SEQ ID NO.4):

5’-ccgtggaataattatttaacatattgggtc-3’。

Further, in kit provided in an embodiment of the present invention, the amino acid sequence of the Uvs X such as SEQ ID Shown in NO.5；It is specific as follows:

> sp | P04529 | UVSX_BPT4Recombination and repair protein OS= Enterobacteria phage T4OX=10665GN=UVSX PE=1SV=2

MSDLKSRLIKASTSKLTAELTASKFFNEKDVVRTKIPMMNIALSGEITGGMQSGLLILAGPSKSFKSNFGLTMVSSY MRQYPDAVCLFYDSEFGITPAYLRSMGVDPERVIHTPVQSLEQLRIDMVNQLDAIERGEKVVVFIDSLGNLASKKET EDALNEKVVSDMTRAKTMKSLFRIVTPYFSTKNIPCIAINHTYETQEMFSKTVMGGGTGPMYSADTVFIIGKRQIKD GSDLQGYQFVLNVEKSRTVKEKSKFFIDVKFDGGIDPYSGLLDMALELGFVVKPKNGWYAREFLDEETGEMIREEKS WRAKDTNCTTFWGPLFKHQPFRDAIKRAYQLGAIDSNEIVEAEVDELINSKVEKFKSPESKSKSAADLETDLEQLSD MEEFNE。

The amino acid sequence of the Uvs Y is specific as follows as shown in SEQ ID NO.6:

> sp | P04537 | UVSY_BPT4Recombination protein uvsY OS=Enterobacteria Phage T4OX=10665GN=uvsY PE=1SV=3

MRLEDLQEELKKDVFIDSTKLQYEAANNVMLYSKWLNKHSSIKKEMLRIEAQKKVALKARLDYYSGRGDGDEFSMDR YEKSEMKTVLSADKDVLKVDTSLQYWGILLDFCSGALDAIKSRGFAIKHIQDMRAFEAGK。

The amino acid sequence of the SSB is specific as follows as shown in SEQ ID NO.7:

> sp | P0AGE0 | SSB_ECOLI Single-stranded DNA-binding protein OS= Escherichia coli (strain K12) OX=83333GN=ssb PE=1SV=2

MASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKATGEMKEQTEWHRVVLFGKLAEVASEYLRKGSQ VYIEGQLRTRKWTDQSGQDRYTTEVVVNVGGTMQMLGGRQGGGAPAGGNIGGGQPQGGWGQPQQPQGGNQFSGGAQS RPQQSAPAAPSNEPPMDFDDDIPF。

The amino acid sequence of the archaeal dna polymerase is specific as follows as shown in SEQ ID NO.8:

> sp | P52026 | DPO1_GEOSE DNA polymerase I OS=Geobacillus Stearothermophilus OX=1422GN=polA PE=1SV=2

MKNKLVLIDGNSVAYRAFFALPLLHNDKGIHTNAVYGFTMMLNKILAEEQPTHILVAFDAGKTTFRHETFQDYKGGR QQTPPELSEQFPLLRELLKAYRIPAYELDHYEADDIIGTMAARAEREGFAVKVISGDRDLTQLASPQVTVEITKKGI TDIESYTPETVVEKYGLTPEQIVDLKGLMGDKSDNIPGVPGIGEKTAVKLLKQFGTVENVLASIDEIKGEKLKENLR QYRDLALLSKQLAAICRDAPVELTLDDIVYKGEDREKVVALFQELGFQSFLDKMAVQTDEGEKPLAGMDFAIADSVT DEMLADKAALVVEVVGDNYHHAPIVGIALANERGRFFLRPETALADPKFLAWLGDETKKKTMFDSKRAAVALKWKGI ELRGVVFDLLLAAYLLDPAQAAGDVAAVAKMHQYEAVRSDEAVYGKGAKRTVPDEPTLAEHLVRKAAAIWALEEPLM DELRRNEQDRLLTELEQPLAGILANMEFTGVKVDTKRLEQMGAELTEQLQAVERRIYELAGQEFNINSPKQLGTVLF DKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVEHILHYRQLGKLQSTYIEGLLKVVHPVTGKVHTMFNQALTQTGRL SSVEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIAEDDNLIEAFRRGLDIHTKTAMDIFHVS EEDVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMDNIVQEAKQKGYVTTLLHRR RYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDLSVRLREERLQARLLLQVHDELILEAPKEEIERLCRL VPEVMEQAVTLRVPLKVDYHYGPTWYDAK。

Further, in kit provided in an embodiment of the present invention, the kit includes amplification buffer (Amplification buffer), the pH=7-10 of the amplification buffer then connect nucleic acid base in DNA if pH is lower than 7 Chemical bond can hydrolyze；If pH is greater than 10, then forming double-stranded dinucleotides key can be destroyed, therefore, in pH=7- In the range of 10, it can stablize and carry out HPV amplification.It is highly preferred that when pH=8.8, the optimal stability of entire HPV amplified reaction.

Further, the amplification buffer contains: Tris-HCl, (NH₄)₂SO₄、KCl、MgSO₄、20。 In mentioned component, Tris-HCl can maintain the pH of amplification buffer to stablize；KCl can be by stablizing primer and DNA profiling knot It closes, K⁺Its net negative charge can be reduced in conjunction with negatively charged phosphate group, so that negatively charged primer will not be with Template is repelled；And MgCl₂As a kind of stabilizer of a kind of confactor and primer in conjunction with DNA profiling；(NH₄)₂SO₄ The mispairing of primer and DNA profiling can be prevented, and in different temperature and MgCl₂Under concentration conditions, contain (NH₄)₂SO₄'s Amplified reaction is all very stable, reaction temperature adjustable in this way and MgCl₂Concentration, so as to preferably optimized expansion system；20 be a kind of nonionic detergent, can not only stablize the activity of enzyme, can also inhibit the formation of secondary structure, from And provide amplification efficiency.Therefore, the above-mentioned amplification buffer in the kit of the embodiment of the present invention, by ingredient therein into Row adjusts proportion, and the amplification system of available suitable constant-temperature amplification HPV finally makes the kit of the embodiment of the present invention can With it is accurate, efficient detect HPV, and reduce the interference of other DNA/ background signals, to improve detection time, significantly reduce Testing cost.Specifically, for the length of target HPV, the concentration of DNA, activity of required various enzymes etc. factor can be into Row optimization obtains suitable amplification buffer.

Preferably, in embodiments of the present invention, for the detection of target HPV16 and HPV18, the amplification of the embodiment of the present invention The pH=8.8 of buffer, and the amplification buffer contains: (the NH of Tris-HCl, 10mM of 20mM₄)₂SO₄, 50mM KCl, The MgSO of 2mM₄With 0.1% (mass percentage)20.Amplification buffer under this condition can be better suited for Detection to target HPV16 and HPV18.

Further, in kit provided in an embodiment of the present invention, the kit further includes positive controls, and institute It states positive controls and contains the positive control primers as shown in SEQ ID NO.9 and SEQ ID NO.10.The positive control primers HPV16 type and HPV18 type can be expanded simultaneously, and particular sequence is as follows:

SEQ ID NO.9:5 '-catagaaataacctgtgtatattgcaagac-3 '；

SEQ ID NO.10:5 '-cacagattcaaaaagacgacctaagttgcc-3 '.

Further, in kit provided in an embodiment of the present invention, the kit is under 37-40 DEG C of amplification condition HPV is detected.It is highly preferred that the time of the kit detection HPV is 10-20min.

The present invention successively carried out test of many times, and it is further detailed as reference pair invention progress now to lift A partial experiment result Thin description, is described in detail combined with specific embodiments below.

Embodiment 1 is used to detect the kit of HPV

A kind of kit based on the high quick amplification detection HPV of recombination, comprising:

For expanding the first primer of HPV16 to (SEQ ID NO.1 and SEQ ID NO.2) and for expanding HPV18's Second primer pair (SEQ ID NO.3 and SEQ ID NO.4)；Uvs X(SEQ ID NO.5),Uvs Y(SEQ ID NO.6), SSB (SEQ ID NO.7) and archaeal dna polymerase (SEQ ID NO.8)；And amplification buffer: 20mM Tris-HCl, 10mM (NH₄)₂SO₄、50mM KCl、2mM MgSO₄With 0.1%20 (pH=8.8).

Embodiment 2DNA extracts (extraction)

The extraction for detecting DNA in sample, includes the following steps:

1. 1ml extraction buffer (100mM is added in cervical tissue (Cervical tissue) cell Tris-HCl, pH=8.0；50mM EDTA, pH=8.0；500mM NaCl ,+0.07% mercaptoethanol), it is sufficiently mixed.

2. 130ul 10%SDS is added, and a small amount of breaks down proteins Enzyme K, shaken several times are inverted, 65 DEG C of water baths are placed in Middle water-bath 1 hour.

3. being added 300ul 5M potassium acetate (potassium acetate), sufficiently mixes, is subsequently placed in 30 minutes on ice, In favor of protein precipitation.

4.12000rpm is centrifuged 30 minutes, is taken out supernatant and is moved on in new centrifuge tube (eppendorf tube).

5. isopropanol two volumes are added, 12000rpm is centrifuged 30 minutes.

6. outwelling supernatant, 70% ethyl alcohol is added, 12000rpm is centrifuged 30 minutes.

7. outwell supernatant, as being air-dried at room temperature, 30 minutes.

8. 50ul distilled water (distilled water) dissolving DNA is added, it is stored in minus 20 degrees.

Using the above method, according to the Infection Status of cervical tissue cell: HPV16 infection, is not felt by HPV HPV18 infection Dye extracts three DNA samples: i.e. HPV16-positive (HPV16 infects DNA sample), HPV18-positive respectively (HPV18 infects DNA sample) and HPV-negative samples (negative control DNA sample).

Embodiment 3HPV detection

Extraction in kit and embodiment 2 in embodiment 1 is obtained into DNA sample according to the ratio in following table 1, is added Enter into microtest tube (final volume are as follows: 50ul), homogeneous rocks 300rpm and reacts 10-20 minutes in the environment of 37-40 DEG C.

Table 1

To after reaction, reaction product be carried out electrophoresis, the primer size of interpretation DNA in 3~4% agar glue (tracking stain being wherein added, compare electrophoresis with control group, and be put into DNA marker and be compared), by attaching plug The positions of positive and negative anodes (pay attention to) is connected to power supply, and in electrophoresis process, negatively charged DNA molecular can shift to anode by cathode, It is run glue about 10 minutes in 100vol.It wears gloves to be placed on strong UV transmitted light source plate agar glue, can take pictures to result deposit, most Whole result figure is as shown in Figure 1.As can be seen from Figure 1: the kit of the present embodiment has the detection of HPV16 and HPV18 good Sensitivity and specificity.

The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.

Sequence table

<110>Shenzhen Xin Si microorganism Science and Technology Ltd.

<120>for detecting the kit of HPV

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

gtttcaggac ccacaggagc gacccagaaa 30

<210> 2

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

aacataacga caagattaca acaaggtagt 30

<210> 3

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

atccaacacg gcgaccctac aagctacctg 30

<210> 4

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

ccgtggaata attatttaac atattgggtc 30

<210> 5

<211> 391

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 5

Met Ser Asp Leu Lys Ser Arg Leu Ile Lys Ala Ser Thr Ser Lys Leu

1 5 10 15

Thr Ala Glu Leu Thr Ala Ser Lys Phe Phe Asn Glu Lys Asp Val Val

20 25 30

Arg Thr Lys Ile Pro Met Met Asn Ile Ala Leu Ser Gly Glu Ile Thr

35 40 45

Gly Gly Met Gln Ser Gly Leu Leu Ile Leu Ala Gly Pro Ser Lys Ser

50 55 60

Phe Lys Ser Asn Phe Gly Leu Thr Met Val Ser Ser Tyr Met Arg Gln

65 70 75 80

Tyr Pro Asp Ala Val Cys Leu Phe Tyr Asp Ser Glu Phe Gly Ile Thr

85 90 95

Pro Ala Tyr Leu Arg Ser Met Gly Val Asp Pro Glu Arg Val Ile His

100 105 110

Thr Pro Val Gln Ser Leu Glu Gln Leu Arg Ile Asp Met Val Asn Gln

115 120 125

Leu Asp Ala Ile Glu Arg Gly Glu Lys Val Val Val Phe Ile Asp Ser

130 135 140

Leu Gly Asn Leu Ala Ser Lys Lys Glu Thr Glu Asp Ala Leu Asn Glu

145 150 155 160

Lys Val Val Ser Asp Met Thr Arg Ala Lys Thr Met Lys Ser Leu Phe

165 170 175

Arg Ile Val Thr Pro Tyr Phe Ser Thr Lys Asn Ile Pro Cys Ile Ala

180 185 190

Ile Asn His Thr Tyr Glu Thr Gln Glu Met Phe Ser Lys Thr Val Met

195 200 205

Gly Gly Gly Thr Gly Pro Met Tyr Ser Ala Asp Thr Val Phe Ile Ile

210 215 220

Gly Lys Arg Gln Ile Lys Asp Gly Ser Asp Leu Gln Gly Tyr Gln Phe

225 230 235 240

Val Leu Asn Val Glu Lys Ser Arg Thr Val Lys Glu Lys Ser Lys Phe

245 250 255

Phe Ile Asp Val Lys Phe Asp Gly Gly Ile Asp Pro Tyr Ser Gly Leu

260 265 270

Leu Asp Met Ala Leu Glu Leu Gly Phe Val Val Lys Pro Lys Asn Gly

275 280 285

Trp Tyr Ala Arg Glu Phe Leu Asp Glu Glu Thr Gly Glu Met Ile Arg

290 295 300

Glu Glu Lys Ser Trp Arg Ala Lys Asp Thr Asn Cys Thr Thr Phe Trp

305 310 315 320

Gly Pro Leu Phe Lys His Gln Pro Phe Arg Asp Ala Ile Lys Arg Ala

325 330 335

Tyr Gln Leu Gly Ala Ile Asp Ser Asn Glu Ile Val Glu Ala Glu Val

340 345 350

Asp Glu Leu Ile Asn Ser Lys Val Glu Lys Phe Lys Ser Pro Glu Ser

355 360 365

Lys Ser Lys Ser Ala Ala Asp Leu Glu Thr Asp Leu Glu Gln Leu Ser

370 375 380

Asp Met Glu Glu Phe Asn Glu

385 390

<210> 6

<211> 137

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 6

Met Arg Leu Glu Asp Leu Gln Glu Glu Leu Lys Lys Asp Val Phe Ile

1 5 10 15

Asp Ser Thr Lys Leu Gln Tyr Glu Ala Ala Asn Asn Val Met Leu Tyr

20 25 30

Ser Lys Trp Leu Asn Lys His Ser Ser Ile Lys Lys Glu Met Leu Arg

35 40 45

Ile Glu Ala Gln Lys Lys Val Ala Leu Lys Ala Arg Leu Asp Tyr Tyr

50 55 60

Ser Gly Arg Gly Asp Gly Asp Glu Phe Ser Met Asp Arg Tyr Glu Lys

65 70 75 80

Ser Glu Met Lys Thr Val Leu Ser Ala Asp Lys Asp Val Leu Lys Val

85 90 95

Asp Thr Ser Leu Gln Tyr Trp Gly Ile Leu Leu Asp Phe Cys Ser Gly

100 105 110

Ala Leu Asp Ala Ile Lys Ser Arg Gly Phe Ala Ile Lys His Ile Gln

115 120 125

Asp Met Arg Ala Phe Glu Ala Gly Lys

130 135

<210> 7

<211> 178

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 7

Met Ala Ser Arg Gly Val Asn Lys Val Ile Leu Val Gly Asn Leu Gly

1 5 10 15

Gln Asp Pro Glu Val Arg Tyr Met Pro Asn Gly Gly Ala Val Ala Asn

20 25 30

Ile Thr Leu Ala Thr Ser Glu Ser Trp Arg Asp Lys Ala Thr Gly Glu

35 40 45

Met Lys Glu Gln Thr Glu Trp His Arg Val Val Leu Phe Gly Lys Leu

50 55 60

Ala Glu Val Ala Ser Glu Tyr Leu Arg Lys Gly Ser Gln Val Tyr Ile

65 70 75 80

Glu Gly Gln Leu Arg Thr Arg Lys Trp Thr Asp Gln Ser Gly Gln Asp

85 90 95

Arg Tyr Thr Thr Glu Val Val Val Asn Val Gly Gly Thr Met Gln Met

100 105 110

Leu Gly Gly Arg Gln Gly Gly Gly Ala Pro Ala Gly Gly Asn Ile Gly

115 120 125

Gly Gly Gln Pro Gln Gly Gly Trp Gly Gln Pro Gln Gln Pro Gln Gly

130 135 140

Gly Asn Gln Phe Ser Gly Gly Ala Gln Ser Arg Pro Gln Gln Ser Ala

145 150 155 160

Pro Ala Ala Pro Ser Asn Glu Pro Pro Met Asp Phe Asp Asp Asp Ile

165 170 175

Pro Phe

<210> 8

<211> 876

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 8

Met Lys Asn Lys Leu Val Leu Ile Asp Gly Asn Ser Val Ala Tyr Arg

1 5 10 15

Ala Phe Phe Ala Leu Pro Leu Leu His Asn Asp Lys Gly Ile His Thr

20 25 30

Asn Ala Val Tyr Gly Phe Thr Met Met Leu Asn Lys Ile Leu Ala Glu

35 40 45

Glu Gln Pro Thr His Ile Leu Val Ala Phe Asp Ala Gly Lys Thr Thr

50 55 60

Phe Arg His Glu Thr Phe Gln Asp Tyr Lys Gly Gly Arg Gln Gln Thr

65 70 75 80

Pro Pro Glu Leu Ser Glu Gln Phe Pro Leu Leu Arg Glu Leu Leu Lys

85 90 95

Ala Tyr Arg Ile Pro Ala Tyr Glu Leu Asp His Tyr Glu Ala Asp Asp

100 105 110

Ile Ile Gly Thr Met Ala Ala Arg Ala Glu Arg Glu Gly Phe Ala Val

115 120 125

Lys Val Ile Ser Gly Asp Arg Asp Leu Thr Gln Leu Ala Ser Pro Gln

130 135 140

Val Thr Val Glu Ile Thr Lys Lys Gly Ile Thr Asp Ile Glu Ser Tyr

145 150 155 160

Thr Pro Glu Thr Val Val Glu Lys Tyr Gly Leu Thr Pro Glu Gln Ile

165 170 175

Val Asp Leu Lys Gly Leu Met Gly Asp Lys Ser Asp Asn Ile Pro Gly

180 185 190

Val Pro Gly Ile Gly Glu Lys Thr Ala Val Lys Leu Leu Lys Gln Phe

195 200 205

Gly Thr Val Glu Asn Val Leu Ala Ser Ile Asp Glu Ile Lys Gly Glu

210 215 220

Lys Leu Lys Glu Asn Leu Arg Gln Tyr Arg Asp Leu Ala Leu Leu Ser

225 230 235 240

Lys Gln Leu Ala Ala Ile Cys Arg Asp Ala Pro Val Glu Leu Thr Leu

245 250 255

Asp Asp Ile Val Tyr Lys Gly Glu Asp Arg Glu Lys Val Val Ala Leu

260 265 270

Phe Gln Glu Leu Gly Phe Gln Ser Phe Leu Asp Lys Met Ala Val Gln

275 280 285

Thr Asp Glu Gly Glu Lys Pro Leu Ala Gly Met Asp Phe Ala Ile Ala

290 295 300

Asp Ser Val Thr Asp Glu Met Leu Ala Asp Lys Ala Ala Leu Val Val

305 310 315 320

Glu Val Val Gly Asp Asn Tyr His His Ala Pro Ile Val Gly Ile Ala

325 330 335

Leu Ala Asn Glu Arg Gly Arg Phe Phe Leu Arg Pro Glu Thr Ala Leu

340 345 350

Ala Asp Pro Lys Phe Leu Ala Trp Leu Gly Asp Glu Thr Lys Lys Lys

355 360 365

Thr Met Phe Asp Ser Lys Arg Ala Ala Val Ala Leu Lys Trp Lys Gly

370 375 380

Ile Glu Leu Arg Gly Val Val Phe Asp Leu Leu Leu Ala Ala Tyr Leu

385 390 395 400

Leu Asp Pro Ala Gln Ala Ala Gly Asp Val Ala Ala Val Ala Lys Met

405 410 415

His Gln Tyr Glu Ala Val Arg Ser Asp Glu Ala Val Tyr Gly Lys Gly

420 425 430

Ala Lys Arg Thr Val Pro Asp Glu Pro Thr Leu Ala Glu His Leu Val

435 440 445

Arg Lys Ala Ala Ala Ile Trp Ala Leu Glu Glu Pro Leu Met Asp Glu

450 455 460

Leu Arg Arg Asn Glu Gln Asp Arg Leu Leu Thr Glu Leu Glu Gln Pro

465 470 475 480

Leu Ala Gly Ile Leu Ala Asn Met Glu Phe Thr Gly Val Lys Val Asp

485 490 495

Thr Lys Arg Leu Glu Gln Met Gly Ala Glu Leu Thr Glu Gln Leu Gln

500 505 510

Ala Val Glu Arg Arg Ile Tyr Glu Leu Ala Gly Gln Glu Phe Asn Ile

515 520 525

Asn Ser Pro Lys Gln Leu Gly Thr Val Leu Phe Asp Lys Leu Gln Leu

530 535 540

Pro Val Leu Lys Lys Thr Lys Thr Gly Tyr Ser Thr Ser Ala Asp Val

545 550 555 560

Leu Glu Lys Leu Ala Pro His His Glu Ile Val Glu His Ile Leu His

565 570 575

Tyr Arg Gln Leu Gly Lys Leu Gln Ser Thr Tyr Ile Glu Gly Leu Leu

580 585 590

Lys Val Val His Pro Val Thr Gly Lys Val His Thr Met Phe Asn Gln

595 600 605

Ala Leu Thr Gln Thr Gly Arg Leu Ser Ser Val Glu Pro Asn Leu Gln

610 615 620

Asn Ile Pro Ile Arg Leu Glu Glu Gly Arg Lys Ile Arg Gln Ala Phe

625 630 635 640

Val Pro Ser Glu Pro Asp Trp Leu Ile Phe Ala Ala Asp Tyr Ser Gln

645 650 655

Ile Glu Leu Arg Val Leu Ala His Ile Ala Glu Asp Asp Asn Leu Ile

660 665 670

Glu Ala Phe Arg Arg Gly Leu Asp Ile His Thr Lys Thr Ala Met Asp

675 680 685

Ile Phe His Val Ser Glu Glu Asp Val Thr Ala Asn Met Arg Arg Gln

690 695 700

Ala Lys Ala Val Asn Phe Gly Ile Val Tyr Gly Ile Ser Asp Tyr Gly

705 710 715 720

Leu Ala Gln Asn Leu Asn Ile Thr Arg Lys Glu Ala Ala Glu Phe Ile

725 730 735

Glu Arg Tyr Phe Ala Ser Phe Pro Gly Val Lys Gln Tyr Met Asp Asn

740 745 750

Ile Val Gln Glu Ala Lys Gln Lys Gly Tyr Val Thr Thr Leu Leu His

755 760 765

Arg Arg Arg Tyr Leu Pro Asp Ile Thr Ser Arg Asn Phe Asn Val Arg

770 775 780

Ser Phe Ala Glu Arg Thr Ala Met Asn Thr Pro Ile Gln Gly Ser Ala

785 790 795 800

Ala Asp Ile Ile Lys Lys Ala Met Ile Asp Leu Ser Val Arg Leu Arg

805 810 815

Glu Glu Arg Leu Gln Ala Arg Leu Leu Leu Gln Val His Asp Glu Leu

820 825 830

Ile Leu Glu Ala Pro Lys Glu Glu Ile Glu Arg Leu Cys Arg Leu Val

835 840 845

Pro Glu Val Met Glu Gln Ala Val Thr Leu Arg Val Pro Leu Lys Val

850 855 860

Asp Tyr His Tyr Gly Pro Thr Trp Tyr Asp Ala Lys

865 870 875

<210> 9

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

catagaaata acctgtgtat attgcaagac 30

<210> 10

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

cacagattca aaaagacgac ctaagttgcc 30

Claims

1. a kind of for detecting the kit of HPV, which is characterized in that the kit includes the primer for expanding HPV, and Uvs X, Uvs Y, SSB and archaeal dna polymerase.

2. kit as described in claim 1, which is characterized in that described for expand the primer of HPV to include for expanding The first primer pair of HPV16, and the nucleotide sequence of the first primer pair is as shown in SEQ ID NO.1 and SEQ ID NO.2； And/or

The primer for expanding HPV includes the second primer pair for expanding HPV18, and the nucleosides of second primer pair Acid sequence is as shown in SEQ ID NO.3 and SEQ ID NO.4.

3. kit as described in claim 1, which is characterized in that the amino acid sequence of the Uvs X such as SEQ ID NO.5 institute Show；And/or

The amino acid sequence of the Uvs Y is as shown in SEQ ID NO.6.

4. kit as described in claim 1, which is characterized in that the amino acid sequence of the SSB such as SEQ ID NO.7 institute Show；And/or

The amino acid sequence of the archaeal dna polymerase is as shown in SEQ ID NO.8.

5. kit as described in claim 1, which is characterized in that the kit includes amplification buffer, and the amplification Buffer contains: Tris-HCl, (NH₄)₂SO₄、KCl、MgSO₄、20。

6. kit as claimed in claim 5, which is characterized in that the pH=7-10 of the amplification buffer.

7. kit as claimed in claim 5, which is characterized in that the amplification buffer contains: the Tris-HCl of 20mM, (the NH of 10mM₄)₂SO₄, 50mM KCl, 2mM MgSO₄With 0.1%20。

8. kit as claimed in claim 2, which is characterized in that the kit further includes positive controls, and the sun Property control group contains the positive control primers as shown in SEQ ID NO.9 and SEQ ID NO.10.

9. such as the described in any item kits of claim 1-8, which is characterized in that amplification item of the kit at 37-40 DEG C HPV is detected under part.

10. kit as claimed in claim 9, which is characterized in that the time of the kit detection HPV is 10-20min.