CN101082064A - Detection method for high risk type human papilloma virus and reagent case - Google Patents
Detection method for high risk type human papilloma virus and reagent case Download PDFInfo
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Abstract
The present invention is high risk human papilomavirus (HPV) detecting process, which includes the following steps: 1. designing and synthesizing proper specific primer, and amplifying the target DNA of the detected sample specifically for the second time by means of one-step nest PCR technology; and 2. hybridizing and selectively typing the target DNA of the detected sample through twice specific selective amplification and the specific segment of high risk HPV by means of combination to low density DNA chip technology, and signal identification through fluorescent detecting or direct color developing. The present invention provides also one corresponding kit. The present invention can amplify specific target DNA segments of 13 kinds of HPV simultaneously and detect several kinds of virus subtypes of HPV through once reaction, and has the advantages of high speed, high efficiency, high sensitivity, high specificity, etc.
Description
Technical field
The present invention relates to biology field, more particularly, relate to a kind of detection method for high risk type human papilloma virus and test kit.
Background technology
Cervical cancer is the malignant tumour of serious threat women life, in case take place, its survival and curative ratio are all very low.Scientific research confirms that human papillomavirus (HPV) is the prerequisite that causes cervical cancer, all finds to have the infection of high-risk HPV virus in 99.7% the cases of cervical cancer.Because causing the high-risk HPV virus of cervical cancer pathology is a class low virus, if can in time find to infect, timely conditioning and treating, but virus Be Controlled or removing reach the purpose of preventing and treating cervical cancer.Therefore, the generation of control cervical cancer focuses on prevention, and key is examination in early days.China is populous, and Economic development is unbalanced, particularly carries out the examination of HPV virus infection in vast rural area, the detection technique that urgtented need equipment is simple, easy and simple to handle, economical and practical.
It is the transmissible disease that a kind of trafficability characteristic life is propagated that HPV infects.HPV virus has more than 100 kind of hypotype, and that finds in female genital has 35 kinds, can cause that the high-risk HPV with cervical cancer has 13 kinds: 16,18,31,33,35,39,45,51,52,56,58,59,68, and wherein HPV 16 and 18 type infection rates are the highest.In all types that pick, HPV 16 accounts for 50%, and HPV 18 accounts for 14%, and HPV 45 accounts for 8.4%, and HPV 31 accounts for 5.3%, and HPV 33 accounts for 2.8%.
Human papillomavirus (HPV) is small double-stranded DNA virus, the about 60nm of virion diameter, and no coating, molecular weight are 5000kD, and involucrum is for being made of 20 bodies of symmetrical deflection 72 shell particulates, and the centre is the double-stranded cyclic DNA that contains 7900bp.Main viral coat protein gene is positioned at the L1 fragment.
Behind the HPV virus infection tesselated epithelium, at first hide in basal cell, its viral DNA is free in host's the nucleus, is restricted but duplicate.Move to the epidermis middle-shallow layer on the differentiation and maturation with the work basal cell, removed inhibition, so the replicability infection has taken place to virus.Virus massive duplication in cell increases nucleus, and cytopathy causes that mitochondrial swelling, destruction or glycogen dissolving disappear, and forms physaliphore, finally causes necrocytosis, forms cervical erosion ulcer.This have virus epithelial cell parasitic, breeding not cancerate.Behind the HPV high-risk-type virus infection epithelium, its DNA then may integrate and cytogene is undergone mutation, and the cell atypia is more remarkable, the degree of atypical hyperplasia increases the weight of, scope just changes carcinoma in situ into, and can be further development of infiltrating carcinoma to the expansion of epidermis middle-shallow layer when involving the epidermis holostrome.
The characteristics that the high-risk virus infection approach of foundation and its DNA may undergo mutation cytogene with infected cellular genome integration, adopting nucleic acid detection technique to detect virogene is the unique selection that detects HPV high-risk-type virus, and the most critical index of measurement detection method is the sensitivity and the specific degree of detection technique.
At present the virus detection techniques of comparative maturity be utilize nucleic acid probe directly and target DNA carry out molecular hybridization, yet if in this scheme tested target DNA signal a little less than, sensitivity and specific degree are all lower.Round pcr also is applied to virus and detects, but the regular-PCR technology is selected amplification owing to only carry out a specificity, and it is not high therefore to detect specific degree, the higher proportion false positive can occur; Because this technology is too sensitive, cause detected result very unstable in addition.
The signal capture technology of utilizing of latest developments detects the technology of HPV virus, be HPVRNA probe and the hybridization of the target DNA in the detected sample with mark, the heterozygote signal is by anti-heterozygote antibody capture, amplify by fluorescence or enzyme mark system again, measure luminous signal with the luminescence assays instrument, determine the content of target DNA with strength of signal.This technological operation level of automation is than higher, but because the amplification of all signals is based on the target DNA direct cross in rna probe and the detected sample, detection specific degree and sensitivity are restricted.
Because high-risk HPV virus is 13 kinds of hypotypes nearly, so the DNA chip technology that gets up of development in recent years also is used to the detection of HPV virus.The viral target gene that the principle of this technology also is based in viral probe and the sample is hybridized, and mark fluorescent is luminous then, yet if the target signal is not strong, the sensitivity of its detection and specific degree are not high.Owing to the restriction of technology and condition, high-density DNA chip stability also can not get guaranteeing simultaneously.
Summary of the invention
The technical problem to be solved in the present invention is,, false positive results low and low density DNA chip technology unstable at above-mentioned existing round pcr detection specificity with and be direct primer hybridization, the defective that the target signal is weak, background interference is strong, resolving power is low provides a kind of new detection method for high risk type human papilloma virus and test kit.
The technical solution adopted for the present invention to solve the technical problems is: construct a kind of detection method for high risk type human papilloma virus, may further comprise the steps:
(a) employing single stage method nest-type PRC technology is carried out the amplification of secondary specificity to the target DNA of detected sample;
(b) adopt low density DNA chip technology to hybridize the selection somatotype through detected sample target DNA and HPV high-risk-type virus specific fragment that twice special selection amplified.
In detection method for high risk type human papilloma virus of the present invention, in described step (a), also comprise design synthetic special primer NP1, NP2, NP3 and NP4, increase the ratio of the single stranded DNA amplification of viral target DNA in the test sample, making in the test sample viral target DNA signal obtain effective specificity amplifies, wherein NP3 and NP4 are labeled primer, comprise in fluorescent mark, biotin labeling, the digoxigenin labeled one or more.
In detection method for high risk type human papilloma virus of the present invention, the sequence of described special primer is respectively: NP1:5 '-aataaaccttattggttaca-3 ', NP2:5 '-actgttgttgatac-3 ', NP3:5 '-gtaaatcatattcctc-3 ', NP4:5 '-tgaaaaataaactgtaaatca-3 '.
In detection method for high risk type human papilloma virus of the present invention, the Tm value of described special primer NP1 and NP4 is significantly higher than the Tm value of NP2 and NP3; And NP1, NP4, NP2, NP3 all have high homology with the distinguished sequence of multiple high-risk HPV virus.
In detection method for high risk type human papilloma virus of the present invention, described step (b) comprising: with high-risk HPV virus specific fragment probe stationary on low density DNA chip, to carry out the specific hybridization selection through the detected sample target DNA specific fragment reactant of twice special selection amplification and with high-risk HPV virus specific fragment probe, if test sample fluorescence is luminous then positive; Perhaps described step (b) comprising: the detected sample target DNA specific fragment reactant transfer that will amplify through twice special selection is to low density DNA chip, carry out the selectivity specific combination with high-risk HPV virus specific fragment probe, test sample develops the color after adding corresponding enzyme reaction substrate, if colour developing is then positive.
In detection method for high risk type human papilloma virus of the present invention, described high-risk HPV viral fragment probe 5 ' end sterically hindered with amino and 8-12 thymus pyrimidine T modification and/or when adding arm molecule and hybridizing with minimizing, described high-risk HPV viral fragment probe gene order is:
HPV16:5 '-CATATCTACTTCAGAAAC-3 ' or 5 '-GCCATATCTACTTCAGAAACTA-3 ';
HPV18:5 '-TACACAGTCTCCTGTACC-3 ' or 5 '-TCTACACAGTCTCCTGTACCTG-3 ';
HPV58:5 '-AGTAPPPACTAAGGAAGG-3 ' or 5 '-GAAGTAPPPACTAAGGAAGGTA-3 ':
HPV31:5 '-AATTGCAAACAGTGATAC-3 ' or 5 '-GCAATTGCAAACAGTGATAC-TA3 ';
HPV45:5 '-TACACAAAATCCTGTGCC-3 ' or 5 '-TCTACACAAAATCCTGTGCCAA-3 ';
HPV51:5 '-CACTGCTGCGGTTTCCCC-3 ' or 5 '-GCCACTGCTGCGGTTTCCCCAA-3 ';
HPV52:5 '-GGTPPPTAAAAAGCAAAG-3 ' or 5 '-GAGGTPPPTAAAAAGCAAAGCA-3 ';
HPV33:5 '-AAGTAACTAGTPPPGACA-3 ' or 5 '-ACAAGTAACTAGTPPPGACACT-3 ';
HPV35:5 '-TGTGTCTTCTAGTGACAG-3 ' or 5 '-GCTGTGTCTTCTAGTGACAGTA-3 ';
HPV39:5 '-TATAGAGTCTTCCATACC-3 ' or 5 '-TCTATAGAGTCTTCCATACCTT-3 ';
HPV56:5 '-TACAGAACAGTPPPTAAG-3 ' or 5 '-GCTACAGAACAGTPPPTAAGTA-3 ';
HPV59:5 '-TACTACTTCTTCTATTCC-3 ' or 5 '-TCTACTACTTCTTCTATTCCTA-3 ';
HPV68:5 '-TACTGAATCAGCTGTACC-3 ' or 5 '-ACTACTGAATCAGCTGTACCAA-3.
The present invention also provides a kind of high-risk human mammilla papillomavirus detection kit, includes the reagent and the device of device that sample to be checked is increased and reagent, low density chip, the reagent of hybridizing and device and enzyme connection and/or fluoroscopic examination.
In high-risk human mammilla papillomavirus detection kit of the present invention, described device and the reagent that sample to be checked is increased comprises device and reagent and synthetic special primer NP1, NP2, NP3 and the NP4 that uses single stage method nest-type PRC technology that detected sample virus target DNA is carried out twice specificity amplification; Described low density chip supervisory system comprises by blank spot, positive internal reference, negative internal reference and forming; Described reagent of hybridizing and device comprise various reaction buffers, washings, marked product, high-risk HPV virus probe, high-risk HPV virus target DNA examination criteria product, DNA chip solid phase carrier substrate, quick crossing system; The reagent of described enzyme connection and/or fluoroscopic examination and device comprise fluorescence detection device and/or integrated enzyme reaction substrate, various reaction buffer, washings, colour developing liquid.
In high-risk human mammilla papillomavirus detection kit of the present invention, described high-risk HPV virus comprises for multiple virus subtype: HPV16, HPV18, HPV58, HPV31, HPV45, HPV51, HPV52, HPV33, HPV35, HPV39, HPV56, HPV59, HPV68.
High-risk HPV method for detecting virus of the present invention adopts single stage method nest-type PRC technology, the 13 kinds of high-risk-type virus specific target dna fragmentations that can increase simultaneously, and primary first-order equation can detect multiple virus subtype.In addition, the present invention amplifies the target DNA signal of detected sample by twice specific selectivity, improved the target signal of positive greatly.
By precision design, a large amount of experiment, the present invention is directed to regular-PCR detection method and DNA chip detection technology and be applied to detect the problem that 13 kinds of high-risk HPV viruses exist, reduced the interference of background signal greatly, overcome the deficiency of regular-PCR; Adopt low density DNA chip technology, not only the multiple high-risk HPV virus of special selection can be carried out hypotype identifies, and can measure a plurality of samples simultaneously, in order to improve hybridization sensitivity, when for the second time the target DNA signal being amplified, adopt uneven primer technology, adjust the ratio of NP2 and NP3 primer dosage, when for the second time special selection was amplified, (mark) specific single-chain target dna fragment molecule number increased considerably, and increases substantially the sensitivity of its detection.Adopt fluorescence and enzyme two kinds of mark hybridization of connection developing technology to identify multiple virus subtype, can adapt to the needs of different user, the needs of adaptation population's examination and different testing conditions, economical and practical.
Embodiment
The present invention is high-risk human mammilla papillomavirus (HPV) detection method that a kind of nest-type PRC (Nest PCR) technology combines with the low density chip technology, it is low to have overcome the regular-PCR detection specificity, false positive results waits defectives such as deficiency and high-density DNA chip technology detected result unstable, background interference be strong more, has advantages such as highly sensitive, high specific degree, good stability.
The present invention at first adopts single stage method nest-type PRC technology that the target DNA of detected sample is carried out the secondary specificity to amplify, adopt asymmetric primer and labeling technique thereof simultaneously, increase the ratio of the single stranded DNA amplification of viral target DNA in the test sample, make in the test sample viral target DNA signal obtain effective specificity and amplify.Adopt low density DNA chip technology then, will hybridize the selection somatotype, both improved sensitivity and specific degree through detected sample target DNA and multiple HPV high-risk-type virus specific fragment that twice special selection amplified, simple and practical again.
In the present invention, can select NP1, NP2, NP3 and the NP4 primer of special design and detected sample target DNA to carry out special selection amplification (wherein NP3 and NP4 are labeled primer, can be fluorescence or biotin labeling).When special primer NP1, NP2, NP3 and NP4 synthesized, the Tm value of NP1 and NP4 was significantly higher than the Tm value of NP2 and NP3, and NP1, NP4, NP2, NP3 all have high homology with the distinguished sequence of multiple high-risk HPV virus.For example, special primer NP1, NP2, NP3 and NP4 sequence can be respectively:
NP1：5’-aataaaccttattggttaca-3’；
NP2：5’-actgttgttgatac-3’；
NP3：5’-gtaaatcatattcctc-3’；
NP4：5’-tgaaaaataaactgtaaatca-3’。
In the present invention, can be with the primer of two pairs of special designs application of sample simultaneously, and, twice special being chosen in the same reaction finished by setting reaction conditions.In addition, also can make and select specific single-chain target dna fragment molecule number to increase considerably, and simultaneously this single-chain fragment be carried out mark by adjusting the ratio of NP2 and NP3 primer dosage.
When using low density DNA chip to hybridize the selection somatotype, can adopt signal to select to discern and amplification system (A): high-risk HPV virus specific fragment probe stationary on Hybond membrane or microwell plate, will be carried out the specific hybridization selection through the detected sample target DNA specific fragment reactant of twice special selection amplification and with high-risk HPV virus specific fragment probe.After unconjugated reactant is removed in washing, test sample fluorescence, luminous positive, not luminous negative.High-risk HPV virus specific fragment probe 5 ' end sterically hindered and/or when adding arm molecule and hybridizing wherein with minimizing with amino and 8-12 thymus pyrimidine T modification, described high-risk HPV viral fragment probe gene order is:
HPV16:5 '-CATATCTACTTCAGAAAC-3 ' or 5 '-GCCATATCTACTTCAGAAACTA-3 ';
HPV18:5 '-TACACAGTCTCCTGTACC-3 ' or 5 '-TCTACACAGTCTCCTGTACCTG-3 ';
HPV58:5 '-AGTAPPPACTAAGGAAGG-3 ' or 5 '-GAAGTAPPPACTAAGGAAGGTA-3 ';
HPV31:5 '-AATTGCAAACAGTGATAC-3 ' or 5 '-GCAATTGCAAACAGTGATAC-TA3 ';
HPV45:5 '-TACACAAAATCCTGTGCC-3 ' or 5 '-TCTACACAAAATCCTGTGCCAA-3 ';
HPV51:5 '-CACTGCTGCGGTTTCCCC-3 ' or 5 '-GCCACTGCTGCGGTTTCCCCAA-3 ';
HPV52:5 '-GGTPPPTAAAAAGCAAAG-3 ' or 5 '-GAGGTPPPTAAAAAGCAAAGCA-3 ';
HPV33:5 '-AAGTAACTAGTPPPGACA-3 ' or 5 '-ACAAGTAACTAGTPPPGACACT-3 ';
HPV35:5 '-TGTGTCTTCTAGTGACAG-3 ' or 5 '-GCTGTGTCTTCTAGTGACAGTA-3 ';
HPV39:5 '-TATAGAGTCTTCCATACC-3 ' or 5 '-TCTATAGAGTCTTCCATACCTT-3 ';
HPV56:5 '-TACAGAACAGTPPPTAAG-3 ' or 5 '-GCTACAGAACAGTPPPTAAGTA-3 ';
HPV59:5 '-TACTACTTCTTCTATTCC-3 ' or 5 '-TCTACTACTTCTTCTATTCCTA-3 ';
HPV68:5 '-TACTGAATCAGCTGTACC-3 ' or 5 '-ACTACTGAATCAGCTGTACCAA-3.
The solid phase carrier substrate of low density DNA chip can be a kind of in Hybond membrane, nitrocellulose filter, nylon membrane, sheet glass, silicon chip and the gel.
In addition, when using low density DNA chip to hybridize the selection somatotype, also can adopt signal to select identification and amplification system (B): the detected sample target DNA specific fragment reactant transfer that will amplify through twice special selection is to Hybond membrane, carry out the selectivity specific combination with high-risk HPV virus specific fragment probe, unconjugated reactant is removed in washing, add corresponding enzyme reaction substrate, the test sample colour developing.If colour developing is then positive, it is then negative not develop the color.
Below be above-mentioned HPV method for detecting virus embodiment:
Embodiment one
(1) preparation of chip
The selection slide is a chip carrier, with high-risk HPV virus probe dilution, is dissolved in the Na of 500mmol/L pH9.0 with deionized water
2CO
3/ NaHCO
3Make the sample that final concentration is 10nmol/L in the solution, sample is added in the point template, fix 48～72h under the room temperature, dry standby.The chip supervisory system is made up of blank spot, positive internal reference and negative internal reference.Male fluorescent signal mean value surpasses the male standard that is judged to be more than three times of negative some signal.
(2) DNA preparation
Adopt the DNA extraction test kit to carry out 4 ℃ of preservations.
(3) sample amplification to be checked
1, gets the 0.2mlEP pipe, under condition of ice bath, add aseptic double-distilled water 16.5ul successively, 10 times of PCR reaction buffer 2.5ul, dNTP 0.5ul, each 1ul of special primer NP1, NP2, NP3 and NP4, HPV standard substance 1ul to be checked, Taq enzyme 0.5ul, wherein primer NP3, NP4 are fluorescent dye primer.The amplification system that does not add template is done negative control.
2, put into the PCR instrument behind the mixing and increase, 4 ℃ of preservations of amplified production.
(4) hybridization
1, get 0.2ml EP pipe, add hybridization solution 10ul successively, fluorescent mark product 2ul, mixing drips in chip surface covered.
2, place in the wet hybridizing box, hybridized 1 hour down for 40-50 ℃.
4, hybridization back chip was washed 5 minutes with SSC, SDS solution, rinsed well with aseptic double-distilled water again.
5, drying at room temperature.
(5) scanning detects
To hybridize dried chip and insert array scanning instrument (for example GMS418 Array Scanner) scanning, with detected result.Wherein scan luminous positive, otherwise negative.
The present invention can detect the multiple virus subtype of high-risk HPV virus, specifically comprises: HPV16, HPV18, HPV58, HPV31, HPV45, HPV51, HPV52, HPV33, HPV35, HPV39, HPV56, HPV59, HPV68 etc.
Detection method corresponding kit of the present invention includes: device and reagent that sample to be checked is increased; Low density chip and reagent of hybridizing and device.When using fluorescent mark, also comprise the device that low density chip is scanned.
Above-mentioned device that sample to be checked is increased and reagent comprise device and the reagent that uses single stage method nest-type PRC technology that detected sample virus target DNA is carried out twice specificity amplification.In addition, the above-mentioned reagent that sample to be checked is increased also comprises special primer NP1, NP2, NP3, NP4, reaction buffer, Tap enzyme etc.
Described low density chip supervisory system comprises by blank spot, positive internal reference, negative internal reference and forming.
Reagent of hybridizing and device then comprise hybridization solution, washings, marked product and high-risk HPV virus probe, high-risk HPV virus target DNA examination criteria product, DNA chip solid phase carrier substrate, quick crossing system etc.
The reagent and the device of described enzyme connection and fluoroscopic examination comprise fluorescence detection device, integrated enzyme reaction substrate, various reaction buffer, washings, colour developing liquid.The device that low density chip is scanned can be array scanning instrument (for example GMS418 Array Scanner).
The novel high-risk HPV virus detection kit of using high-risk HPV method for detecting virus preparation provided by the invention adopts built-up type application of sample and quick, easy crossing system, make target DNA signal carry out hybridization with multiple high-risk HPV virus probe fast, make trace routine easy, quick through twice specific selectivity amplification.
The above; only for the preferable embodiment of the present invention, but protection scope of the present invention is not limited thereto, and anyly is familiar with those skilled in the art in the technical scope that the present invention discloses; the variation that can expect easily or replacement all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain of claim.
Claims (10)
1, a kind of detection method for high risk type human papilloma virus is characterized in that, may further comprise the steps:
(a) employing single stage method nest-type PRC technology is carried out the amplification of secondary specificity to the target DNA of detected sample;
(b) adopt low density DNA chip technology to hybridize the selection somatotype through detected sample target DNA and HPV high-risk-type virus specific fragment probe that twice special selection amplified.
2, detection method for high risk type human papilloma virus according to claim 1, it is characterized in that, in described step (a), also comprise design synthetic special primer NP1, NP2, NP3 and NP4, increase the ratio of the single stranded DNA amplification of viral target DNA in the test sample, make in the test sample viral target DNA signal obtain effective specificity and amplify, wherein NP3 and NP4 are labeled primer, comprise in fluorescent mark, biotin labeling, the digoxigenin labeled one or more.
3, detection method for high risk type human papilloma virus according to claim 2, it is characterized in that, the sequence of described special primer is respectively: NP1:5 '-aataaaccttattggttaca-3 ', NP2:5 '-actgttgttgatac-3 ', NP3:5 '-gtaaatcatattcctc-3 ', NP4:5 '-tgaaaaataaactgtaaatca-3 '.
4, detection method for high risk type human papilloma virus according to claim 2 is characterized in that, the Tm value of described special primer NP1 and NP4 is significantly higher than the Tm value of NP2 and NP3; And NP1, NP4, NP2, NP3 all have high homology with the distinguished sequence of multiple high-risk HPV virus.
5, detection method for high risk type human papilloma virus according to claim 1, it is characterized in that, described step (b) comprising: with high-risk HPV virus specific fragment probe stationary on low density DNA chip, to carry out the specific hybridization selection through detected sample target DNA specific fragment reactant and the probe that twice special selection amplified, if test sample fluorescence is luminous then positive.
6, detection method for high risk type human papilloma virus according to claim 1, it is characterized in that, described step (b) comprising: the detected sample target DNA specific fragment reactant transfer that will amplify through twice special selection is to low density DNA chip, carry out the selectivity specific combination with high-risk HPV virus specific fragment probe, test sample develops the color after adding corresponding enzyme reaction substrate, if colour developing is then positive.
7, according to claim 5 or 6 described detection method for high risk type human papilloma virus, it is characterized in that, described high-risk HPV viral fragment probe 5 ' end sterically hindered with amino and 8-12 thymus pyrimidine T modification and/or when adding arm molecule and hybridizing with minimizing, described high-risk HPV viral fragment probe gene order is:
HPV16:5 '-CATATCTACTTCAGAAAC-3 ' or 5 '-GCCATATCTACTTCAGAAACTA-3 ';
HPV18:5 '-TACACAGTCTCCTGTACC-3 ' or 5 '-TCTACACAGTCTCCTGTACCTG-3 ';
HPV58:5 '-AGTAPPPACTAAGGAAGG-3 ' or 5 '-GAAGTAPPPACTAAGGAAGGTA-3 ';
HPV31:5 '-AATTGCAAACAGTGATAC-3 ' or 5 '-GCAATTGCAAACAGTGATAC-TA3 ';
HPV45:5 '-TACACAAAATCCTGTGCC-3 ' or 5 '-TCTACACAAAATCCTGTGCCAA-3 ';
HPV51:5 '-CACTGCTGCGGTTTCCCC-3 ' or 5 '-GCCACTGCTGCGGTTTCCCCAA-3 ';
HPV52:5 '-GGTPPPTAAAAAGCAAAG-3 ' or 5 '-GAGGTPPPTAAAAAGCAAAGCA-3 ';
HPV33:5 '-AAGTAACTAGTPPPGACA-3 ' or 5 '-ACAAGTAACTAGTPPPGACACT-3 ';
HPV35:5 '-TGTGTCTTCTAGTGACAG-3 ' or 5 '-GCTGTGTCTTCTAGTGACAGTA-3 ';
HPV39:5 '-TATAGAGTCTTCCATACC-3 ' or 5 '-TCTATAGAGTCTTCCATACCTT-3 ';
HPV56:5 '-TACAGAACAGTPPPTAAG-3 ' or 5 '-GCTACAGAACAGTPPPTAAGTA-3 ';
HPV59:5 '-TACTACTTCTTCTATTCC-3 ' or 5 '-TCTACTACTTCTTCTATTCCTA-3 ';
HPV68:5 '-TACTGAATCAGCTGTACC-3 ' or 5 '-ACTACTGAATCAGCTGTACCAA-3.
8, a kind of high-risk human mammilla papillomavirus detection kit is characterized in that, includes the reagent and the device of device that sample to be checked is increased and reagent, low density chip, the reagent of hybridizing and device and enzyme connection and/or fluoroscopic examination.
9, high-risk human mammilla papillomavirus detection kit according to claim 8, it is characterized in that described device and the reagent that sample to be checked is increased comprises device and reagent and synthetic special primer NP1, NP2, NP3 and the NP4 that uses single stage method nest-type PRC technology that detected sample virus target DNA is carried out twice specificity amplification; Described low density chip supervisory system comprises by blank spot, positive internal reference, negative internal reference and forming; Described hybridize and reagent that the result detects and device comprise various reaction buffers, washings, marked product and high-risk HPV virus probe, high-risk HPV virus target DNA examination criteria product, DNA chip solid phase carrier substrate, quick crossing system; The reagent of described enzyme connection and/or fluoroscopic examination and device comprise fluorescence detection device and/or integrated enzyme reaction substrate, various reaction buffer, washings, colour developing liquid.
10, a kind of detection method for high risk type human papilloma virus, it is characterized in that described high-risk HPV virus comprises for multiple virus subtype: HPV16, HPV18, HPV58, HPV31, HPV45, HPV51, HPV52, HPV33, HPV35, HPV39, HPV56, HPV59, HPV68.
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| CN101503741B (en) * | 2009-03-04 | 2011-11-30 | 上海之江生物科技有限公司 | Reagent kit for detecting high-risk human mammilla papillomavirus, as well as preparation and application thereof |
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| CN101709335B (en) * | 2009-12-10 | 2011-12-21 | 浙江大学 | Quadruple fluorescence quantitive PCR typing detection method for common human papillomavirus |
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