CN105400901A - Kit for detecting human papilloma virus and application of kit - Google Patents
Kit for detecting human papilloma virus and application of kit Download PDFInfo
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- CN105400901A CN105400901A CN201410415013.5A CN201410415013A CN105400901A CN 105400901 A CN105400901 A CN 105400901A CN 201410415013 A CN201410415013 A CN 201410415013A CN 105400901 A CN105400901 A CN 105400901A
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Abstract
The invention discloses a kit for detecting human papilloma virus (HPV). The kit comprises the following components: a pre-treating fluid, a primary treating fluid, a hybridizing fluid, and a post-treating fluid, wherein the hybridizing fluid comprises alkaline-phosphatase-marked streptavidin, and a biotin-marked DNA probe for detecting human papilloma virus. In the kid, alkaline-phosphatase-marked streptavidin is directly added into the hybridizing fluid, so that a process of DNA molecular hybridization and a process of alkaline phosphatase coupling are combined into one process, and the detection efficiency of HPV in a sample is improved; and a surface of a solid holder before and after a one-step reaction is subjected to synergic cooperative treatment through the pre-treating fluid, the primary treating fluid, and the post-treating fluid, thereby improving specificity of HPV molecular hybridization on the surface of the holder, and effectively avoiding a false positive detection result.
Description
Technical field
The present invention relates to the detection of human papillomavirus, be specifically related to the test kit and the application thereof that detect human papillomavirus.
Background technology
Human papillomavirus (humanpapillomavirus, HPV) belongs to Papovaviridae (papovaviridae) Papillomavirus, is a kind of virus causing the optimum and malignant tumour of mankind's mucous membrane tissue.It was found in 1949 by Strauss under Electronic Speculum, by DNA core and protein coat form without coating double-stranded cyclic DNA virus, its core is made up of with covalent linkage 7800 ~ 7900 base pairs, close circular double stranded DNA containing genetic information, be outward that 72 capsomeres hold, form 20 bodies of symmetric form.Virus is without coating, and diameter is 55nm, and molecular weight is 54000 dalton.It infects mainly epithelium, has the host specificity of height, has avidity to the epithelial cell of human body specific regions.HPV is the easiest growing multiplication in the gentle condition of human body, and property contact is the main route of infection, and stadium is the strongest about 3 months person's infectivity.Under a few cases, likely infected by contact stain article, as propagation such as underpants, bathtub, towels.In birth process, fetus all can infect with the female close contact of trouble after the infection birth canal or birth of mother HPV.
HPV very easily Spreading and diffusion in crowd, by directly or indirectly contacting cross infection, its infection site is hidden, incidence of occult, not easily early discovery, can cause multiple proliferative lesion, and the modal malignant tumour caused by female reproductive system is cervical cancer.The whole world about has 500,000 woman uterus cancer new cases every year, and wherein Asia accounts for 380,000; 230,000 women are about had to die from cervical cancer every year.In different areas, the M & M of the national cervical cancer of different economic situation has marked difference, and wherein the case of 80% occurs in developing country.China about has 150,000 cervical cancer new cases every year, and about have 80,000 people to die from cervical cancer every year, its mortality ratio occupies the second of gynecological tumor.In recent years, Patients with Cervical Cancer number is rising gradually, and the trend in rejuvenation.The detection that high-risk HPV infects has very important meaning for prevention and early discovery cervical cancer.
Because HPV still can not cultivate so far in vitro, again without suitable laboratory animal, thus morphological method and Protocols in Molecular Biology are depended on to its detection.Current HPV detection method mainly contains cytology method, spot hybridization, hybridization in situ, fluorescence quantitative PCR method or belong to Digene house journal of the U.S. two generation hybrid capture analytical method etc.Cytology susceptibility and specificity lower; Hybridization in situ is more complicated, and unsuitable larger scale clinical uses; It is few that fluorescence quantitative PCR method detects genotype, easily produces false positive and pollute; Two generations hybrid capture analyze rule because expensive, detection time is long, there will be the reasons such as false positive results, larger scale clinical examination cannot be used for.Although traditional dot hybridization method is highly sensitive, specificity good, owing to having radioactivity and complex operation, larger scale clinical cannot be realized always and use.
For this consideration, the present inventor is studied, and object solves the problem that association area prior art comes out, expect to provide a kind of highly sensitive, specificity good and the test kit of detection human papillomavirus easy and simple to handle.
Summary of the invention
The object of this invention is to provide a kind of test kit detecting human papillomavirus.
The object of the invention is to realize by following technical solution:
The invention provides a kind of test kit detecting human papillomavirus, it comprises following component: pretreatment fluid, pretreatment liquid, hybridization solution and aftertreatment fluid; Wherein, described hybridization solution comprises alkali phosphatase enzyme mark Streptavidin and the biotin labeled DNA probe for detecting human papillomavirus.
On the one hand, test kit of the present invention directly adds alkali phosphatase enzyme mark Streptavidin (hereinafter referred to as SA-AP) and DNA probe in hybridization solution, make DNA molecule hybridize process can join process implementation with alkaline phosphatase to unite two into one and carry out, improve the detection efficiency of HPV in sample; On the other hand, the solid support surface that pretreatment fluid in test kit of the present invention, pretreatment liquid and aftertreatment fluid can be used for before and after to single step reaction carries out coordinated process, improve the specificity of solid support surface HPV viruses molecule hybridization, effectively prevent the false positive of detected result.
According to a specific embodiment of the present invention, the concentration of described alkali phosphatase enzyme mark Streptavidin in hybridization solution is 5ng/ml ~ 50ng/ml, is preferably 10ng/ml ~ 40ng/ml, is more preferably 15ng/ml ~ 30ng/ml.
In test kit of the present invention, directly joined in hybridization solution by alkali phosphatase enzyme mark Streptavidin, the concentration of described alkali phosphatase enzyme mark Streptavidin in hybridization solution is the importance that the present invention needs to consider.The present inventor is found by lot of experiments and creative work, and the concentration of alkali phosphatase enzyme mark Streptavidin is too low, affects the sensitivity that target DNA detects; The excessive concentration of alkali phosphatase enzyme mark Streptavidin, easily brings non-specific adsorption phenomenon, affects the accuracy of target DNA detected result.The example for the concentration of described alkali phosphatase enzyme mark Streptavidin of the present invention in hybridization solution that can enumerate includes but not limited to: 5ng/ml, 15ng/ml, 16ng/ml, 17ng/ml, 18ng/ml, 19ng/ml, 20ng/ml, 21ng/ml, 22ng/ml, 23ng/ml, 24ng/ml, 25ng/ml, 26ng/ml, 27ng/ml, 28ng/ml, 29ng/m, 30ng/ml or 50ng/ml.
According to a specific embodiment of the present invention, the concentration of described biotin labeled DNA probe in hybridization solution is 0.1 ~ 5pmol/ml, is preferably 0.2 ~ 2pmol/ml, is more preferably 0.25 ~ 1pmol/ml.
The present inventor is found by lot of experiments and creative work, and the example for the concentration of described biotin-labelled DNA probe of the present invention in hybridization solution that can enumerate includes but not limited to: 0.1pmol/ml, 0.2pmol/ml, 0.3pmol/ml, 0.4pmol/ml, 0.5pmol/ml, 0.6pmol/ml, 0.7pmol/ml, 0.8pmol/ml, 0.9pmol/ml or 1.0pmol/ml.
In test kit of the present invention, described alkaline phosphatase is a kind of heterodimeric protein, is a kind of metalloenzyme containing zinc.Per molecule enzyme is at least containing 2 Zn atoms.Enzyme comprises the metal-binding sites of 3 types, i.e. so-called catalyzed combination site, structure binding site and adjustment binding site.Wherein the combination in two catalyzed combination sites then only can cause the phosphorylation of a subunit, namely interacts between negative cooperation subunit.
In test kit of the present invention, described biology have two ring texture I and II.Wherein, I ring is imidazolone ring, is the main portions that it is combined with Streptavidin; II ring is thiphene ring, C2 has a pentanoic acid side chain; Biotin molecule is connected with target DNA of the present invention by the carboxyl of its end, thus is marked on target DNA molecule.
In test kit of the present invention, described Streptavidin is a kind of protein secreted by streptomycete, and its molecule is made up of 4 identical peptide chains, and every bar peptide chain can in conjunction with a vitamin H, and therefore each Streptavidin molecular energy is in conjunction with 4 biotin molecules.In addition, in the amino acid composition of every bar peptide chain, the content of glycine and L-Ala is comparatively large, and the tryptophan residue in peptide chain is the active group connecting vitamin H.The constant (K) of the affine combination of both described Streptavidin and vitamin H is 1015L/mol.In the method for the invention, while target DNA and DNA probe are hybridized, Streptavidin and vitamin H also carry out affine combination, the alkali phosphatase enzyme mark Streptavidin molecule therefore in hybridization solution and the biotin labeled target DNA of DNA probe molecule competitive binding in single step reaction being fixed on solid support surface.
Present invention also offers a kind of test kit detecting human papillomavirus, it comprises following component: pretreatment fluid, pretreatment liquid, hybridization solution, aftertreatment fluid, alkali phosphatase enzyme mark Streptavidin and the biotin labeled DNA probe solution for detecting human papillomavirus.
Alkali phosphatase enzyme mark Streptavidin and DNA probe solution in use directly join in hybridization solution and use by test kit of the present invention, make DNA molecule hybridize process can join process implementation with alkaline phosphatase to unite two into one and carry out, improve the detection efficiency of HPV in sample; On the other hand, the solid support surface that pretreatment fluid in test kit of the present invention, pretreatment liquid and aftertreatment fluid can be used for before and after to hybridization carries out coordinated process, improve the specificity of solid support surface HPV viruses molecule hybridization, effectively prevent the false positive of detected result.
According to a specific embodiment of the present invention, described pretreatment fluid comprises encapsulant and nonionic surface active agent; Wherein, described encapsulant is casein and/or bovine serum albumin, and described nonionic surface active agent is tween and/or Triton; In described pretreatment fluid, encapsulant accounts for 1 ~ 5% (w/v) of pretreatment fluid, and nonionic surface active agent accounts for 0.05% ~ 1% (v/v) of pretreatment fluid; Preferably, encapsulant accounts for 2 ~ 4% (w/v) of pretreatment fluid, and nonionic surface active agent accounts for 0.05% ~ 0.2% (v/v) of pretreatment fluid.
In test kit of the present invention, the avtive spot that the composite use of described encapsulant and nonionic surface active agent can be used for solid support surface can be combined with DNA is closed.Described tween is selected from polysorbas20 (TWEEN-20), tween 21 (TWEEN-21), polysorbate40 (TWEEN-40), polysorbate60 (TWEEN-60), Tween61 (TWEEN-61), tween 80 (TWEEN-80), sorbimacrogol oleate100 (TWEEN-81) and polysorbate85 (TWEEN-85), and wherein polysorbas20 is particularly preferred.Described Triton is selected from triton x-100 (TritonX-100), Triton X-114 (TritonX-114) and Triton X-200 (TritonX-200), and wherein, triton x-100 is particularly preferred.
According to a specific embodiment of the present invention, described pretreatment fluid also comprises anionic dispersing agent and anion-polyacrylamide; Wherein, described anionic dispersing agent is selected from sulfonated lignin, preferred sodium lignosulfonate; In described pretreatment fluid, anionic dispersing agent accounts for 0.05% ~ 0.2% (w/v) of pretreatment fluid, and anion-polyacrylamide accounts for the 0.02-0.5% (w/v) of pretreatment fluid; Preferably, anionic dispersing agent accounts for 0.1% ~ 0.2% (w/v) of pretreatment fluid, and anion-polyacrylamide accounts for the 0.1-0.3% (w/v) of pretreatment fluid.
The present inventor is found by lot of experiments and creative work, in test kit of the present invention, contriver with nonionogenic tenside together with carries out composite with anion-polyacrylamide with encapsulant by the anionic dispersing agent added in pretreatment fluid, effectively improve encapsulant and nonionogenic tenside at the adsorption effect of hydrophobic interfaces, greatly improve the specificity of detected result.
In test kit of the present invention, described anionic dispersing agent can produce enough energy barriers to encapsulant, ensures that encapsulant disperses more stable in pretreatment fluid.Described anionic dispersing agent and encapsulant produce adsorption, can reduce encapsulant because separating coalescent required mechanical work.Described anionic dispersing agent and nonionic surface active agent are carried out composite, further increases the sealing effect of nonionogenic tenside.Described anion-polyacrylamide can reduce the Stern current potential of solid/liquid interfaces, reduces electric energy and builds, and promotes that the encapsulant that is dispersed in pretreatment fluid and nonionogenic tenside active adsorption are to the surface of solid support.Because the hydrophilic group of polyacrylamide can adsorb by preferred direction at solid support surface, hydrophobic group points to aqueous phase, thus make the tension force of solid/liquid interfaces become large, the water repellency of solid support surface strengthens, consequently when encapsulant contacts with solid support surface, stretch to the hydrophobic chain of aqueous phase interactional make encapsulant occur to flocculate from pretreatment fluid simultaneously and active adsorption to the surface of solid support.
In a preferred embodiment of the invention, described anionic dispersing agent is preferably sodium lignosulfonate, and it is a kind of natural polymers, has very strong dispersiveness, can be adsorbed on the surface of solid particle; Because there is various active group in its weave construction, thus can with encapsulant generation hydrogen bond action.
According to a specific embodiment of the present invention, the PH of described pretreatment fluid is 7.0 ~ 8.0.
The present inventor is found by lot of experiments and creative work, and in test kit of the present invention, the pH value of pretreatment fluid affects the chargeding performance of Multiple components in pretreatment fluid.In a preferred embodiment of the invention, the PH of preferred described pretreatment fluid is 7.0 ~ 8.0, now encapsulant neutral, anionic dispersing agent and the interactional resistance of encapsulant lower, the dispersing property of encapsulant in pretreatment fluid reaches more excellent, and now under the effect of polyacrylamide, be more easily adsorbed onto solid/liquid interfaces, and the adsorptive power of encapsulant and solid support is stronger.
According to a specific embodiment of the present invention, described pretreatment liquid is the buffered soln of PH7.5 ~ 9.0, and described buffered soln is selected from Veronal sodium-hydrochloric acid buffer solution, Tris-hydrochloric acid buffer solution, Glycine-NaOH buffered soln and boric acid-borax buffer solution.
According to a specific embodiment of the present invention, described aftertreatment fluid comprises C
8~ C
18alkyl-glucoside, preferred C
9~ C
13alkyl-glucoside; Described alkyl-glucoside accounts for 0.1 ~ 5% (w/v) of aftertreatment fluid, preferably 0.5 ~ 2% (w/v).
The present inventor is found by lot of experiments and creative work, it is very important that solid support surface after test kit of the present invention utilizes aftertreatment fluid to complete single step reaction carries out washing, on the one hand can by do not hybridize with target DNA and the biotin-labelled DNA probe molecule of non-specific hybridization wash away from solid support surface, and specific hybrid is retained in solid support surface; The activity of alkaline phosphatase can be kept on the other hand, ensure the efficiency of colour developing, aberration better effects if.
According to a specific embodiment of the present invention, described hybridization solution also comprises zine ion, magnesium ion, nonionic surface active agent and cation type polymer; Wherein, described nonionic surface active agent is tween and/or Triton, and described cation type polymer is selected from cationic polyacrylamide, poly-lysine and polymerize aluminum chloride.
According to a specific embodiment of the present invention, in described hybridization solution, zine ion is 0.001 ~ 0.1mol/L, magnesium ion is 0.001 ~ 0.1mol/L, nonionic surface active agent accounts for 0.1 ~ 0.5% (v/v) of hybridization solution, and cation type polymer accounts for 0.01 ~ 0.1% (w/v) of hybridization solution; Preferably, zine ion is 0.004 ~ 0.05mol/L, magnesium ion is 0.004 ~ 0.05mol/L, and nonionic surface active agent accounts for 0.2 ~ 0.4% (v/v) of hybridization solution, and cation type polymer accounts for 0.02 ~ 0.06% (w/v) of hybridization solution.
According to a specific embodiment of the present invention, described hybridization solution does not comprise edetate, inorganic phosphate and thanomin.
The present inventor finds through great many of experiments and creative work, in test kit of the present invention, the change of acid, alkali, salt ion or temperature condition all may change even makes alkaline phosphatase lose activity completely, therefore in order to realize object of the present invention, the selection of hybridization solution composition is very important.Test kit of the present invention by adding zine ion, magnesium ion and nonionic surface active agent in hybridization solution, contribute on the one hand improving nucleic acid hybridization efficiency, prevent on the other hand the alkali phosphatase enzyme mark Streptavidin sex change because of absorption in hybridization solution, then prevent the polymerization sex change that causes because of interaction between alkali phosphatase enzyme mark Streptavidin molecule on the one hand.
In test kit of the present invention, described cation type polymer can produce electrostatic adsorption with biotin labeled DNA probe in hybridization solution, single strand dna (being with many negative charges) is made to bring a positive charge part, because alkali phosphatase enzyme mark Streptavidin is also with many negative charges, the resistance that alkali phosphatase enzyme mark Streptavidin is combined with biotin-labelled DNA probe so just can be reduced further.In addition, described cation type polymer can make alkali phosphatase enzyme mark Streptavidin in hybridization solution, form even suspension, be marked at after single step reaction is completed in nucleic acid conjugates alkaline phosphatase keep active condition, thus make the balance of alkaline phosphatase conformation change move towards native state.
According to a specific embodiment of the present invention, described zine ion can be selected from the soluble salt containing zine ion.The example that can be used as the described soluble salt containing zine ion of the present invention comprises: zinc sulfate, zinc chloride and other various salt that can dissociate zine ion at solution state.
According to a specific embodiment of the present invention, described magnesium ion can be selected from the soluble salt containing magnesium ion.The example that can be used as the described soluble salt containing magnesium ion of the present invention comprises: magnesium sulfate, magnesium acetate, magnesium chloride and other various salt that can dissociate magnesium ion at solution state.
In test kit of the present invention, described hybridization solution can also comprise other compositions, and in the hybridization solution that can enumerate, the example of other compositions includes but not limited to: sodium-chlor, hybridization buffer, Denhardt ' s solution, sarcosyl or sodium laurylsulfonate.
In test kit of the present invention, hybridization promoter can also be comprised in described hybridization solution, described hybridization promoter is known to the person skilled in the art in itself, and the example that can be used as hybridization promoter of the present invention includes but not limited to: T 500, polyoxyethylene glycol, phenol or guanidine thiocyanate.
According to a specific embodiment of the present invention, described biotin labeled DNA probe is according to the L1 gene design of HPV virus.HPV encoding viral 6 ~ 8 early protein E and 2 late protein L; Wherein, L1 and L2 gene is encoded Major capsid protein L1 and secondary capsid protein L2 respectively, and both form the protein coat of virus; And L1 protein sequence is quite conservative, be HPV essential species genus-specific antigen, the L1 full length gene 1.5kb of its correspondence, HPV virus can be divided into multiple genotype by its polymorphism and conservative property.
According to a specific embodiment of the present invention, described biotin labeled DNA probe comprises:
For detecting the genotypic DNA probe of HPV16, its nucleotide sequence is selected from SEQIDNOs:1 ~ 3;
For detecting the genotypic DNA probe of HPV18, its nucleotide sequence is selected from SEQIDNOs:4 ~ 6;
For detecting the genotypic DNA probe of HPV31, its nucleotide sequence is selected from SEQIDNOs:7 ~ 9;
For detecting the genotypic DNA probe of HPV33, its nucleotide sequence is selected from SEQIDNOs:10 ~ 11;
For detecting the genotypic DNA probe of HPV35, its nucleotide sequence is selected from SEQIDNOs:12 ~ 14;
For detecting the genotypic DNA probe of HPV39, its nucleotide sequence is selected from SEQIDNOs:15 ~ 17;
For detecting the genotypic DNA probe of HPV45, its nucleotide sequence is selected from SEQIDNOs:18 ~ 20;
For detecting the genotypic DNA probe of HPV51, its nucleotide sequence is selected from SEQIDNOs:21 ~ 23;
For detecting the genotypic DNA probe of HPV52, its nucleotide sequence is selected from SEQIDNOs:24 ~ 26;
For detecting the genotypic DNA probe of HPV56, its nucleotide sequence is selected from SEQIDNOs:27 ~ 29;
For detecting the genotypic DNA probe of HPV58, its nucleotide sequence is selected from SEQIDNOs:30 ~ 32;
For detecting the genotypic DNA probe of HPV59, its nucleotide sequence is selected from SEQIDNOs:33 ~ 35;
For detecting the genotypic DNA probe of HPV68, its nucleotide sequence is selected from SEQIDNOs:36 ~ 38.
In test kit of the present invention, contriver have selected clinically of paramount importance 13 HPV genotype (16,18,31,33,35,39,45,51,52,56,58,59,68) as detected object, by great many of experiments and creative work, have studied a large amount of HPV nucleic acid sequences, devise 38 oligonucleotide probes of high special and sensitivity respectively for these 13 HPV genotype.Because the nucleotide sequence of the L1 gene between HPV virus different genotype is closely similar, verify so the present inventor has carried out a large amount of experiments to the oligonucleotide probe just selected pointedly, and filter out probe SEQIDNOs:1 ~ 38 of available high degree of specificity.
According to a specific embodiment of the present invention, described biotin labeled DNA probe comprises:
For detecting the genotypic DNA probe of HPV16, its nucleotide sequence is as shown in SEQIDNO:1;
For detecting the genotypic DNA probe of HPV18, its nucleotide sequence is as shown in SEQIDNO:4;
For detecting the genotypic DNA probe of HPV31, its nucleotide sequence is as shown in SEQIDNO:7;
For detecting the genotypic DNA probe of HPV33, its nucleotide sequence is as shown in SEQIDNO:10;
For detecting the genotypic DNA probe of HPV35, its nucleotide sequence is as shown in SEQIDNO:12;
For detecting the genotypic DNA probe of HPV39, its nucleotide sequence is as shown in SEQIDNO:15;
For detecting the genotypic DNA probe of HPV45, its nucleotide sequence is as shown in SEQIDNO:18;
For detecting the genotypic DNA probe of HPV51, its nucleotide sequence is as shown in SEQIDNO:21;
For detecting the genotypic DNA probe of HPV52, its nucleotide sequence is as shown in SEQIDNO:24;
For detecting the genotypic DNA probe of HPV56, its nucleotide sequence is as shown in SEQIDNO:27;
For detecting the genotypic DNA probe of HPV58, its nucleotide sequence is as shown in SEQIDNO:30;
For detecting the genotypic DNA probe of HPV59, its nucleotide sequence is as shown in SEQIDNO:33;
For detecting the genotypic DNA probe of HPV68, its nucleotide sequence is as shown in SEQIDNO:36.
In test kit of the present invention, contriver considers that some HPV genotype also exists some common gene hypotypes simultaneously, the types such as such as HPV45, HPV52 and HPV59, for these HPV genotype, devise the DNA probe of degeneracy type, it can detect these genotypic various gene hypotypes simultaneously and can not cause undetected.Consider specificity that DNA probe detects, sensitivity and the level of coverage to some genotypic different genes hypotype, contriver finally optimize sequence as SEQIDNOs:1,4,7,10,12,15,18,21,24,27,30, the DNA probe shown in 33 and 36.
According to a specific embodiment of the present invention, described DNA probe is selected from the oligonucleotide probe that length is 15 ~ 40 bases, is preferably selected from the oligonucleotide probe that length is 16 ~ 25 bases.
Test kit of the present invention, by the adjustment length of biotin-labelled DNA probe and composition, reduces the temperature that nucleic acid probe and target DNA are hybridized, thus makes hybridization and integrated enzyme reaction can be unified into a reaction process to carry out.The present inventor finds through great many of experiments, and described nucleic acid probe is selected from when length is the oligonucleotide probe of 15 ~ 40 bases proper, is preferably selected from the oligonucleotide probe that length is 16 ~ 25 bases.The length of particularly preferred DNA probe comprises 16,17,18,19,20,21,22,23,24 and 25 bases.
In test kit of the present invention, the foundation of the selection of DNA probe length is: if DNA probe length is too low, although can improve the specificity of probe hybridization, significantly can reduce the sensitivity of probe hybridization; If DNA probe length is long, the sensitivity of probe hybridization can be improved further, but probe hybridization specificity significantly can reduce, especially be difficult to distinguish the just closely similar multiple HPV genotype of these original sequences.For long DNA probe, the specificity of probe hybridization can not be improved by improving hybridization temperature, because too high temperature can make the alkali phosphatase enzyme mark Streptavidin inactivation in hybridization system.Comprehensive various factors above, consider that the GC content of probe is worth impact to hybridization Tm, the oligonucleotide probe of 16 ~ 25 bases is preferred simultaneously.
According to a specific embodiment of the present invention, described biotin labeled DNA probe 5 ' holds biotin labeled oligonucleotide probe.
According to a specific embodiment of the present invention, test kit of the present invention also comprises chromophoric solution, substrate is comprised in described chromophoric solution, it can by alkaline phosphatase dephosphorylation, namely alkaline phosphatase by hydrolyzes phosphomonoesters by the phosphate group removing on substrate molecule, and can generate phosphate anion and hydroxyl freely.That can enumerate includes but not limited to for the example of substrate in test kit of the present invention: nitroblue tetrazolium (NBT) (NBT) and the chloro-mixture of 3-indyl-phosphoric acid (BCIP) of the bromo-4-of 5-or the mixture of fast red and naphthols ASMX.Under the catalysis of alkaline phosphatase, BCIP can be hydrolyzed the product producing strong reactivity, and the meeting of this product and NBT react, and forms insoluble mazarine to hepatic NBT-formazan.
According to a specific embodiment of the present invention, test kit of the present invention also comprises solid support, and described solid support is selected from nylon membrane, nitrocellulose filter or polypropylene screen.Wherein, nylon membrane and nitrocellulose filter are preferred, and nitrocellulose filter is particularly preferred.
In test kit of the present invention, described target DNA can by non covalent bond or covalently immobolization at described solid support surface.The non covalent bond of target DNA is fixed and can be attracted by positive and negative charge by the phosphate radical negative ion in target DNA and positively charged solid support surface or hydrophobic interaction and make DNA be fixed on solid support surface.The covalently immobolization of target DNA can pass through covalent linkage, as amido linkage, ester bond or ehter bond etc. make target DNA be fixed on solid support surface.
According to a specific embodiment of the present invention, can also comprise positive control sample and negative control sample in test kit of the present invention, described positive control can select HPV16 type full-length genome plasmid, and described negative control can select salmon sperm dna.
According to a specific embodiment of the present invention, test kit of the present invention can use conventional DNA extraction method or commercially available DNA extraction kit to extract target DNA from the cervical exfoliated cell sample deriving from experimenter.
According to a specific embodiment of the present invention, test kit of the present invention allows differentiate high-risk HPV types and not it may be noted that to there is which kind of concrete high-risk type in the sample to which.
The kind of high-risk type (always find in High grade SIL [infringement in tesselated epithelium] and carcinoma in situ those) includes but not limited to: HPV16,18,31,33,35,39,45,51,52,56,58,59,66 and 68;
The kind of low danger type (mainly finding in low SIL) includes but not limited to: HPV6,11,34,40,42,43,44,53,70 and 74.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing used required in describing embodiment is done and introduce simply, obviously, accompanying drawing in brief description is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 represents the colour developing result figure of embodiments of the invention 11.
Fig. 2 represents the colour developing result figure of embodiments of the invention 12.
Fig. 3 represents the colour developing result figure of embodiments of the invention 13.
Fig. 4 represents the colour developing result figure of embodiments of the invention 14.
Fig. 5 represents the colour developing result figure of embodiments of the invention 15.
Fig. 6 represents the colour developing result figure of embodiments of the invention 16.
Fig. 7 represents the colour developing result figure of embodiments of the invention 17.
Fig. 8 represents the colour developing result figure of embodiments of the invention 18.
Fig. 9 represents the colour developing result figure of embodiments of the invention 19.
Figure 10 represents the colour developing result figure of embodiments of the invention 20.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturers suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
In the examples below, the concentration of bovine serum albumin (hereinafter referred to as BSA), anion-polyacrylamide (hereinafter referred to as APAM), alkali phosphatase enzyme mark Streptavidin (hereinafter referred to as SA-AP), sodium lignosulfonate (hereinafter referred to as SLS), poly-lysine (hereinafter referred to as PLL), cationic-type polyacrylamide (hereinafter referred to as CPAM), PEG 8000 and alkyl-glucoside refers to quality volume percent (w/v) concentration in g/ml; The concentration of Tween-20 and TritonX-100 refers to volume percent (v/v) concentration.
Below for detecting the preparation embodiment of the test kit of human papillomavirus
Embodiment 1
Main raw material and reagent
Nitrocellulose filter, is produced by Millipore Corp. (Millipore), 0.45 μm of aperture specification;
The preparation process of 20 × SSC buffered soln is as follows: take 88.2g trisodium citrate (purchased from the raw work in Shanghai) and 175.3gNaCl is dissolved in 800ml pure water, abundant mixing, with dense HCl regulator solution pH value to 7.0 ± 0.1, add pure water to 1000ml.
Tris, zinc chloride, six water and magnesium chloride, Tween20, poly-lysine (referred to as PLL), PEG 8000, anion-polyacrylamide (referred to as APAM), ndodecyl glucoside, nitroblue tetrazolium (NBT) (referred to as NBT) and the bromo-4-of 5-chloro-3-indyl-phosphoric acid (referred to as BCIP) are all direct purchased from Sangon Biotech (Shanghai) Co., Ltd..
Tween-20, bovine serum albumin and sodium lignosulfonate are all direct purchased from Sigma-Aldrich company.
Alkali phosphatase enzyme mark Streptavidin is directly purchased from Gibcol company.
(1) preparation of solid support
Nitrocellulose filter is cut into 2cm × 1cm for subsequent use.Then get nitrocellulose filter with tweezers and be placed on immersion 15min in 15 × SSC (diluted by 20 × SSC buffered soln and form), take out, be placed on filter paper, dry 1.5 hours for 60 DEG C.
(2) preparation of pretreatment fluid
Table 1
| Material name | Every 1000ml consumption |
| NaCl | 58.4g |
| Tris | 12.1g |
| Pure water | 800ml |
| Tween-20 | 1ml |
| BSA | 20g |
| SLS | 1.5g |
| APAM | 2g |
Take 58.4gNaCl and 12.1gTris respectively, add 800ml pure water, abundant dissolving mixing, with dense HCl regulator solution pH value to 7.5 ± 0.1, then the APAM of SLS and 2g of BSA, 1.5g of Tween-20,20g of 1ml is added respectively, abundant dissolving, mixing, add pure water and can obtain pretreatment fluid composed as follows to 1000ml:
PH7.5,0.1mol/LTris-HCl, 1mol/LNaCl, 2%BSA, 0.1%Tween-20,0.15%SLS and 0.2%APAM, surplus is water.
(3) preparation of hybridization solution
Table 2
| Material name | Every 1000ml consumption |
| 20×SSC | 150ml |
| ZnCl 2 | 1.36g |
| MgCl 2·6H 2O | 2.03g |
| Tween-20 | 3ml |
| PLL | 0.4g |
| PEG 8000 | 50g |
| SA-AP | 20μg |
| 13 kinds of HPV DNA probe solution | 50pmol/ml |
| Pure water | 700ml |
Student on commission's work biotechnology (Shanghai) limited-liability company synthesizes 13 kinds for what detect human papillomavirus and 5 ' holds biotin labeled DNA probe, and the nucleotides sequence of described DNA probe is classified as SEQIDNOs:1,4,7,10,12,15,18,21,24,27,30,33 and 36.Dissolve the dry powder of above-mentioned 13 kinds of probes with sterilizing pure water respectively, be mixed with the probe solution that concentration is 50pmol/ml.
Measure 20 × SSC damping fluid of 150ml and the pure water of 650ml respectively, fully mix; Then the ZnCl of 1.36g is added wherein respectively
2, the MgCl of 2.03g
26H
2often kind of HPVDNA probe solution of the PEG 8000 of the PLL of the Tween-20 of O, 3ml, 0.4g, 50g and SA-AP and 10ml of 20 μ g, fully dissolves, mixes, add pure water to 1000ml, can obtain hybridization solution composed as follows:
PH7.0,3 × SSC, 20ng/mlSA-AP, 0.5pmol/mlDNA probe, 10mMZnCl
2, 10mMMgCl
2, 0.3%Tween-20,0.04%PLL and 5% PEG 8000, surplus is water.
(4) preparation of aftertreatment fluid
Table 3
| Material name | Every 1000ml consumption |
| NaCl | 5.8g |
| MgCl 2·6H 2O | 10.2g |
| Tris | 12.1g |
| Ndodecyl glucoside | 10g |
| Pure water | 900ml |
Each raw material in table 3 is fully dissolved mixing according to consumption, with dense HCl regulator solution pH value to 9.5 ± 0.1, transfers in volumetric flask and add pure water and be settled to 1000ml, above-mentioned solution is transferred in 1000ml reagent bottle, obtains aftertreatment fluid composed as follows:
PH9.5,0.1mol/LNaCl, 0.1mol/LTris-HCl, 50mMMgCl
2and 1% ndodecyl glucoside, surplus is water.
Therefore, the concrete component that the present embodiment a kind of detects the test kit of human papillomavirus is as shown in the table:
Table 4
Embodiment 2:
With embodiment 1, difference is the composition of the pretreatment fluid liquid in embodiment 1 to be adjusted to: PH7.0,0.1mol/LTris-HCl, 1mol/LNaCl, 3%BSA, 0.05%Tween-20,0.1%SLS and 0.1%APAM, surplus is water.Other conditions are constant.
Embodiment 3:
With embodiment 1, difference is the composition of the pretreatment fluid liquid in embodiment 1 to be adjusted to: PH8.0,0.1mol/LTris-HCl, 1mol/LNaCl, 4%BSA, 0.2%TritonX-100,0.2%SLS and 0.3%APAM, surplus is water.Other conditions are constant.
Embodiment 4:
With embodiment 2, difference is the part of the hybridization solution in embodiment 2 composition to be adjusted to: 15ng/mlSA-AP, 0.25pmol/mlHPVDNA probe, 3 × SSC, 4mMZnCl
2, 4mMMgCl
2, 0.2%Tween-20,0.02%PLL and 5% PEG 8000, surplus is water.Other conditions are constant.
Embodiment 5:
With embodiment 2, difference is the part of the hybridization solution in embodiment 2 composition to be adjusted to: 30ng/mlSA-AP, 1pmol/mlHPVDNA probe, 3 × SSC, 50mMZnCl
2, 50mMMgCl
2, 0.4%TritonX-100,0.06%CPAM and 8% PEG 8000, surplus is water.Other conditions are constant.
Embodiment 6:
With embodiment 1, difference is the composition of the aftertreatment fluid in embodiment 1 to be adjusted to: PH9.5,0.1mol/LNaCl, 0.1mol/LTris-HCl, 50mMMgCl
2and 0.5% ndodecyl glucoside, surplus is water.Other conditions are constant.
Embodiment 7:
With embodiment 1, difference is the composition of the aftertreatment fluid in embodiment 1 to be adjusted to: PH9.5,0.1mol/LNaCl, 0.1mol/LTris-HCl, 50mMMgCl
2and 2% ndodecyl glucoside, surplus is water.Other conditions are constant.
Embodiment 8:
With embodiment 1, difference is the composition of the aftertreatment fluid in embodiment 1 to be adjusted to: PH9.0,0.1mol/LNaCl, 0.1mol/LTris-HCl, 50mMMgCl
2and 3% n-hexadecyl glucoside, surplus is water.Other conditions are constant.
Embodiment 9:
With embodiment 1, difference is the composition of the aftertreatment fluid in embodiment 1 to be adjusted to: PH10.0,0.1mol/LNaCl, 0.1mol/LTris-HCl, 50mMMgCl
2and 0.2% n-Octylglucoside, surplus is water.Other conditions are constant.
Embodiment 10:
With embodiment 1, difference is the solid support in embodiment 1 to replace with nylon membrane, is adjusted to by the composition of pretreatment liquid simultaneously: PH9.0,0.1mol/LNaCl and 0.1mol/L Veronal sodium-hydrochloric acid, and surplus is water.Other conditions are constant.
Below for detecting the Application Example of the test kit of human papillomavirus
Embodiment 11 test kit of embodiment 1 detects clinical sample
Clinical sample in the present embodiment is the detachment of cervix cell-preservation liquid sample of ASCUS or more patient from clinical cytology check result, and totally 6 examples, are numbered L1, L2, L3, L4, L5 and L6 respectively.
(1) extraction of target DNA
" blood tissues cellular genome extracts test kit " that utilize sky root biochemical reagents (Beijing) company to provide extracts the nucleic acid extraction liquid that may comprise target DNA in above-mentioned 6 routine clinical detachment of cervix cell-preservation liquid samples.
(2) target DNA fixing at solid support surface
2.1 point samples are fixed: get the nucleic acid extraction liquid of clinical sample L1, L2, L3, L4, L5 and L6 of obtaining in the step () of 1 μ L with the pipettor of 2.5 μ l flow processs respectively, salmon sperm dna solution (negative control) that HPV16 type plasmid standard (positive control) that 1ul concentration is 1pg/ul and 1ul concentration are 1pg/ μ l puts on nitrocellulose filter respectively, room temperature is dried.
The layout viewing of target DNA on nitrocellulose filter surface is all as shown in the table:
Table 5
| L1 | L2 | L3 | Positive control |
| L4 | L5 | L6 | Negative control |
2.2 sex change: the nitrocellulose filter putting sample is immersed in 10min in sex change liquid, object is that genomic double-strand is become strand, to hybridize with DNA probe below.
2.3 neutralizations: the nitrocellulose filter after sex change is immersed 10min in neutralizer again.
2.4 dry: from neutralizer, take out nitrocellulose filter, then suck excessive moisture with filter paper, dry after 1 hour for subsequent use for 80 DEG C.
(3) pre-treatment
The nitrocellulose filter that surface in step (two) is fixed with target DNA is immersed in 30min in the pretreatment fluid of 37 DEG C, and period is stirred once.
(4) pre-treatment
Utilize pretreatment liquid to wash the surface 3 times of pretreated nitrocellulose filter in step (three), wash 5min at every turn.
(5) single step reaction
The nitrocellulose filter of pre-treatment in step (four) is placed in hybridization solution, in temperature 37 DEG C of water-baths, reacts 10min.
(6) aftertreatment
Utilize aftertreatment fluid to wash the surface 3 times of the nitrocellulose filter in the above-mentioned steps (five) after single step reaction, wash 2min at every turn.
(7) color reaction
7.1 add substrate 1 and each 33 μ l of substrate 2 respectively in colorbuffer, are mixed with the chromophoric solution that 10ml component is as follows: PH9.5,0.1mol/LTris-HCl, 0.1mol/LNaCl, 50mMMgCl
2, 0.33mg/mlNBT and 0.17mg/mlBCIP, surplus is water.
7.2 by above-mentioned in fruit step (six) reprocessed nitrocellulose filter be immersed in chromophoric solution, colour developing 8min, observe colour developing result.
The colour developing result of the present embodiment as shown in Figure 1.
Embodiment 12
With embodiment 11, difference is to detect clinical sample with the test kit of embodiment 2, and other conditions remain unchanged.
The colour developing result of the present embodiment as shown in Figure 2.
Embodiment 13
With embodiment 11, difference is to detect clinical sample with the test kit of embodiment 3, and other conditions remain unchanged.
The colour developing result of the present embodiment as shown in Figure 3.
Embodiment 14
With embodiment 11, difference is to detect clinical sample with the test kit of embodiment 4, and other conditions remain unchanged.
The colour developing result of the present embodiment as shown in Figure 4.
Embodiment 15
With embodiment 11, difference is to detect clinical sample with the test kit of embodiment 5, and other conditions remain unchanged.
The colour developing result of the present embodiment as shown in Figure 5.
Embodiment 16
With embodiment 11, difference is to detect clinical sample with the test kit of embodiment 6, and other conditions remain unchanged.
The colour developing result of the present embodiment as shown in Figure 6.
Embodiment 17
With embodiment 11, difference is to detect clinical sample with the test kit of embodiment 7, and other conditions remain unchanged.
The colour developing result of the present embodiment as shown in Figure 7.
Embodiment 18
With embodiment 11, difference is to detect clinical sample with the test kit of embodiment 8, and other conditions remain unchanged.
The colour developing result of the present embodiment as shown in Figure 8.
Embodiment 19
With embodiment 11, difference is to detect clinical sample with the test kit of embodiment 9, and other conditions remain unchanged.
The colour developing result of the present embodiment as shown in Figure 9.
Embodiment 20
With embodiment 11, difference is to detect clinical sample with the test kit of embodiment 10, and other conditions remain unchanged.
The colour developing result of the present embodiment as shown in Figure 10.
Comparative example 1
This comparative example uses the commercialization of German Qiagen company, commercially available HCII high-risk HPV is caught hybridization kit and detected the detachment of cervix cell-preservation liquid sample being ASCUS or more patient from clinical cytology check result in embodiment 8, totally 6 examples, be numbered L1 respectively, L2, L3, L4, L5 and L6.Detected result shows, L1, L2, L4 sample is positive, and L3, L5 and L6 sample is negative.
From Fig. 1 ~ 10, this test kit can detect accurately to the HPV virus in clinical sample, wherein clinical sample L2 is strong positive, L1 and L4 is the positive of medium tenacity, illustrate in clinical L1, L2 and L4 sample containing SEQIDNOs:1,4,7,10,12,15,18,21,24,27,30, one or more high-risk HPVs viruses in 13 kinds of different genotype of 33 and 36.Clinical sample L3, L5 and L6 are detected as feminine gender.There is not any non-specific result in the target DNA that this test kit detects in clinical sample, completely the same with the HCII detected result in comparative example 1.Positive control sample (HPV16 type plasmid) is significantly positive, negative control (salmon sperm dna) is negative, Quality Control result shows further, this test kit not only clinical sample testing process is completely normal, and the colour developing background of solid support is very low, contribute to the interpretation of result, significantly improve the specificity of detection.
In sum, alkali phosphatase enzyme mark Streptavidin and HPVDNA probe is directly contained in the hybridization solution of this test kit, target DNA and biotin labeled DNA probe carry out in the process of molecular hybridization on the surface of solid support, synchronously achieve the process that alkali phosphatase enzyme mark Streptavidin is combined with vitamin H, pass through pretreatment fluid simultaneously, pretreatment liquid and aftertreatment fluid carry out coordinated process to the solid support surface before and after single step reaction, not only eliminate in existing molecular hybridization the step needing to carry out separately integrated enzyme reaction after hybridization, significantly improve the detection efficiency of target DNA in sample, and positive test symbol is very clear, detection background is extremely low, to the point of each target HPVDNA, except to should type HPVDNA probe colour developing except, the DNA probe of other type does not all develop the color, the detection indicating test kit has the feature of significantly high special and low background, effectively prevent the false positive of detected result.
It should be noted that above-described embodiment only for explaining the present invention, not forming any limitation of the invention.By referring to exemplary embodiments, invention has been described, but to should be understood to word wherein used be descriptive and explanatory vocabulary, instead of limited vocabulary.Can modify the present invention by the scope being defined in the claims in the present invention, and the present invention be revised not deviating from scope and spirit of the present invention.Although the present invention wherein described relates to specific method, material and embodiment, and do not mean that the present invention is limited to particular case disclosed in it, on the contrary, easily extensible of the present invention is to other all methods and applications with identical function.
Claims (15)
1. detect a test kit for human papillomavirus, it comprises following component: pretreatment fluid, pretreatment liquid, hybridization solution and aftertreatment fluid; Wherein, described hybridization solution comprises alkali phosphatase enzyme mark Streptavidin and the biotin labeled DNA probe for detecting human papillomavirus.
2. test kit according to claim 1, is characterized in that, the concentration of described alkali phosphatase enzyme mark Streptavidin in hybridization solution is 5ng/ml ~ 50ng/ml, is preferably 10ng/ml ~ 40ng/ml, is more preferably 15ng/ml ~ 30ng/ml.
3. test kit according to claim 1 and 2, is characterized in that, the concentration of described biotin labeled DNA probe in hybridization solution is 0.1 ~ 5pmol/ml, is preferably 0.2 ~ 2pmol/ml, is more preferably 0.25 ~ 1pmol/ml.
4. detect a test kit for human papillomavirus, it comprises following component: pretreatment fluid, pretreatment liquid, hybridization solution, aftertreatment fluid, alkali phosphatase enzyme mark Streptavidin and the biotin labeled DNA probe solution for detecting human papillomavirus.
5. according to the test kit in Claims 1 to 4 described in any one, it is characterized in that, described pretreatment fluid comprises encapsulant and nonionic surface active agent; Wherein, described encapsulant is casein and/or bovine serum albumin, and described nonionic surface active agent is tween and/or Triton; In described pretreatment fluid, encapsulant accounts for 1 ~ 5% (w/v) of pretreatment fluid, and nonionic surface active agent accounts for 0.05% ~ 1% (v/v) of pretreatment fluid; Preferably, encapsulant accounts for 2 ~ 4% (w/v) of pretreatment fluid, and nonionic surface active agent accounts for 0.05% ~ 0.2% (v/v) of pretreatment fluid.
6. according to the test kit in Claims 1 to 5 described in any one, it is characterized in that, described pretreatment fluid also comprises anionic dispersing agent and anion-polyacrylamide; Wherein, described anionic dispersing agent is selected from sulfonated lignin, preferred sodium lignosulfonate; In described pretreatment fluid, anionic dispersing agent accounts for 0.05% ~ 0.2% (w/v) of pretreatment fluid, and anion-polyacrylamide accounts for the 0.02-0.5% (w/v) of pretreatment fluid; Preferably, anionic dispersing agent accounts for 0.1% ~ 0.2% (w/v) of pretreatment fluid, and anion-polyacrylamide accounts for the 0.1-0.3% (w/v) of pretreatment fluid.
7. according to the test kit in claim 1 ~ 6 described in any one, it is characterized in that, the PH of described pretreatment fluid is 7.0 ~ 8.0.
8. according to the test kit in claim 1 ~ 7 described in any one, it is characterized in that, described pretreatment liquid is the buffered soln of PH7.5 ~ 9.0, and described buffered soln is selected from Veronal sodium-hydrochloric acid buffer solution, Tris-hydrochloric acid buffer solution, Glycine-NaOH buffered soln and boric acid-borax buffer solution.
9. according to the test kit in claim 1 ~ 8 described in any one, it is characterized in that, described aftertreatment fluid comprises C
8~ C
18alkyl-glucoside, preferred C
9~ C
13alkyl-glucoside; Described alkyl-glucoside accounts for 0.1 ~ 5% (w/v) of aftertreatment fluid, preferably 0.5 ~ 2% (w/v).
10. according to the test kit in claim 1 ~ 9 described in any one, it is characterized in that, the PH of described aftertreatment fluid is 9.0 ~ 10.0.
11., according to the test kit in claim 1 ~ 10 described in any one, is characterized in that, described hybridization solution also comprises zine ion, magnesium ion, nonionic surface active agent and cation type polymer; Wherein, described nonionic surface active agent is tween and/or Triton, and described cation type polymer is selected from cationic polyacrylamide, poly-lysine and polymerize aluminum chloride.
12. according to the test kit in claim 1 ~ 11 described in any one, it is characterized in that, in described hybridization solution, zine ion is 0.001 ~ 0.1mol/L, magnesium ion is 0.001 ~ 0.1mol/L, nonionic surface active agent accounts for 0.1 ~ 0.5% (v/v) of hybridization solution, and cation type polymer accounts for 0.01 ~ 0.1% (w/v) of hybridization solution; Preferably, zine ion is 0.004 ~ 0.05mol/L, magnesium ion is 0.004 ~ 0.05mol/L, and nonionic surface active agent accounts for 0.2 ~ 0.4% (v/v) of hybridization solution, and cation type polymer accounts for 0.02 ~ 0.06% (w/v) of hybridization solution.
13., according to the test kit in claim 1 ~ 12 described in any one, is characterized in that, described biotin labeled DNA probe 5 ' holds biotin labeled oligonucleotide probe.
14., according to the test kit in claim 1 ~ 13 described in any one, is characterized in that, described biotin labeled DNA probe comprises:
For detecting the genotypic DNA probe of HPV16, its nucleotide sequence is selected from SEQIDNOs:1 ~ 3;
For detecting the genotypic DNA probe of HPV18, its nucleotide sequence is selected from SEQIDNOs:4 ~ 6;
For detecting the genotypic DNA probe of HPV31, its nucleotide sequence is selected from SEQIDNOs:7 ~ 9;
For detecting the genotypic DNA probe of HPV33, its nucleotide sequence is selected from SEQIDNOs:10 ~ 11;
For detecting the genotypic DNA probe of HPV35, its nucleotide sequence is selected from SEQIDNOs:12 ~ 14;
For detecting the genotypic DNA probe of HPV39, its nucleotide sequence is selected from SEQIDNOs:15 ~ 17;
For detecting the genotypic DNA probe of HPV45, its nucleotide sequence is selected from SEQIDNOs:18 ~ 20;
For detecting the genotypic DNA probe of HPV51, its nucleotide sequence is selected from SEQIDNOs:21 ~ 23;
For detecting the genotypic DNA probe of HPV52, its nucleotide sequence is selected from SEQIDNOs:24 ~ 26;
For detecting the genotypic DNA probe of HPV56, its nucleotide sequence is selected from SEQIDNOs:27 ~ 29;
For detecting the genotypic DNA probe of HPV58, its nucleotide sequence is selected from SEQIDNOs:30 ~ 32;
For detecting the genotypic DNA probe of HPV59, its nucleotide sequence is selected from SEQIDNOs:33 ~ 35;
For detecting the genotypic DNA probe of HPV68, its nucleotide sequence is selected from SEQIDNOs:36 ~ 38.
15., according to the test kit in claim 1 ~ 13 described in any one, is characterized in that, described biotin labeled DNA probe comprises:
For detecting the genotypic DNA probe of HPV16, its nucleotide sequence is as shown in SEQIDNO:1;
For detecting the genotypic DNA probe of HPV18, its nucleotide sequence is as shown in SEQIDNO:4;
For detecting the genotypic DNA probe of HPV31, its nucleotide sequence is as shown in SEQIDNO:7;
For detecting the genotypic DNA probe of HPV33, its nucleotide sequence is as shown in SEQIDNO:10;
For detecting the genotypic DNA probe of HPV35, its nucleotide sequence is as shown in SEQIDNO:12;
For detecting the genotypic DNA probe of HPV39, its nucleotide sequence is as shown in SEQIDNO:15;
For detecting the genotypic DNA probe of HPV45, its nucleotide sequence is as shown in SEQIDNO:18;
For detecting the genotypic DNA probe of HPV51, its nucleotide sequence is as shown in SEQIDNO:21;
For detecting the genotypic DNA probe of HPV52, its nucleotide sequence is as shown in SEQIDNO:24;
For detecting the genotypic DNA probe of HPV56, its nucleotide sequence is as shown in SEQIDNO:27;
For detecting the genotypic DNA probe of HPV58, its nucleotide sequence is as shown in SEQIDNO:30;
For detecting the genotypic DNA probe of HPV59, its nucleotide sequence is as shown in SEQIDNO:33;
For detecting the genotypic DNA probe of HPV68, its nucleotide sequence is as shown in SEQIDNO:36.
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| CN201410415013.5A CN105400901A (en) | 2014-08-20 | 2014-08-20 | Kit for detecting human papilloma virus and application of kit |
| PCT/CN2015/087681 WO2016026453A2 (en) | 2014-08-20 | 2015-08-20 | Test kit and method for testing target nucleic acid in sample |
| US15/504,764 US10494665B2 (en) | 2014-08-20 | 2015-08-20 | Test kit and method for testing target nucleic acid in sample |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106645717A (en) * | 2017-01-04 | 2017-05-10 | 广州华弘生物科技有限公司 | Kit for rapidly detecting chlamydia trachomatis and gonorrhea and application of kit |
| CN106680506A (en) * | 2017-01-04 | 2017-05-17 | 广州华弘生物科技有限公司 | ProANP (pro-atrial natriuretic peptide) rapid test paper and related application |
| CN106771192A (en) * | 2017-01-04 | 2017-05-31 | 广州华弘生物科技有限公司 | A kind of quick diagnosis reagent kit of monocytosis,mononucleosis |
| CN108676797A (en) * | 2018-06-07 | 2018-10-19 | 迈基诺（重庆）基因科技有限责任公司 | Reagent set for detecting human papilloma virus and method |
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