CN109112184A - A kind of HPV genetic chip and its preparation method and application - Google Patents

A kind of HPV genetic chip and its preparation method and application Download PDF

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CN109112184A
CN109112184A CN201810946620.2A CN201810946620A CN109112184A CN 109112184 A CN109112184 A CN 109112184A CN 201810946620 A CN201810946620 A CN 201810946620A CN 109112184 A CN109112184 A CN 109112184A
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陈腾祥
江银辉
徐澍
兰金芝
禹文峰
张金娟
王欢
肖俊
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Guizhou Medical University
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Abstract

The invention discloses a kind of HPV genetic chips, the genetic chip individually or simultaneously can accurately detect one or more HPV hypotypes, screen and parting, and have human genome beta globin and HPV primer amplification Quality Control point to monitor entire detection process, which can have highly sensitive and specificity；The invention also discloses the preparation methods of the HPV genetic chip, utilize the probe of PCR revert dot blot hybridization screening high specificity, by the deposition probe concentration optimization to HPV genetic chip, the quality and efficiency of hybridization are improved, and further demonstrates the genetic chip of the condition of optimization with clinical sample.HPV genetic chip of the invention has preferable potential applicability in clinical practice.

Description

A kind of HPV genetic chip and its preparation method and application
Technical field
The present invention relates to field of biomedicine technology, and in particular to a kind of HPV genetic chip and its preparation method and application.
Background technique
Cervical carcinoma is common one of the malignant tumour of women's diseases, and disease incidence accounts for global female tumor the 4th.According to close Data statistics is found over year, the increasingly rejuvenation of China's cervical cancer patient, and its morbidity and mortality is remained at compared with Gao Shui It is flat, therefore the early screening of precancerous lesions of uterine cervix, there is great role to the prevention of cervical carcinoma, so as to cause researcher to uterine neck The high attention of cancer early screening.Nineteen eighty-three, Germany scientist Hausen propose the theory that HPV is the cervical carcinoma cause of disease, this Many researchers both at home and abroad have done the largely relationship research about cervical carcinoma and HPV infection afterwards, and achieve a large amount of harvest. Mass data is studies have shown that high-risk HPV persistent infection is the necessary factor for leading to uterine neck carcinogenesis.Existing research result is aobvious Show, about 30 kinds of high-risk HPVs can cause cervical lesions, especially HPV16 hypotype and HPV18 hypotype.
HPV is a kind of nonencapsulated DNA virus of double-stranded circular, belongs to the nipple of papovavirus (papovaviridae) Tumor vacuolating virus A belongs to, and genome is about 8000bp.It is 3 regions according to HPV function division, is long control area respectively (LCR), early transcription area (area E) and late transcription area (area L).The area E includes E1~E7, is responsible for coding viral associated proteins；The area L Including L1 and L2, the major capsid protein (L1) and secondary capsid protein (L2), L1 for encoding virus account for about the 80% of capsid protein, Sequence is highly conserved, and L2 content is less, makes a variation more；Contain the replication orgin of genome and the regulation member that expression is required in the area LCR Part participates in the transcription and duplication of regulation virus.
Since the region HPV L1 is one section of sequence the most conservative in HPV genome, can be read according to viral open The homology difference of frame gene coding nucleotide sequence to carry out parting to virus, and the height of cervical carcinoma risk is caused according to HPV Be classified as high-risk HPV (HRHPV) and low risk HPV (LRHPV), high-risk HPV mainly has with Types Below, as HPV16, 18, the types such as 26,31,33,35,39,45,51,52,53,56,58,59,66,67,68,69,73；Low risk HPV includes The types such as HPV6,11,40,42,43,44,54,55,57,61,71,81,83, wherein low risk HPV mainly results in condyloma class disease Become, and high-risk HPV may cause the generation of cervical carcinoma.A large number of studies show that HPV infection and cervical carcinogenesis have close pass System.Since HPV can hole up in the body 10 years even for more time, and do not occur any clinical symptoms, people are easy to ignore HPV Detection, the process for developing to cervical carcinoma which results in precancerous lesions of uterine cervix are extremely very long.Therefore, clinically HPV is detected As a part of particularly significant of routine screening, HPV early detective rate is improved by early detection, to improve uterine neck precancerosis The cure rate of change, the final generation for preventing cervical carcinoma, reduces the disease incidence of cervical carcinoma.HPV detection and parting cervical carcinoma screening, Early prevention, clinical diagnosis and anaphase etc. have highly important value.
Not yet there is the method for HPV in vitro culture at present, HPV detection method is mainly cytology and molecular Biological Detection Method.Traditional cytology detection method for example conventional smear detection accuracy rate, sensitivity and repetitive rate it is low, and false negative rate and False positive rate is high, cannot be to HPV parting.As people are to HPV detection and the further investigation of classifying method, HPV DNA/RNA Detection increasingly shows the effect of cervical lesions early screening, more sensitive, accurate compared to traditional cytology detection method.Mesh Preceding HPV molecular biological testing can be divided into 3 classes: nucleic acid hybridization, signal amplification technique and nucleic acid amplification technologies.Fluorescence Hybridization in situ can position, and false positive rate is low, but its detection sensitivity is low, cumbersome, time-consuming effort, be not appropriate for logical greatly Measure the screening of clinical sample.Hybrid capture II technology (HC II) approval in 1999 is detected for HPV, and the technology is by the U.S. Digene company releases, and is that earliest obtain Food and Drug Administration (FDA) ratifies, the most widely used HPV detection in the world Technology, although HC II is detected, specificity is good and high sensitivity, HC II are detected in the presence of not can be carried out parting, and expense cost is high, And the defects of without interior Quality Control.U.S. FDA in 2011 has approved a line primary dcreening operation of Cobas 4800HPV technology, which can be qualitative The HPV of 14 kinds of high-risk-types is detected, and has beta globin as interior Quality Control, but it detects HPV type and is only limitted to this 14 kinds of high-risk classes Type.In recent years, the application of HPV detection method clinically still has some defects, such as cannot carry out one to more kinds of hypotypes of HPV Secondary property detection, operating process is cumbersome, and testing cost is high, and takes time and effort.Although many companies are about cervical carcinoma screening development Many HPV detection kits are gone out, but how much these kits have some defects, so ensuring that HPV detection is comprehensive same When, guarantee that the quality of HPV detection kit is even more important.Since HPV genotype has different potential carcinogenicities, so to facing The suspicious sample of bed, which carries out specific parting, to be helped accurately to analyze and treat.Therefore, a kind of novel HPV genetic chip is established, Detection rapidly and accurately can be carried out to HPV and parting is particularly significant.
Biochip (biochip or bioarray) is integrated in discontinuous analytic process solid using shrink technology The micro-analysis system of phase carrier can be realized the high throughput detection to sample component.Genetic chip and DNA microarray technology are all It belongs to biochip, is set on planar substrate carrier using array manner, multiple genes in parallel processing biological sample The microprocessing unit of information, biochip have the feature of micromation and a large amount of clinical samples of parallel processing.In view of genetic chip The advantage of technology realizes a large amount of clinical sample Parallel testings and high throughput analysis using biochip technology.Genetic chip work Principle be by specific probe point on film, prepare genetic chip, with by specific markers substance markers DNA cloning product carry out it is miscellaneous It hands over and develops the color, be scanned analysis detection sample HPV type.Biochip technology is current clinical diagnosis with widest skill Art, it plays the role of critical in HPV detection method, brings Gospel for the diagnosis of cervical cancer patient.Base in recent years Because chip technology is as low consumption, high sensitivity, a high-flux detection method, medical research neck has been widely used in it Domain, detection and parting to HPV have very wide potential applicability in clinical practice, therefore, research and develop a kind of novel HPV detection method Detection and accurate parting to HPV have very important meaning.
Summary of the invention
It is an object of the invention to prepare a kind of novel HPV genetic chip, the gene by using biochip technology Chip can disposably detect 30 kinds of HPV hypotypes, wherein covering 19 kinds of high-risk HPVs and 11 kinds of low risk HPV, and have people's gene Group beta globin monitors entire detection process as Quality Control point, which is had highly sensitive and special Property, and can be with clinically.
In order to achieve the above objectives, the present invention adopts the following technical scheme:
A kind of HPV genetic chip includes more than one HPV hypotype test point and two on film using Biodyne C film as carrier A positive quality control point, two of them positive quality control point detect human genome beta globin genes and HPV primer amplification respectively.
Detection human genome beta globin genes in HPV genetic chip above-mentioned, in two positive quality control points In probe sequence such as SEQ ID NO:1~SEQ ID NO:5 it is any shown in, and in 5 ' end label amino, i.e. PC (Positive Control) positive quality control point；It detects any in the probe sequence such as SEQ ID NO:6~SEQ ID NO:10 of HPV primer amplification It is shown, and in 5 ' end label amino, i.e. QC (Quality Control) positive quality control point.
In HPV genetic chip above-mentioned, the HPV hypotype test point includes high-risk HPV and/or low risk HPV detection Point.
In HPV genetic chip above-mentioned, the high-risk HPV include HPV16,18,26,31,33,35,39,45,51, 52, one or more of 53,56,58,59,66,67,68,69 and 73 hypotypes；The low risk HPV include HPV6,11,34, 40, one or more of 42,43,44,54,55,57 and 62 hypotypes.
In HPV genetic chip above-mentioned, the probe sequence of the HPV6 hypotype such as SEQ ID NO:11~SEQ ID NO: It is any shown in 15；It is any shown in the probe sequence such as SEQ ID NO:16~SEQ ID NO:20 of the HPV11 hypotype；Institute It states any shown in the probe sequence such as SEQ ID NO:21~SEQ ID NO:25 of HPV16 hypotype；The spy of the HPV18 hypotype In needle sequence such as SEQ ID NO:26~SEQ ID NO:30 it is any shown in；The probe sequence of the HPV26 hypotype such as SEQ ID It is any shown in NO:31~SEQ ID NO:35；The probe sequence of the HPV31 hypotype such as SEQ ID NO:36~SEQ ID It is any shown in NO:40；The probe sequence of the HPV33 hypotype such as any institute in SEQ ID NO:41~SEQ ID NO:45 Show；It is any shown in the probe sequence such as SEQ ID NO:46~SEQ ID NO:50 of the HPV34 hypotype；The HPV35 is sub- It is any shown in the probe sequence such as SEQ ID NO:51~SEQ ID NO:55 of type；The probe sequence of the HPV39 hypotype is such as It is any shown in SEQ ID NO:56~SEQ ID NO:60；The probe sequence of the HPV40 hypotype such as SEQ ID NO:61~ It is any shown in SEQ ID NO:65；In the probe sequence such as SEQ ID NO:66~SEQ ID NO:70 of the HPV42 hypotype Shown in any；It is any shown in the probe sequence such as SEQ ID NO:71~SEQ ID NO:75 of the HPV43 hypotype；It is described It is any shown in the probe sequence such as SEQ ID NO:76~SEQ ID NO:80 of HPV44 hypotype；The probe of the HPV45 hypotype In sequence such as SEQ ID NO:81~SEQ ID NO:85 it is any shown in；The probe sequence of the HPV51 hypotype such as SEQ ID It is any shown in NO:86~SEQ ID NO:90；The probe sequence of the HPV52 hypotype such as SEQ ID NO:91~SEQ ID It is any shown in NO:95；The probe sequence of the HPV53 hypotype such as any institute in SEQ ID NO:96~SEQ ID NO:100 Show；The probe sequence of the HPV54 hypotype is as shown in SEQ ID NO:101~SEQ ID NO:105；The HPV55 hypotype In probe sequence such as SEQ ID NO:106~SEQ ID NO:110 it is any shown in；The probe sequence such as SEQ of the HPV56 hypotype It is any shown in ID NO:111~SEQ ID NO:115；The probe sequence of the HPV57 hypotype such as SEQ ID NO:116~ It is any shown in SEQ ID NO:120；The probe sequence of the HPV58 hypotype such as SEQ ID NO:121~SEQ ID NO:125 In it is any shown in；It is any shown in the probe sequence such as SEQ ID NO:126~SEQ ID NO:130 of the HPV59 hypotype；Institute It states any shown in the probe sequence such as SEQ ID NO:131~SEQ ID NO:135 of HPV62 hypotype；The HPV66 hypotype In probe sequence such as SEQ ID NO:136~SEQ ID NO:140 it is any shown in；The probe sequence such as SEQ of the HPV67 hypotype It is any shown in ID NO:141~SEQ ID NO:145；The probe sequence of the HPV68 hypotype such as SEQ ID NO:146~ It is any shown in SEQ ID NO:150；The probe sequence of the HPV69 hypotype such as SEQ ID NO:151~SEQ ID NO:155 In it is any shown in；The probe sequence of the HPV73 hypotype is as shown in SEQ ID NO:156~SEQ ID NO:160；And 5 ' End marks amino.
The preparation method of HPV genetic chip above-mentioned, comprising the following steps: (1) preparation of HPV examination criteria product；(2) The amplification of the area HPV L1 DNA fragmentation target；(3) HPV primer and probe design and synthesis；(4) preparation of HPV genetic chip.
In the preparation method of HPV genetic chip above-mentioned, in the amplification of the area step (2) HPV L1 DNA fragmentation target 4 forward primers used are det-1A, det-1B, det-1C and det-1D, the wherein sequence of det-1A such as SEQ ID NO: It is any shown in 161~SEQ ID NO:165, it is any in the sequence such as SEQ ID NO:166~SEQ ID NO:170 of det-1B It is shown, any shown in the sequence such as SEQ ID NO:171~SEQ ID NO:175 of det-1C, the sequence of det-1D such as SEQ In ID NO:176~SEQ ID NO:180 it is any shown in, 4 reverse primers are GP-1A, GP-1B, GP-1C and GP-1D, wherein It is any shown in the sequence such as SEQ ID NO:181~SEQ ID NO:185 of GP-1A, the sequence of GP-1B such as SEQ ID NO: It is any shown in 186~SEQ ID NO:190, it is any in the sequence such as SEQ ID NO:191~SEQ ID NO:195 of GP-1C It is shown, it is any shown in the sequence such as SEQ ID NO:196~SEQ ID NO:200 of GP-1D, wherein 45 ' ends of reverse primers Biotin labeling is used at end, and human genome beta globin genes amplimer is added and expands together, HPV amplimer and people's base Because the concentration ratio of group beta globin genes amplimer is 1:6~1:4, the human genome beta globin genes amplimer PC04 With GH20 sequence as shown in SEQ ID NO:201 and SEQ ID NO:202, and the end PC045 ' biotin labeling.
In the preparation method of HPV genetic chip above-mentioned, the preparation step of step (4) the HPV genetic chip is as follows:
(4.1) Biodyne C film is cut into film forming item；
(4.2) the film item of cutting is placed after being impregnated in 0.1mol/L HCl solution, transfer membrane item is into 20%EDC solution Activation, is simply rinsed, room temperature is dried with aseptic double-distilled water；
(4.3) Na of 0.1mol/L pH=8.4 is used2CO3/NaHCO3Buffer is by the HPV probe of amino labeled, amino mark The HPV primer amplification probe dilution of beta globin genes probe in the human genome of note and amino labeled, takes probe dilution liquid to have It is fixed on to sequence on Biodyne C film, makes the carboxyl Covalent bonding together activated on amino labeled probe and Biodyne C film；
(4.4) after Biodyne C film item air-dries, 0.1mol/L NaOH is taken to neutralize, sterile water wash is used later, in room Warm air dried overnight is up to HPV genetic chip.
In the preparation method of HPV genetic chip above-mentioned, the preparation step of step (4) the HPV genetic chip is as follows:
(4.1) Biodyne C film is cut into film forming item；
(4.2) the film item of cutting is placed after impregnating 1min in 0.1mol/L HCl solution, transfer membrane item is molten to 20%EDC 15min is activated in liquid, simply rinses 30s with aseptic double-distilled water, room temperature dries 1h；
(4.3) Na of 0.1mol/L pH=8.4 is used2CO3/NaHCO3Buffering, by the HPV probe and amino mark of amino labeled Beta globin genes probe dilution in the human genome of note to 0.5~8pmol/L and amino labeled HPV primer amplification probe It is diluted to 0.05pmol/L, takes each 3 μ L of probe dilution liquid to be fixed on Biodyne C film in an orderly manner, makes amino labeled probe With the carboxyl Covalent bonding together activated on Biodyne C film；
(4.4) it after Biodyne C film item air-dries 15min, takes in 0.1mol/L NaOH and 10min, uses sterile water later Cleaning 4 times, it is every all over 30s, it stays overnight in being air-dried at room temperature up to HPV genetic chip.
Application of the HPV genetic chip above-mentioned in HPV detection and parting.
Application of the HPV genetic chip above-mentioned as cervical disease or condyloma class disorder in screening or forecasting tool.
The application of HPV genetic chip above-mentioned, includes the following steps:
(S1) PCR reverse dot blot hybridization:
(S1.1) prepared by sample to be tested: extracting sample DNA and is expanded, obtains PCR product；
(S1.2) hybridization solution I and hybridization solution II are placed be preheated in water-bath 45 DEG C it is spare；
(S1.3) setting constant temperature shaker temperature is 45 DEG C in advance, the most suitable hybridization temperature for making the temperature in shaking table reach 45 DEG C Degree；
(S1.4) by PCR product obtained by step (S1.1) it is denatured by boiling after, be immediately placed on and be incubated on ice；
(S1.5) it is put into centrifuge tube with aseptic nipper tweezer HPV gene chip film item, preheating hybridization solution I is added, it is spare；
(S1.6) PCR product of step (S1.4) resulting denaturation is added to the centrifugation that film item is placed with obtained by step (S1.5) Guan Zhong hybridizes in 100rpm, 45 DEG C of constant-temperature tables；
(S1.7) hybridization solution I is abandoned, film item is taken to be put into centrifuge tube, 10mL is added and preheats hybridization solution II, in 100rpm, 45 DEG C Film is washed in constant-temperature table；
(S1.8) hybridization solution II is abandoned, centrifuge tube is inverted on blotting paper, blots tube bottom liquid, 20mL HRP dilution is added Liquid, at room temperature in 50rpm being protected from light 10min on oscillation shaking table；
(S1.9) it abandons and combines liquid, centrifuge tube is inverted on blotting paper, blot tube bottom liquid, I Room of 20mL hybridization solution is added Temperature 0 under in oscillation shaking table on wash film；
(S1.10) hybridization solution I is abandoned, blotting paper is shelved on desk, centrifuge tube is upside down in above, blots tube bottom liquid 20mL TMB developing solution, reacting at normal temperature without light is added in body；After colour developing, developing solution is abandoned immediately, and the sterile distillation of 20mL is added Water takes out after washing film on shaking table, sucks film surface moisture with blotting paper and dry in room temperature；
(S2) result interpretation: in scanning chip on scanner after film is dry, to the positive hybridization signal on HPV genetic chip Carry out signal strength collection analysis.
The application of HPV genetic chip above-mentioned, includes the following steps:
(S1) PCR reverse dot blot hybridization:
(S1.1) prepared by sample to be tested: extracting sample DNA and using 4 forward primers described in claim 7 and 4 Reverse primer and human genome beta globin genes amplimer are expanded, HPV amplimer and human genome beta globin The concentration ratio of gene magnification primer is 1:6~1:4, obtains PCR product；
(S1.2) hybridization solution I and hybridization solution II are placed be preheated in water-bath 45 DEG C it is spare, wherein I ingredient of hybridization solution is 2 × SSC and 0.1%SDS, II ingredient of hybridization solution are 0.5 × SSC and 0.1%SDS；
(S1.3) setting constant temperature shaker temperature is 45 DEG C in advance, the most suitable hybridization temperature for making the temperature in shaking table reach 45 DEG C Degree；
(S1.4) by PCR product obtained by step (S1.1) after 98 DEG C are denatured by boiling, be immediately placed on be incubated on ice 2min with On；
(S1.5) it is put into centrifuge tube with aseptic nipper tweezer HPV gene chip film item, 5mL is added and preheats hybridization solution I, it is standby With；
(S1.6) PCR product of step (S1.4) resulting denaturation is added to the centrifugation that film item is placed with obtained by step (S1.5) Guan Zhong hybridizes 15min~1h in 100rpm, 45 DEG C of constant-temperature table；
(S1.7) hybridization solution I is abandoned, film item is taken to be put into centrifuge tube, 10mL is added and preheats hybridization solution II, in 100rpm, 45 DEG C Constant-temperature table in wash 10~30min of film, wash in total twice；
(S1.8) hybridization solution II is abandoned, centrifuge tube is inverted on blotting paper, blots tube bottom liquid, 20mL HRP dilution is added Liquid, wherein HRP dilution be volume ratio HRP: abandon hybridization solution I=1:2000~8000 now match, at room temperature in oscillation shaking table on The revolving speed of 50rpm is protected from light 10min；
(S1.9) it abandons and combines liquid, centrifuge tube is inverted on blotting paper, blot tube bottom liquid, I Room of 20mL hybridization solution is added In washing film twice on oscillation shaking table under temperature, film 2min is washed every time；
(S1.10) hybridization solution I is abandoned, blotting paper is shelved on desk, centrifuge tube is upside down in above, blots tube bottom liquid 20mL TMB developing solution is added in body, and wherein TMB developing solution is the mixing of 20 × TMB-A:1 of volume ratio × TMB-B=1:30~100 It is made, reacting at normal temperature without light 5min, after colour developing, abandons developing solution immediately, 20mL sterile distilled water is added, washes film on shaking table It is taken out after 3min, repetition is washed film one time, is sucked film surface moisture with blotting paper and is dried in room temperature；
(S2) result interpretation: in scanning chip on scanner after film is dry, to the positive hybridization signal on HPV genetic chip Carry out signal strength collection analysis.
It is carried out for the specificity, detection quality and testing result, inventor of HPV genetic chip obtained by the verifying present invention It tests below:
1, gene chip hybridization atopic is detected with the target DNA that HPV recombinant plasmid expands
Using the resulting 30 kinds of HPV recombinant plasmid standard items DNA of abovementioned steps (1) to HPV gene core prepared by the present invention Piece carries out specific detection, in scanning chip on scanner after film is dry, using Image J software on HPV genetic chip Positive hybridization signal carries out signal strength acquisition.Positive hybridization signal intensity target spot signal Intensity Value value (INS Value) it indicates.Specificity, sensitivity and the coincidence rate of HPV genetic chip are calculated using 22.0 software of IBM SPSS.Calculation formula: Specificity=true negative number of sites/(number of sites+false positive number of sites of true negative) × 100%, sensitivity=true positives Number of sites/(true positives number of sites+false negative number of sites) × 100%.
The hybridization figure of chip is as shown in Fig. 1 result.A is 30 kinds of HPV type probe spotting matrix schematic diagrames, B, C, D difference It is that the HPV gene chip hybridization of HPV16, HPV18, HPV11 target DNA standard items and preparation develops the color as a result, remaining 27 kinds of HPV Recombinant plasmid results of hybridization generates positive hybridization signal, as shown in Fig. 2, and every kind of HPV standard items all only in HPV chip dot matrix It develops the color in upper corresponding site.Same probe site is after 3 hybridization on genetic chip, and 3 times positive hybridization signal intensity is presented Consistency.Results of hybridization illustrates that probe has preferable specificity and sensitivity, calculates the sensitivity of HPV genetic chip, specificity, As a result be specificity 93.5%, sensitivity 100%, and 3 times it is independent repeat experiment and can get stable and consistent as a result, explanation 30 The repeatability of kind hypotype HPV probe is good.
2, with the detection effect of HPV recombinant plasmid dna verifying genetic chip
The true detection situation of simulation, by HPV recombinant plasmid dna obtained by abovementioned steps (1) and people's beta globin genes DNA Coamplification is carried out, amplified production and the present invention prepare the colour developing of HPV gene chip hybridization, as a result as shown in figure 3, A is HPV base Because of chip, B, C, D are that HPV16 recombinant plasmid, HPV58 recombinant plasmid and HPV11 recombinant plasmid are separately added into beta globin base respectively Because DNA carries out coamplification, PCR product and genetic chip dot blot is taken to develop the color, results of hybridization is shown, HPV test point, PC and QC Quality Control point generates positive hybridization signal, other HPV test points do not generate positive hybridization signal.As a result, illustrating HPV genetic chip Under the monitoring of positive quality control point, there is high specific and high sensitivity.With beta globin genes widest in human genome work For the positive quality control point of genetic chip, the whole experiment process of DNA extraction, PCR amplification and molecule hybridization can be monitored, ensure that HPV genechip detection quality.
The beneficial effects of the present invention are:
The present invention uses biochip technology, is prepared for a kind of novel HPV parting detection chip.Genetic chip work Principle is by HPV specific probe point sample and to be solidificated on Biodyne C film, is prepared into genetic chip according to matrix point sample, into When row detection, with the DNA of biotin labeling PCR primer amplified sample, gene core then is loaded to using DNA amplification product On piece carries out hybridization incubation, and specific amplified production DNA can connect into double-strand with corresponding probes complementary, and parent is then added It is coupled with the biotin on the horseradish peroxidase (HRP) and amplified production of element label, the aobvious of horseradish peroxidase is added Color substrate, chromogenic substrate form the cationic product of blue under the catalytic action of enzyme, to generate at the position of hybridization reaction Colour developing precipitating is scanned acquisition colour developing site, compares with the array of chip design, to analyze sample HPV type.
The design of HPV primer and probe of the present invention is the selected region HPV genome L1 conserved region as target gene area, according to Existing constant region and variable region design primer and probe between HPV sequence in the region L1, using sequence alignment program to each Asia The gene order in the region HPV L1 of type is compared, so that constant region be selected to design PCR primer；Select each hypotype The area HPV L1 variation large area designs the probe of various HPV.Using primer amplification HPV target-gene sequence, by each of design Hypotype HPV probe points prepare genetic chip on Biodyne C film.HPV standard is prepared using the DNA cloning of clinical sample HPV Product can search DNA sequence dna in NCBI nucleic acid database, then be synthesized if lacking the clinical sample of certain hypotype HPV It obtains.By the DNA standard items of HPV target, hybridization check is carried out, evaluates the specificity of chip detection, as the result is shown respective target There is positive hybridization signal at dot blot, and nonspecific target spot amixia signal, as a result illustrate the independent design tool of HPV probe There is high specific.Using the method for the HPV area genome L1 as target gene area design primer and probe meet genetic chip it is extensive, High-throughput, parallelization detection requirement, can will design successful probe spotting into chip, be verified with the product of primer amplification The specificity of probe.Good technological reserve is provided for the research and development of HPV detection and genotyping and detection chip.
In order to guarantee the reliability of microarray data, also needed in genetic chip plus house-keeping gene as control.This Invention devises positive quality control point on chip, selects to be widely present in the gene of people's beta globin in human body cell as sun Property control, selection dilution probe NaHCO3As blank control.Positive quality control point can monitor DNA extraction, PCR amplification and divide Each experimentation of son hybridization is conducive to the quality control of chip detection.If positive hybridization signal is presented in positive quality control point, And the signal strength is consistent with the positive hybridization signal intensity of HPV test point, illustrates that this hybrid process does not pollute, as a result Accurately；On the contrary, if positive hybridization signal does not occur in positive quality control point, and positive hybridization signal, explanation is presented in HPV test point This time results of hybridization is false positive；If positive hybridization signal is presented in positive quality control point but HPV test point does not have hybridization signal to go out It is existing, illustrate that this results of hybridization is false negative, it is likely to which pollution occurs in hybrid process.Pass through the whole process to positive quality control point Monitoring can exclude false positive and false negative result in HPV detection, improve the reliability of genechip detection result.
Since there are dual and multiple HPV infections for clinical sample, after directly the clinical sample of multiple infection is expanded with core Piece hybridization, to the screening inaccuracy of HPV specific probe.Although each HPV monoclonal of multiple infection is carried out sequencing point Analysis can theoretically be accomplished, but there is very big difficulty, thus the present invention by design to the various hypotype primers of HPV and Gene cloning constructs HPV recombinant plasmid, is prepared into HPV standard items.A kind of HPV is contained only due to constructing successful recombinant plasmid 2 kinds or two or more other HPV hypotype are not present in hypotype, and the HPV standard items being successfully prepared are expanded, are hybridized with chip, Testing result would not be influenced by other kinds of HPV.HPV testing result theoretically only has test point to have positive hybridization letter Number, other points do not develop the color.If only having HPV test point to have positive hybridization signal on chip, illustrate that the HPV probe of the hypotype is special It is anisotropic good, if illustrating that the HPV probe specificity of the hypotype is bad there is also the positive hybridization signal other than test point, consider weight New design probe.Verified, HPV genetic chip obtained by the present invention has good specificity.
The present invention passes through the independent design of HPV amplimer and HPV specific probe, HPV probe spotting concentration and PCR The optimization of reverse dot blot hybridization colour developing optimum condition, so that HPV genetic chip has sufficiently high sensitivity and specificity, clinical sample Originally detection and parting can be carried out to 30 kinds of HPV types by further demonstrating HPV genetic chip.30 kinds of successful collection in the present invention HPV hypotype clinical sample successfully constructs 30 kinds of HPV type recombinant plasmids by the amplification and gene cloning of HPV universal primer. Using HPV recombinant plasmid as template, high efficiency expands the short genetic fragment in the area HPV genome L1.According to 30 kinds of HPV genome design HPV specific probe prepares HPV genetic chip, utilizes PCR revert dot blot hybridization by 30 kinds of HPV specific probe points on film Screen the probe of high specificity.By the deposition probe concentration optimization to HPV genetic chip, the quality and effect of hybridization are improved Rate, and further demonstrate with clinical sample the genetic chip of the condition of optimization.In actual use, it can according to need selection HPV genetic chip is prepared into using the same preparation method of a certain or several use in 30 kinds of HPV specific probes, and Targetedly carry out the detection and screening or the detection and screening of smaller range HPV hypotype of certain HPV hypotype.
In conclusion HPV genetic chip high sensitivity of the invention, high specificity can be individually or simultaneously to a kind of or more Kind of HPV hypotype accurately detect, screens and parting, with preferable potential applicability in clinical practice.
Detailed description of the invention
Fig. 1 is the HPV gene chip hybridization colour developing result of HPV16, HPV18, HPV11 target DNA standard items and preparation.
Fig. 2 is remaining 27 kinds of HPV recombinant plasmid and HPV gene chip hybridization result.
Fig. 3 is the detection effect results of hybridization that genetic chip is verified with the recombinant plasmid dna of HPV11,16 and 58.
Fig. 4 is the area various HPV L1 DNA cloning product electrophoretogram.
Fig. 5 is various HPV colony PCR product electrophoretogram.
Fig. 6 is various HPV plasmid electrophoretogram.
Fig. 7 is HPV standard items electrophoretogram.
Fig. 8 is influence of the HPV probe spotting concentration to chip colour developing gray value.
Specific embodiment
Technical solution of the present invention is further detailed below with reference to embodiment.
1. experimental material
1.1 Specimen origin
40 female outpatient HPV infection patient samples (cervical smear cast-off cells) have drawn from first people of Kweiyang Qingzhen City Hospital, age are 21-67 years old, and average age is 37.9 years old.40 samples are by subtype of human papilomavirus gene detection reagent Box (Da'an Gene Company, Zhongshan University) or gene sequencing detection, it has been determined that the HPV type of 40 samples.
In 40 HPV clinical samples, the HPV of 30 kinds of hypotypes is detected, amplification produces testing result as shown in figure 4,30 Kind HPV testing goal gene about 500bp size, stripe size fulfills the expectation, and the amplification of 30 kinds of HPV clinical samples is prompted to produce Object can be used for studying in next step.
1.2 key instrument
Micropipettor: German Eppendorf；
Electrophoresis apparatus power supply: Liuyi Instruments Plant, Beijing, model are DYY-6C；
PCR amplification instrument: Applied biosystems, model are ABI9700；
Gel imager: Britain Syngene, model are BOXEF2；
Supercentrifuge: Anhui Zhong Kezhongjia scientific instrument Co., Ltd, model are HC-2062；
Constant-temperature incubation shaking table: Hangzhou meter Ou Instrument Ltd., model are ES-60；
Horizontal electrophoresis tank: 61 Biotechnology Co., Ltd of Beijing, model are DYCP-31DN；
1.3 main agents
C film: Pall Life Science；
HPV probe: Tian Yihuiyuan Biotechnology Co., Ltd；
HPV primer: Tian Yihuiyuan Biotechnology Co., Ltd；
Streptavidin-POD: green skies Biotechnology Co., Ltd；
PMD18-T simple vector: precious biology (Dalian) Co., Ltd；
Taq archaeal dna polymerase: precious biology (Dalian) Co., Ltd；
DNA Marker series: precious biology (Dalian) Co., Ltd；
Horseradish peroxidase (HRP): health is century Biotechnology Co., Ltd
TMB developing solution: health is century Biotechnology Co., Ltd；
DNA Clean-up Kit: health is century Biotechnology Co., Ltd；
Plasmid (a small amount of) extraction agent box: Tiangeng biochemical technology Co., Ltd；
EDC: hundred Lin Wei Science and Technology Ltd. of Beijing；
The preparation of 1.4 main agents
I 2 × SSC of hybridization solution (0.1%SDS): sodium citrate 1.76g, SDS 0.2g, NaCl 3.5g are weighed, 100mL is added Ultrapure water dissolution is settled to 200mL, room temperature preservation with ultrapure water after dissolution completely.
II 0.5 × SSC of hybridization solution (0.1%SDS): sodium citrate 0.44g, SDS 0.2g, NaCl 0.875g are weighed, is added The dissolution of 100mL ultrapure water is settled to 200mL, room temperature preservation with ultrapure water after dissolution completely.
DNA extracting solution: weighing EDTA 9.3g, SDS 5g, NaCl 14.6g, Tris 24.2g, adds 800mL ultrapure water-soluble Solution is settled to 1L, room temperature preservation with ultrapure water after dissolution completely.
20%EDC: weighing 1g EDC, is dissolved in the cooling ultrapure water of 5mL high pressure, ready-to-use.
E.colistraindh5α is provided by molecular biology key lab of Guizhou medical university.
2. preparing HPV genetic chip
The preparation of 2.1HPV examination criteria product
2.1.1HPV the extraction of clinical sample DNA
1) HPV infection clinical sample is provided by Qingzhen City First People's Hospital, by the cervical exfoliated cell of collection with 10000rpm revolving speed is centrifuged 5min in centrifuge, abandons supernatant；
2) it takes 50 μ L DNA extracting solutions in centrifuge tube with liquid-transfering gun, gently blows and beats tube bottom sediment to mixing, place 100 10min is incubated in DEG C boiling water bath；
3) 5min is centrifuged with 10000rpm revolving speed in centrifuge after the completion of being incubated for, retains supernatant, the DNA sample of extraction Immediately using or be placed in -20 DEG C of preservations.
2.1.2HPV the amplification of clinical sample
The improved PGMY09 primer (Rahman in the conserved region of the region HPV genome L1 is used with reference to Gravitt etc. M,Sasagawa T,Yamada R,et al.High prevalence of intermediate‐risk human papillomavirus infection in uterine cervices of kenyan women infected with human immunodeficiency virus[J].Journal of Medical Virology,2011,83(11):1988- 1996.) HPV universal primer, is designed, HPV clinical sample is expanded.PCR amplification is carried out by 1 system of following table and 2 condition of table:
The area HPV gene L1 DNA cloning PCR reaction system in 1 clinical sample of table
2 area clinical sample HPV L1 DNA cloning PCR reaction condition of table
It is detected by agarose gel electrophoresis；After electrophoresis, it is put into EB color pond and dyes；After dyeing, it will coagulate Glue is put into nucleic acid ultraviolet imagery system and takes pictures, and analyzes result.
2.1.3PCR the purifying of product
Positive PCR product is purified according to operating instruction using DNA Clean-up Kit kit.
2.1.4 the conversion of recombinant plasmid and coated plate
1) HPV purifying DNA is mixed by 3 linked system of table (10 μ L), places and is connected in 16 DEG C of water-baths overnight:
The linked system of table 3 HPV Insert Fragment DNA and plasmid vector
2) competent cell DH5 α is thawed on ice from -80 DEG C of taking-up placements, draws 50 μ L competence bacterium solutions with sample injector Into connection product, careful piping and druming places after mixing and is incubated for 30min on ice；
3) above-mentioned solution is put into 42 DEG C of water-baths after water-bath 75s to take out to be placed in rapidly and is incubated for 5min on ice；
4) thermal shock converted product is transferred in the 1.5mL centrifuge tube equipped with 600 μ L LB culture mediums, in 37 DEG C, 200rpm Under the conditions of constant-temperature table in recovery 30min；
6) converted product is placed in centrifuge and 3min is centrifuged with 4000rpm revolving speed, abandoned most of supernatant, retain 100 μ L Left and right supernatant resuspended bacterium solution, resuspended bacterium solution are transferred on the LB plate containing Amp, and bacterium is resuspended with sterile spreading rod even spread Liquid is inverted above-mentioned plate overnight incubation in 37 DEG C of constant incubators；
7) observation is incubated overnight whether plate grows bacterium colony, if not growing bacterium colony, considers whether competent cell goes wrong Or target gene and carrier are not connected with, and convert coated plate again；If growing monoclonal colonies, further to monoclonal colonies PCR It is detected with agarose gel electrophoresis, whether testing goal gene converts success.
Picking monoclonal carries out bacterium colony PCR and agarose gel electrophoresis detection after the HPV product connection pMD18-T of purifying, Testing result is as shown in figure 5, the length of 30 kinds of HPV type colony PCR products prompts HPV L1 area DNA and sky in 500bp or so Vector plasmid successful connection.
2.1.7 monoclonal plasmid identification
1) using the plasmid of monoclonal DH5 α as template, using the primer (M13-47 and M13-RM) in plasmid kit into The site of row PCR amplification, amplification originates in the upstream and downstream of insertion point.If HPV L1 area DNA and the successful connection of empty carrier plasmid, The length of PCR product is about in 500bp or so；If it is empty plasmid, PCR product is only 70-80bp；
2) it takes and is incubated overnight plate, using the monoclonal on sterile toothpick picking plate, sterile toothpick is in following PCR systems (10 μ L) is put into equipped in LB culture medium of the 600 μ L containing Amp after dipping in, vibrates toothpick, the bacterium on toothpick is made to fall entrance In culture medium, the shaken cultivation in 37 DEG C, the constant-temperature table of 200rpm condition, by bacterium solution shaken cultivation to logarithmic phase mid-term (OD =0.4-0.6)；
3) PCR is carried out by 4 system of following table and 5 condition of table:
4 PCR reaction system of table
5 PCR reaction condition of table
It is detected by agarose gel electrophoresis；After electrophoresis, gel is put into EB color pond and is dyed；Dyeing terminates Afterwards, gel is put into nucleic acid ultraviolet imagery system and is taken pictures, analyze result.
Will test result is that positive monoclonal bacterium solution expands culture, extracts plasmid and carries out agarose gel electrophoresis detection, Testing result as shown in fig. 6,30 kinds of HPV type recombinant plasmid stripe sizes with it is expected that size be consistent；Further verify HPV The area L1 DNA and the successful connection of pMD18T carrier.
2.1.8 gene sequencing is verified
For further verify building monoclonal plasmid correctness, select the corresponding bacteria liquid sample of positive findings and surveyed Sequence retains and correct monoclonal is sequenced in glycerol stock, as standard items, -20 DEG C of preservations according to bacterium solution sequencing result.
Picking monoclonal is sequenced after the HPV product connection pMD18-T of purifying, and the whole genome sequence length of HPV insertion is about For 500bp.HPV recombinant plasmid sequencing result and HPV gene chip results coincidence rate reach 96.7%.It is tested through recombinant clone sequencing Card successfully constructs 30 kinds of HPV recombinant plasmids.
2.1.9HPV the extraction of recombinant plasmid
Positive HPV, which is extracted, according to operating instruction by (centrifugal column type) using the small extraction reagent kit of plasmid recombinates bacterium solution.
The amplification of the area 2.2HPV L1 DNA fragmentation target
The DNA of target fragments is the critical sites for identifying HPV parting, the probe and primer of genetic chip in the area L1 Design is all from herein.
2.2.1 the preparation of recombinant plasmid dna
1) after sequence verification recombinant plasmid is correct, glycerol stock is inoculated into equipped with 600 μ L benzyls containing ammonia with aseptic inoculation ring LB culture medium in, in 37 DEG C, 200rpm condition constant-temperature table recovery overnight；
2) above-mentioned HPV is extracted according to operating instruction by (centrifugal column type) using the small extraction reagent kit of plasmid and recombinate bacterium solution, with this HPV recombinant plasmid is that template is expanded.
2.2.2HPV the amplification of recombinant plasmid
1) in order to improve the quality of PCR amplification efficiency and results of hybridization, 4 forward directions are designed in the area L1 of HPV genome Primer and 4 reverse primers, biotin labeling is used in 4 designed 5 ' ends of reverse primer, as shown in table 8, as colour developing The connection molecule of group.Using degenerate primer principle, the target DNA sequence dna in the area HPV recombinant plasmid L1, PCR system and item are expanded Part is as shown in table 6 and table 7:
The PCR reaction system of 6 HPV target DNA fragmentation of table
The PCR reaction condition of 7 HPV standard items target DNA fragmentation of table
Pass through agarose gel electrophoresis；Gel is put into EB color pond after electrophoresis and is dyed；After dyeing, it will coagulate Glue is put into nucleic acid ultraviolet imagery system and takes pictures, and analyzes result.
The DNA of target fragments is the critical sites for identifying HPV parting, the probe and primer of genetic chip in the area L1 Design is all from herein.The present invention utilizes degenerate primer principle, and high efficiency expands the area HPV L1 DNA target mark segment, the target The theoretical length of segment is in 160-200bp or so.It is detected with agarose gel electrophoresis, it is found that the segment of amplification is about The amplified band of 180bp size, size fulfill the expectation, as shown in fig. 7, containing the area HPV L1 target in prompt recombinant plasmid DNA sequence dna, and the length of PCR product is accurate, can be used for hybridizing, the standard items as detection genetic chip.
2.3HPV primer and probe design
Universal primer is designed according to the constant region in the area HPV genome L1, guarantees the specificity of the area HPV L1 amplified production；Again All types of probes are designed according to the variable region of the target fragments DNA sequence dna in the area HPV L1, guarantee the specificity of hypotype probe；Root (PC) is visited according to the DNA sequence dna design internal reference in people's beta globin characteristic region, guarantees the validity and PCR reaction of sample source Validity；Amino is marked at 5 ' ends of probe, connect and solidifies with solid-phase matrix for probe, as shown in table 8, wherein sequence Column are identical as SEQ ID NO:1~SEQ ID NO:168170, and 1,2 be PC and QC probe sequence in table 8, and 3~32 be HPV in 30 Hypotype probe sequence, 33~40 be that 4 forward primers used in the amplification of the area HPV L1 DNA fragmentation target and 4 reversely draw Object sequence, and in 4 end of reverse primer 5 ' label biotins, 41 and 42 be human genome beta globin genes amplimer sequence, And synthetic primer and probe.
Table 8HPV probe sequence, HPV type amplification forward primer and reverse primer and the amplification of human genome beta globin genes Primer sequence
The preparation of 2.4HPV genetic chip
The array for being designed as 3 × 11 of HPV genetic chip, probe lattice array include 32 points altogether, 2 positive quality control points and 30 HPV type test points, 32 kinds of probes put in order as shown in figure 1 shown in A.Respectively there are PC and QC two in the upper left corner and the lower right corner of array A, PC (Positive Control) is positive quality control point, detects the beta globin genes in human genome, monitors sample DNA Extraction and PCR amplification process validity；QC (Quality Control) is positive quality control point, monitors HPV primer amplification； Remaining 30 point is 30 kinds of HPV hypotype test points.
1) before HPV probe spotting, Biodyne C film is cut into film forming item；
2) the film item of cutting is placed after impregnating 1min in 0.1mol/L HCl solution, transfer membrane item to 20%EDC solution Middle activation 15min simply rinses 30s with aseptic double-distilled water, and room temperature dries 1h；
3) 0.1mol/L Na is used2CO3/NaHCO3Buffer (pH=8.4), by the HPV probe and people's gene of amino labeled For beta globin genes probe dilution in group to 0.5~8pmol/L, each probe takes 3 μ L dilutions to be fixed in an orderly manner On Biodyne C film, make the carboxyl Covalent bonding together activated on amino labeled probe and Biodyne C film, each sample room every 1cm；
4) it after Biodyne C film item air-dries 15min, takes in 0.1mol/L NaOH and 10min, it is clear with sterile water later It washes 4 times, it is every all over 30s, it stays overnight in being air-dried at room temperature to get HPV genetic chip.
The point sample concentration of probe influences whether sensitivity of the genetic chip to HPV sample P CR product detection, uses 0.1mol/L NaHCO3Solution respectively by the HPV probe dilution of 30 kinds of hypotypes at 0.5pmol/L, 1pmol/L, 2pmol/L, 4pmol/L and The concentration of 8pmol/L, control group are 0.1mol/L NaHCO3Solution.According to the dot matrix arrangement of design, by different probe concentration with Identical point sample amount point sample is hybridized in being prepared into genetic chip on Biodyne C film with corresponding HPV standard items.To difference The HPV genetic chip of probe spotting concentration carries out hybridization check and gray value analysis, converts INS data for colour developing image, obtains To the relational graph of concentration and probe concentration and detection gray value, as shown in Figure 8.The intensity of HPV hybridization signal can be with probe as the result is shown The increase of concentration and enhance, when the point sample concentration of the HPV probe of most hypotypes reaches 2pmol/L, hybridization colour developing gray scale Value curve tends towards stability, and illustrates that the best point sample concentration of HPV probe is about 2pmol/L.
3.PCR reverse dot blot hybridization
1) it extracts sample DNA and is expanded using 4 forward primers described in table 8 and 4 reverse primers, obtained PCR product, it is spare；
2) by I (2 × SSC of hybridization solution；0.1%SDS) and II (0.5 × SSC of hybridization solution；0.1%SDS) place in water-bath Be preheated to 45 DEG C it is spare；
3) setting constant temperature shaker temperature is 45 DEG C in advance, and presses acknowledgement key, and the temperature in shaking table is made to reach 45 DEG C most suitable Hybridization temperature.
4) PCR product is immediately placed on incubation 2min or more on ice after 98 DEG C are boiled 8min denaturation；
5) it is put into 15mL centrifuge tube with the Biodyne C film item of aseptic nipper tweezer prehybridization, 5mL preheating hybridization is added Liquid I, it is spare；
6) the PCR product addition of denaturation is placed in the centrifuge tube of film item, is hybridized in 100rpm, 45 DEG C of constant-temperature table 15min~1h；
7) hybridization solution I is abandoned, tweezer film item is put into 50mL centrifuge tube, and 10mL is added and preheats hybridization solution II, in 100rpm, 45 DEG C constant-temperature table in wash 10~30min of film, wash in total twice；
8) hybridization solution II is abandoned, centrifuge tube is inverted on blotting paper, blots tube bottom liquid, 20mL HRP dilution is added (HRP: abandoning hybridization solution I=1:2000~8000 now match), at room temperature in being protected from light on oscillation shaking table with the revolving speed of 50rpm 10min；
9) it abandons and combines liquid, centrifuge tube is inverted on blotting paper, blot tube bottom liquid, 20mL hybridization solution I is added at room temperature In washing film twice on oscillation shaking table, film 2min is washed every time；
10) hybridization solution I is abandoned, blotting paper is shelved on desk, centrifuge tube is upside down in above, tube bottom liquid is blotted, adds Enter 20mL TMB developing solution (now matching, 20 × TMB-A:1 of volume ratio × TMB-B=1:30~100), reacting at normal temperature without light 5min； After colour developing, developing solution to be abandoned immediately, and 20mL sterile distilled water is added, takes out after washing film 3min on shaking table, repetition is washed film one time, Film surface moisture is sucked with blotting paper and is dried in room temperature.
4 result interpretations
In scanning chip on scanner after film is dry, using Image J software to the positive hybridization letter on HPV genetic chip Number carry out signal strength acquisition.Positive hybridization signal intensity is indicated with target spot signal Intensity Value value (INS value).Knot When fruit interpretation, HPV test point is high-visible bluish violet spot, while QC and PC positive quality control point is positive, experiment knot Fruit is effective.
Sequence table
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<213>artificial sequence (unknown)
<400> 31
tttttttttt tttttcatta tctgcagcat ctgcatccac tccat 45
<210> 32
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 32
tttttttttt tttttcatta tctgcagcat ctgcatccac tcca 44
<210> 33
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 33
tttttttttt ttttttacat tatctgcagc atctgcatcc actcc 45
<210> 34
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 34
tttttttttt tttttgtaca ttatctgcag catctgcatc cactc 45
<210> 35
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 35
tttttttttt tttttacatt atctgcagca tctgcatcca ctcc 44
<210> 36
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 36
tttttttttt ttttttgcaa ttgcaaacag tgatactaca tttaa 45
<210> 37
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 37
tttttttttt tttttcaatt gcaaacagtg atactacatt taaaa 45
<210> 38
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 38
tttttttttt ttttttgcaa ttgcaaacag tgatactaca ttta 44
<210> 39
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 39
tttttttttt tttttgctgc aattgcaaac agtgatacta cattt 45
<210> 40
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 40
tttttttttt tttttgctgc aattgcaaac agtgatacta cattt 45
<210> 41
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 41
tttttttttt tttttttatg tgcatccgta actacatctt ccaca 45
<210> 42
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 42
tttttttttt tttttttatg cacacaagta actagtgaca gtac 44
<210> 43
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 43
tttttttttt ttttttatgt gcatccgtaa ctacatcttc caca 44
<210> 44
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 44
tttttttttt tttttatgtg catccgtaac tacatcttcc acat 44
<210> 45
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 45
tttttttttt ttttttgtgc atccgtaact acatcttcca cata 44
<210> 46
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 46
tttttttttt tttttcacaa tccacaagta caactgcacc atatg 45
<210> 47
<211> 42
<212> DNA
<213>artificial sequence (unknown)
<400> 47
tttttttttt tttttacaca atccacaagt acaactgcac ca 42
<210> 48
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 48
tttttttttt tttttacaat ccacaagtac aactgcacca tatgc 45
<210> 49
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 49
tttttttttt tttttcaatc cacaagtaca actgcaccat atgca 45
<210> 50
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 50
tttttttttt ttttttacac aatccacaag tacaactgca ccata 45
<210> 51
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 51
tttttttttt tttttgtctg tgtgttctgc tgtgtctact agtg 44
<210> 52
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 52
tttttttttt ttttttctgt gtgttctgct gtgtctacta gtgac 45
<210> 53
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 53
tttttttttt tttttgtctg tgtgttctgc tgtgtcttct agtga 45
<210> 54
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 54
tttttttttt ttttttgtgt gttctgctgt gtctactagt gaca 44
<210> 55
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 55
tttttttttt tttttagtct gtgtgttctg ctgtgtctac tagt 44
<210> 56
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 56
tttttttttt ttttttatct acctctatag agtcttccat acctt 45
<210> 57
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 57
tttttttttt ttttttctac ctctatagag tcttccatac cttct 45
<210> 58
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 58
tttttttttt ttttttacct ctatagagtc ttccatacct tcta 44
<210> 59
<211> 46
<212> DNA
<213>artificial sequence (unknown)
<400> 59
tttttttttt tttttatcta cctctataga gtcttccata ccttct 46
<210> 60
<211> 46
<212> DNA
<213>artificial sequence (unknown)
<400> 60
tttttttttt tttttacctc tatagagtct tccatacctt ctacat 46
<210> 61
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 61
tttttttttt tttttgctgc cacacagtcc cccacaccaa cccca 45
<210> 62
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 62
tttttttttt tttttgccac acagtccccc acaccaaccc cata 44
<210> 63
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 63
tttttttttt tttttccaca cagtccccca caccaacccc atat 44
<210> 64
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 64
tttttttttt tttttgctgc cacacagtcc cccacaccaa cccc 44
<210> 65
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 65
tttttttttt tttttgtgct gccacacagt cccccacacc aaccc 45
<210> 66
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 66
tttttttttt tttttactgc aacatctggt gatacatata cagc 44
<210> 67
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 67
tttttttttt tttttcactg caacatctgg tgatacatat acagc 45
<210> 68
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 68
tttttttttt ttttttgcaa catctggtga tacatataca gctgc 45
<210> 69
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 69
tttttttttt tttttcaaca tctggtgata catatacagc tgcta 45
<210> 70
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 70
tttttttttt tttttctgca acatctggtg atacatatac agctg 45
<210> 71
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 71
tttttttttt tttttctcta ctgaccctac tgtgcccagt acata 45
<210> 72
<211> 46
<212> DNA
<213>artificial sequence (unknown)
<400> 72
tttttttttt tttttcctct actgacccta ctgtgcccag tacata 46
<210> 73
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 73
tttttttttt tttttctact gaccctactg tgcccagtac atatg 45
<210> 74
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 74
tttttttttt ttttttctac tgaccctact gtgcccagta catat 45
<210> 75
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 75
tttttttttt ttttttactg accctactgt gcccagtaca tatga 45
<210> 76
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 76
tttttttttt tttttgccac tacacagtcc cctccgtcta cata 44
<210> 77
<211> 43
<212> DNA
<213>artificial sequence (unknown)
<400> 77
tttttttttt tttttcacta cacagtcccc tccgtctaca tat 43
<210> 78
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 78
tttttttttt tttttcacta cacagtcccc tccgtctaca tatac 45
<210> 79
<211> 43
<212> DNA
<213>artificial sequence (unknown)
<400> 79
tttttttttt tttttgccac tacacagtcc cctccgtcta cat 43
<210> 80
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 80
tttttttttt tttttgctgc cactacacag tcccctccgt ctac 44
<210> 81
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 81
tttttttttt tttttcacaa aatcctgtgc caactccata tgatc 45
<210> 82
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 82
tttttttttt tttttacaca aaatcctgtg ccaagtacat atgac 45
<210> 83
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 83
tttttttttt tttttcacac aaaatcctgt gccaactcca tatg 44
<210> 84
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 84
tttttttttt tttttacaaa atcctgtgcc aactccatat gatcc 45
<210> 85
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 85
tttttttttt tttttcaaaa tcctgtgcca actccatatg atcct 45
<210> 86
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 86
tttttttttt tttttactgc tgcagtttcc ccaacattta ctcca 45
<210> 87
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 87
tttttttttt ttttttgctg cagtttcccc aacatttact ccaa 44
<210> 88
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 88
tttttttttt ttttttgctg cagtttcccc aacatttact ccaag 45
<210> 89
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 89
tttttttttt tttttcactg ctgcggtttc cccaacattt actcc 45
<210> 90
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 90
tttttttttt tttttccact gctgcagttt ccccaacatt tact 44
<210> 91
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 91
tttttttttt tttttgctga ggttaaaaag gaaagcacat ataa 44
<210> 92
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 92
tttttttttt tttttgaggt taaaaaggaa agcacatata aaaa 44
<210> 93
<211> 43
<212> DNA
<213>artificial sequence (unknown)
<400> 93
tttttttttt tttttggtta aaaaggaaag cacatataaa aat 43
<210> 94
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 94
tttttttttt ttttttgctg aggttaaaaa ggaaagcaca tataa 45
<210> 95
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 95
tttttttttt tttttgtgct gaggttaaaa aggaaagcac atat 44
<210> 96
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 96
tttttttttt ttttttcttt ccgcaaccac acagtctatg tcta 44
<210> 97
<211> 46
<212> DNA
<213>artificial sequence (unknown)
<400> 97
tttttttttt ttttttcttt ccgcaaccac acagtctatg tctaca 46
<210> 98
<211> 46
<212> DNA
<213>artificial sequence (unknown)
<400> 98
tttttttttt tttttgactc tttccgcaac cacacagtct atgtct 46
<210> 99
<211> 46
<212> DNA
<213>artificial sequence (unknown)
<400> 99
tttttttttt ttttttgact ctttccgcaa ccacacagtc tatgtc 46
<210> 100
<211> 46
<212> DNA
<213>artificial sequence (unknown)
<400> 100
tttttttttt tttttacatg actctttccg caaccacaca gtctat 46
<210> 101
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 101
tttttttttt ttttttacag catccacgca ggatagcttt aataa 45
<210> 102
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 102
tttttttttt tttttcagca tccacgcagg atagctttaa taatt 45
<210> 103
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 103
tttttttttt ttttttacag catccacgca ggatagcttt aata 44
<210> 104
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 104
tttttttttt tttttgctac agcatccacg caggatagct ttaat 45
<210> 105
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 105
tttttttttt tttttgtgct acagcatcca cgcaggatag cttta 45
<210> 106
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 106
tttttttttt ttttttacaa ctcagtctcc atctacaaca tata 44
<210> 107
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 107
tttttttttt ttttttgcta caactcagtc tccatctaca acata 45
<210> 108
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 108
tttttttttt tttttctaca actcagtctc catctacaac atata 45
<210> 109
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 109
tttttttttt tttttacaac tcagtctcca tctacaacat ataat 45
<210> 110
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 110
tttttttttt tttttctaca actcagtctc catctacaac atata 45
<210> 111
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 111
tttttttttt tttttactgc tacagaacag ttaagtaaat atga 44
<210> 112
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 112
tttttttttt tttttctgct acagaacagt taagtaaata tgatg 45
<210> 113
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 113
tttttttttt tttttgtact gctacagaac agttaagtaa atatg 45
<210> 114
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 114
tttttttttt tttttgtact gctacagaac agttaagtaa atat 44
<210> 115
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 115
tttttttttt ttttttagta ctgctacaga acagttaagt aaata 45
<210> 116
<211> 46
<212> DNA
<213>artificial sequence (unknown)
<400> 116
tttttttttt ttttttcttt gtgtgccact gtaaccacag aaacta 46
<210> 117
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 117
tttttttttt tttttctctt tgtgtgccac tgtaaccaca gaaac 45
<210> 118
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 118
tttttttttt ttttttgtct ctttgtgtgc cactgtaacc acag 44
<210> 119
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 119
tttttttttt ttttttctct ttgtgtgcca ctgtaaccac agaa 44
<210> 120
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 120
tttttttttt ttttttttgt gtgccactgt aaccacagaa actaa 45
<210> 121
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 121
tttttttttt tttttacgac tgaagtaact aaggaaggta catat 45
<210> 122
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 122
tttttttttt tttttgactg aagtaactaa ggaaggtaca tataa 45
<210> 123
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 123
tttttttttt ttttttgaag taactaagga aggtacatat aaaa 44
<210> 124
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 124
tttttttttt tttttactga agtaactaag gaaggtacat ataaa 45
<210> 125
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 125
tttttttttt tttttaagta actaaggaag gtacatataa aaatg 45
<210> 126
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 126
tttttttttt tttttaagtt ctattcctaa tgtacacaca ccta 44
<210> 127
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 127
tttttttttt tttttttcta ttcctaatgt atacacacct accag 45
<210> 128
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 128
tttttttttt tttttattcc taatgtacac acacctacca gttt 44
<210> 129
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 129
tttttttttt ttttttattc ctaatgtaca cacacctacc agttt 45
<210> 130
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 130
tttttttttt tttttgttct attcctaatg tacacacacc tacca 45
<210> 131
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 131
tttttttttt tttttggtct gctgcagcag aatacaaggc tacca 45
<210> 132
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 132
tttttttttt ttttttctgc tgcagcagaa tacaaggcta ccaac 45
<210> 133
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 133
tttttttttt tttttgctgc agcagaatac aaggctacca acttt 45
<210> 134
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 134
tttttttttt tttttctgca gcagaataca aggctaccaa cttt 44
<210> 135
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 135
tttttttttt tttttctgct gcagcagaat acaaggctac caact 45
<210> 136
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 136
tttttttttt tttttttaac taaatatgat gcccgtgaaa tcaat 45
<210> 137
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 137
tttttttttt tttttaacta aatatgatgc acgtgaaatc aatca 45
<210> 138
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 138
tttttttttt tttttattaa ctaaatatga tgcacgtgaa atca 44
<210> 139
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 139
tttttttttt tttttacatt aactaaatat gatgcacgtg aaatc 45
<210> 140
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 140
tttttttttt tttttctaaa tatgatgcac gtgaaatcaa tcaa 44
<210> 141
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 141
tttttttttt tttttaaaaa tcagaggcta catacaaaaa tgaa 44
<210> 142
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 142
tttttttttt tttttaaatc agaggctaca tacaaaaatg aaaa 44
<210> 143
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 143
tttttttttt tttttcagag gctacataca aaaatgaaaa cttta 45
<210> 144
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 144
tttttttttt tttttaatca gaggctacat acaaaaatga aaact 45
<210> 145
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 145
tttttttttt ttttttcaga ggctacatac aaaaatgaaa actt 44
<210> 146
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 146
tttttttttt tttttctact actactgaat cagctgtacc aaata 45
<210> 147
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 147
tttttttttt tttttctact actgaatcag ctgtaccaaa tatt 44
<210> 148
<211> 46
<212> DNA
<213>artificial sequence (unknown)
<400> 148
tttttttttt tttttgtcta ctactactga atcagctgta ccaaat 46
<210> 149
<211> 46
<212> DNA
<213>artificial sequence (unknown)
<400> 149
tttttttttt ttttttgtct actactactg aatcagctgt accaaa 46
<210> 150
<211> 46
<212> DNA
<213>artificial sequence (unknown)
<400> 150
tttttttttt ttttttttgt ctactactac tgaatcagct gtacca 46
<210> 151
<211> 43
<212> DNA
<213>artificial sequence (unknown)
<400> 151
tttttttttt tttttatctg catctgccac ttttaaacca tca 43
<210> 152
<211> 43
<212> DNA
<213>artificial sequence (unknown)
<400> 152
tttttttttt tttttaatct gcatctgcca cttttaaacc atc 43
<210> 153
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 153
tttttttttt tttttctgca tctgccactt ttaaaccatc agat 44
<210> 154
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 154
tttttttttt tttttgcatc tgccactttt aaaccatcag atta 44
<210> 155
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 155
tttttttttt tttttatctg ccacttttaa accatcagat tata 44
<210> 156
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 156
tttttttttt ttttttgtaa tcagaggcta catacaaaaa tgaa 44
<210> 157
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 157
tttttttttt tttttaggct acatacaaaa atgaaaacta attga 45
<210> 158
<211> 44
<212> DNA
<213>artificial sequence (unknown)
<400> 158
tttttttttt tttttagagg ctacatacaa aaatgaaaac taat 44
<210> 159
<211> 45
<212> DNA
<213>artificial sequence (unknown)
<400> 159
tttttttttt tttttaatca gaggctacat acaaaaatga aaact 45
<210> 160
<211> 43
<212> DNA
<213>artificial sequence (unknown)
<400> 160
tttttttttt ttttttaatc agaggctaca tacaaaaatg aaa 43
<210> 161
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 161
gcncagggnc acaacaatgg 20
<210> 162
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 162
gcncagggnc acaagaatgg 20
<210> 163
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 163
gcncagggnc acaataatgg 20
<210> 164
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 164
gcncagggnc agaaaaatgg 20
<210> 165
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 165
gcncagggnc agaacaatgg 20
<210> 166
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 166
gcncagggnc ataacaatgg 20
<210> 167
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 167
gcncagggnc aaaacaatgg 20
<210> 168
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 168
gcncagggnc ataagaatgg 20
<210> 169
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 169
gcncagggnc aaaataatgg 20
<210> 170
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 170
gcncagggnc aaaagaatgg 20
<210> 171
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 171
gcncagggnc ataaaaatgg 20
<210> 172
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 172
gcncagggnc acaaaaatgg 20
<210> 173
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 173
gcncagggnc agaagaatgg 20
<210> 174
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 174
gcncagggnc ataataatgg 20
<210> 175
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 175
gcncagggnc agaataatgg 20
<210> 176
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 176
gcncaaggnc acaaaaatgg 20
<210> 177
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 177
gcncaaggnc acaataatgg 20
<210> 178
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 178
gcncaaggnc agaataatgg 20
<210> 179
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 179
gcncaaggnc acaagaatgg 20
<210> 180
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n=i
<220>
<221> misc_feature
<222> (9)..(9)
<223> n=i
<400> 180
gcncaaggnc ataataatgg 20
<210> 181
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 181
gaaaaataaa ctgtaaatca tattcgtc 28
<210> 182
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 182
gaaaaataaa ctgtaactca tattcttc 28
<210> 183
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 183
gaaaaataaa ctgtaaatca tattcttc 28
<210> 184
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 184
gaaaaataaa ctgtaagtca tattcttc 28
<210> 185
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 185
gaaaaataaa ctgtaaatca tattcatc 28
<210> 186
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 186
gaaaaataaa ctgtaattca aattcttc 28
<210> 187
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 187
gaaaaataaa ctgtaattca tattcatc 28
<210> 188
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 188
gaaaaataaa ctgtaattca aattcatc 28
<210> 189
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 189
gaaaaataaa ctgtaattca cattcttc 28
<210> 190
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 190
gaaaaataaa ctgtaattca tattcttc 28
<210> 191
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 191
gaaaaataaa ctgtaaatca cattcctc 28
<210> 192
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 192
gaaaaataaa ctgtaaatca tattcctc 28
<210> 193
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 193
gaaaaataaa ctgtaaatca cattcgtc 28
<210> 194
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 194
gaaaaataaa ctgtaaatca gattcttc 28
<210> 195
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 195
gaaaaataaa ctgtaattca tattcctc 28
<210> 196
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 196
gaaaaataaa ctgtaaatca aattcatc 28
<210> 197
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 197
gaaaaataaa ctgtaaatca cattcttc 28
<210> 198
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 198
gaaaaataaa ctgtaaatca gattcctc 28
<210> 199
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 199
gaaaaataaa ctgtaattca gattcttc 28
<210> 200
<211> 28
<212> DNA
<213>artificial sequence (unknown)
<400> 200
gaaaaataaa ctgtaaatca aattcttc 28
<210> 201
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<400> 201
caacttcatc cacgttcacc 20
<210> 202
<211> 20
<212> DNA
<213>artificial sequence (unknown)
<400> 202
gaagagccaa ggacaggtac 20

Claims (13)

1. a kind of HPV genetic chip, which is characterized in that include more than one on film using Biodyne C film transfer film as carrier HPV hypotype test point and two positive quality control points, two of them positive quality control point detect respectively human genome beta globin genes and HPV primer amplification.
2. HPV genetic chip according to claim 1, which is characterized in that the detection in two positive quality control points It is any shown in the probe sequence such as SEQ ID NO:1~SEQ ID NO:5 of human genome beta globin genes, detect HPV primer It is any shown in the probe sequence such as SEQ ID NO:6~SEQ ID NO:10 of amplification, and amino is marked at 5 ' ends.
3. HPV genetic chip according to claim 1 or 2, which is characterized in that the HPV hypotype test point includes high-risk Type HPV and/or low risk HPV test point.
4. HPV genetic chip according to claim 3, which is characterized in that the high-risk HPV include HPV16,18,26, 31, one or more of 33,35,39,45,51,52,53,56,58,59,66,67,68,69 and 73 hypotypes；The low risk HPV includes one or more of hypotype of HPV6,11,34,40,42,43,44,54,55,57 and 62.
5. HPV genetic chip according to claim 4, which is characterized in that the probe sequence such as SEQ of the HPV6 hypotype It is any shown in ID NO:11~SEQ ID NO:15；The probe sequence of the HPV11 hypotype such as SEQ ID NO:16~SEQ It is any shown in ID NO:20；It is any in the probe sequence such as SEQ ID NO:21~SEQ ID NO:25 of the HPV16 hypotype It is shown；It is any shown in the probe sequence such as SEQ ID NO:26~SEQ ID NO:30 of the HPV18 hypotype；The HPV26 It is any shown in the probe sequence such as SEQ ID NO:31~SEQ ID NO:35 of hypotype；The probe sequence of the HPV31 hypotype As shown in any in SEQ ID NO:36~SEQ ID NO:40；The probe sequence of the HPV33 hypotype such as SEQ ID NO:41 It is any shown in~SEQ ID NO:45；The probe sequence of the HPV34 hypotype such as SEQ ID NO:46~SEQ ID NO:50 In it is any shown in；It is any shown in the probe sequence such as SEQ ID NO:51~SEQ ID NO:55 of the HPV35 hypotype；It is described It is any shown in the probe sequence such as SEQ ID NO:56~SEQ ID NO:60 of HPV39 hypotype；The probe of the HPV40 hypotype In sequence such as SEQ ID NO:61~SEQ ID NO:65 it is any shown in；The probe sequence of the HPV42 hypotype such as SEQ ID It is any shown in NO:66~SEQ ID NO:70；The probe sequence of the HPV43 hypotype such as SEQ ID NO:71~SEQ ID It is any shown in NO:75；The probe sequence of the HPV44 hypotype such as any institute in SEQ ID NO:76~SEQ ID NO:80 Show；It is any shown in the probe sequence such as SEQ ID NO:81~SEQ ID NO:85 of the HPV45 hypotype；The HPV51 is sub- It is any shown in the probe sequence such as SEQ ID NO:86~SEQ ID NO:90 of type；The probe sequence of the HPV52 hypotype is such as It is any shown in SEQ ID NO:91~SEQ ID NO:95；The probe sequence of the HPV53 hypotype such as SEQ ID NO:96~ It is any shown in SEQ ID NO:100；The probe sequence of the HPV54 hypotype such as SEQ ID NO:101~SEQ ID NO:105 It is shown；It is any shown in the probe sequence such as SEQ ID NO:106~SEQ ID NO:110 of the HPV55 hypotype；It is described It is any shown in the probe sequence such as SEQ ID NO:111~SEQ ID NO:115 of HPV56 hypotype；The spy of the HPV57 hypotype In needle sequence such as SEQ ID NO:116~SEQ ID NO:120 it is any shown in；The probe sequence such as SEQ of the HPV58 hypotype It is any shown in ID NO:121~SEQ ID NO:125；The probe sequence of the HPV59 hypotype such as SEQ ID NO:126~ It is any shown in SEQ ID NO:130；The probe sequence of the HPV62 hypotype such as SEQ ID NO:131~SEQ ID NO:135 In it is any shown in；It is any shown in the probe sequence such as SEQ ID NO:136~SEQ ID NO:140 of the HPV66 hypotype；Institute It states any shown in the probe sequence such as SEQ ID NO:141~SEQ ID NO:145 of HPV67 hypotype；The HPV68 hypotype In probe sequence such as SEQ ID NO:146~SEQ ID NO:150 it is any shown in；The probe sequence such as SEQ of the HPV69 hypotype It is any shown in ID NO:151~SEQ ID NO:155；The probe sequence of the HPV73 hypotype such as SEQ ID NO:156~ Shown in SEQ ID NO:160；And amino is marked at 5 ' ends.
6. the preparation method of any HPV genetic chip of Claims 1 to 5, which comprises the following steps: (1) The preparation of HPV examination criteria product；(2) amplification of the area HPV L1 DNA fragmentation target；(3) HPV primer and probe design and synthesis； (4) preparation of HPV genetic chip.
7. the preparation method of HPV genetic chip according to claim 6, which is characterized in that the area step (2) HPV L1 4 forward primers used in the amplification of DNA fragmentation target are det-1A, det-1B, det-1C and det-1D, wherein det-1A Sequence such as SEQ ID NO:161~SEQ ID NO:165 in it is any shown in, the sequence of det-1B such as SEQ ID NO:166~ It is any shown in SEQ ID NO:170, it is any shown in the sequence such as SEQ ID NO:171~SEQ ID NO:175 of det-1C, Any shown in the sequence such as SEQ ID NO:176~SEQ ID NO:180 of det-1D, 4 reverse primers are GP-1A, GP- 1B, GP-1C and GP-1D, it is wherein any in the sequence of GP-1A such as SEQ ID NO:181~SEQ ID NO:185 shown in, GP-1B Sequence such as SEQ ID NO:186~SEQ ID NO:190 in it is any shown in, the sequence of GP-1C such as SEQ ID NO:191~ It is any shown in SEQ ID NO:195, it is any shown in the sequence such as SEQ ID NO:196~SEQ ID NO:200 of GP-1D, Wherein biotin labeling is used in 45 ' ends of reverse primer, and human genome beta globin genes amplimer is added and expands together Increase, the concentration ratio of HPV amplimer and human genome beta globin genes amplimer is 1:6~1:4, the human genome β pearl Protein gene amplimer PC04 and GH20 sequence is as shown in SEQ ID NO:201 and SEQ ID NO:202, and PC04 5 ' End biotin labeling.
8. the preparation method of HPV genetic chip according to claim 6, which is characterized in that step (4) the HPV gene The preparation step of chip is as follows:
(4.1) Biodyne C film is cut into film forming item；
(4.2) the film item of cutting is placed after being impregnated in 0.1mol/L HCl solution, transfer membrane item swashs into 20%EDC solution It is living, it is simply rinsed with aseptic double-distilled water, room temperature is dried；
(4.3) Na of 0.1mol/L pH=8.4 is used2CO3/NaHCO3Buffer is by the HPV probe of amino labeled, amino labeled The HPV primer amplification probe dilution of beta globin genes probe in human genome and amino labeled, takes probe dilution liquid in an orderly manner It is fixed on transfer film, makes the carboxyl Covalent bonding together activated on amino labeled probe and Biodyne C film；
(4.4) after Biodyne C film item air-dries, 0.1mol/L NaOH is taken to neutralize, sterile water wash is used later, in room temperature wind It is dry to stay overnight up to HPV genetic chip.
9. the preparation method of HPV genetic chip according to claim 8, which is characterized in that step (4) the HPV gene The preparation step of chip is as follows:
(4.1) Biodyne C film is cut into film forming item；
(4.2) the film item of cutting is placed after impregnating 1min in 0.1mol/L HCl solution, transfer membrane item is into 20%EDC solution 15min is activated, simply rinses 30s with aseptic double-distilled water, room temperature dries 1h；
(4.3) Na of 0.1mol/L pH=8.4 is used2CO3/NaHCO3Buffering, by the HPV probe of amino labeled and amino labeled Beta globin genes probe dilution in human genome to 0.5~8pmol/L and amino labeled HPV primer amplification probe dilution To 0.05pmol/L, each 3 μ L of probe dilution liquid is taken to be fixed on Biodyne C film in an orderly manner, make amino labeled probe with The carboxyl Covalent bonding together activated on Biodyne C film；
(4.4) it after Biodyne C film item air-dries 15min, takes in 0.1mol/L NaOH and 10min, uses sterile water wash later It is 4 times, every all over 30s, it stays overnight in being air-dried at room temperature up to HPV genetic chip.
10. application of the HPV genetic chip described in Claims 1 to 5 in HPV detection and parting.
11. HPV genetic chip described in Claims 1 to 5 is as cervical disease or condyloma class disorder in screening or forecasting tool Using.
12. the application of HPV genetic chip described in 0 or 11 according to claim 1, which comprises the steps of:
(S1) PCR reverse dot blot hybridization:
(S1.1) prepared by sample to be tested: extracting sample DNA and is expanded, obtains PCR product；
(S1.2) hybridization solution I and hybridization solution II are placed be preheated in water-bath 45 DEG C it is spare；
(S1.3) setting constant temperature shaker temperature is 45 DEG C in advance, and the temperature in shaking table is made to reach 45 DEG C of most suitable hybridization temperature；
(S1.4) by PCR product obtained by step (S1.1) it is denatured by boiling after, be immediately placed on and be incubated on ice；
(S1.5) it is put into centrifuge tube with aseptic nipper tweezer HPV gene chip film item, preheating hybridization solution I is added, it is spare；
(S1.6) PCR product of step (S1.4) resulting denaturation is added to the centrifuge tube that film item is placed with obtained by step (S1.5) In, hybridize in 100rpm, 45 DEG C of constant-temperature tables；
(S1.7) hybridization solution I is abandoned, film item is taken to be put into centrifuge tube, 10mL is added and preheats hybridization solution II, in 100rpm, 45 DEG C of constant temperature Film is washed in shaking table；
(S1.8) hybridization solution II is abandoned, centrifuge tube is inverted on blotting paper, blots tube bottom liquid, 20mL HRP dilution is added, At room temperature in 50rpm being protected from light 10min on oscillation shaking table；
(S1.9) it abandons and combines liquid, centrifuge tube is inverted on blotting paper, blot tube bottom liquid, be added under I room temperature 0 of 20mL hybridization solution Film is washed on shaking table in vibrating；
(S1.10) hybridization solution I is abandoned, blotting paper is shelved on desk, centrifuge tube is upside down in above, tube bottom liquid is blotted, adds Enter 20mL TMB developing solution, reacting at normal temperature without light；After colour developing, developing solution is abandoned immediately, and 20mL sterile distilled water, shaking table is added On wash film after take out, suck film surface moisture with blotting paper and dried in room temperature；
(S2) result interpretation: in scanning chip on scanner after film is dry, the positive hybridization signal on HPV genetic chip is carried out Signal strength collection analysis.
13. the application of HPV genetic chip according to claim 12, which comprises the steps of:
(S1) PCR reverse dot blot hybridization:
(S1.1) prepared by sample to be tested: extraction sample DNA is simultaneously reversed using 4 forward primers described in claim 7 and 4 Primer and human genome beta globin genes amplimer are expanded, HPV amplimer and human genome beta globin genes The concentration ratio of amplimer is 1:6~1:4, obtains PCR product；
(S1.2) hybridization solution I and hybridization solution II are placed be preheated in water-bath 45 DEG C it is spare, wherein I ingredient of hybridization solution be 2 × SSC and 0.1%SDS, II ingredient of hybridization solution are 0.5 × SSC and 0.1%SDS；
(S1.3) setting constant temperature shaker temperature is 45 DEG C in advance, and the temperature in shaking table is made to reach 45 DEG C of most suitable hybridization temperature；
(S1.4) by PCR product obtained by step (S1.1) after 98 DEG C denatured by boiling, it is immediately placed on incubation 2min or more on ice；
(S1.5) it is put into centrifuge tube with the HPV gene chip film item of aseptic nipper tweezer prehybridization, 5mL is added and preheats hybridization solution I, it is spare；
(S1.6) PCR product of step (S1.4) resulting denaturation is added to the centrifuge tube that film item is placed with obtained by step (S1.5) In, 15min~1h is hybridized in 100rpm, 45 DEG C of constant-temperature table；
(S1.7) hybridization solution I is abandoned, film item is taken to be put into centrifuge tube, 10mL is added and preheats hybridization solution II, in 100rpm, 45 DEG C of perseverance 10~30min of film is washed in warm shaking table, is washed in total twice；
(S1.8) hybridization solution II is abandoned, centrifuge tube is inverted on blotting paper, blots tube bottom liquid, 20mL HRP dilution is added, Wherein HRP dilution be volume ratio HRP: abandon hybridization solution I=1:2000~8000 now match, at room temperature in oscillation shaking table on The revolving speed of 50rpm is protected from light 10min；
(S1.9) it abandons and combines liquid, centrifuge tube is inverted on blotting paper, blot tube bottom liquid, 20mL hybridization solution I is added at room temperature In washing film twice on oscillation shaking table, film 2min is washed every time；
(S1.10) hybridization solution I is abandoned, blotting paper is shelved on desk, centrifuge tube is upside down in above, tube bottom liquid is blotted, adds Enter 20mL TMB developing solution, wherein TMB developing solution is that the mixing of 20 × TMB-A:1 of volume ratio × TMB-B=1:30~100 is made, Reacting at normal temperature without light 5min after colour developing, abandons developing solution immediately, 20mL sterile distilled water is added, after washing film 3min on shaking table It takes out, repetition is washed film one time, is sucked film surface moisture with blotting paper and is dried in room temperature；
(S2) result interpretation: in scanning chip on scanner after film is dry, the positive hybridization signal on HPV genetic chip is carried out Signal strength collection analysis.
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CN111172328A (en) * 2020-03-06 2020-05-19 杭州博日科技有限公司 Complete set of primers, complete set of probes, kit and method for HPV nucleic acid typing detection
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