CN101397590A - Typing method for human papilloma virus gene - Google Patents
Typing method for human papilloma virus gene Download PDFInfo
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Abstract
The invention discloses a genotyping method of human papilloma virus, which carries out permutation and combination on the sequences of 25bp behind GP5 of 40 common HPV subtypes by designed software, and the detailed steps are as follows: 1. the sequences of 25bp behind GP5 of a single type of the 40 common HPV subtypes; 2. mixed sequence mode of 25bp behind GP5 after arbitrary two types of the 40 common HPV subtypes are combined; 3. mixed sequence mode of 25bp behind GP5 after arbitrary three types of the 40 common HPV subtypes are combined; 4. mixed sequence mode of 25bp behind GP5 after arbitrary four types of the 40 common HPV subtypes are combined; 5. mixed sequence mode of 25bp behind GP5 after arbitrary five types of the 40 common HPV subtypes are combined; and 6. mixed sequence mode of 25bp behind GP5 after arbitrary six types of the 40 common HPV subtypes are combined. All the modes carry out pairing with the bases following GP5 by the ATCG sequence, and the pairing number is recorded, circulation is carried out in this way until the sequences of 25bp behind GP5 are all paired, then a pyrosequencing mode database infected by 1 to 6 types of different subtypes is created, the sample is carried out DNA extraction and MY09/11 amplification, the amplification product is taken as a template and then carried out amplification by GP5+/6+, then GP5 is taken as a sequencing primer, and the sequencing result is compared with the pyrosequencing mode database to determine the type. The genotyping method has the advantages of low cost, convenient operation, strong specificity and synchronously detecting a plurality of types of inflection.
Description
Technical field
What the present invention relates to is a kind of typing method for human papilloma virus gene, belongs to cma gene diagnostics technical field.
Background of invention
Cervical cancer is one of common gynecologic malignant tumor.According to the world wide statistics, its sickness rate occupies second in women's malignant tumour, be only second to mammary cancer.The cervical cancer new cases in the whole world annual nearly 49.3 ten thousand, 27.4 ten thousand women die from this disease, and wherein 83% case occurs in developing country, accounts for 15% of developing country's female tumor, and only accounts for 3.6% of female tumor in developed country's cervical cancer.The annual new cases 13.15 ten thousand of China account for 1/4th of world's cervical cancer new cases.Through extensively carrying out gynaecology generaI investigation decades, it is nearly 68% that the sickness rate of cervical cancer and mortality ratio have reduced, but from the whole country, the morbidity of cervical cancer still occupies first of gynecologic malignant tumor.And the trend that the year mild case that cervical cancer occurred increases year by year.Therefore the control of cervical cancer becomes the focal point of China's gynecological tumor.Cervical cancer be one multifactor, multistep is rapid, the coefficient result of polygene, wherein high-risk HPV (human papillomavirus) infects WHO and announced it is the primary factor that causes cervical carcinogenesis in 1992.International cancer research institution in 1993 symposium is thought: HPV infect be cervical cancer main diseases because of, and be the important evidence of handling cervical lesions to HPV INFECTION IN DETECTION and somatotype, also be indispensable content in the cervical cancer examination.S-generation hybrid capture (hubrid capture II, HC-II) test is that only up to now acquisition FDA Food and Drug Administration (FDA) authentication is used for clinical HPV detection means, be widely used in clinical, but its shortcoming is can not be specifically to the HPV genotyping, high-risk-type and low risk can not detect simultaneously, can not judge multiple infection.And the HPV somatotype detects the judgement disease type, following up a case by regular visits to after the treatment, and the analysis of the popular situation of HPV and the development of gene vaccine are all significant.At present, surpass 100 kinds of HPV types and differentiated that at least 40 kinds of HPV types are found in anus and reproductive tract mucous membrane, 30 kinds of types are found in cervical cancer.HPV is that standard is divided into high-risk-type and low risk with the pass that whether causes malignant change and plant between system, and high-risk-type comprises HPV16,18,31,33,35,39,45,51,52,56,58,59,68,73,82; Low risk comprises HPV6,11,40,42,43,44,54,61,70,72,81, and CP6108.In addition, HPV26,53,66 may be high-risk-type.Different high-risk-types interrelates with different pathologies, for example, in cervical squamous cancer based on the HPV16 infection, and in gland cancer based on the HPV18 infection.Because methodological restriction, the nosetiology of different type HPV and persistent infection situation are not clear.In addition, the HPV multiple infection is very common in pathology, but its effect in cancer development is also unclear.At the specific type of HPV in the developing vital role of cervix lesion, consider the diversity of HPV type spectrum and the rate occurred frequently of multiple HPV co-infection, HPV detection accurately and typing method seem most important to the treatment of clinical cervix lesion and the prevention of cancer.
Various gene order and its biological behaviour of virus exist high correlation, and the HPV of different genotype has different pathogenic risk.The right decorrelation state of an illness of the detection of HPV DNA and somatotype, judging prognosis and guidance treatment have important value, and be particularly significant to the cancer feelings forecast of female genital tract tumour.Owing to lack vitro culture system and reliable sero-reaction thing, so the classification of HPV almost completely depends on its molecules characteristic.And the reorganization between the different HPV genotype is considerably less, is reliable and stable so analyze as genotype classification with a virus genomic part.The selection that is used for the gene location of viral somatotype is strict, and this position needs sufficient separating capacity and distinguishes all different genotype even hypotypes.According to definition, if specific 291bp has 10% difference just to belong to different HPV types in the L1 open reading frame.At present, a lot of for the research of human papillomavirus genotypes classifying method, mainly contain hybrid method and with the polymerase chain reaction (polymerase chain reaction, PCR) be the basis detection method two big classes.
The hybrid method that hybrid method 1. is traditional: mainly contain southern blotting technique method (Sout hernblot) and in situ hybridization method (in situhybridization, ISH) etc.Southern blot is the gold standard of HPV genetic analysis, this technology once was used for the early stage research of HPV, but this method susceptibility is low, consuming time, need a large amount of highly purified DNA, and the fresh sample of needs, can not be used for the tissue that DNA has degraded, use so be not suitable for the detection of clinical HPV somatotype.ISH can be in histopathology estimation purpose nucleic acid or expression of gene, but that ISH measures the susceptibility of HPV sequence is very low.And the branched DNA that grows up on this basis (branchedDNA, b2DNA) the more common in situ hybridization method of the susceptibility of in situ hybridization method height, when being arranged in cell, the DNA of 1~2 copy just can be detected, very HPV216 and HPV218 are told in the sensitive area, and the accurate localization function is arranged.This method can detect DNA and the mRNA in whole cell and the tissue samples, but sensing range is narrower.
2. novel hybrid method: hybrid capture method: hybrid capture method II (hybridcapt ure II, HC II) is a kind of HPV DNA detection technology of Digene company, HC II method be at present only by the FDA approval can be in the HPVDNA of clinical use detection technique.This technique sensitive is better, and repeatability is high, in the U.S. as the auxiliary examination of Pasteur's method examination cervical cancer, and in some country as the basic skills of cervical cancer examination, can use separately or unite use with Pasteur's method.But it is high that this method requires test set, and the reagent testing cost is more expensive, and can not detect a certain specific type of HPV, can not detect multiple infection.The disadvantage of HC II detection method is to measure concrete HPV type.In addition, there is the problem of cross hybridization in HC II as hybridization, promptly plays cross reaction with the HPV of other in mixed probe type not and causes false positive results and reduce specificity.
Detection method based on PCR
Different with hybrid method is, is to carry out the amplification of goal gene earlier with PCR method based on the detection method of PCR, and then ins all sorts of ways and detect.Think that at present the detection method based on PCR is the best method of carrying out HPV DNA detection and somatotype.1.HPV the pcr amplification of DNA: the amplification to HPV DNA mainly can be divided into type specific PCR and regular-PCR.(1) type specific PCR: the type specific PCR is with type specific PCR primer, and at the maximum zone of variation between type, normally the E5/E7 district is designed to the single HPV genotype of the specific amplification of energy.With can determining other HPV of ad hoc type behind the type specificity PCR, but this method effort in order to detect the existence of HPV DNA in the clinical sample, need be finished the special PCR reaction of a plurality of types respectively.(2) universal primer PCR: universal primer PCR is to use the increase HPV of wide spectrum of universal primer, and primer is at the conservative region between the different HPV types.Determining of this conserved sequence need be from the genotypic many sequences of each HPV.Because the L1 district is the most conservative district in the HPV genome, some normally used universal primers are just at this district at present, as PGMY (about amplification 450bp), the SPF10 (about amplification 65bp) of GP5+/6+ (amplification about 150bp), MY09/11 and improvement thereof, universal primer also had and was located at the E1 district in the past.With the regular-PCR somatotype detection that the back just can in all sorts of ways and carry out HPV the male amplified production of increasing.For improving the susceptibility of regular-PCR, nest-type PRC also commonly used as making template again with GP5+/6+ amplification with MY09/11 amplification back with its amplified production earlier, can detect lower viral level.(3) real-time fluorescence quantitative PCR (real2time quantitativePCR, RQ2PCR): the somatotype for HPV detects, general real-time fluorescence quantitative PCR type specificity primer, be equivalent to the type specific PCR, only many quantitative functions, and can also detect multitype hpv simultaneously based on the real-time fluorescence quantitative PCR of molecular beacon.Two general probes that designed at present can detect the HPV of more than 40 kind of type, and its effect is similar to HC II, can detect the infection of one group of type, but can not determine concrete type, can be applicable to the examination of great amount of samples.But also have and think and can not detect all types fully with 1~2 fluorescent probe, need be than the mixing of multiprobe, but the different qualities of probe causes being difficult for stdn.The nucleic acid crossed contamination can be effectively eliminated in the operation of PCR in real time stopped pipe, has real-time, highly sensitive advantage, and possesses quantitative function.But the somatotype that is used for HPV, the workload of the special PCR in real time of type is too big, and need a large amount of checking and improvement based on the scorpion primer of the PCR in real time of molecular probe and the validity of probe, and the real-time fluorescence PCR experimental cost has also limited its widespread use than higher at present.2.PCR the somatotype of product detects: the product of universal primer PCR need carry out somatotype with various detection methods and detect, and mainly contains direct sequencing, restriction fragment length polymorphism analysis, hybrid method and has very much the gene chips of influence power at present.(1) direct sequencing of PCR product: direct sequencing can be measured the sequence between a pair of primer in the PCR product.Through after the order-checking during, can compare definite type with the known array in the GeneBank database when unidentified sequence<5%; When with other sequence similarity of all known type<90%, can be considered to new type.PCR product direct sequencing can detect all types, and can avoid the inherent cross reaction of hybridization institute.It is a kind of reliable method that the HPV type specificity is detected, particularly to detecting rare type and finding that new type has important effect, but direct sequencing is not high for the not homotactic susceptibility of synchronous detection in the polyinfection, sometimes because thereby other existence of mixed type makes the order-checking time series can't read and can't judge type, poor type is left in the basket easily and only detects prevailing genotype in the sample.Though present quick sequencing can be carried out high-throughout conventional sense to clinical sample, this step of sequential analysis is still time-consuming relatively, expensive.(2) the restriction fragment length polymorphism analysis of PCR product (PCR2restrictionf ragment lengt h polymorp hism, PCR2RFL P): different HPV has different sequences, if it is variant on restriction site, endonuclease then can not be discerned and can not specificity enzymolysis DNA, thereby produce different restriction fragments, produce different electrophoretic band patterns.Select suitable restriction endonuclease, or select the combination of several restriction endonucleases, just can judge the type of HPV according to specific band pattern.This method is relatively simple, does not need the plant and instrument of special expensive, and expense is low.The best of breed of selection restriction endonuclease can detect the concrete type of HPV, particularly can detect the rare type that HC II is not comprised, and when enzyme cut out the band pattern that now can not judge, the binding sequence analysis can be found new type.When the band pattern that produces with the method for PCR RFL P was judged type, software assistance that can simplicity of design was judged, thereby the somatotype of optimizing this method is judged.But RFLP requires than higher the specificity of PCR product, uncertainty after the appearance of non-specific band can cause enzyme to be cut on the somatotype, so the specificity that needs the segmental universal primer of amplification purpose is than higher, and needs to optimize the PCR reaction conditions, increase the specificity of purpose band.(3) hybridization analysis of PCR product: hybridization analysis is the method for the most frequently used detection PCR product sequence.Type specific hybridization method susceptibility is higher, once can detect a plurality of PCR products, and it is higher to the detectivity of multiple infection, but its shortcoming is to be used to detect known certain common type, HPV to unknown type and anomaly can not detect, and can not get rid of hybridization inherent problem, the possibility that promptly has cross hybridization to exist.
Pyrosequencing (tetra-sodium order-checking) technology is a dna sequence analysis technology of new generation, and this technology need not be carried out electrophoresis, and dna fragmentation also need not fluorescent mark, operates very easy.The Pyrosequencing technology is by the enzyme cascade chemiluminescence reaction in 4 kinds of enzymatic same reaction systems, take turns in the sequencing reaction at each, only add a kind of dNTP, if this dNTP and template are matched, polysaccharase just can be incorporated into it in primer strand and the tetra-sodium group (PPi) of mole number such as discharge.PPi can finally be converted into visible light signal, and is converted into a peak value by PyrogramTM.The height of each peak value is directly proportional with the Nucleotide number that mixes in the reaction.Add a kind of dNTP down then, continue the synthetic of DNA chain.Gharizadeh etc. (2001) adopt the tetra-sodium sequencing technologies to 67 routine human papillomavirus (human loapillomaviruses, HPV) sample has carried out evaluation and somatotype, and with tradition order-checking checking mutually, the result proves that this technology also is highly suitable for the research of extensive evaluation, somatotype and the sudden change of pathogenic agent such as HPV.For improving the susceptibility that HPV detects, adopt nest-type PRC, make template again with GP5+/6+ amplification with MY09/11 amplification back with its amplified production earlier, use GP5 as sequencing primer then, the known array in sequencing result and the GeneBank database compares definite type; For solving the multiple infection problem, when the order-checking pattern of using the tetra-sodium sequence measurement to produce is judged type, can assist to judge by design software, thereby the somatotype of optimization this method is judged.Compare with traditional sequence measurement, this method has with low cost, and is easy to operate, can detect the advantage of multiple infection; Compare with hybridizing method, have with low cost, easy to operate, the characteristics of high specificity; Compare for the additive method on basis with PCR, have with low costly, detect the advantage of multiple type simultaneously.
Summary of the invention
The objective of the invention is to overcome the deficiency that prior art exists, improve on the prior art basis and provide a kind of, easy to operate, high specificity can detect the typing method for human papilloma virus gene of multiple infection simultaneously.
The objective of the invention is to finish by following technical solution, it mainly combines the tetra-sodium sequencing technologies and based on the detection method of PCR, by design software the sequence of the common 40 kinds of hypotype GP5 back 25bp of HPV has been carried out permutation and combination, concrete grammar is as follows: the sequence of the single type GP5 back 25bp of 1 common 40 kinds of HPV hypotypes; The mixed sequence pattern of GP5 back 25bp after any two kinds of combinations of 2 common 40 kinds of HPV hypotypes; The mixed sequence pattern of GP5 back 25bp after any three kinds of combinations of 3 common 40 kinds of HPV hypotypes; The mixed sequence pattern of GP5 back 25bp after any four kinds of combinations of 4 common 40 kinds of HPV hypotypes; The mixed sequence pattern of GP5 back 25bp after any five kinds of combinations of 5 common 40 kinds of HPV hypotypes; The mixed sequence pattern of GP5 back 25bp after any six kinds of combinations of 6 common 40 kinds of HPV hypotypes.More than various patterns all with the ATCG order with immediately following the base pairing of GP5 back, and record paired number, so circulation is carried out, and all matches up to the sequence of GP5 back 25bp, has produced the tetra-sodium order-checking pattern database that the heavy different subtype of 1-6 infects; Sample is made template again with GP5+/6+ amplification through DNA extracting, MY09/11 amplification, its amplified production, use GP5 as sequencing primer then, type is judged in sequencing result and the comparison of tetra-sodium order-checking pattern database.
This method can once sequencing be distinguished common 40 hypotypes of HPV and other polyinfection of different shaped, can be used for the clinical diagnosis that HPV infects somatotype; It has with low cost, and is easy to operate, and high specificity can detect the advantage of multiple infection simultaneously.
Embodiment
Below will the present invention will be described in detail by specific embodiment: typing method for human papilloma virus gene of the present invention, it is:
1) by design software the sequence of the common 40 kinds of hypotype GP5 back 25bp of HPV has been carried out permutation and combination, concrete grammar is as follows: the sequence of the single type GP5 back 25bp of 1 common 40 kinds of HPV hypotypes; The mixed sequence pattern of GP5 back 25bp after any two kinds of combinations of 2 common 40 kinds of HPV hypotypes; The mixed sequence pattern of GP5 back 25bp after any three kinds of combinations of 3 common 40 kinds of HPV hypotypes; The mixed sequence pattern of GP5 back 25bp after any four kinds of combinations of 4 common 40 kinds of HPV hypotypes; The mixed sequence pattern of GP5 back 25bp after any five kinds of combinations of 5 common 40 kinds of HPV hypotypes; The mixed sequence pattern of GP5 back 25bp after any six kinds of combinations of 6 common 40 kinds of HPV hypotypes.More than various patterns all with the ATCG order with immediately following the base pairing of GP5 back, and record paired number, so circulation is carried out, and all matches up to the sequence of GP5 back 25bp, has produced the tetra-sodium order-checking pattern database that the heavy different subtype of 1-6 infects;
2) sample DNA extracting;
3) with the human papilloma virus gene group for touching plate, design universal primer MY09/11 and GP5+/6+ respectively, GP5+ biotin labeling wherein, L1 district in the nest-type PRC amplification HPV genome;
4) PCR product strand purifying;
5) check order as the sequencing primer tetra-sodium with GP5;
6) type is judged in the comparison of sequencing result and tetra-sodium order-checking pattern database.
Embodiment 1
1, sample process: the sample on the abundant wash-out Uterine neck bush, and after extracting on the tube wall, abandon Uterine neck bush; Elutriant is all transferred in the 1.5ml Eppendorf tube, and centrifugal 10 minutes of 13000rpm abandons supernatant, keeps the pipe cell lump at the end; Add the 50ul lysate precipitation that suspends, 100 ℃ of heating in water bath 10 minutes.Centrifugal 10 minutes of 13000rpm, it is stand-by to keep supernatant;
2, pcr amplification: pcr amplification adopts nest-type PRC, promptly for the first time with the MY09/MY11 primer to increasing; For the second time with the first time PCR product be masterplate with the GP5+/GP6+ primer to increasing; Concrete PCR system and PCR condition are as follows:
3, sample pretreatment, the strand separation and purification
● before use, guarantee that all solution all reach room temperature;
● add 45 μ l annealing buffer in PSQ 96 plates, every then hole adds GP5+ sequencing primer (10uM) 1ul;
● use Vertex mixing Sepharose beads, the sepharoe beads total amount (every sample 3 μ l) that needs use is transferred in the Eppendorf pipe, in sepharose bead, add binding buffer, make average each sample that the volume of 50 μ l be arranged approximately, with the mixture mixing;
● above mixture is added in the PCR product (50 μ l reaction volume), and every sample 50 μ l with PCR product mixing 10 minutes at normal temperatures, make beads combine with vitamin H;
● in Vacuum prep workstation, add 180ml high purity water, 70% ethanol, washingbuffer and 120ml Denaturation buffer in four sample panel successively;
● open the pump of vacuum prep workstation, vacuum prep tool was cleaned in high purity water 30 seconds, then vacuum prep tool is moved on in the PCR plate, grasp sepharose beads, vacuum prep tool was put into 70% ethanol 5 seconds, moved on to then among the denatureation buffer 5 seconds, move on to again among the washing buffer and cleaned 10 seconds, suction nozzle is placed on the corresponding top of containing the plate hole of sequencing primer, do not contact liquid level, turn off pump, vacuum preptool is put into the plate that contains sequencing primer, shake, discharge sepharose beads);
● use high purity water to clean vacuum prep tool.With PSQ 96 plates that are placed with sample be placed on be heated on the ThermoPlate 80 ℃ 2 minutes, put into sequenator behind the cool to room temperature again;
4, operation pyrosequencing program is clicked Run, carries out the tetra-sodium order-checking;
5, analytical results: type is judged in sequencing result and the comparison of tetra-sodium order-checking pattern database.
The present invention can once sequencing distinguish common 40 hypotypes of HPV and other polyinfection of different shaped, is used for the clinical diagnosis that HPV infects somatotype.
Claims (1)
1, a kind of typing method for human papilloma virus gene, it mainly combines the tetra-sodium sequencing technologies and based on the detection method of PCR, it is characterized in that by design software the sequence of the common 40 kinds of hypotype GP5 back 25bp of HPV having been carried out permutation and combination, concrete grammar is as follows: the sequence of the single type GP5 back 25bp of 1 common 40 kinds of HPV hypotypes; The mixed sequence pattern of GP5 back 25bp after any two kinds of combinations of 2 common 40 kinds of HPV hypotypes; The mixed sequence pattern of GP5 back 25bp after any three kinds of combinations of 3 common 40 kinds of HPV hypotypes; The mixed sequence pattern of GP5 back 25bp after any four kinds of combinations of 4 common 40 kinds of HPV hypotypes; The mixed sequence pattern of GP5 back 25bp after any five kinds of combinations of 5 common 40 kinds of HPV hypotypes; The mixed sequence pattern of GP5 back 25bp after any six kinds of combinations of 6 common 40 kinds of HPV hypotypes.More than various patterns all with the ATCG order with immediately following the base pairing of GP5 back, and record paired number, so circulation is carried out, and all matches up to the sequence of GP5 back 25bp, has produced the tetra-sodium order-checking pattern database that the heavy different subtype of 1-6 infects; Sample is made template again with GP5+/6+ amplification through DNA extracting, MY09/11 amplification, its amplified production, use GP5 as sequencing primer then, type is judged in sequencing result and the comparison of tetra-sodium order-checking pattern database.
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Application publication date: 20090401 |