CN101435002A - Method for detecting human papilloma virogene type - Google Patents
Method for detecting human papilloma virogene type Download PDFInfo
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Abstract
The invention provides a method for detecting the HPV genotypes, with the gene to be detected being one or several types of the type 17 HPV, comprising the following steps: according to the variant sites of the selected HPV genotype universal primer sequence to be detected, an amplification primer aiming at each type is designed; the specific extension primer of each type is designed; (2) PCR amplification is conducted; (3) SAP enzyme treatment is conducted; (4) extension reaction is conducted, among the extending products and the extending primers, the difference of the molecular weight among the extending products of each type is not less than 9D; (5) resin is used to purify extension reaction products; and (6) mass spectrometry detection is conducted, and the type of HPV gene to be detected is determined. By using the method, the invention solves the problems of some of existing detection methods that the typing can not be realized, the multiple infections can not be detected, the accuracy is limited, the throughput is low, the cost is high, and the stability of reaction may be affected as the probe is RNA.
Description
Technical field
The present invention relates to a kind of method that detects human papilloma virogene type.
Background technology
HPV is a kind of small-sized no coating dna virus, the DNA genome with double-stranded closed loop, 7.2-8kb, relative molecular weight 5 * 10
6, the HPV genome can be divided into 3 zones: noncoding upstream regulation district, and early stage opening code-reading frame comprises E6, E7, E1, E2, E4, E5, late period, the open sign indicating number of reading was distinguished L1 (main capsid protein) and L2 (little capsid protein).Opening code-reading frame (ORF) gene order difference according to the coding capsid protein L 1 is carried out somatotype, the difference of L1 district ORF is divided into different genus less than 60%, between 60-70%, be divided into different kinds, difference is positioned at 71-89% and then is divided into different types, it is 130 many types of that the HPV that has identified at present has approximately, according to viral virulence size, be divided into high-risk-type, as HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 etc., can cause cervical intraepithelial neoplasia pathology (cervical intraepithelial, CN), be more common in the women at advanced age, be difficult for natural remission, with being related closely of cervical cancer, low risk HPV has HPV6, HPV11, HPV42, HPV43, HPV44 etc. are more common in young woman, natural remission rate height.
By to the HPV genotype tests, different HPV types can be infected and study more deeply with the relation of cervical lesions, very high research value is arranged.At present HPV detects and mostly is to adopt the s-generation hybrid capture method (HC2) that authenticates by food and drug administration, 13 kinds of high-risk HPVs of this kind method detection, but can not somatotype, can not detect multiple infection.Existing testing product based on the HPV nucleic acid hybridization technique, accuracy rate is limited, and flux is low, the cost height, and most probes is RNA, may influence the stability of reaction.
Summary of the invention
Embodiment of the invention technical problem to be solved is to provide a kind of method that detects human papilloma virogene type, being intended to solve existing detection method can not somatotype, can not detect multiple infection, accuracy rate is limited, and flux is low, the cost height, and, may influence the problem of the stability of reaction because probe is RNA.
The embodiment of the invention is to realize like this, a kind of method that detects human papilloma virogene type, human papillomavirus's gene to be detected is one or more among HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68 and the HPV73, comprises the steps:
(1) according to the variant sites of selected HPV gene type universal primer sequence to be detected, revise the GP5+/GP6+ universal primer, design amplimer at every type, the coupling fully that 12 bases are arranged in other amplimer 3 ' position of each Idiotype, and this amplimer is all with the sequence label of 10 base acgttggatg; Design extension primer, the length of this extension primer are the 17-28 base, comprise in 4 bases of extending base in the position, 3 ' end end of extending primer, forgive a special polymorphic site mark of type; The position of extending the primer selection is zone conservative relatively in the GP5+/GP6+ sequence;
(2), obtain to contain the target sequence amplified production in described mutational site by pcr amplification;
(3) handle by the SAP enzyme, remove the dNTPs that contains in the described amplified production;
(4) by extension, connect a base at the 3 ' end that extends primer, molecular weight difference is not less than 9D between extension products that obtains and the described extension primer, and the molecular weight difference between the extension products of each type is not less than 9D;
(5) adopt resin purification extension product;
(6) product behind the purifying is adopted matrix-assisted laser desorption/ionization flight time mass spectrum system carry out mass spectrometric detection, determine selected HPV gene type to be detected.
Compared with prior art, technique scheme adopts matrix-assisted laser desorption/ionization flight time mass spectrum (MALDI-TOF-MS) system to detect, the reagent consumptive material that uses in mass-spectrometric technique is simple relatively and relatively stable, do not need fluorescence dye, special expensive reagent such as enzyme, reaction can be carried out in micro-system, reduces the use of sample and various running stores.Because mass-spectrometric technique directly detects the molecular weight (mass-to-charge ratio) of DNA, directly determine the type of base, do not need through any type of conversion of signals, as long as there is the SNP segment of a copy to be amplified and can to discern in theory, stopped the possibility that false positive takes place.Mass-spectrometric technique also has automatization, high throughput testing characteristics in addition, mass-spectrometric technique combines with many primer extensions technology, can in a reaction system, detect multitype hpv simultaneously, the PCR reaction can be carried out in micro-system, automatically extract equipment, multiple PCR primer design software and data analysis software in conjunction with DNA, alleviate workload greatly, improved the detection flux.
In addition, the mass spectrum classification system is fit to carry out the HPV detection from the angle of design of primers.The product of PCR be best in the scope of 100-180bp before mass spectrometer system required, and HPV is at the L1 district universal primer GP5+/GP6+ fragment product of 140bp that approximately increases, and the validity that increases is confirmed.The product length of amended primer GP5+/GP6+ amplification satisfies the requirement of mass spectrum to preceding PCR product sheet segment length in this method.Require in the mass spectrometer system extension to extend between primer and the extension products, molecular weight difference between the extension products of each type is not less than 9D, and the special extension primer of above-mentioned 17 type HPV that designs in this method can be distinguished from each other in mass spectrometer system, also can satisfy the requirement of mass spectrometer system, therefore, can detect above-mentioned 17 type HPV genes simultaneously.
Description of drawings
Fig. 1 represents that the HPV of HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV 52, HPV 56, HPV 58, HPV 59, HPV 66, HPV68 and HPV 73 types extends primer peak figure;
Fig. 2 represents HPV6 analytical results figure;
Fig. 3 represents HPV11 analytical results figure;
Fig. 4 represents HPV16 analytical results figure;
Fig. 5 represents HPV18 analytical results figure;
Fig. 6 represents HPV31 analytical results figure;
Fig. 7 represents HPV33 analytical results figure;
Fig. 8 represents HPV35 analytical results figure;
Fig. 9 represents HPV39 analytical results figure;
Figure 10 represents HPV45 analytical results figure;
Figure 11 represents HPV51 analytical results figure;
Figure 12 represents HPV52 analytical results figure;
Figure 13 represents HPV56 analytical results figure;
Figure 14 represents HPV58 analytical results figure;
Figure 15 represents HPV59 analytical results figure;
Figure 16 represents HPV66 analytical results figure;
Figure 17 represents HPV68 analytical results figure;
Figure 18 represents HPV73 analytical results figure;
Figure 19 represents HBB analytical results figure.
Embodiment
In order to make the technical problem to be solved in the present invention, technical scheme and beneficial effect clearer,, the present invention is further elaborated below in conjunction with embodiment.Should be appreciated that specific embodiment described herein only in order to explanation the present invention, and be not used in qualification the present invention.
The method of the detection human papilloma virogene type that the embodiment of the invention provides, human papillomavirus's gene to be detected is one or more among HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV 52, HPV 56, HPV 58, HPV 59, HPV 66, HPV68 and the HPV 73, comprises the steps:
(1) according to the variant sites of selected HPV gene type universal primer sequence to be detected, revise the GP5+/GP6+ universal primer, design amplimer at every type, the coupling fully that 12 bases are arranged in other amplimer 3 ' position of each Idiotype, and this amplimer is all with the sequence label of 10 base acgttggatg; The design extension primer, the length of extending primer is the 17-28 base, comprises in 4 bases of extending base in the position, 3 ' end end of extending primer, forgives a special polymorphic site mark of type; The position of extending the primer selection is zone conservative relatively in the GP5+/GP6+ sequence; Between described extension primer and the extension products, the molecular weight difference between the extension products of each type is not less than 9D.Various HPV amplimer sequence sees Table 1.Corresponding HPV extends primer sequence and quality sees Table 2.Amplimer is the PAGE purifying, and extending primer is the HPLC purifying.
Table 1
| Sequence numbering | Primer sequence | The HPV type |
| SEQ ID NO.1 | acgttggatgTTTGTTACTGTGGTGGAATCTAC | The HPV18/66 forward primer |
| SEQ ID NO.2 | acgttggatgTTTGTTACCGTTGTTGATGCTAC | The HPV16 forward primer |
| SEQ ID NO.3 | acgttggatgTTTGTTACTGTGGTGCATACCTC | The HPV31/33/52 forward primer |
| SEQ ID NO.4 | acgttggatgTTTGTTACTGTTGTGGATAGATC | The HPV35/68 forward primer |
| SEQ ID NO.5 | acgttggatgTTTGTTACTGTTGTGGACACTAC | The HPV39/45 forward primer |
| SEQ ID NO.6 | acgttggatgTTTGTTACTGAGGTAGAATAGGAC | The HPV11/6 forward primer |
| SEQ ID NO.7 | acgttggatgTTTGTTACTGTTCTAGATACTTC | The HPV59/73 forward primer |
| SEQ ID NO.8 | acgttggatgTTTATTACCTGTGTAGATACTAC | The HPV51 forward primer |
| SEQ ID NO.9 | acgttggatgTTTGTTACCGTGGTTGATTCCAC | The HPV58 forward primer |
| SEQ ID NO.10 | acgttggatgTTTGTTACAATAGTAGATACATC | The HPV56 forward primer |
| SEQ ID NO.12 | acgttggatgGAAAAATAAACTGTAAATGATAGTCCT | The HPV16/18/45/68 reverse primer |
| SEQ ID NO.13 | acgttggatgGAAAAATAAATTGTAAATCATAGTC | The HPV39/6 reverse primer |
| SEQ ID NO.14 | acgttggatgGAAATATAAATTGTAAATCATATAC | The HPV52 reverse primer |
| SEQ ID NO.15 | acgttggatgGAAAAATAAACTGTAAATCATATAC | The HPV35 reverse primer |
| SEQ ID NO.16 | acgttggatgGAAATATAAATTGTAAATCGTATTC | The HPV31 reverse primer |
| SEQ ID NO.17 | acgttggatgGAAAAATAAACTGTAAATCATACTC | The HPV73/11 reverse primer |
| SEQ ID NO.18 | acgttggatgGAAAAACAGACTGTAGATGTTATTC | The HPV33 reverse primer |
| SEQ ID NO.19 | acgttggatgGAAAAATAACATGCAATTCTTACTC | The HPV51 reverse primer |
| SEQ ID NO.20 | acgttggatgGAAATATAATCTGCAAATCTATTTC | The HPV59 reverse primer |
| SEQ ID NO.21 | acgttggatgGAAACACAAAGTGTAACGCATCTTC | The HPV58 reverse primer |
| SEQ ID NO.22 | acgttggatgGAAACACAAACTGTGGTGCAAATTC | The HPV66 reverse primer |
| SEQ ID NO.23 | acgttggatgGAAAAACAATATGTAATGCATCTTC | The HPV56 reverse primer |
Table 2
| Sequence numbering | The HPV type | Extend primer sequence | Extend base |
| SEQ ID NO.25 | HPV16 | TTATAGTAAGTTTAATTATATTTGGT | T |
| SEQ ID NO.26 | HPV18 | TAGTACTTTATGATTAAGCTGCA | T |
| SEQ ID NO.27 | HPV31 | ATCACATCTCTGAACTTA | T |
| SEQ ID NO.28 | HPV33 | CTATAAGTCTGCTAGTT | A |
| SEQ ID NO.29 | HPV35 | CTGTGGTGGATTATTATCCTAAG | G |
| SEQ ID NO.30 | HPV39 | TACTCATCTAACCCTTATAGAATTCT | C |
| SEQ ID NO.31 | HPV45 | ATTAGTATCACTACTCTCATAG | G |
| SEQ ID NO.32 | HPV51 | GGGTGAACCCACAGC | A |
| SEQ ID NO.33 | HPV52 | TTTATATTTGCTTTCGCCCCTAG | C |
| SEQ ID NO.34 | HPV56 | TTCAATTCTCCGTGTTCTAATGAACTT | T |
| SEQ ID NO.35 | HPV58 | TGGAAGTCTAACTTTTCTCTTCC | T |
| SEQ ID NO36 | HPV59 | ACCGATTTCCCACAGT | C |
| SEQ ID NO.37 | HPV66 | GGATGTGTTAGACTGTAATTG | C |
| SEQ ID NO.38 | HPV68 | CTACCTCGCAAAATCTCTAAT | C |
| SEQ ID NO.39 | HPV73 | GCTGTAAAGCTAGCGTGTA | T |
| SEQ ID NO.40 | HPV6 | TCACACTTACCTGAATGCGC | T |
| SEQ ID NO.41 | HPV11 | AACATCTACTCTGCTA | A |
(2), obtain to contain the target sequence amplified production in described mutational site by pcr amplification.The configuration of pcr amplification reaction reagent sees Table 3.
Table 3
Reaction conditions is 94 ℃, 15min; 94 ℃ of sex change 20s, 56 ℃ of annealing 30s, 72 ℃ are extended 1min, coamplification 45 circulations; Final 72 ℃ are extended 3min.
(3) handle by SAP digestive ferment (shrimp alkaline phosphotase), remove the dNTPs that contains in the described amplified production.SAP digestive ferment reaction system sees Table 4.
Table 4
| Reagent | Volume/reaction |
| H2O (HPLE level) | 1.53ul |
| SAP enzyme buffer liquid | 0.17ul |
| The SAP enzyme | 0.30ul |
| Volume | 2.0ul |
Reaction conditions is 37 ℃ hatched 40 minutes, removed remaining dNTP in the reaction; 85 ℃ made the SAP enzyme deactivation in 5 minutes.
(4) by extension, connect a base at the 3 ' end that extends primer, molecular weight difference is not less than 9D between extension products that obtains and the described extension primer, and the molecular weight difference between the extension products of each type is not less than 9D.The extension system sees Table 5.
Table 5
*Wherein extension mix carries out the linear relationship adjustment according to the molecular weight size of various primer.
Reaction conditions is 94 ℃, 30s; 94 ℃ of sex change 5s, 52 ℃ of annealing 5s, 80 ℃ are extended 5s, coamplification 40 circulations, 5 partial circulatings are carried out in annealing and extension in each circulation; Final 72 ℃ are extended 3min.
(5) adopt resin purification extension product.
(6) product behind the purifying is adopted matrix-assisted laser desorption/ionization flight time mass spectrum system carry out mass spectrometric detection, determine HPV gene type to be detected.Among Fig. 1-19, X-coordinate is represented the molecular weight size of various extension primer and product among the figure, wherein the dotted line on the left side represents to extend (because the completely consumed of primer molecule amount position, so do not have the peak type), the right dotted line is represented extension products molecular weight position (because extension is arranged, all have the peak type); Ordinate zou is represented the strength of signal of primer.Read peak figure and derived data with TYPE4.0 software.Do not having under the situation about infecting, each type HPV has the peak figure that the extension primer is arranged at the different quality place of mass spectrum with confidential reference items HBB; Under the situation that has HPV to infect, relative HPV has the peak figure of extension products.In a sample, can detect multiple infection.Peak figure reports that the result is divided into 4 kinds: A: reliable results; B: moderate is reliable; C: generally reliable; D: low reliable.It is effective that first three kind is considered as this extension, has corresponding HPV to infect, and the 4th kind because mass spectra peak figure baseline is uneven causes, and is considered as not having the HPV infection.Will be in the concrete sample according to sample information and mass spectra peak figure concrete analysis.
Also comprise the preparation of HPV plasmid standard in the present embodiment.
After using L1 district special primer will comprise the amplified production purifying of L1 district gene fragment of HPV6,11,16,18,31,33,35,39,45,51,52,56,58,59,66,68,73 types, be connected respectively to PMD-T carrier (Takara PMD-T connects test kit), be converted into bacillus coli DH 5 alpha again, the dull and stereotyped coating of LB solid medium was cultivated 12～14 hours, select mono-clonal secondary enlarged culturing by blue hickie screening, use plasmid extraction kit (AXYGEN) to extract and plasmid purification, be accredited as the male plasmid by dna sequencing.
With the HPV 6,11,16,18,31,33,35,39,45,51,52,56,58,59,68,66 for preparing, 73 plasmids are measured the OD value respectively, according to the OD value plasmid are diluted 500pg again, 5pg, the storage liquid of three kinds of concentration of 0.5pg.Then according to the mole formula: the copy number ≈ [1ng*10 of 1ng plasmid
-9/ 2*330* (carrier molecule amount+insertion fragmental molecule amount)] * 6.02*10
23Calculating the copy number of 1ng, is 10 according to this value with the plasmid gradient dilution
5, 10
4, 5000,10
3, 10
2, 10
16 concentration gradients of copies/ul are carried out the sensitivity checking.
The sensitivity of HPV standard substance detects.The dilution of HPV standard substance is 10
5, 10
4, 5000,10
3, 10
2, 6 concentration gradients of 10copies/ul are carried out the sensitivity checking.HPV11,33,35,45,51,52,58,66,68 sensitivity are 50copies/ul; HPV16,18,31 sensitivity are 50copies/ul; HPV 6,39, and 56,59,73 sensitivity are 100copies/ul.
Comprise also in the present embodiment that the HPV reference substance is selected and preparation.
The positive contrast of HPV18 recombinant plasmid (250copies/ul), and mix 10ng/ul do not have HPV infected person genomic dna background after testing.The HPV negative control is aseptic double-distilled water and mouse liver.The positive control and the negative control that in reaction, add design.Quality control standard is referring to table 6.
Table 6
| Quality control product | The result |
| The HPV18 positive control | Only HPV18 has the extension peak |
| Distilled water | There is not the peak of extension |
| The mouse liver | There is not the peak of extension |
Three Quality Control projects are just often in all detections, and it is effective to look the result, otherwise invalid.
In the present embodiment HPV genotype detection confidential reference items with betaglobulin gene (HBB) as the internal reference gene, the quality of monitoring of DNA template and the quality of pcr amplification reaction.The universal primer sequence is:
SEQ ID NO.11acgttggatgACACAACTGTGTTCACTAG
SEQ ID NO.24acgttggatgCAACTTCACCCACGTTCACC，
Extending primer sequence is:
SEQ ID NO.42GGTAGGGCAGATTTCC。
Matrix-assisted laser desorption/ionization flight time mass spectrum of the present invention has highly sensitive, high specific and high-throughout characteristics, and low with respect to other detection method cost.The present invention is adapted to: HPV infects the research of genotype and cancer-related, the monitoring that large-scale epidemiology survey that HPV infects and cervical disease take place and develop.
The above only is preferred embodiment of the present invention, not in order to restriction the present invention, all any modifications of being done within the spirit and principles in the present invention, is equal to and replaces and improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1, a kind of method that detects human papilloma virogene type, human papilloma virogene type to be detected is one or more among HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV 52, HPV 56, HPV58, HPV 59, HPV 66, HPV 68 and the HPV 73, comprises the steps:
(1) according to the variant sites of selected HPV gene type universal primer sequence to be detected, revise the GP5+/GP6+ universal primer, design amplimer at every type, the coupling fully that 12 bases are arranged in other amplimer 3 ' position of each Idiotype, and this amplimer is all with the sequence label of 10 base acgttggatg; The design extension primer, the length of extending primer is the 17-28 base, comprises in 4 bases of extending base in the position, 3 ' end end of extending primer, forgives a special polymorphic site mark of type; The position of extending the primer selection is zone conservative relatively in the GP5+/GP6+ sequence;
(2), obtain to contain the target sequence amplified production in described mutational site by pcr amplification;
(3) handle by the SAP enzyme, remove the dNTPS that contains in the described amplified production;
(4) by extension, connect a base at the 3 ' end that extends primer, molecular weight difference is not less than 9D between extension products that obtains and the described extension primer, and the molecular weight difference between the extension products of each type is not less than 9D;
(5) adopt resin purification extension product;
(6) product behind the purifying is adopted matrix-assisted laser desorption/ionization flight time mass spectrum system carry out mass spectrometric detection, determine HPV gene type to be detected.
2, detect the method for human papilloma virogene type according to claim 1, it is characterized in that, the sequence of other corresponding amplimer of HPV genotype to be detected is in the described step (1):
SEQ ID NO.1 HPV18/66 forward primer
SEQ ID NO.2 HPV16 forward primer
SEQ ID NO.3 HPV31/33/52 forward primer
SEQ ID NO.4 HPV35/68 forward primer
SEQ ID NO.5 HPV39/45 forward primer
SEQ ID NO.6 HPV11/6 forward primer
SEQ ID NO.7 HPV59/73 forward primer
SEQ ID NO.8 HPV51 forward primer
SEQ ID NO.9 HPV58 forward primer
SEQ ID NO.10 HPV56 forward primer
SEQ ID NO.12 HPV16/18/45/68 reverse primer
SEQ ID NO.13 HPV39/6 reverse primer
SEQ ID NO.14 HPV52 reverse primer
SEQ ID NO.15 HPV35 reverse primer
SEQ ID NO.16 HPV31 reverse primer
SEQ ID NO.17 HPV73/11 reverse primer
SEQ ID NO.18 HPV33 reverse primer
SEQ ID NO.19 HPV51 reverse primer
SEQ ID NO.20 HPV59 reverse primer
SEQ ID NO.21 HPV58 reverse primer
SEQ ID NO.22 HPV66 reverse primer
SEQ ID NO.23 HPV56 reverse primer
Extending primer sequence is:
SEQ ID NO.25 HPV16
SEQ ID NO.26 HPV18
SEQ ID NO.27 HPV31
SEQ ID NO.28 HPV33
SEQ ID NO.29 HPV35
SEQ ID NO.30 HPV39
SEQ ID NO.31 HPV45
SEQ ID NO.32 HPV51
SEQ ID NO.33 HPV52
SEQ ID NO.34 HPV56
SEQ ID NO.35 HPV58
SEQ ID NO.36 HPV59
SEQ ID NO.37 HPV66
SEQ ID NO.38 HPV68
SEQ ID NO.39 HPV73
SEQ ID NO.40 HPV6
SEQ ID NO.41 HPV11
3, as the method for detection human papilloma virogene type as described in the claim 2, it is characterized in that, in the described step (1) with the betaglobulin gene as the internal reference gene, the amplimer sequence that adopts is SEQ ID NO.11 and SEQ ID NO.24, and extending primer sequence is SEQ ID NO.42.
4, as the method for detection human papilloma virogene type as described in claim 2 or 3, it is characterized in that described amplimer is the PAGE purifying, described extension primer is the HPLC purifying.
5, detect the method for human papilloma virogene type according to claim 1, it is characterized in that, also be included in and add positive control and negative control in the reaction, described positive control HPV18 recombinant plasmid, and mix and do not have HPV infected person genomic dna background after testing, described negative control is aseptic double-distilled water and mouse liver DNA.
6, detect the method for human papilloma virogene type according to claim 1, it is characterized in that, the preparation process that also comprises the HPV plasmid standard, and the step that adopts these standard substance that primer specificity and sensitivity are tested, described HPV plasmid standard is for inserting the PMD plasmid of human papillomavirus MY09/MY11 sequence.
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