CN105483249A - Method for absolute quantitative detection of number of copies of type-16 and type-18 HPV (human papillomavirus) virus genes - Google Patents
Method for absolute quantitative detection of number of copies of type-16 and type-18 HPV (human papillomavirus) virus genes Download PDFInfo
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Abstract
The invention provides a method for absolute quantitative detection of the number of copies of type-16 and type-18 HPV (human papillomavirus) virus genes. The method comprises the following steps: retrieving type-16 and type-18 HPV genome sequences in the NCBI (National Center for Biotechnology Information) to obtain E6/E7 gene sequence information of HPV; constructing E6/E7 standard plasmids of type-16 and type-18 HPV in 31-934 site areas and 41-981 site areas respectively; designing E6 and E7 fluorescent quantitative PCR (polymerase chain reaction) specific primers of the type-16 and type-18 HPV respectively in reference to the gene sequences of the obtained E6/E7 orders; and detecting the number of copies of the type-16 and type-18 HPV viruses of tissue samples of cervical carcinoma patients respectively by using the obtained E6/E7 standard plasmids of the type-16 and type-18 HPV. The establishment of the detection system can achieve the aim of performing absolute quantification on the number of copies of HPV DNA in tissues of the cervical carcinoma patients by detecting E6 genes or detecting E7 gene, thereby providing references for early prevention and early treatment of cervical carcinoma.
Description
Technical field
The present invention relates to viral diagnosis technical field, be specifically related to the absolute quantitation detection method of 16 types, 18 type HPV virogene copy numbers.
Background technology
Human papillomavirus (humanpapillomavirus, HPV) is the important factor causing cervical cancer.In the world, annual new cases about 500,000 people of cervical cancer, dead number of cases about 250,000 people, in gynecological tumor, sickness rate occupies the 2nd.
HPV is spherical DNA virus, its full-length genome is about 8000bp, comprise 6 open reading frame (open-readingframe of early gene (E1, E2, E4, E5, E6 and E7), ORF), 2 open reading frames of late gene (L1, L2) and 1 non-coding LCR (non-codinglongcontrolregion, LCR), wherein, E6, E7 gene is two main proto-oncogenes.
HPV main infection skin and mucous membrane tesselated epithelium, be divided into 2 large class, skin-type and mucosal patterns.The mucosal epithelium of mucosal pattern main infection people urogenital tract, causes the various diseases relevant to urogenital organ.According to the relation size occurred with cancer, mucosal pattern HPV is divided into high-risk-type (high-risk, HR) and low risk (low-risk, LR).High-risk-type mainly contain HPV-16,18,31,33,35,39,45,51,52,56,58,59 and 66 types; Low risk mainly contain HPV-6,11,40,42,43,44,54,61,70,72 and 81 types.Molecular epidemiology and virological investigation find, HPV infects uterine neck can develop into cervical cancer, and relevant with HPV type, in Cervix Squamous Cell cancer patient, HPV-16 type and HPV-18 type are main infection hypotype.
At present, polymerase chain reaction (PolymeraseChainReaction, PCR) is mainly contained to the conventional pathogeny detection method of human papillomavirus, nucleic acid hybridization detects (comprising nucleic acid blot in situ hybridization, dot hybridization, original flavor hybridization, hybrid capture etc.).Detection method of nucleic acid hybridization complicated operation, and PCR detection method is easy and simple to handle and sensitivity is higher, can carry out HPV somatotype, PCR in real time detects tentatively can carry out relative quantification to viral copy number.But the method is not owing to designing standard control reference product and the copy number counting system of viral copy number, and the specifying information thus for the absolute copy number of virus fails to know.
Summary of the invention
The present invention is intended to for 16 types, the gene constructed standard substance plasmid of 18 type HPVE6/E7, set up 16 types, 18 type HPV quantitative fluorescent PCR absolute quantitation systems, the foundation of this detection system both can by having detected E6 gene, again can by detecting E7 gene, conclude that cervical cancer patient is subject to HPV virus infection fast for clinical, and absolute quantitation is carried out to HPVDNA copy number in cervical cancer patient tissue, thus provide technical support for preventing the morning of cervical cancer, early controlling.
The present invention is realized by following technical proposal: the absolute quantitation detection method of a kind of 16 types, 18 type HPV virogene copy numbers, through following each step:
(1) 16 types, 18 type HPVE6/E7 standard substance plasmids are built:
A. according to 16 type HPV (NCBIReferenceSequence:NC_001526.2) in ncbi database and 18 type HPV (GenBank:AY262282.1) gene order, for E6/E7 district, by following design Auele Specific Primer:
The forward and reverse primer of PCR of 16 type HPVE6/E7:
HPV16E6/E7For：5'-CGTAACCGAAATCGGTTGAAC-3'
HPV16E6/E7Rev：5'-CAGCCTCTACATAAAACCATC-3'
The forward and reverse primer of PCR of 18 type HPVE6/E7:
HPV18E6/E7For：5'-AACCGAAAACGGTCGGGACC-3'
HPV18E6/E7Rev：5'-TAGCTTGTACATAAAACCAGC-3'；
B. the extraction of STb gene in cervical cancer tissues: first 1mLHPV tissue preserration liquid is sucked in the EP pipe of 1.5mL sterilizing, with 13000rpm centrifugal 5min at 4 DEG C; Abandon supernatant, in precipitation, add 1mLTris-HCl lavation buffer solution (pH8.1), with 13000rpm centrifugal 5min at 4 DEG C; Abandon supernatant, then add 1mLTris-HCl lavation buffer solution, with 13000rpm centrifugal 5min at 4 DEG C, abandon supernatant; In precipitation, add the Proteinase K lysate that 200 μ L concentration are 0.1mg/mL, digest at 50 DEG C and spend the night; Then with 95 DEG C of denatured by boiling 10min of boiling water; Again with 13000rpm centrifugal 5min at 4 DEG C, supernatant is transferred in 0.5mLEP pipe, frozen at-70 DEG C, obtain 16 types, 18 type HPV genomic dnas;
C. by step b gained 16 type, 18 type HPV genomic dnas by the method for PCR, the Auele Specific Primer of step a is utilized to increase respectively the E6/E7 of 16 types, 18 types, obtain corresponding amplified production respectively, again amplified production is connected respectively on pMD-18TVector cloning vector, transform DH5 α competent cell, choose mono-clonal and carry out order-checking qualification, obtain 16 types, positive strain that 18 type HPV goal gene Sequence Identification are correct respectively;
D. positive strain correct for step c gained Sequence Identification is carried out cellar culture respectively, obtain 16 types, 18 types and 58 type HPV standard substance plasmids respectively, and the concentration of 16 types, 18 type HPV standard substance plasmids is measured respectively with ultraviolet spectrophotometer, then press the copy number of following formulae discovery standard substance plasmid:
Numberofcopies(copies/μl)＝(Amount×6.022×10
23)/(Length×1×10
9×650)
Wherein, Amount is surveyed content (ng/ μ l) by ultraviolet spectrophotometer records standard substance plasmid, and Length represents template DNA length;
(2) the quantitative fluorescent PCR Auele Specific Primer of following 16 types, 18 type HPVE6 and E7 is designed respectively with reference to step (1) c gained 16 type, 18 type HPV goal gene sequences:
16 types: HPV16E6For:5'-TGCGACGTGAGGTATATGACTTTG-3'
HPV16E6Rev：5'-ACGGTTTGTTGTATTGCTGTTCTA-3'
16 types: HPV16E7For:5'-TTAGATTTGCAACCAGAGACA-3'
HPV16E7Rev：5'-GCACAACCGAAGCGTAGAGTC-3'
18 types: HPV18E6For:5'-ACATTGGAAAAACTAACTAAC-3'
HPV18E6rev：5'-TGCAGCACGAATGGCACTGG-3'
18 types: HPV18E7For:5'-AATTCCGGTTGACCTTCTATG-3'
HPV18E7Rev：5'-GCTCAATTCTGGCTTCACACTT-3'
(3) utilize step (1) d gained 16 type, 18 type HPV standard substance plasmids detect E6 and E7 copy number in cervical cancer tissues respectively, concrete detection method is as follows:
A. step (1) d gained 16 type, 18 type HPV standard substance plasmids are pressed gradient dilution to 10 with distilled water respectively
-6by PCR reaction system with SYBR method real-time fluorescence quantitative PCR examination criteria product plasmid amplification efficiency and specificity, 16 types, the amplification curve of 18 type HPV standard substance plasmids and solubility curve is directly obtained by fluorescence quantitative PCR detection, simultaneously according to fluorescence quantitative PCR detection gained standard substance Ct value as X-axis, standard substance copy number log
10draw the typical curve of 16 types, 18 type HPV standard substance plasmids as Y-axis respectively, and then obtain the typical curve equation of 16 types, 18 type HPV standard substance plasmids:
Described PCR reaction system is as follows:
Table 1 quantitative fluorescent PCR reaction system (reaction cumulative volume is 20 μ l)
The response procedures adopted is as follows:
B. the PCR reaction system provided by step (3) a detects cervical cancer tissues DNA with SYBR method real-time fluorescence quantitative PCR, utilize standard substance plasmid to obtain typical curve and typical curve equation according to step (3) a respectively, and then obtain the copy number of HPVDNA in cervical cancer tissues.
Gradient dilution to 10 is pressed with distilled water in described step (3) a
-6refer to and 16 types, 18 type HPV standard substance plasmids are diluted to Gradient with distilled water respectively: 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6.
The present invention is by retrieval NCBI16 type, 18 type HPV genome sequences, for 16 types, 18 type HPVE6/E7 genes, standard substance plasmid is built respectively with 31-934 site areas and 41-980 site areas, by 16 types, 18 type HPVE6/E7 gene regions are building up on a carrier respectively, set up 16 types and 18 type HPV quantitative fluorescent PCR absolute quantitation systems, the foundation of this detection system both can by having detected E6 gene, again can by detecting E7 gene, conclude that cervical cancer patient is subject to HPV virus infection fast for clinical, and absolute quantitation is carried out to HPV viral copy number in cervical cancer patient tissue, thus the morning of cervical cancer is prevented, early control and reference is provided.
The advantage that the present invention possesses and effect: the present invention be directed to 16 types, 18 type HPVE6/E7 gene regions build standard substance plasmid, be building up on a carrier respectively by 16 types and 18 type HPVE6/E7 gene regions, set up 16 types and 18 type HPV quantitative fluorescent PCR absolute quantitation systems, the foundation of this detection system both can by having detected E6 gene, again can by detecting E7 gene, reach the object of the HPVDNA copy number in cervical cancer patient tissue being carried out to absolute quantitation, thus provide reference to preventing the morning of cervical cancer, early controlling.
Accompanying drawing explanation
Fig. 1 is 16 types, 18 type HPVE6/E7 regional gene pcr amplified fragments; In figure, M:markerDL1200;
Fig. 2 is the amplification curve of the 16 type HPV standard substance plasmid E6 genes that embodiment 1 fluorescence quantitative PCR detection directly obtains;
Fig. 3 is the solubility curve of the 16 type HPV standard substance plasmid E6 genes that embodiment 1 fluorescence quantitative PCR detection directly obtains
Fig. 4 is the typical curve of 16 type HPV standard substance plasmid E6 genes of embodiment 1;
Fig. 5 is the amplification curve of the 16 type HPV standard substance plasmid E7 genes that embodiment 1 fluorescence quantitative PCR detection directly obtains;
Fig. 6 is the solubility curve of the 16 type HPV standard substance plasmid E7 genes that embodiment 1 fluorescence quantitative PCR detection directly obtains
Fig. 7 is the typical curve of 16 type HPV standard substance plasmid E7 genes of embodiment 1;
Fig. 8 be embodiment 1 fluorescence quantitative PCR detection cervical cancer tissues sample directly obtain 16 type HPVE6, E7 amplification curve;
Fig. 9 be embodiment 1 fluorescence quantitative PCR detection cervical cancer tissues sample directly obtain 16 type HPVE6, E7 solubility curve;
Figure 10 is the amplification curve of the 18 type HPV standard substance plasmid E6 genes that embodiment 2 fluorescence quantitative PCR detection directly obtains;
Figure 11 is the solubility curve of the 18 type HPV standard substance plasmid E6 genes that embodiment 2 fluorescence quantitative PCR detection directly obtains
Figure 12 is the typical curve of 18 type HPV standard substance plasmid E6 genes of embodiment 2;
Figure 13 is the amplification curve of the 18 type HPV standard substance plasmid E7 genes that embodiment 2 fluorescence quantitative PCR detection directly obtains;
Figure 14 is the solubility curve of the 18 type HPV standard substance plasmid E7 genes that embodiment 2 fluorescence quantitative PCR detection directly obtains
Figure 15 is the typical curve of 18 type HPV standard substance plasmid E7 genes of embodiment 2;
Figure 16 be embodiment 2 fluorescence quantitative PCR detection cervical cancer tissues sample directly obtain 18 type HPVE6, E7 amplification curve;
Figure 17 be embodiment 2 fluorescence quantitative PCR detection cervical cancer tissues sample directly obtain 18 type HPVE6, E7 solubility curve.
Embodiment
Below by embodiment, the present invention is described further.
Embodiment 1
(1) 16 type HPVE6/E7 standard substance plasmids are built:
A. according to the sequence (NCBIReferenceSequence:NC_001526.2) of 16 type HPVE6/E7 in ncbi database, Auele Specific Primer is designed by table 2, amplification E6/E7 district gene order:
The PCR primer in table 216 type HPVE6/E7 region
B. total DNA is extracted in cervical cancer tissues:
First 1mLHPV tissue preserration liquid is sucked in the EP pipe of 1.5mL sterilizing, 13000rpm4 DEG C of centrifugal 5min; Abandon supernatant, in precipitation, add 1mLTris-HCl lavation buffer solution (pH8.1), 13000rpm4 DEG C of centrifugal 5min; Abandon supernatant, then add 1mLTris-HCl lavation buffer solution, 13000rpm4 DEG C of centrifugal 5min, as far as possible residual to the greatest extent liquid; In precipitation, add 200 μ l0.1mg/mL Proteinase K lysates, 50 DEG C of digestion are spent the night; 95 DEG C of denatured by boiling 10min of boiling water; The centrifugal 5min of 13000rpm, be transferred to by supernatant in 0.5mLEP pipe ,-70 DEG C are frozen, obtain 16 types, 18 type HPV genomic dnas;
C. by the method for step b gained 16 type HPV genome by PCR, utilize the primer amplified 16 type HPVE6/E7 gene of step (1) a, obtain corresponding amplified production, again amplified production is connected on pMD-18TVector cloning vector, transform DH5 α competent cell, choose mono-clonal and carry out order-checking qualification, obtain the positive strain that 16 type HPV goal gene Sequence Identification are correct;
D. positive strain correct for step (1) c gained Sequence Identification is carried out cellar culture, obtain 16 type HPVE6/E7 standard substance plasmids, and the concentration of 16 type HPVE6/E7 standard substance plasmids is measured with ultraviolet spectrophotometer, then press the copy number of following formulae discovery standard substance plasmid:
Numberofcopies(copies/μl)＝(Amount×6.022×10
23)/(Length×1×10
9×650)
Wherein, Amount is surveyed content (ng/ μ l) by ultraviolet spectrophotometer records standard substance plasmid, and Length represents template DNA length, and 16 type HPVE6/E7 standard substance plasmid DNALength are 3596bp.In this embodiment, it is 180ng/ μ l that ultraviolet spectrophotometer records 16 type HPVE6/E7 standard substance plasmid Amount, and calculating 16 type HPVE6/E7 standard substance plasmid copy numbers is 4.64 × 10
10copies/ μ l.
(2) with reference to the quantitative fluorescent PCR Auele Specific Primer of E6 and E7 of step (1) c gained 16 type HPVE6/E7 gene goal gene sequences Design table 3:
Table 316 type HPVE6, E7 fluorescence quantification PCR primer
(3) utilize 16 type HPVDNA copy numbers in step (1) d gained 16 type HPVE6/E7 standard substance plasmids detection cervical cancer tissues, concrete detection method is as follows:
A. step (1) d gained 16 type HPVE6/E7 standard substance plasmid distilled water is pressed gradient dilution to 10
-6, namely with Gradient: 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6use 16 type HPVE6, E7 fluorescence quantification PCR primer respectively, by table 4PCR reaction system with SYBR method real-time fluorescence quantitative PCR examination criteria product plasmid amplification efficiency and specificity, the amplification curve (see Fig. 2 and Fig. 5) of 16 type HPVE6/E7 standard substance plasmids detection E6, E7 genes and solubility curve (see Fig. 3 and Fig. 6) is obtained respectively by fluorescence quantitative PCR detection, simultaneously according to fluorescence quantitative PCR detection gained standard substance Ct value as X-axis, standard substance copy number log
10(see table 5, table 6) draws the typical curve (see Fig. 4 and Fig. 7) of 16 type HPVE6/E7 standard substance plasmids as Y-axis, and then obtains the typical curve equation of 16 type HPVE6/E7 standard substance plasmids detection E6, E7 genes respectively: y=-0.3011x+12.003 and y=-0.3183x+12.323.
Described PCR reaction system is as table 4:
Table 4 quantitative fluorescent PCR reaction system (reaction cumulative volume is 20 μ l)
The response procedures adopted is as follows:
Table 516 type HPV standard substance E6 gene test result
Table 616 type HPV standard substance E7 gene test result
B. the PCR reaction system provided by step (3) a detects the STb gene extracted in cervical cancer tissues with SYBR method real-time fluorescence quantitative PCR, obtain amplification curve (see Fig. 8) and the solubility curve (see Fig. 9) of 16 type HPVE6, E7 in cervical cancer tissues sample, utilize standard substance plasmid to obtain typical curve and typical curve equation (see Fig. 4 and Fig. 7) according to step (3) a, and then obtain 16 type HPVDNA copy numbers (see table 7 and table 8) in cervical cancer tissues:
16 type HPVE6 gene test results in table 7 cervical cancer tissues sample
16 type HPVE7 gene test results in table 8 cervical cancer tissues sample
Embodiment 2
(1) 18 type HPVE6/E7 standard substance plasmids are built:
A. according to the sequence (GenBank:AY262282.1) of 18 type HPVE6/E7 in ncbi database, Auele Specific Primer is designed by table 9, amplification E6/E7 district gene order:
The PCR primer in table 918 type HPVE6/E7 region
B. the extraction of STb gene in cervical cancer tissues: first 1mLHPV tissue preserration liquid is sucked in the EP pipe of 1.5mL sterilizing, 13000rpm4 DEG C of centrifugal 5min; Abandon supernatant, in precipitation, add 1mLTris-HCl lavation buffer solution (pH8.1), 13000rpm4 DEG C of centrifugal 5min; Abandon supernatant, then add 1mLTris-HCl lavation buffer solution, 13000rpm4 DEG C of centrifugal 5min, as far as possible residual to the greatest extent liquid; In precipitation, add 200 μ l0.1mg/mL Proteinase K lysates, 50 DEG C of digestion are spent the night; 95 DEG C of denatured by boiling 10min of boiling water; The centrifugal 5min of 13000rpm, be transferred to by supernatant in 0.5mLEP pipe ,-70 DEG C are frozen, obtain 16 types, 18 type HPV genomic dnas;
C. by the method for step b gained 18 type HPV genome by PCR, utilize the primer amplified 18 type HPVE6/E7 gene of step (1) a, obtain corresponding amplified production, again amplified production is connected on pMD-18TVector cloning vector, transform DH5 α competent cell, choose mono-clonal and carry out order-checking qualification, obtain the positive strain that 18 type HPV goal gene Sequence Identification are correct;
D. positive strain correct for step (1) c gained Sequence Identification is carried out cellar culture, obtain 18 type HPVE6/E7 standard substance plasmids, and the concentration of 18 type HPVE6/E7 standard substance plasmids is measured with ultraviolet spectrophotometer, then press the copy number of following formulae discovery standard substance plasmid:
Numberofcopies(copies/μl)＝(Amount×6.022×10
23)/(Length×1×10
9×650)
Wherein, Amount is surveyed content (ng/ μ l) by ultraviolet spectrophotometer records standard substance plasmid, and Length represents template DNA length, and 18 type HPVE6/E7 standard substance plasmid DNALength are 3632.In this embodiment, it is 276ng/ μ l that ultraviolet spectrophotometer records 18 type HPVE6/E7 standard substance plasmid Amount, and calculating 18 type HPVE6/E7 standard substance plasmid copy numbers is 7.04 × 10
10copies/ μ l.
(2) design the quantitative fluorescent PCR Auele Specific Primer of E6 and E7 by table 10 with reference to step (1) c gained 18 type HPVE6, E7 district goal gene sequence:
Table 1018 type HPVE6, E7 fluorescence quantification PCR primer
(3) utilize 18 type HPV viral copy number in step (1) d gained 18 type HPVE6/E7 standard substance plasmids detection cervical cancer tissues, concrete detection method is as follows:
A. step (1) d gained 18 type HPVE6, E7 standard substance plasmid distilled water are pressed gradient dilution to 10
-6, namely with Gradient: 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6use 18 type HPVE6, E7 fluorescence quantification PCR primer in table 10 respectively, by table 11PCR reaction system with the amplification efficiency of SYBR method real-time fluorescence quantitative PCR examination criteria product plasmid E6, E7 gene and specificity, the amplification curve (see Figure 10 and Figure 13) of 18 type HPVE6/E7 standard substance plasmids detection E6, E7 genes and solubility curve (see Figure 11 and Figure 14) is obtained respectively by fluorescence quantitative PCR detection, simultaneously according to fluorescence quantitative PCR detection gained standard substance Ct value as X-axis, standard substance copy number log
10(see table 12 and table 13) draws the typical curve (see Figure 12 and Figure 15) of 18 type HPVE6/E7 standard substance plasmids as Y-axis, and then obtains the typical curve equation of 18 type HPVE6/E7 standard substance plasmids detection E6, E7 genes respectively: y=-0.1785x+12.652 and y=-0.4807x+13.525.
Described PCR reaction system is as table 11:
Table 11 quantitative fluorescent PCR reaction system (reaction cumulative volume is 20 μ l)
The response procedures adopted is as follows:
Table 1218 type HPV standard substance E6 gene test result
Table 1318 type HPV standard substance E7 gene test result
B. the PCR reaction system provided by step (3) a detects the STb gene extracted in cervical cancer tissues with SYBR method real-time fluorescence quantitative PCR, obtain amplification curve (see Figure 16) and the solubility curve (see Figure 17) of 18 type HPVE6, E7 in cervical cancer tissues sample, utilize standard substance plasmid to obtain typical curve and typical curve equation (see Figure 12 and Figure 15) according to step (3) a, and then obtain 18 type HPVDNA copy numbers (see table 14 and table 15) in cervical cancer tissues sample:
18 type HPVE6 gene test results in table 14 cervical cancer tissues sample
Sequence table
The PCR forward primer (HPV16E6/E7For) in 16 type HPVE6/E7 regions:
CGTAACCGAAATCGGTTGAAC21
The PCR reverse primer (HPV16E6/E7Rev) in 16 type HPVE6/E7 regions:
CAGCCTCTACATAAAACCATC21
16 type HPVE6/E7 regions goal gene PCR total length (HPV16E6/E7):
The quantitative fluorescent PCR forward primer (HPV16E6For) in 16 type HPVE6 regions:
TGCGACGTGAGGTATATGACTTTG24
The quantitative fluorescent PCR reverse primer (HPV16E6Rev) in 16 type HPVE6 regions:
ACGGTTTGTTGTATTGCTGTTCTA24
16 type HPVE6 regions goal gene PCR total length (HPV16E6):
The quantitative fluorescent PCR forward primer (HPV16E7For) in 16 type HPVE7 regions:
TTAGATTTGCAACCAGAGACA21
The quantitative fluorescent PCR reverse primer (HPV16E7Rev) in 16 type HPVE7 regions:
GCACAACCGAAGCGTAGAGTC21
16 type HPVE7 regions goal gene PCR total length (HPV16E7):
The PCR forward primer (HPV18E6/E7For) in 18 type HPVE6/E7 regions:
AACCGAAAACGGTCGGGACC20
The PCR reverse primer (HPV18E6/E7Rev) in 18 type HPVE6/E7 regions:
TAGCTTGTACATAAAACCAGC21
18 type HPVE6/E7 regions goal gene PCR total length (HPV18E6/E7):
The quantitative fluorescent PCR forward primer (HPV18E6For) in 18 type HPVE6 regions:
ACATTGGAAAAACTAACTAAC21
The quantitative fluorescent PCR reverse primer (HPV18E6Rev) in 18 type HPVE6 regions:
TGCAGCACGAATGGCACTGG20
18 type HPVE6 regions goal gene PCR total length (HPV18E6):
The quantitative fluorescent PCR forward primer (HPV18E7For) in 18 type HPVE7 regions:
AATTCCGGTTGACCTTCTATG21
The quantitative fluorescent PCR reverse primer (HPV18E7Rev) in 18 type HPVE7 regions:
GCTCAATTCTGGCTTCACACTT22
18 type HPVE7 regions goal gene PCR total length (HPV18E7):
。
Claims (2)
1. an absolute quantitation detection method for 16 types, 18 type HPV virogene copy numbers, is characterized in that through following each step:
(1) 16 types, 18 type HPVE6/E7 standard substance plasmids are built:
A. according to 16 type HPV(NCBIReferenceSequence:NC_001526.2 in ncbi database) and 18 type HPV(GenBank:AY262282.1) gene order, for E6/E7 gene regions, by following design Auele Specific Primer:
The forward and reverse primer of PCR of 16 type HPVE6/E7:
HPV16E6/E7For：5'-CGTAACCGAAATCGGTTGAAC-3'
HPV16E6/E7Rev：5'-CAGCCTCTACATAAAACCATC-3'
The forward and reverse primer of PCR of 18 type HPVE6/E7:
HPV18E6/E7For：5'-AACCGAAAACGGTCGGGACC-3'
HPV18E6/E7Rev：5'-TAGCTTGTACATAAAACCAGC-3'；
B. the extraction of STb gene in cervical cancer tissues: first 1mLHPV tissue preserration liquid is sucked in the EP pipe of 1.5mL sterilizing, with 13000rpm centrifugal 5min at 4 DEG C; Abandon supernatant, in precipitation, add 1mLTris-HCl lavation buffer solution, with 13000rpm centrifugal 5min at 4 DEG C; Abandon supernatant, then add 1mLTris-HCl lavation buffer solution, with 13000rpm centrifugal 5min at 4 DEG C, abandon supernatant; In precipitation, add the Proteinase K lysate that 200 μ L concentration are 0.1mg/mL, digest at 50 DEG C and spend the night; Then with 95 DEG C of denatured by boiling 10min of boiling water; Again with 13000rpm centrifugal 5min at 4 DEG C, supernatant is transferred in 0.5mLEP pipe, frozen at-70 DEG C, obtain 16 types, 18 type HPV genomic dnas;
C. by step b gained 16 type, 18 type HPV genomic dnas by the method for PCR, the Auele Specific Primer of step a is utilized to increase respectively 16 types, 18 type HPVE6/E7 genes, obtain corresponding amplified production respectively, again amplified production is connected respectively on pMD-18TVector cloning vector, transform DH5 α competent cell, choose mono-clonal and carry out order-checking qualification, obtain 16 types, positive strain that 18 type HPV goal gene Sequence Identification are correct respectively;
D. positive strain correct for step (1) c gained Sequence Identification is carried out cellar culture respectively, obtain 16 types, 18 type HPV standard substance plasmids respectively, and the concentration of 16 types, 18 type HPV standard substance plasmids is measured respectively with ultraviolet spectrophotometer, then press the copy number of following formulae discovery standard substance plasmid:
Numberofcopies（copies/μl）=(Amount×6.022×10
23)/（Length×1×10
9×650）
Wherein, Amount is surveyed content (ng/ μ l) by ultraviolet spectrophotometer records standard substance plasmid, and Length represents template DNA length;
(2) the quantitative fluorescent PCR Auele Specific Primer of following 16 types, 18 type HPVE6 and E7 is designed respectively with reference to step (1) c gained 16 type, 18 type HPV goal gene sequences:
16 types: HPV16E6For:5'-TGCGACGTGAGGTATATGACTTTG-3'
HPV16E6Rev：5'-ACGGTTTGTTGTATTGCTGTTCTA-3'
16 types: HPV16E7For:5'-TTAGATTTGCAACCAGAGACA-3'
HPV16E7Rev：5'-GCACAACCGAAGCGTAGAGTC-3'
18 types: HPV18E6For:5'-ACATTGGAAAAACTAACTAAC-3'
HPV18E6Rev：5'-TGCAGCACGAATGGCACTGG-3'
18 types: HPV18E7For:5'-AATTCCGGTTGACCTTCTATG-3'
HPV18E7Rev：5'-GCTCAATTCTGGCTTCACACTT-3'
(3) utilize step (1) d gained 16 type, 18 type HPV standard substance plasmids detect E6 and E7 copy number in cervical cancer tissues respectively, concrete detection method is as follows:
A. step (1) d gained 16 type, 18 type HPV standard substance plasmids are pressed gradient dilution to 10 with distilled water respectively
-6by PCR reaction system with SYBR method real-time fluorescence quantitative PCR examination criteria product plasmid amplification efficiency and specificity, 16 types, the amplification curve of 18 type HPV standard substance plasmids and solubility curve is directly obtained by fluorescence quantitative PCR detection, simultaneously according to fluorescence quantitative PCR detection gained standard substance Ct value as X-axis, standard substance copy number log
10draw the typical curve of 16 types, 18 type HPV standard substance plasmids as Y-axis respectively, and then obtain the typical curve equation of 16 types, 18 type HPV standard substance plasmids:
Described PCR reaction system is as table 1:
Table 1 quantitative fluorescent PCR reaction system (reaction cumulative volume is 20 μ l)
The response procedures adopted is as follows:
B. the PCR reaction system provided by step (3) a detects cervical cancer tissues DNA with SYBR method real-time fluorescence quantitative PCR, utilize standard substance plasmid to obtain typical curve and typical curve equation according to step (3) a respectively, and then obtain the copy number of HPVDNA in cervical cancer tissues.
2. absolute quantitation detection method according to claim 1, is characterized in that: in described step (3) a with distilled water by gradient dilution to 10
-6refer to and 16 types, 18 type HPV standard substance plasmids are diluted to Gradient with distilled water respectively: 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755102A (en) * | 2016-12-21 | 2017-05-31 | 中国医学科学院医学生物学研究所 | A kind of absolute quantitation detection method of 58 type HPV viruse E6/E7 oncogene copy numbers |
| CN114410837A (en) * | 2021-12-22 | 2022-04-29 | 广州白云山拜迪生物医药有限公司 | Reagent and kit for detecting HPV18 and application |
| CN114410838A (en) * | 2021-12-22 | 2022-04-29 | 广州白云山拜迪生物医药有限公司 | Reagent and kit for detecting HPV16 and application |
| CN116286982A (en) * | 2022-09-09 | 2023-06-23 | 广州凯普医药科技有限公司 | HPV genotyping detection positive reference, preparation method and application thereof |
| CN117757991A (en) * | 2023-12-27 | 2024-03-26 | 海宁奥羚医学检验实验室有限公司 | POCT detection method for DNA and mRNA of HPV16/18 virus gene in cervical exfoliated cells |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101781686A (en) * | 2009-01-16 | 2010-07-21 | 上海裕隆基因科技中心有限公司 | Fluorescent PCR (Polymerase Chain Reaction) kit for quantitively detecting HPV16/18 type infection |
| WO2010129941A1 (en) * | 2009-05-08 | 2010-11-11 | Becton, Dickinson And Company | Correlation of hpv e6 and e7 expression with progression of cervical disease |
| CN104020291A (en) * | 2014-06-26 | 2014-09-03 | 南京大学 | HPV nucleic acid detection kit based on enzyme-linked immunosorbent assay and application of HPV nucleic acid detection kit |
| CN105018640A (en) * | 2014-04-17 | 2015-11-04 | 兰州安康伯乐生物技术有限公司 | Fluorescent PCR detecting kit for high-risk human papilloma viruses HPV16 and 18 |
-
2015
- 2015-12-29 CN CN201511018981.3A patent/CN105483249A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101781686A (en) * | 2009-01-16 | 2010-07-21 | 上海裕隆基因科技中心有限公司 | Fluorescent PCR (Polymerase Chain Reaction) kit for quantitively detecting HPV16/18 type infection |
| WO2010129941A1 (en) * | 2009-05-08 | 2010-11-11 | Becton, Dickinson And Company | Correlation of hpv e6 and e7 expression with progression of cervical disease |
| CN105018640A (en) * | 2014-04-17 | 2015-11-04 | 兰州安康伯乐生物技术有限公司 | Fluorescent PCR detecting kit for high-risk human papilloma viruses HPV16 and 18 |
| CN104020291A (en) * | 2014-06-26 | 2014-09-03 | 南京大学 | HPV nucleic acid detection kit based on enzyme-linked immunosorbent assay and application of HPV nucleic acid detection kit |
Non-Patent Citations (2)
| Title |
|---|
| FENG WANG-JOHANNING等: "Quantitation of Human Papillomavirus 16 E6 and E7 DNA and RNA in Residual Material from ThinPrep Papanicolaou Tests Using Real-Time Polymerase Chain Reaction Analysis", 《CANCER》 * |
| 陈悦悦等: "实时荧光定量PCR和RT—PCR检测***细胞株HPV一16 E6和E7基因的实验研究", 《四川大学学报（医学版）》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755102A (en) * | 2016-12-21 | 2017-05-31 | 中国医学科学院医学生物学研究所 | A kind of absolute quantitation detection method of 58 type HPV viruse E6/E7 oncogene copy numbers |
| CN114410837A (en) * | 2021-12-22 | 2022-04-29 | 广州白云山拜迪生物医药有限公司 | Reagent and kit for detecting HPV18 and application |
| CN114410838A (en) * | 2021-12-22 | 2022-04-29 | 广州白云山拜迪生物医药有限公司 | Reagent and kit for detecting HPV16 and application |
| CN116286982A (en) * | 2022-09-09 | 2023-06-23 | 广州凯普医药科技有限公司 | HPV genotyping detection positive reference, preparation method and application thereof |
| CN116286982B (en) * | 2022-09-09 | 2024-01-30 | 广州凯普医药科技有限公司 | HPV genotyping detection positive reference, preparation method and application thereof |
| CN117757991A (en) * | 2023-12-27 | 2024-03-26 | 海宁奥羚医学检验实验室有限公司 | POCT detection method for DNA and mRNA of HPV16/18 virus gene in cervical exfoliated cells |
| CN117757991B (en) * | 2023-12-27 | 2024-05-28 | 海宁奥羚医学检验实验室有限公司 | POCT detection method for DNA and mRNA of HPV16/18 virus gene in cervical exfoliated cells |
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