CN104020291B - A kind of HPV nucleic acid detection kit based on enzyme-linked immuno assay and application thereof - Google Patents
A kind of HPV nucleic acid detection kit based on enzyme-linked immuno assay and application thereof Download PDFInfo
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Abstract
The invention belongs to molecular biology, immunology and nucleic acid chemistry field, be specifically related to a kind of HPV nucleic acid detection kit based on enzyme-linked immuno assay and application thereof.This kit detects the immunologic detection method of high-risk HPV16 type E6, E7RNA based on a kind of Sdr of utilization monoclonal antibody, testing process does not need the technology such as reverse transcription and PCR to increase to target sequence, but utilization does not need the DNA probe of mark and HPV16 type E6, E7RNA to hybridize, form DNA:RNA heterozygote, then utilize Sdr monoclonal antibody to carry out identifying and the amplification of follow-up signal to heterozygote.Detection method has fast, low cost, highly sensitive, do not need complicated amplification and detecting instrument, and the advantage of original viral carrying capacity can be detected.
Description
Technical field
The invention belongs to molecular biology, immunology and nucleic acid chemistry field.Be specifically related to a kind of HPV nucleic acid detection kit based on enzyme-linked immuno assay and application thereof.
Background technology
Human infectious warts virus (hereinafter referred to as HPV) is circular double stranded DNA virus, belongs to Papillomaviridae, α Papillomavirus.From 20 century 70s by since Late Cambrian, now announce existing more than 100 kinds of the genotype of HPV.Some of them genotype has been proved relevant with the malignant change of multiple epithelium or mucosal tissue, is especially cervix cancer.In the women in the whole world, cervix cancer is the ubiquitous malignant tumour being only second to breast cancer.In the U.S., 1.2 ten thousand women are had to be diagnosed as cervical carcinoma every year.Generally acknowledge at present, HPV is the factor causing global nearly all cervical carcinoma.HPV result in the cervical carcinoma of 99%, and high-risk HPV genotypes 16 and 18 has accounted for 70%.The more important thing is, E6 and the E7 gene in this virus and transcription and translation product thereof are the key factors determining its invasion and attack and ability of curing the disease.
In decades, women relies on the instrument whether cytolgical examination exists as detection cervix cancer always.Women needs to obtain better screening instruments, comprises the elementary examination of HPV, to reduce the risk of developing cervical cancer.The method that present diagnosis HPV infects is a lot, such as: histology, serology and molecular biology etc.But so far based on the feature of HPV virus itself, can't carry out in vitro culture, therefore cultivation is also unrealistic; In addition, owing to lacking enough sensitivity and specificity for the immunoassay of HPV, serum or Protein Detection are also failed to be applied to clinical.Therefore, the method detected for HPV at present also depends on its nucleic acid (DNA or RNA), such as, detection for DNA has the direct method based in situ hybridization, DNA sequencing method, based on the signal amplification method of the high-risk HPV detection method of branch DNA analysis, hybrid capture system and Cervista, based on Real-TimePCR, the target Amplification Analysis integrating HPV sequence PCR (DIPS-PCR); Detection for RNA has amplification (TMA) method etc. of reverse transcription PCR, nucleic acid sequence based amplification (NASBA) method and transcriptive intermediate.Wherein, it should be noted that the cobasHPVTest method of the HybridCapture2 method of the Qiagen company of FDA approval in 2003 and Roche (Roche) company of FDA approval in 2014.
But the above detection method for HPV nucleic acid also has its weak point, such as, some method operation relative complex, difficulty cause cost higher, and some method can't reflect initial virus load really except needing corresponding amplification and detecting instrument.And virus load propagates the relevant key factor of the extent of injury to the course of disease of patient and virus.Current research shows, with high risk HPV gene group DNA>1pg/ml (100,000HPV copies) be threshold value, the interim positive rate of epithelium of cervix uteri sarcomatous change (CIN) II-III is 97.5%, the interim positive rate of CINIII is 100%, and the positives rate of cervical carcinoma is 100%.Therefore, the detection method of desirable HPV should be do not need to increase the target nucleic acid of HPV based on the hybridization analysis of nucleic acid.
Monoclonal antibody Sdr be mouse source for the high specific of DNA:RNA heterozygote and the antibody of affinity.Namely our patented technology is establish the immunologic detection method that a kind of Sdr of utilization monoclonal antibody detects high-risk HPV16 type E6, E7RNA, the method does not need reverse transcription and round pcr to increase to target sequence to be checked, but utilization does not need the DNA probe of mark and HPV16 type E6, E7RNA to hybridize, form DNA:RNA heterozygote, then utilize Sdr monoclonal antibody to carry out identifying and the amplification of follow-up signal.Detection method has fast, low cost, highly sensitive, do not need complicated amplification and detecting instrument, and the advantage of original viral carrying capacity can be detected.
Summary of the invention
The present invention needs the problem solved: for the weak point of the detection method of existing HPV nucleic acid, such as operate relative complex, cost that difficulty causes is higher, need corresponding amplification and detecting instrument, can not reflect initial virus load really.Method and the reagent of a kind of quick detection HPV are provided, and be assembled into detection kit, and determine the using method of kit, can carry out fast high-risk HPV16 type E6, E7RNA, low cost, highly sensitive detection, testing process does not need complicated amplification and detecting instrument, can detect original viral carrying capacity.
General technical route of the present invention: the collection of sample, process, extracts HPV16 type DNA or RNA from sick sample (blood, tissue, cervical secretions, cell detachment thing etc.); Primer according to the standard sequence design of GenBank announcement is cloned, sequential analysis; The standard sequence design of announcing according to GenBank and target nucleic acid carry out the oligonucleotide fragment (probe) of hybridizing; Under specific hybridization conditions, oligonucleotide probe and nucleic acid to be checked are hybridized; Hybrid product is incorporated into PLL and wraps in advance in the ELISA Plate of quilt; Add the monoclonal antibody for DNA-RNA heterozygote; Add the ELIAS secondary antibody of anti-monoclonal antibody; Add the substrate colour developing of enzyme, after cessation reaction, survey absorbance.Be optimized key reaction condition, agent formulations and concentration in experiment flow, by probe mixed liquor, annealing buffer, primary antibodie, two anti-dilutions, substrate nitrite ion, reaction terminating liquid is developed to the kit detecting HPV16 type E6, E7RNA.
Use HPV16 type E6, the E7RNA of this kit to in-vitro transcription to detect, and carry out methodological study.The detectability that this detection method can detect HPV16E6 and E7RNA is respectively 0.923pg/mL and 0.424pg/mL, and the concentration range of linearity is from 92.3pg/mL to 0.923pg/mL and from 42.4pg/mL to 0.424pg/mL respectively.
Technical scheme of the present invention: 1. extract the genomic DNA in sick sample; 2., according to the standard sequence design in-vitro transcription primer that GenBank announces, target sequence is cloned, sequential analysis; 3. the standard sequence announced according to GenBank designs the oligonucleotide fragment (probe) carrying out with target nucleic acid hybridizing; 4. set up the immunologic detection method of HPV16 type E6, E7RNA; 5. optimize key reaction condition, agent formulations and concentration, determine its using method; The sensitivity experiment of the immunologic detection method of 6.HPV16 type E6, E7RNA.Details are as follows:
1. extract the genomic DNA in sick sample or RNA
Phenol/chloroform extraction method or genomic DNA or RNA extract kit directly extracts the sick sample of HPV16 type DNA or RNA from sick sample (blood, tissue, cervical secretions, cell detachment thing etc.).Measure nucleic acid concentration, adjustment nucleic acid concentration is to 10ng/uL.
2., according to the standard sequence design in-vitro transcription primer that GenBank announces, target sequence is cloned, sequential analysis
Disclosed in NCBI, HPV16 type complete genomic sequence (reference sequences: NC_001526.2) design is for the in-vitro transcription primer of the whole genetic fragment of E6, E7, and primer sequence (5 '-3 '): HPV16E6 upstream region of gene primer
tAATACGACTCACTATAGGGaTGCACCAAAAGAGAACTGCAATGTTTCAG, HPV16E6 downstream of gene primer TTACAGCTGGGTTTCTCTACGTGTTCTTG; HPV16E7 upstream region of gene primer
tAATACGACTCACTATAGGGaTGCATGGAGATACACCTACATTGCAT, HPV16E7 downstream of gene primer TTATGGTTTCTGAGAACAGATGGGGCACAC.Pcr amplification said extracted nucleic acid-templated; Glue reclaims positive PCR primer; Be transformed into competent cell after being connected to carrier, expand after picking positive colony and cultivate, extract plasmid and carry out sequencing analysis after identifying the positive.Determine use template be HPV16 type E6, E7 genetic fragment.
3. the standard sequence announced according to GenBank designs the oligonucleotide fragment (probe) carrying out with target nucleic acid hybridizing
Disclosed in NCBI, HPV16 type complete genomic sequence (reference sequences: NC_001526.2) design is for the oligonucleotide probe of E6, E7 genetic fragment, and sequence is as follows:
4. set up the immunologic detection method of HPV16 type E6, E7RNA
In-vitro transcription is carried out through the PCR primer with the primer amplification of vitro transcription promoters by step 2; In probe in transcription product and step 3 one or several be combined in the conditions such as annealing temperature in certain annealing buffer, certain under hybridize; After hybrid product and the pretreated ELISA Plate of PLL are hatched, add the mouse resource monoclonal antibody for DNA-RNA heterozygote, then add the ELIAS secondary antibody for mouse source monoclonal antibody, finally add substrate colour developing, after cessation reaction, detect absorbance.
5. optimize key reaction condition, agent formulations and concentration, determine its using method
After this system of checking can be used for detecting the RNA in sample, to concentration and probe concentration, quantity, annealing buffer, annealing time, annealing temperature, the indexs such as confining liquid and primary antibodie, two anti-concentration and dilution thereof are optimized.Under optimal conditions, the component in detection system is assembled into kit, the ingredient of kit is:
Pre-coated ELISA Plate: the polystyrene micropore plate of PLL process, BSA closes, and storage condition is 4 DEG C, half a year shelf-life.
Probe mixed liquor 1: for the probe combinations of HPV16E6RNA fragment, E6DNA1, E6DNA2, E6DNA3, concentration is 10uM.
Probe mixed liquor 2: for the probe combinations of HPV16E7RNA fragment, E7DNA1, E7DNA2, concentration is 10uM.
10 × annealing buffer: Tris-HCl100mM, pH7.5, EDTA10mM, NaCl1M.
For the mouse resource monoclonal antibody Sdr solution of DNA-RNA.
ELIAS secondary antibody solution for Sdr: HRP-Goatanti-mouseIgG (H+L) solution.
Substrate chromophoric solution: TMB solution.
Lavation buffer solution: 10 × PBS solution.
Reaction terminating liquid: 2MH
2sO
4.
Using the hybridization reaction condition optimized and immunoassay process as the instructions of this kit, details are as follows:
(1) in-vitro transcription RNA template or the RNA that directly extracts from sick sample are hybridized with probe mixed liquor 1 or 2, hybridization system: 10uL10 × annealing buffer, 3uL or 2uL probe mixed liquor, RNA template solution 10uL, supplementary DEPCH
2o to 100uL; Hybridization conditions: 95 DEG C of 5min, 65 DEG C of 2h, cooled on ice 5min.
(2) in pre-coated ELISA Plate, every hole adds 100uL hybrid product, and incubated at room 1h, 300uLPBS (pH7.4, containing 1 ‰ Tween-20) wash 5 times; Every hole adds 100uLSdr monoclonal antibody solution, and incubated at room 1h as above washs; Every hole adds 100uL ELIAS secondary antibody solution, and incubated at room 1h, as above washs; Add 100uLTMB substrate chromophoric solution, hatch 20min for 37 DEG C; Every hole adds 50uL reaction terminating liquid cessation reaction, surveys Abs value, measures wavelength 450nm, reference wavelength 570nm.
The sensitivity experiment of the immunologic detection method of 6.HPV16 type E6, E7RNA
Detect after the HPV16 type E6 of in-vitro transcription, E7RNA template 10 times of gradient dilutions.
Beneficial effect of the present invention: for the weak point of the detection method of existing HPV nucleic acid, namely patented technology of the present invention is establish the immunologic detection method that a kind of Sdr of utilization monoclonal antibody detects high-risk HPV16 type E6, E7RNA, the method does not need reverse transcription and round pcr to increase to target sequence to be checked, but utilization does not need the DNA probe of mark and HPV16 type E6, E7RNA to hybridize, form DNA:RNA heterozygote, then utilize Sdr monoclonal antibody to carry out identifying and the amplification of follow-up signal.The detectability that this detection method can detect HPV16E6 and E7RNA is respectively 0.923pg/mL and 0.424pg/mL, and the concentration range of linearity is from 92.3pg/mL to 0.923pg/mL and from 42.4pg/mL to 0.424pg/mL respectively.Detection method has fast, low cost, highly sensitive, do not need complicated amplification and detecting instrument, and the advantage of original viral carrying capacity can be detected.
Accompanying drawing explanation
Fig. 1 inventive principle schematic diagram
1. more than PLL 2.DNA:RNA heterozygote 3.Sdr monoclonal antibody 4. goat anti-mouse antibody 5. horseradish peroxidase 6.TMB substrate 7. color product
The PCR primer (A) of Fig. 2 HPV16E6, E7ORF fragment and in-vitro transcription product electrophoretogram (B) thereof
A1.HPV16E62.HPV16E7M.DL2000DNAMarker
B1.HPV16E62.HPV16E6RNA3.HPV16E74.HPV16E7RNAM.DL2000DNAMarker
This inventive method of Fig. 3 detects HPV16 type E6, E7RNA sensitivity
The hybrid product of the E6RNA fragment of A.E6DNA probe 1/2/3 potpourri and gradient dilution
B.E7DNA probe 1/2 potpourri and gradient dilution E7RNA hybrid product
Embodiment
There is provided specific embodiment to set forth technical scheme of the present invention further below, but the application of the technology of the present invention is not limited to embodiment.
The clone of 1HPV16 type E6, E7 genetic fragment, sequential analysis and in-vitro transcription
Extract the genomic DNA in disease sample, pcr amplification HPV16 type E6, E7 gene (296bp-477bp length range), with the HPV16 type E6 of T7RNAPolyase promoter (underscore part), E7ORF primer sequence (5 '-3 '): HPV16E6 upstream region of gene primer
tAATACGACTCACTATAGGGaTGCACCAAAAGAGAACTGCAATGTTTCAG, HPV16E6 downstream of gene primer TTACAGCTGGGTTTCTCTACGTGTTCTTG; HPV16E7 upstream region of gene primer
tAATACGACTCACTATAGGGaTGCATGGAGATACACCTACATTGCAT, HPV16E7 downstream of gene primer TTATGGTTTCTGAGAACAGATGGGGCACAC.
PCR reaction system: single reaction is containing 2.5uL10 × PCRBuffer, 1uL2.5mMeachdNTPMix, 2uL25mMMgCl
2, 0.4uLTaq polymerase (5U/ μ L), 1uLSenseprimer (10uM), 1uLAnti-senseprimer (10uM), 5uL template DNA, adds ddH
2o supplies 25uL; Amplification condition: 95 DEG C of denaturation 5min, 1 circulation; 95 DEG C of sex change 40s, HPV16E6 annealing temperature is 62.5 DEG C, HPV16E7 annealing temperature is 71.0 DEG C of 1min that all anneal, and 72 DEG C extend 1min, and above three steps carry out 40 circulations; 72 DEG C are supplemented extension 8min, 1 circulation.Run 1% Ago-Gel and reclaim HPV16E6, E7 positive fragment.Glue is reclaimed fragment and is connected to pMD-19TVector; PMD-19T-HPV16E6, E7 are transformed into DH5 α competent cell, are coated with on the LB agar plate containing ampicillin (Amp), be inverted cultivation 12 ~ 16h for 37 DEG C and formed to single bacterium colony.Choose single colony inoculation in the test tube of the LB nutrient culture media containing Amp, 37 DEG C, 200rpm shakes cultivation 12 ~ 16h.Extract pMD-19T-HPV16E6, E7 plasmid.Through PCR, plasmid identifies that (system condition is as front) is for carrying out sequencing analysis after the positive.After sequence and standard sequence comparison, the genetic fragment consistent with standard sequence is selected to carry out In vitro transcription.
In-vitro transcription is carried out to above-mentioned PCR primer, PCR primer concentration HPV16E6, HPV16E7 are 0.2ug/uL, in-vitro transcription system, each reaction contains 2uL10 × TranscriptionBuffer, ATP, GTP, CTP, UTPSolution each 2uL, 0.5uLRNaseInhibitor, 2uLT7RNAPolymerase, 5uLHPV16E6 or E7DNA template, supplements DEPCH
2o to 20uL.Reaction conditions is: 42 DEG C, reaction 2h.It is for subsequent use that product puts-80 DEG C of refrigerators.
The foundation of the immunologic detection method of 2HPV16 type E6, E7RNA
2.1 anneal
According to NCBI announce HPV16 type E6, E7 gene order designing probe sequence as shown in Table 1.
Table one probe sequence
Hybridized by RNA in above-mentioned probe system and in-vitro transcription template or the sick sample of extracting directly, hybridization system: 10uL10 × annealing buffer, 3uL or 2uL probe mixed liquor, RNA template solution 10uL, supplements DEPCH
2o to 100uL; Hybridization conditions: 95 DEG C of 5min, 65 DEG C of 2h, cooled on ice 5min.
2.2 enzyme linked immunosorbent detection
In pre-coated ELISA Plate, every hole adds 100uL hybrid product, and incubated at room 1h, 300uLPBS (pH7.4, containing 1 ‰ Tween-20) wash 5 times; Every hole adds 100uLSdr monoclonal antibody solution, and incubated at room 1h as above washs; Every hole adds 100uL ELIAS secondary antibody solution, and incubated at room 1h, as above washs; Add 100uLTMB substrate chromophoric solution, hatch 20min for 37 DEG C; Every hole adds 50uL reaction terminating liquid cessation reaction, surveys Abs value, measures wavelength 450nm, reference wavelength 570nm.
Each experiment obtains more than twice repetition, is considered as repeatability, has good stability.
<110> Nanjing University
<120> detection kit based on the HPV nucleic acid of enzyme-linked immuno assay and application thereof
<160>9
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>1
taatacgactcactatagggatgcaccaaaagagaactgcaatgtttcag
<210>2
<211>29
<212>DNA
<213> artificial sequence
<400>2
ttacagctgggtttctctacgtgttcttg
<210>3
<211>47
<212>DNA
<213> artificial sequence
<400>3
taatacgactcactatagggatgcatggagatacacctacattgcat
<210>4
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<212>DNA
<213> artificial sequence
<400>4
ttatggtttctgagaacagatggggcacac
<210>5
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<212>DNA
<213> artificial sequence
<400>5
gctctgtgcataactgtggtaactttctgggtcg
<210>6
<211>40
<212>DNA
<213> artificial sequence
<400>6
tcccgaaaagcaaagtcatatacctcacgtcgcagtaact
<210>7
<211>40
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<400>7
cagctgggtttctctacgtgttcttgatgatctgcaacaa
<210>8
<211>32
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<213> artificial sequence
<400>8
tacgcacaaccgaagcgtagagtcacacttgc
<210>9
<211>49
<212>DNA
<213> artificial sequence
<400>9
agtgtgcccattaacaggtcttccaaagtacgaatgtctacgtgtgtgc
Claims (2)
1., based on HPV16 type E6, the E7RNA detection kit of enzyme-linked immuno assay, it is characterized in that the ingredient of kit is:
(1) pre-coated ELISA Plate: the polystyrene micropore plate of PLL process, BSA closes, and storage condition is 4 DEG C, half a year shelf-life;
(2) probe mixed liquor 1: for the probe combinations of HPV16E6RNA fragment, E6DNA1, E6DNA2, E6DNA3, concentration is 10 μMs;
Probe mixed liquor 1 probe E6DNA1:SEQIDNO5, and sequence (5 '-3 '): GCTCTGTGCATAACTGTGGTAACTTTCTGGGTCG;
Probe mixed liquor 1 probe E6DNA2:SEQIDNO6, and sequence (5 '-3 '): TCCCGAAAAGCAAAGTCATATACCTCACGTCGCAGTAACT;
Probe mixed liquor 1 probe E6DNA3:SEQIDNO7, and sequence (5 '-3 '): CAGCTGGGTTTCTCTACGTGTTCTTGATGATCTGCAACAA;
(3) probe mixed liquor 2: for the probe combinations of HPV16E7RNA fragment, E7DNA1, E7DNA2, concentration is 10 μMs;
Probe mixed liquor 2E7DNA1:SEQIDNO8, and sequence (5 '-3 '): TACGCACAACCGAAGCGTAGAGTCACACTTGC;
Probe mixed liquor 2E7DNA2:SEQIDNO9, and sequence (5 '-3 '): AGTGTGCCCATTAACAGGTCTTCCAAAGTACGAATGTCTACGTGTGTGC;
(4) 10 × annealing buffers: Tris-HCl100mM, pH7.5, EDTA10mM, NaCl1M;
(5) for the mouse resource monoclonal antibody Sdr solution of DNA-RNA;
(6) for the ELIAS secondary antibody solution of Sdr;
(7) substrate chromophoric solution: TMB solution;
(8) lavation buffer solution: 10 × PBS solution;
(9) reaction terminating liquid: 2MH
2sO
4.
2. kit is used for the detection method of in-vitro transcription RNA template according to claim 1, it is characterized in that being made up of following steps:
(1) in-vitro transcription RNA template and probe mixed liquor 1 or 2 are hybridized, hybridization system: 10 μ L10 × annealing buffers, 3 μ L or 2 μ L probe mixed liquors, RNA template solution 10 μ L, supplements DEPCH
2o to 100 μ L; Hybridization conditions: 95 DEG C of 5min, 65 DEG C of 2h, cooled on ice 5min;
(2), in pre-coated ELISA Plate, every hole adds 100 μ L hybrid product, incubated at room 1h, and 300 μ LPBST wash 5 times; Every hole adds 100 μ LSdr monoclonal antibody solutions, and incubated at room 1h, as above washs; Every hole adds 100 μ L ELIAS secondary antibody solution, and incubated at room 1h, as above washs; Add 100 μ LTMB substrate chromophoric solutions, hatch 20min for 37 DEG C; Every hole adds 50 μ L reaction terminating liquid cessation reactions, surveys Abs value, measures wavelength 450nm, reference wavelength 570nm.
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| CN104726606B (en) * | 2015-04-09 | 2017-07-04 | 南京爱佩捷生物科技有限公司 | A kind of enzyme-linked double cross method detection the pathogenic microorganism examination methods of PCR |
| CN105483249A (en) * | 2015-12-29 | 2016-04-13 | 中国医学科学院医学生物学研究所 | Method for absolute quantitative detection of number of copies of type-16 and type-18 HPV (human papillomavirus) virus genes |
| CN106610378A (en) * | 2016-02-26 | 2017-05-03 | 弗雷米德生物医药技术（天津）有限公司 | Detection kit for HPV16-E7 protein |
| CN107523611A (en) * | 2016-06-22 | 2017-12-29 | 南京大学 | A kind of non-amplification type nucleic acid hybrid capture system and its application in detection of nucleic acids |
| CN109206511B (en) * | 2018-09-13 | 2021-03-16 | 东南大学 | Nano antibody capable of specifically binding HPVL6-E6 protein, and coding sequence, preparation method and application thereof |
| CN109613236B (en) * | 2018-12-12 | 2022-04-15 | 安邦（厦门）生物科技有限公司 | Nucleic acid hybridization capture immunofluorescence detection method, immunofluorescence chromatography test strip and kit |
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| 人***状瘤病毒E6/E7 mRNA检测宫颈高度病变的临床评价;卢文波 等;《中华医院感染学杂志》;20091120;第19卷(第22期);摘要，第3145页左栏第1段－右栏第1段，3讨论 * |
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