CN104020291A - HPV nucleic acid detection kit based on enzyme-linked immunosorbent assay and application of HPV nucleic acid detection kit - Google Patents
HPV nucleic acid detection kit based on enzyme-linked immunosorbent assay and application of HPV nucleic acid detection kit Download PDFInfo
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Abstract
The invention belongs to the technical fields of molecular biology, immunology and nucleic acid chemistry, and particularly relates to an HPV nucleic acid detection kit based on enzyme-linked immunosorbent assay and application of the HPV nucleic acid detection kit. The kit is based on an immunoassay method for detecting high-risk HPV16 type E6 and E7 RNA by using an Sdr monoclonal antibody; a detection process is realized as follows: amplification of a target sequence by using technologies such as inverse transcription and PCR is not needed, an unlabelled DNA probe is used for being hybridized with the HPV16 type E6 and E7 RNA to form a DNA:RNA heterozygote, and then the heterozygote is recognized by using the Sdr monoclonal antibody, and subsequent signal multiplication is performed. A detection method has the advantages of high speed, low cost, high sensitivity, and no need of complicated amplification and detection instruments, and is capable of detecting an initial viral load.
Description
Technical field
The invention belongs to molecular biology, immunology and nucleic acid chemistry technical field.Be specifically related to a kind of HPV kit for detecting nucleic acid and application thereof based on enzyme-linked immuno assay.
Background technology
Human infectious warts virus (hereinafter to be referred as HPV) is circular double stranded DNA virus, belongs to Papillomaviridae, α Papillomavirus.Since 20 century 70s are found first, the genotype of now having announced HPV is existing more than 100 kinds.It is relevant with the malignant change of multiple epithelium or mucosal tissue that some of them genotype has been proved, and is especially cervix cancer.In global women, cervix cancer is the ubiquitous malignant tumour that is only second to breast cancer.In the U.S., there are every year 1.2 ten thousand women to be diagnosed as cervical carcinoma.Generally acknowledge at present, HPV is the factor that causes global nearly all cervical carcinoma.HPV has caused 99% cervical carcinoma, and high-risk HPV genotype 16 and 18 has accounted for 70%.The more important thing is, the E6 in this virus and E7 gene and transcription and translation product thereof are the key factors that determines its invasion and attack and the ability of curing the disease.
In decades, women relies on the instrument whether cytolgical examination exists as detection cervix cancer always.Women need to obtain better screening instruments, comprises the elementary examination of HPV, to reduce the risk of suffering from cervix cancer.The method that diagnosis HPV infects is now a lot, such as: histology, serology and molecular biology etc.But the feature based on HPV virus itself, can't carry out in vitro culture, so cultivation is also unrealistic so far; In addition, because the immunoassay for HPV lacks enough sensitivity and specificity, serum or Protein Detection also fail to be applied to clinical.Therefore, the method detecting for HPV at present also depends on its nucleic acid (DNA or RNA), for example, detection for DNA has the direct method based in situ hybridization, DNA sequencing method, signal amplification method based on branch DNA analysis, hybrid capture system and the high-risk HPV detection method of Cervista, the target Amplification Analysis based on Real-Time PCR, integration HPV sequence PCR (DIPS-PCR); Detection for RNA has amplification (TMA) method of reverse transcription PCR, nucleic acid sequence based amplification (NASBA) method and transcriptive intermediate etc.Wherein, it should be noted that the Hybrid Capture2 method of Qiagen company of FDA approval in 2003 and the cobas HPV Test method of Roche (Roche) company of FDA approval in 2014.
Yet 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 is except needing corresponding amplification and detecting instrument can't reflect really initial virus load.And virus load is to propagate to patient's the course of disease and virus the key factor that the extent of injury is relevant.Research at present shows, with high-risk HPV genomic DNA >1pg/ml (100,000HPV copy), for threshold value, the interim positive rate of epithelium of cervix uteri sarcomatous change (CIN) II-III is 97.5%, the interim positive rate of CIN III is 100%, and in cervical carcinoma, positive rate is 100%.Therefore, the detection method of desirable HPV should be hybridization analysis based on nucleic acid do not need the to increase target nucleic acid of HPV.
Monoclonal antibody Sdr be mouse source for the high specific of DNA:RNA heterozygote and the antibody of affinity.Our patented technology is to have set up 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 utilize, do not need the DNA probe of mark and HPV16 type E6, E7RNA to hybridize, form DNA:RNA heterozygote, then utilize Sdr monoclonal antibody to identify and follow-up signal amplification.Detection method has fast, low-cost, highly sensitive, do not need complicated amplification and detecting instrument and the advantage that can detect initial virus load.
Summary of the invention
The problem that the present invention need to solve: for the weak point of the detection method of existing HPV nucleic acid, the cost causing such as operation relative complex, difficulty is higher, needs corresponding amplification and detecting instrument, can not reflect really initial virus load.Method and the reagent of a kind of fast detecting HPV are provided, and be assembled into detection kit, and the using method of definite 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 initial virus load.
General technical route of the present invention: the collection of sample, processing, from sick sample (blood, tissue, cervical secretions, cell detachment thing etc.), extract HPV16 type DNA or RNA; The primer of the standard sequence design of announcing according to GenBank is cloned, sequential analysis; The oligonucleotide fragment (probe) that standard sequence design and the target nucleic acid of announcing according to GenBank hybridized; Under specific hybridization conditions, oligonucleotide probe and nucleic acid to be checked are hybridized; Hybridization product is incorporated into PLL in advance in coated ELISA Plate; Add the monoclonal antibody for DNA-RNA heterozygote; The ELIAS secondary antibody that adds anti-monoclonal antibody; Add the substrate colour developing of enzyme, after cessation reaction, survey absorbance.Key reaction condition in experiment flow, reagent composition and concentration are optimized, by probe mixed liquor, annealing buffer, primary antibodie, two anti-dilutions, substrate nitrite ion, reaction terminating liquid is developed to the kit that detects HPV16 type E6, E7RNA.
Use this kit to detect HPV16 type E6, the E7RNA of in-vitro transcription, 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 respectively from 92.3pg/mL to 0.923pg/mL and from 42.4pg/mL to 0.424pg/mL.
Technical scheme of the present invention: 1. extract the genomic DNA in sick sample; 2. the standard sequence design in-vitro transcription primer of announcing according to GenBank, clones sequential analysis to target sequence; 3. the oligonucleotide fragment (probe) that standard sequence design and the target nucleic acid of announcing according to GenBank hybridized; 4. set up the immunologic detection method of HPV16 type E6, E7RNA; 5. optimize key reaction condition, reagent composition 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 genomic DNA or the RNA in sick sample
Phenol/chloroform extraction method or genomic DNA or RNA extract kit and directly from sick sample (blood, tissue, cervical secretions, cell detachment thing etc.), extract DNA or the RNA of the sick sample of HPV16 type.Measure nucleic acid concentration, adjust nucleic acid concentration to 10ng/uL.
2. the standard sequence design in-vitro transcription primer of announcing according to GenBank, clones sequential analysis to target sequence
According to the disclosed HPV16 type of NCBI complete genome group sequence (reference sequences: NC_001526.2) design is for the in-vitro transcription primer of E6, the whole genetic fragment of E7, primer sequence (5 '-3 '): HPV16E6 upstream region of gene primer
tAATACGACTCACTATAGGGaTGCACCAAAAGAGAACTGCAATGTTTCAG, HPV16E6 gene downstream primer TTACAGCTGGGTTTCTCTACGTGTTCTTG; HPV16E7 upstream region of gene primer
tAATACGACTCACTATAGGGaTGCATGGAGATACACCTACATTGCAT, HPV16E7 gene downstream primer TTATGGTTTCTGAGAACAGATGGGGCACAC.Pcr amplification said extracted nucleic acid-templated; Glue reclaims positive PCR product; After being connected to carrier, be transformed into competent cell, after picking positive colony, expand and cultivate, extract plasmid and after identifying the positive, carry out sequencing analysis.Determine that the template of using is HPV16 type E6, E7 genetic fragment.
3. the oligonucleotide fragment (probe) that standard sequence design and the target nucleic acid of announcing according to GenBank hybridized
According to the disclosed HPV16 type of NCBI complete genome group 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
PCR product through the primer amplification with in-vitro transcription promoter in step 2 is carried out to in-vitro transcription; Under being combined in certain annealing buffer of one or several in probe in transcription product and step 3, certain conditions such as annealing temperature, hybridize; After hybridization 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, reagent composition and concentration, determine its using method
In this system of checking, can be used for detecting after 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 the condition of optimizing, the component in detection system is assembled into kit, the ingredient of kit is:
The polystyrene micropore plate that pre-coated ELISA Plate: PLL processes, BSA sealing, storage condition is 4 ℃, 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.
Mouse resource monoclonal antibody Sdr solution for DNA-RNA.
ELIAS secondary antibody solution for Sdr: HRP-Goat anti-mouse IgG (H+L) solution.
Substrate chromophoric solution: TMB solution.
Lavation buffer solution: 10 * PBS solution.
Reaction terminating liquid: 2M H
2sO
4.
Instructions using the hybridization reaction condition of optimizing and immunoassay process as this kit, details are as follows:
(1) in-vitro transcription RNA template or direct RNA and the probe mixed liquor 1 or 2 extracting are hybridized to hybridization system from sick sample: 10uL10 * annealing buffer, 3uL or 2uL probe mixed liquor, RNA template solution 10uL, supplements DEPC H
2o to 100uL; Hybridization conditions: 95 ℃ of 5min, 65 ℃ of 2h, cooled on ice 5min.
(2), in pre-coated ELISA Plate, every hole adds 100uL hybridization product, incubated at room 1h, and 300uL PBS (pH7.4, containing 1 ‰ Tween-20) washes 5 times; Every hole adds 100uL Sdr monoclonal antibody solution, incubated at room 1h, as above washing; Every hole adds 100uL ELIAS secondary antibody solution, incubated at room 1h, as above washing; Add 100uL tmb substrate chromophoric solution, hatch 20min for 37 ℃; 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
After the HPV16 type E6 of in-vitro transcription, 10 times of gradient dilutions of E7RNA template, detect.
Beneficial effect of the present invention: for the weak point of the detection method of existing HPV nucleic acid, patented technology of the present invention is to have set up 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 utilize, do not need the DNA probe of mark and HPV16 type E6, E7RNA to hybridize, form DNA:RNA heterozygote, then utilize Sdr monoclonal antibody to identify and follow-up signal amplification.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 respectively from 92.3pg/mL to 0.923pg/mL and from 42.4pg/mL to 0.424pg/mL.Detection method has fast, low-cost, highly sensitive, do not need complicated amplification and detecting instrument and the advantage that can detect initial virus load.
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 substrates 7. colour developing products
The PCR product (A) of Fig. 2 HPV16E6, E7ORF fragment and in-vitro transcription product electrophoretogram (B) thereof
A1.HPV16E62.HPV16E7M.DL2000DNA?Marker
B1.HPV16E62.HPV16E6RNA3.HPV16E74.HPV16E7RNA?M.DL2000DNA?Marker
This inventive method of Fig. 3 detects HPV16 type E6, E7RNA sensitivity
The hybridization 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 hybridization product
Embodiment
Provide specific embodiment further to set forth technical scheme of the present invention below, but the application of the technology of the present invention is not limited to embodiment.
Clone, sequential analysis and the in-vitro transcription of 1HPV16 type E6, E7 genetic fragment
Extract the genomic DNA in disease sample, pcr amplification HPV16 type E6, E7 gene (296bp-477bp length range), with HPV16 type E6, the E7ORF primer sequence (5 '-3 ') of T7RNA Polyase promoter (underscore part): HPV16E6 upstream region of gene primer
tAATACGACTCACTATAGGGaTGCACCAAAAGAGAACTGCAATGTTTCAG, HPV16E6 gene downstream primer TTACAGCTGGGTTTCTCTACGTGTTCTTG; HPV16E7 upstream region of gene primer
tAATACGACTCACTATAGGGaTGCATGGAGATACACCTACATTGCAT, HPV16E7 gene downstream primer TTATGGTTTCTGAGAACAGATGGGGCACAC.
PCR reaction system: single reaction is containing 2.5uL10 * PCR Buffer, 1uL2.5mM each dNTP Mix, 2uL25mM MgCl
2, 0.4uL Taq polymerase (5U/ μ L), 1uL Sense primer (10uM), 1uL Anti-sense primer (10uM), 5uL template DNA, adds dd H
2o supplies 25uL; Amplification condition: 95 ℃ of denaturation 5min, 1 circulation; 95 ℃ of sex change 40s, HPV16E6 annealing temperature is that 62.5 ℃, HPV16E7 annealing temperature are 71.0 ℃ of 1min that all anneal, and 72 ℃ are extended 1min, and above three steps are carried out 40 circulations; 72 ℃ are supplemented extension 8min, 1 circulation.Run 1% Ago-Gel and reclaim HPV16E6, E7 positive fragment.Glue is reclaimed to fragment and be connected in pMD-19T Vector; PMD-19T-HPV16E6, E7 are transformed into DH5 α competent cell, are coated with containing on the LB agar plate of ampicillin (Amp), be inverted for 37 ℃ and cultivate 12～16h to single bacterium colony formation.Choose single colony inoculation in the test tube of the LB nutrient culture media containing Amp, 37 ℃, 12～16h is cultivated in 200rpm concussion.Extract pMD-19T-HPV16E6, E7 plasmid.Plasmid identifies that through PCR (system condition is as front) carries out sequencing analysis after positive.After sequence and standard sequence are compared, select the genetic fragment consistent with standard sequence to carry out In vitro transcription.
Above-mentioned PCR product is carried out to in-vitro transcription, PCR production concentration HPV16E6, HPV16E7 are 0.2ug/uL, in-vitro transcription system, each reaction contains 2uL10 * Transcription Buffer, ATP, GTP, CTP, each 2uL of UTP Solution, 0.5uL RNase Inhibitor, 2uL T7RNA Polymerase, 5uL HPV16E6 or E7DNA template, supplement DEPC H
2o to 20uL.Reaction conditions is: 42 ℃, and reaction 2h.It is standby that product is put-80 ℃ of refrigerators.
The foundation of the immunologic detection method of 2HPV16 type E6, E7RNA
2.1 annealing hybridization
The HPV16 type E6 announcing according to NCBI, E7 gene order designing probe sequence are as shown in Table 1.
Table one probe sequence
Above-mentioned probe system and in-vitro transcription template or the RNA that directly extracts in disease sample are hybridized to hybridization system: 10uL10 * annealing buffer, 3uL or 2uL probe mixed liquor, RNA template solution 10uL, supplements DEPC H
2o to 100uL; Hybridization conditions: 95 ℃ of 5min, 65 ℃ of 2h, cooled on ice 5min.
2.2 enzyme linked immunosorbent detection
In pre-coated ELISA Plate, every hole adds 100uL hybridization product, incubated at room 1h, and 300uL PBS (pH7.4, containing 1 ‰ Tween-20) washes 5 times; Every hole adds 100uL Sdr monoclonal antibody solution, incubated at room 1h, as above washing; Every hole adds 100uL ELIAS secondary antibody solution, incubated at room 1h, as above washing; Add 100uL tmb substrate chromophoric solution, hatch 20min for 37 ℃; Every hole adds 50uL reaction terminating liquid cessation reaction, surveys Abs value, measures wavelength 450nm, reference wavelength 570nm.
Each experiment is considered as repeatability, has good stability more than obtaining twice repetition.
<110> Nanjing University
Detection kit and the application thereof of mono-kind of the <120> HPV nucleic acid based on enzyme-linked immuno assay
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<213> artificial sequence
<400>1
taatacgact?cactataggg?atgcaccaaa?agagaactgc?aatgtttcag
<210>2
<211>29
<212>DNA
<213> artificial sequence
<400>2
ttacagctgg?gtttctctac?gtgttcttg
<210>3
<211>47
<212>DNA
<213> artificial sequence
<400>3
taatacgact?cactatag?ggatgcatgg?agatacacct?acattgcat
<210>4
<211>30
<212>DNA
<213> artificial sequence
<400>4
ttatggtttc?tgagaacaga?tggggcacac
<210>5
<211>34
<212>DNA
<213> artificial sequence
<400>5
gctctgtgca?taactgtggt?aactttctgg?gtcg
<210>6
<211>40
<212>DNA
<213> artificial sequence
<400>6
tcccgaaaag?caaagtcata?tacctcacgt?cgcagtaact
<210>7
<211>40
<212>DNA
<213> artificial sequence
<400>7
cagctgggtt?tctctacgtg?ttcttgatga?tctgcaacaa
<210>8
<211>32
<212>DNA
<213> artificial sequence
<400>8
tacgcacaac?cgaagcgtag?agtcacactt?gc
<210>9
<211>49
<212>DNA
<213> artificial sequence
<400>9
agtgtgccca?ttaacaggtc?ttccaaagta?cgaatgtcta?cgtgtgtgc
Claims (3)
1. HPV16 type E6, the E7RNA detection kit based on enzyme-linked immuno assay, is characterized in that the ingredient of kit is:
(1) polystyrene micropore plate that pre-coated ELISA Plate: PLL processes, BSA sealing, storage condition is 4 ℃, half a year shelf-life;
(2) probe mixed liquor 1: for the probe combinations of HPV16E6RNA fragment, and E6DNA1, E6DNA2, E6DNA3, concentration is 10uM;
(3) probe mixed liquor 2: for the probe combinations of HPV16E7RNA fragment, and E7DNA1, E7DNA2, concentration is 10uM;
(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: 2M H
2sO
4.
2. kit, for the detection method of in-vitro transcription RNA template or the direct RNA extracting from sick sample, is characterized in that consisting of following steps according to claim 1:
(1) in-vitro transcription RNA template or direct RNA and the probe mixed liquor 1 or 2 extracting are hybridized to hybridization system from sick sample: 10uL10 * annealing buffer, 3uL or 2uL probe mixed liquor, RNA template solution 10uL, supplements DEPC H
2o to 100uL; Hybridization conditions: 95 ℃ of 5min, 65 ℃ of 2h, cooled on ice 5min;
(2), in pre-coated ELISA Plate, every hole adds 100uL hybridization product, incubated at room 1h, and 300uL PBST washes 5 times; Every hole adds 100uL Sdr monoclonal antibody solution, incubated at room 1h, as above washing; Every hole adds 100uL ELIAS secondary antibody solution, incubated at room 1h, as above washing; Add 100uL tmb substrate chromophoric solution, hatch 20min for 37 ℃; Every hole adds 50uL reaction terminating liquid cessation reaction, surveys Abs value, measures wavelength 450nm, reference wavelength 570nm.
3. the application of kit in high-risk HPV16 type Human infectious warts virus detects according to claim 1.
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Cited By (6)
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| CN104726606A (en) * | 2015-04-09 | 2015-06-24 | 南京爱佩捷生物科技有限公司 | Method for detecting pathogenic microorganisms by using PCR (polymerase chain reaction) enzyme-linked double-cross method |
| 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 |
| CN109206511A (en) * | 2018-09-13 | 2019-01-15 | 东南大学 | It is capable of the nano antibody and its coded sequence, preparation method and application of specific bond HPVl6-E6 albumen |
| CN109613236A (en) * | 2018-12-12 | 2019-04-12 | 安邦（厦门）生物科技有限公司 | A kind of nucleic acid hybrid capture immunofluorescent detection method, immunofluorescence chromatography strip and kit |
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| CN104726606A (en) * | 2015-04-09 | 2015-06-24 | 南京爱佩捷生物科技有限公司 | Method for detecting pathogenic microorganisms by using PCR (polymerase chain reaction) enzyme-linked double-cross method |
| 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 |
| CN109206511A (en) * | 2018-09-13 | 2019-01-15 | 东南大学 | It is capable of the nano antibody and its coded sequence, preparation method and application of specific bond HPVl6-E6 albumen |
| CN109613236A (en) * | 2018-12-12 | 2019-04-12 | 安邦（厦门）生物科技有限公司 | A kind of nucleic acid hybrid capture immunofluorescent detection method, immunofluorescence chromatography strip and kit |
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