CN102888457B - Detection technology of molecular beacon strand displacement isothermal amplifying gene chip and kit - Google Patents
Detection technology of molecular beacon strand displacement isothermal amplifying gene chip and kit Download PDFInfo
- Publication number
- CN102888457B CN102888457B CN201210361822.3A CN201210361822A CN102888457B CN 102888457 B CN102888457 B CN 102888457B CN 201210361822 A CN201210361822 A CN 201210361822A CN 102888457 B CN102888457 B CN 102888457B
- Authority
- CN
- China
- Prior art keywords
- molecular beacon
- cane
- strand displacement
- district
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 238000006073 displacement reaction Methods 0.000 title claims abstract description 36
- 238000001514 detection method Methods 0.000 title claims abstract description 27
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 17
- 238000005516 engineering process Methods 0.000 title abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 28
- 230000003321 amplification Effects 0.000 claims abstract description 12
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 12
- 238000005406 washing Methods 0.000 claims abstract description 6
- 230000008569 process Effects 0.000 claims abstract description 5
- 239000000523 sample Substances 0.000 claims description 79
- 238000010791 quenching Methods 0.000 claims description 37
- 230000000171 quenching effect Effects 0.000 claims description 37
- 239000007787 solid Substances 0.000 claims description 23
- 108020000999 Viral RNA Proteins 0.000 claims description 19
- 238000011901 isothermal amplification Methods 0.000 claims description 19
- 239000007788 liquid Substances 0.000 claims description 16
- 238000002493 microarray Methods 0.000 claims description 14
- 238000013461 design Methods 0.000 claims description 11
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 11
- 238000012360 testing method Methods 0.000 claims description 7
- 238000004458 analytical method Methods 0.000 claims description 6
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 claims description 5
- 108091034117 Oligonucleotide Proteins 0.000 claims description 4
- 238000003745 diagnosis Methods 0.000 claims description 3
- 239000011324 bead Substances 0.000 claims description 2
- 201000010099 disease Diseases 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 239000002985 plastic film Substances 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- 239000013615 primer Substances 0.000 claims description 2
- 239000002987 primer (paints) Substances 0.000 claims description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 abstract description 16
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 abstract description 5
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 abstract description 5
- 230000008901 benefit Effects 0.000 abstract description 3
- 102000053602 DNA Human genes 0.000 abstract 5
- 108020004414 DNA Proteins 0.000 abstract 5
- 102000040650 (ribonucleotides)n+m Human genes 0.000 abstract 2
- 239000013614 RNA sample Substances 0.000 description 19
- 241000725303 Human immunodeficiency virus Species 0.000 description 16
- 238000009396 hybridization Methods 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 8
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 241000700721 Hepatitis B virus Species 0.000 description 6
- 239000002585 base Substances 0.000 description 6
- 238000000018 DNA microarray Methods 0.000 description 5
- 230000000295 complement effect Effects 0.000 description 5
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 4
- 239000006059 cover glass Substances 0.000 description 4
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 description 4
- 230000002209 hydrophobic effect Effects 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 229910000077 silane Inorganic materials 0.000 description 4
- 239000007790 solid phase Substances 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- ZTHYODDOHIVTJV-UHFFFAOYSA-N Propyl gallate Chemical class CCCOC(=O)C1=CC(O)=C(O)C(O)=C1 ZTHYODDOHIVTJV-UHFFFAOYSA-N 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 238000011897 real-time detection Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- CBCKQZAAMUWICA-UHFFFAOYSA-N 1,4-phenylenediamine Chemical compound NC1=CC=C(N)C=C1 CBCKQZAAMUWICA-UHFFFAOYSA-N 0.000 description 2
- 102000004594 DNA Polymerase I Human genes 0.000 description 2
- 108010017826 DNA Polymerase I Proteins 0.000 description 2
- -1 JOE Chemical compound 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 125000003368 amide group Chemical group 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 238000002966 oligonucleotide array Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000004153 renaturation Methods 0.000 description 2
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 241000711549 Hepacivirus C Species 0.000 description 1
- 241000721701 Lynx Species 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 239000002262 Schiff base Substances 0.000 description 1
- 150000004753 Schiff bases Chemical class 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- GYDJEQRTZSCIOI-UHFFFAOYSA-N Tranexamic acid Chemical compound NCC1CCC(C(O)=O)CC1 GYDJEQRTZSCIOI-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000007596 consolidation process Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012973 diazabicyclooctane Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 235000010388 propyl gallate Nutrition 0.000 description 1
- 229940075579 propyl gallate Drugs 0.000 description 1
- 239000000473 propyl gallate Substances 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000015607 signal release Effects 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- IMNIMPAHZVJRPE-UHFFFAOYSA-N triethylenediamine Chemical compound C1CN2CCN1CC2 IMNIMPAHZVJRPE-UHFFFAOYSA-N 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a detection technology of molecular beacon strand displacement isothermal amplifying gene chip and a kit. The detection technology disclosed by the invention combines the advantages of strand displacement isothermal amplifying technology and molecular beacon technology. According to the detection technology, DNA (Deoxyribose Nucleic Acid) is amplified under the isothermal condition; fluorescence signals of the molecular beacon are continuously released along with the amplification of the DNA, and the released target DNA are combined to new molecular beacon, so that the amplification and releasing of the fluorescence signals are carried out in turn. According to the detection technology, the purpose of amplifying the signals is achieved based on the strand displacement reaction of DNA polymerase; and the purpose of detecting signals is achieved based on open loop and closed loop of the molecular beacon. With the adoption of the detection technology disclosed by the invention, the amplification of the DNA/RNA (Ribose Nucleic Acid) and the detection of signals are integrated, the processes such as additional marketing, hybridizing and washing are saved, so that, the operation steps are simplified, the efficiency is improved; and moreover, the chip can be detected on real time, and accurate and reliable results are obtained.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of molecular beacon strand displacement isothermal duplication gene chip detecting technique.
Background technology
Gene chip is also DNA chip, DNA microarray (DNA microarray), oligonucleotide arrays (oligonucleotide array), this technology means that a large amount of probe molecules are fixed on to sample molecule rear and mark on upholder hybridizes, by detecting the hybridization signal intensity of each probe molecule and then obtaining quantity and the sequence information of sample molecule, realize biological sample quick, parallel, detect efficiently or medical diagnosis, because conventional silicon is as solid support, and use the technology of preparing of computer chip in preparation process, so be referred to as biochip technology.
Biochip technology is owing to a large amount of probes are fixed on upholder simultaneously, so can disposablely carry out determination and analysis to a large amount of sequences of sample, thereby solve traditional mRNA blot (Southern Blotting and Northern Blotting etc.) deficiencies such as technological operation is numerous and diverse, level of automation is low, operating sequence quantity is few, detection efficiency is low.And, by designing different probe arrays, using specific analytical procedure can make this technology there is multiple different using value, as gene expression profile mensuration, consolidation detection, polymorphism analysis, genomic library mapping and sequencing by hybridization etc.
But gene chip, before detecting, need to must increase to improve to the DNA/mRNA sample obtaining from sample and read sensitivity conventionally.In pcr amplification process, must carry out sample mark simultaneously, marking method has fluorescent marker method, biotin labeling method, isotope-labelling method etc.And due to the sequence difference of DNA, the fluorescently-labeled dCTP mixing is inconsistent in amplification procedure, under similarity condition, the DNA signal that G/C content is high is obviously better than the DNA that G/C content is low.Before chip detection, the probe on PCR product and chip is hybridized, and required time is long, and the required hybridization conditions of differing molecular is inconsistent, and error is large, and because hybridization is positioned at solid phase surface, so there is steric restriction effect to a certain degree.In sample preparation, current most company all will carry out amplification to a certain degree to improve the sensitivity detecting to sample at mark with before measuring, but still have many people attempting walking around this problem, this comprises the Solid phase PCR amplification system of Mosaic Technologies company and a large amount of parallel solid phase cloning process that Lynx Therapeutics company proposes, two kinds of methods respectively have relative merits, but not yet obtain practical application at present.
Strand displacement isothermal amplification technique is the external isothermal amplification method of a kind of enzymatic DNA growing up in recent years,, there is the archaeal dna polymerase of strand displacement activity, under steady temperature, untie DNA double chain by the method for non-thermally denature, a kind of new amplification mode of in extending new chain, downstream old chain being peeled off.
Molecular beacon is that one designs fluorescent probe cleverly.The two ends that are 15-30 mer oligonucleotide probe in length add respectively the cane district of 5-8 mer sequence complementation.In the time of free state, because making probe molecule, the combination of cane district complementary sequence forms hairpin like fold, so the hairpin probe that is otherwise known as.The 5' end of probe and 3' end mark fluorescent element molecule and quencher molecules respectively.When free state, two ends of hairpin structure are close, make fluorescence molecule and quencher molecule near (being about 7-10 nm), now there is FRET (fluorescence resonance energy transfer), the fluorescence that fluorescence molecule sends is quenched molecular absorption and distributes with hot form, fluorescence is almost completely by quencher, and this sole of fluorescence is low.When molecular beacon is combined the double-stranded crossbred of formation with the target molecules of sequence complete complementary, the complementary district of beacon cane is opened, and fluorescence molecule and quencher molecule are apart from increase, and the fluorescence that fluorescence molecule produces can not be quenched molecule and absorb, emitting fluorescence.And detected fluorescence intensity and the solution target amount that hits is directly proportional.Molecular beacons technology is simple to operate, highly sensitive with it, high specificity, can nucleic acid be carried out real-time quantitative mensuration, even can not only in biological study, be had a wide range of applications for features such as in-vivo analysis, and detects in and clinical study basic with the biomedicine such as diagnosis and also will serve as important role at disease gene.
Summary of the invention
The object of the present invention is to provide a kind of molecular beacon strand displacement isothermal duplication gene chip detecting technique
The technical solution adopted in the present invention is:
A kind of method that adopts molecular beacon strand displacement isothermal duplication genechip detection target dna/RNA, comprises the steps:
(1) design of molecular beacon: molecular beacon comprises ring-shaped area, 5 ' cane district and 3 ' cane district; Wherein, ring-shaped area can with target dna/RNA specific combination, the sequence in 5 ' cane district goes out 8～25 bases than 3 ' the cane head of district, is labeled as 5 ' cane district extension, the sequence of 5 ' connection ring-shaped area, cane district part and 3 ' cane region sequence complementation, make to form hairpin structure; Molecular beacon 3 ' is terminal modified fluorescence molecule;
(2) chip point sample and strand displacement isothermal amplification: molecular beacon 5 ' end is fixed to solid support surface, form molecular beacon micro-array chip, then quenching probes and sample DNA/RNA and chip are hybridized, washing, then strand displacement isothermal amplification liquid, primer are added on micro-array chip to isothermal duplication goal gene;
Or: molecular beacon is mixed with quenching probes, point sample is fixed to solid support surface, form molecular beacon micro-array chip, then strand displacement isothermal amplification liquid, primer and sample DNA/RNA are added on micro-array chip to isothermal duplication goal gene;
Described quenching probes is the 5 ' terminal modified oligonucleotide sequence that has fluorescent quenching group, has 8 bases and the extension complementation of 5 ' cane district at least, is preferably 12 bases; Described primer and 3 ' cane district complementation;
(3) amplification is carried out to fluorescence numerical analysis, according to fluorescent value, determine whether sample DNA is target dna and content thereof.
The ring-shaped area of molecular beacon is the sequence of 15～30 bases of length.
Long 5～11 bases of complementary sequence in 5 ' cane district and 3 ' cane district.
Approach chip surface very much and produce sterically hindered impact reaction for fear of molecular beacon, can connect 10～30 thymus pyrimidines or VITAMIN B4 at 5 ' end of molecular beacon.
For realizing, molecular beacon is fixed on solid support, need carry out chemically modified to 5 ' end of molecular beacon, its modification group should react with the chemical group generation covalent chemical of solid support finishing, so that by the stable molecular beacon DNA solid support surface that is connected to securely, amino as end modified in molecular beacon 5 ', solid support finishing aldehyde radical, can form Schiff base, makes the stable solid support surface that is connected to securely of molecular beacon.
The fluorescence molecule of molecular beacon 3 ' end mark is any in the inorganic dyestuffs such as organic fluorescent dye or quantum dot such as FAM, HEX, TET, FITC, Cy3, Cy5, Cy5.5, PE, TAMRA, ROX, Texas Red, AMCA, JOE, rhodamine, bodipy, Alexa dye; Fluorescent quenching group on quenching probes is P-phenylenediamine (Para-phenylene diamine, PPD), n-propyl gallate salt (propyl gallate, NPG), 1,4-bis-azo dicyclos (2,2,2)-octane (1,4-diazobicyelo (2,2,2)-octane, DABCO), any in TAMRA, BHQ-1, BHQ-2, Eclipse, Iowa Black, nm gold particles.
Solid support is any in slide, plastic sheet, microballoon, magnetic bead.
Preferably, in strand displacement isothermal duplication process, add and use fluorescently-labeled dCTP, can improve the sensitivity of its detection.
A kind of molecular beacon strand displacement isothermal duplication genechip detection test kit, comprises the molecular beacon, quenching probes, primer, the strand displacement isothermal amplification liquid that are fixed on solid support surface, wherein:
Described molecular beacon comprises ring-shaped area, 5 ' cane district and 3 ' cane district; Wherein ring-shaped area can with target dna/RNA specific combination, the sequence in 5 ' cane district goes out 8～25 Nucleotide than 3 ' the cane head of district, be labeled as 5 ' cane district extension, 5 ' cane district, near sequence and the complementation of 3 ' cane region sequence and 3 ' cane region sequence complementation of ring-shaped area part, makes to form hairpin structure; The 5 ' terminal modified have-NH of molecular beacon
2, 3 ' terminal modifiedly has a fluorescence molecule;
Described quenching probes is the 5 ' terminal modified oligonucleotide sequence that has fluorescent quenching group, at least 8 bases and the extension complementation of 5 ' cane district;
Described primer and 3 ' cane district complementation.
Consisting of of strand displacement isothermal amplification liquid: 50mM Tris-HCl pH 7.0 ~ 8.0,1.5 mM MgSO
4, 10 mM dithiothreitol (DTT) (DTT), every kind of dNTP of 50～400 μ M, 20 μ g/ml BSA, 25～100 U/ml archaeal dna polymerases.
The advantage of the comprehensive strand displacement isothermal amplification technique of the present invention and molecular beacons technology, DNA is increased under isothermal condition, and along with the amplification of DNA, the fluorescent signal of molecular beacon is constantly released, d/d target dna is attached to again on new molecular beacon, thereby carries out amplification and the fluorescent signal release of next round.The strand replacement reaction of dependence archaeal dna polymerase reaches the object of amplification amplifying signal, reaches the object of signal detection by the Open loop and closed loop of molecular beacon.Due to the strand replacement reaction of archaeal dna polymerase, eliminate quenching group due to the impact luminous on fluorophor of fluorescent energy resonance transfer.In the time there is no additional and DNA that ring is complementary, molecular beacon is in closure state, and fluorophor and quenching group are adjacent and not luminous, encircle and be opened and add after this DNA, between the group of mark, distance increases, and makes effectively cancellation fluorescence of quenching group, thereby sends detection signal.
beneficial effect of the present invention is:
The present invention is integrated DNA/RNA amplification and signal detection, and do not need additional markers, hybridization and washing process, both simplified operation steps, raise the efficiency, reaction can be carried out under constant temperature again, thereby avoided the use of PCR instrument.Also can survey by gene amplification edges frontier inspection, realized the real-time detection of chip, make result more accurately and reliably.Meanwhile, in sample, the hybridization of target dna and molecular beacon occurs on the ring of molecular beacon, has avoided traditional biological chip because hybridization is positioned at the sterically hindered impact that solid phase surface produces.
Therefore, molecular beacon strand displacement isothermal duplication of the present invention detects the development of gene chip in real time, combine the advantage of strand replacement reaction and molecular beacon, overcome the shortcoming of traditional gene chip, realize the real-time high-throughout detection of DNA and RNA biological expression level, for DNA and RNA correlative study in the fields such as functional genomics, proteomics, clinical diagnosis and drug screening provide novel method.
Brief description of the drawings
Fig. 1 is the structural representation (1: ring-shaped area, 2:3 ' cane district, 3:5 ' cane district, 4:5 ' cane district extension) of molecular beacon of the present invention;
Fig. 2 is the schematic diagram that is related between molecular beacon, general quenching probes and universal primer;
Fig. 3 is know-why and scheme schema;
Fig. 4 is that hepatitis C virus detects gene chip real-time fluorescence scanning result (being from left to right followed successively by: HCV RNA sample sets, hepatitis B virus DNA sample sets and HIV viral RNA sample sets);
Fig. 5 is HIV virus detection gene chip real-time fluorescence scanning result (being from left to right followed successively by HIV viral RNA sample sets 1, HIV viral RNA sample sets 2, hepatitis B virus DNA sample sets, HCV RNA sample sets).
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated, but be not limited to this.
embodiment 1
The present embodiment is only taking aldehyde slide as solid support, and detection HCV RNA is example, and development of the present invention and application thereof are described.
(1) design of probe and synthetic
Probe related in test is synthetic by Shanghai Sheng Gong Bioisystech Co., Ltd,
For the detection of HCV RNA, design 1 molecular beacon, sequence is as follows:
MB:NH-T
20-
cATCATCATCATCAT tCTTGGACACA aCTAACGCCATGGCTAGACC tGTGTCCAAGA-FAM(SEQ ID NO:1) (T
20for connecting 20 T of molecular beacon and solid support, underscore is ring-shaped area sequence, and italic is cane region sequence, and overstriking is 5 ' cane district extension);
General quenching probes: GTAGTAGTAGTA-TAMRA(SEQ ID NO:2);
Universal primer: CTTTCTATCTTTCTATCTTGGAC(SEQ ID NO:3);
Wherein, molecular beacon structure is shown in Fig. 1, the relation between molecular beacon structure and quenching probes and primer, as shown in Figure 2.
The special complementation of 5 ' cane district extension of general quenching probes and molecular beacon, 3 ' the cane district specificity complementation of universal primer and molecular beacon.
(2) design of chip and point sample
Slide: aldehyde radical slide (purchased from Bo Ao bio tech ltd)
(i), by above-mentioned amido modified molecular beacon, with 4 × SSC dissolving, configuration concentration is 200 nM, and 95 DEG C of sex change 5 minutes, is slowly down to afterwards room temperature, keeps 1 hour, makes molecular beacon renaturation, and is folded into correct structure;
(ii) after the DMSO that gets product obtained in the previous step and equivalent volumes mixes, adopt point sample instrument, according to putting in order of design in advance, on above-mentioned aldehyde slide, start point sample;
(iii) after point sample, chip is placed in wet box, the wet box of sealing, spends the night in 42 DEG C of reactions.Then use 4 × SSC to clean, remove not in conjunction with upper molecular beacon.
(3) hybridization of sample DNA/RNA and quenching probes and chip
By sample DNA/RNA(1 nM) and quenching probes (200 nM) mixed solution join on the slide that is fixed with molecular beacon, cover solution and microarray with the cover glass of hydrophobic silane treatment, be positioned in the wet box of sealing 42 DEG C of hybridizations 1 hour; After hybridization, slide contains 0.1%SDS with 4 × SSC() solution washing twice, finally clean 2 times with ultrapure aqua sterilisa, be placed in centrifuge tube centrifugal, remove surface liquid natural drying at room temperature.
(4) the real-time detection of the reaction of the isothermal strand displacement on chip and chip
Strand displacement isothermal amplification liquid containing universal primer (10 nM) (200 μ L) is added on the micro-array chip in step (3), with cover glass covering solution and the microarray of hydrophobic silane treatment, 42 DEG C of isothermal duplication 1h, and under chip scanner, carry out real time scan, recording fluorescent signal, according to Robust Multiple-array Average(RMA) method carries out data analysis.
Strand displacement isothermal amplification liquid (200 μ L) consists of: 5 U polymerase Klenow fragment exo-(Fermentas), 50 μ M each dNTPs, 40 mM Tris-HCl pH7.8,10 mM DTT, 10 mM MgCl
2.
Three experimental group (HCV RNA sample sets, hepatitis B virus DNA sample sets and HIV viral RNA sample sets) are established in this experiment, all establish a blank (substituting molecular beacon with deionized water) for each group.Experimental result is as shown in Figure 4: only HCV RNA sample sets has fluorescent signal, fluorescence intensity is 8.27855, analyze according to RMA, its nucleic acid concentration is 1.0524nM, hepatitis B virus DNA sample sets and HIV viral RNA sample sets and each blank group all do not have fluorescent signal, the detected result of present method are described accurately and reliably.
embodiment 2
The present embodiment is only taking aldehyde slide as solid support, and detecting HIV viral RNA is example, and development of the present invention and application thereof are described.
(1) design of probe and synthetic
Probe related in test is synthetic by Shanghai Sheng Gong Bioisystech Co., Ltd,
For the detection of HIV viral RNA, design 1 molecular beacon, sequence is as follows:
MB:NH-T
20-
cATCATCATCATCAT tCTTGGACACA gAACCAAGGGGAACTGAC tGTGTCCAAGA-FAM(SEQ ID NO:4) (T
20for connecting 20 T of molecular beacon and solid support, underscore is ring-shaped area sequence, and italic is cane region sequence, and overstriking is 5 ' cane district extension);
General quenching probes: GTAGTAGTAGTA-TAMRA(SEQ ID NO:5);
Universal primer: TCATCAATCAATCTTTCTTGGAC(SEQ ID NO:6);
Wherein, the relation between molecular beacon structure and quenching probes and primer, as shown in Figure 2.
The special complementation of 5 ' cane district extension of general quenching probes and molecular beacon, 3 ' the cane district specificity complementation of universal primer and molecular beacon.
(2) design of chip, quenching probes hybridization and point sample
Slide: aldehyde radical slide (purchased from Bo Ao bio tech ltd)
(i) by above-mentioned amido modified molecular beacon and quenching probes, with 4 × SSC mixed dissolution, configuration concentration is 200 nM, and 95 DEG C of sex change 5 minutes, slowly be down to afterwards room temperature, keep 1 hour, make molecular beacon renaturation, and being folded into correct structure, quenching probes is attached on molecular beacon simultaneously;
(ii) after the DMSO that gets product obtained in the previous step and equivalent volumes mixes, adopt point sample instrument, according to putting in order of design in advance, on above-mentioned aldehyde slide, start point sample;
(iii) after point sample, chip is placed in wet box, the wet box of sealing, spends the night in 42 DEG C of reactions.Then use 4 × SSC to clean, remove not in conjunction with upper molecular beacon and probe.
(3) hybridization of sample RNA and chip
By sample RNA(1 nM) join on the slide that is fixed with molecular beacon, cover solution and microarray with the cover glass of hydrophobic silane treatment, be positioned in the wet box of sealing 42 DEG C of hybridizations 1 hour; After hybridization, slide contains 0.1%SDS with 4 × SSC() solution washing twice, finally clean 2 times with ultrapure aqua sterilisa, be placed in centrifuge tube centrifugal, remove surface liquid natural drying at room temperature.
(4) the real-time detection of the reaction of the isothermal strand displacement on chip and chip
Strand displacement isothermal amplification liquid containing universal primer (10 nM) (200 μ L) is added on the micro-array chip in step (3), with cover glass covering solution and the microarray of hydrophobic silane treatment, 42 DEG C of isothermal duplication 1h, and under chip scanner, carry out real time scan, recording fluorescent signal, according to Robust Multiple-array Average(RMA) method carries out data analysis.
Strand displacement isothermal amplification liquid (200 μ L) consists of: 5 U polymerase Klenow fragment exo-(Fermentas), 50 μ M dNTPs, 40 mM Tris-HCl pH7.8,10 mM DTT, 10 mM MgCl
2.
Four experimental group (dCTP in HIV viral RNA sample sets 1, HIV viral RNA sample sets 2(strand displacement isothermal amplification liquid replaces with isocyatic FAM-11-dCTP) are established in this experiment), hepatitis B virus DNA sample sets, HCV RNA sample sets, all establish a blank (substituting molecular beacon with deionized water) for each group.Experimental result is as shown in Figure 5: hepatitis B virus DNA sample sets and HCV RNA sample sets, and each blank group does not all have fluorescent signal, only HIV viral RNA sample sets and HIV viral RNA sample sets 2 have fluorescent signal, HIV viral RNA sample sets fluorescence intensity is respectively 7.7218, analyze according to RMA, its nucleic acid concentration is 0.9986nM, HIV viral RNA sample sets 2 fluorescence intensities are 10.63853, the fluorescent value of HIV viral RNA sample sets 2 is higher than the fluorescent value of HIV viral RNA sample sets, prove that FAM-11-dUTP can play the effect that improves fluorescent signal to the replacement of dCTP, the detected result of present method is described accurately and reliably.
Above embodiment is only for introducing preferred case of the present invention, and to those skilled in the art, any apparent changes and improvements of carrying out in the scope that does not deviate from spirit of the present invention, all should be regarded as a part of the present invention.
<110> Jiangyin Tianrui Biotech LLC
<120> molecular beacon strand displacement isothermal duplication gene chip detecting technique and test kit
<130>
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 57
<212> DNA
<213> artificial sequence
<400> 1
catcatcatc atcattcttg gacacaacta acgccatggc tagacctgtg tccaaga 57
<210> 2
<211> 12
<212> DNA
<213> artificial sequence
<400> 2
gtagtagtag ta 12
<210> 3
<211> 23
<212> DNA
<213> artificial sequence
<400> 3
ctttctatct ttctatcttg gac 23
<210> 4
<211> 55
<212> DNA
<213> artificial sequence
<400> 4
catcatcatc atcattcttg gacacagaac caaggggaac tgactgtgtc caaga 55
<210> 5
<211> 12
<212> DNA
<213> artificial sequence
<400> 5
gtagtagtag ta 12
<210> 6
<211> 23
<212> DNA
<213> artificial sequence
<400> 6
tcatcaatca atctttcttg gac 23
Claims (7)
1. non-medical diagnosis on disease object adopts a method of molecular beacon strand displacement isothermal duplication genechip detection target RNA, comprises the steps:
(1) design of molecular beacon: molecular beacon comprises ring-shaped area, 5 ' cane district and 3 ' cane district; Wherein, ring-shaped area can with target RNA specific combination, the sequence in 5 ' cane district goes out 8～25 bases than 3 ' the cane head of district, is labeled as 5 ' cane district extension, the sequence of 5 ' connection ring-shaped area, cane district part and 3 ' cane region sequence complementation, make to form hairpin structure; Molecular beacon 3 ' is terminal modified fluorescence molecule;
(2) chip point sample and strand displacement isothermal amplification: molecular beacon 5 ' end is fixed to solid support surface, form molecular beacon micro-array chip, then quenching probes and sample RNA and chip are hybridized, washing, then strand displacement isothermal amplification liquid, primer are added on micro-array chip to isothermal duplication goal gene;
Or: molecular beacon is mixed with quenching probes, and point sample is fixed to solid support surface, forms molecular beacon micro-array chip, then strand displacement isothermal amplification liquid, primer and sample RNA is added on micro-array chip to isothermal duplication goal gene;
Described quenching probes is the 5 ' terminal modified oligonucleotide sequence that has fluorescent quenching group, has 8 bases and the extension complementation of 5 ' cane district at least; Described primer and 3 ' cane district complementation;
(3) amplification is carried out to fluorescence numerical analysis, according to fluorescent value, determine whether sample RNA is target RNA and content thereof;
It is characterized in that: described molecular beacon, quenching probes and primer are for the detection of HCV RNA or for the detection of HIV viral RNA, and wherein, the sequence detecting for HCV RNA is as follows:
Molecular beacon:
5 '-NH-T
20-CATCATCATCATCATTCTTGGACACA
aCTAACGCCATGGCTAGACCtGTGTCCAAGA-FAM-3 ', wherein T
20for connecting 20 T of molecular beacon and solid support;
Quenching probes: 3 '-GTAGTAGTAGTA-TAMRA-5 ';
Primer: 5 '-CTTTCTATCTTTCTATCTTGGAC-3 ';
The sequence detecting for HIV viral RNA is as follows:
Molecular beacon:
5 '-NH-T
20-CATCATCATCATCATTCTTGGACACA
gAACCAAGGGGAACTGACt GTGTCCAAGA-FAM-3 ', wherein T
20for connecting 20 T of molecular beacon and solid support;
Quenching probes: 3 '-GTAGTAGTAGTA-TAMRA-5 ';
Primer: 5 '-TCATCAATCAATCTTTCTTGGAC-3 '.
2. method according to claim 1, is characterized in that, the 5 ' end modified amino of described molecular beacon, solid support finishing aldehyde radical.
3. method according to claim 1, is characterized in that, described solid support is any in slide, plastic sheet, microballoon, magnetic bead.
4. method according to claim 1, is characterized in that, the consisting of of described strand displacement isothermal amplification liquid: 50mMTris-HClpH7.0～8.0,1.5mMMgSO
4, 10mM dithiothreitol (DTT) (DTT), every kind of dNTP of 50～400 μ M, 20 μ g/mlBSA, 25～100U/mlDNA polysaccharase.
5. method according to claim 1, is characterized in that, in strand displacement isothermal duplication process, and the dCTP fluorescent mark adding.
6. a molecular beacon strand displacement isothermal duplication genechip detection test kit, comprises the molecular beacon, quenching probes, primer, the strand displacement isothermal amplification liquid that are fixed on solid support surface, wherein:
Described molecular beacon comprises ring-shaped area, 5 ' cane district and 3 ' cane district; Wherein ring-shaped area can with target RNA specific combination, the sequence in 5 ' cane district goes out 8～25 Nucleotide than 3 ' the cane head of district, be labeled as 5 ' cane district extension, 5 ' cane district, near sequence and the complementation of 3 ' cane region sequence and 3 ' cane region sequence complementation of ring-shaped area part, makes to form hairpin structure; 3 ' of molecular beacon terminal modifiedly has a fluorescence molecule;
Described quenching probes is the 5 ' terminal modified oligonucleotide sequence that has fluorescent quenching group, at least 8 bases and the extension complementation of 5 ' cane district;
Described primer and 3 ' cane district complementation;
It is characterized in that, described molecular beacon, quenching probes and primer are for the detection of HCV RNA or for the detection of HIV viral RNA, and wherein, the sequence detecting for HCV RNA is as follows:
Molecular beacon:
5 '--NH-T
20-CATCATCATCATCATTCTTGGACACA
aCTAACGCCATGGCTAGAC ctGTGTCCAAGA-FAM-3 ', wherein T
20for connecting 20 T of molecular beacon and solid support;
Quenching probes: 3 '-GTAGTAGTAGTA-TAMRA-5 ';
Primer: 5 '-CTTTCTATCTTTCTATCTTGGAC-3 ';
The sequence detecting for HIV viral RNA is as follows:
Molecular beacon:
5 '-NH-T
20-CATCATCATCATCATTCTTGGACACA
gAACCAAGGGGAACTGACt GTGTCCAAGA-FAM-3 ', wherein T
20for connecting 20 T of molecular beacon and solid support;
Quenching probes: 3 '-GTAGTAGTAGTA-TAMRA-5 ';
Primer: 5 '-TCATCAATCAATCTTTCTTGGAC-3 '.
7. test kit according to claim 6, is characterized in that, the consisting of of described strand displacement isothermal amplification liquid: 50mMTris-HClpH7.0～8.0,1.5mMMgSO
4, 10mM dithiothreitol (DTT) (DTT), every kind of dNTP of 50～400 μ M, 20 μ g/mlBSA, 25～100U/mlDNA polysaccharase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210361822.3A CN102888457B (en) | 2012-09-25 | 2012-09-25 | Detection technology of molecular beacon strand displacement isothermal amplifying gene chip and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210361822.3A CN102888457B (en) | 2012-09-25 | 2012-09-25 | Detection technology of molecular beacon strand displacement isothermal amplifying gene chip and kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102888457A CN102888457A (en) | 2013-01-23 |
| CN102888457B true CN102888457B (en) | 2014-09-03 |
Family
ID=47532139
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210361822.3A Expired - Fee Related CN102888457B (en) | 2012-09-25 | 2012-09-25 | Detection technology of molecular beacon strand displacement isothermal amplifying gene chip and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102888457B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104745727B (en) * | 2015-03-25 | 2017-04-26 | 江苏省原子医学研究所 | Probe based on double signal amplification and application of probe |
| CN105039519B (en) * | 2015-06-12 | 2018-01-02 | 青岛科技大学 | A kind of DNA based on the hybridization of foot point domain determines new method |
| CN106987519A (en) * | 2017-03-28 | 2017-07-28 | 博奥生物集团有限公司 | A kind of micro-array chip, application method and purposes |
| CN107893101B (en) * | 2017-12-22 | 2021-06-15 | 郑州大学 | Kit and method for early diagnosis of tumor diseases and application |
| CN109055500A (en) * | 2018-09-13 | 2018-12-21 | 中国人民解放军疾病预防控制所 | A kind of fluorescence ring mediated isothermal amplification method based on molecular beacon |
| CN109321669B (en) * | 2018-10-29 | 2022-03-15 | 江南大学 | Method for fluorescence detection of staphylococcus aureus based on chimera sequence design and molecular beacon |
| CN109517903A (en) * | 2018-11-28 | 2019-03-26 | 江南大学 | Strand displacement type archaeal dna polymerase induces sex taboo object in pig source in isothermal circulation amplified reaction detection halal food |
| CN109593863A (en) * | 2018-11-28 | 2019-04-09 | 江南大学 | A kind of method of strand displacement type archaeal dna polymerase induction isothermal circulation amplified reaction detection meat sources |
| CN109991203B (en) * | 2019-04-18 | 2020-08-18 | 上海大学 | Kit and method for detecting ochracin A |
| CN113624724A (en) * | 2020-05-07 | 2021-11-09 | 廖世奇 | Multi-element detection and analysis method of aptamer molecular beacon for target molecule |
| CN112941154B (en) * | 2021-02-21 | 2022-09-09 | 中南大学 | Molecular beacon probe, application of molecular beacon probe in preparation of circBART2.2 detection preparation and kit |
| CN114397343B (en) * | 2022-03-25 | 2022-06-14 | 南京邮电大学 | Tumor marker activity detection kit, detection method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1526827A (en) * | 2003-03-03 | 2004-09-08 | �廪��ѧ | Laboratory nucleic acid analyzing chip system and its application |
| WO2011050173A1 (en) * | 2009-10-21 | 2011-04-28 | Brandeis University | Methods, kits and reaction mixtures for analyzing single-stranded nucleic acid sequences |
| CN102373267A (en) * | 2010-08-09 | 2012-03-14 | 北京泰格瑞分子检验有限公司 | Taqman real-time fluorescence PCR (polymerase chain reaction) capable of reducing polymerization with primers |
-
2012
- 2012-09-25 CN CN201210361822.3A patent/CN102888457B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1526827A (en) * | 2003-03-03 | 2004-09-08 | �廪��ѧ | Laboratory nucleic acid analyzing chip system and its application |
| WO2011050173A1 (en) * | 2009-10-21 | 2011-04-28 | Brandeis University | Methods, kits and reaction mixtures for analyzing single-stranded nucleic acid sequences |
| CN102373267A (en) * | 2010-08-09 | 2012-03-14 | 北京泰格瑞分子检验有限公司 | Taqman real-time fluorescence PCR (polymerase chain reaction) capable of reducing polymerization with primers |
Non-Patent Citations (4)
| Title |
|---|
| Qiuping Guo, et al.Sensitive fluorescence detection of nucleic acids based on isothermal circular strand-displacement polymerization reaction.《Nucleic Acids Research》.2009,第37卷(第3期),e20. |
| Sensitive fluorescence detection of nucleic acids based on isothermal circular strand-displacement polymerization reaction;Qiuping Guo, et al;《Nucleic Acids Research》;20090107;第37卷(第3期);e20 * |
| 核酸分子探针结合信号放大技术用于核酸的检测;鲍艳艳;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20120815(第8期);E060-20 * |
| 鲍艳艳.核酸分子探针结合信号放大技术用于核酸的检测.《中国优秀硕士学位论文全文数据库 医药卫生科技辑》.2012,(第8期),E060-20. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102888457A (en) | 2013-01-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102888457B (en) | Detection technology of molecular beacon strand displacement isothermal amplifying gene chip and kit | |
| Shen et al. | Recent advances and perspectives of nucleic acid detection for coronavirus | |
| ES2895184T3 (en) | Nucleic Acid Sequencing Compositions | |
| TWI324182B (en) | Detection of nucleic acid variation by cleavage-amplification method | |
| CN105821138B (en) | A kind of method that double loop-stem structure DNA profiling detection nucleic acid are built based on coupled reaction | |
| US20060292616A1 (en) | Single molecule miRNA-based disease diagnostic methods | |
| CN108431235A (en) | The method of the fluoroscopic examination of the isothermal loop mediated amplification (LAMP) of target nucleic acid, oligonucleotides and its kit | |
| Lee et al. | Direct, sequence-specific detection of dsDNA based on peptide nucleic acid and graphene oxide without requiring denaturation | |
| CN107208140B (en) | Method for detecting and quantifying biological materials by using the activity of nucleic acid polymerases modulated by target materials | |
| CN100417730C (en) | DNA sequence measurement based on primer extension | |
| JP2005537030A (en) | Methods for analyzing nucleic acids | |
| TWI701337B (en) | Pna probes, kits and methods for detecting genotypes of human papillomavirus | |
| CN104946634A (en) | Double-labelling fluorescence activation type probe and method for detecting DNA by adopting same | |
| US20100248980A1 (en) | Method for Selective Labeling and Detection of Target Nucleic Acids Using Immobilized Peptide Nucleic Acid Probes | |
| CN109797201A (en) | A kind of original position multiple nucleic acid detection method | |
| CN107532213A (en) | Method for detecting multiple nucleotide sequences in sample simultaneously | |
| WO2012077819A1 (en) | Method and kit for detecting target nucleic acid | |
| Xu et al. | New molecular beacon for p53 gene point mutation and significant potential in serving as the polymerization primer | |
| Zhang et al. | Sensitive detection of transcription factors in cell nuclear extracts by using a molecular beacons based amplification strategy | |
| CN104726603B (en) | Graphene quantum dot based molecular beacon sensor as well as preparation method and application of sensor | |
| WO2007002375A2 (en) | Methods and compositions for analysis of microrna | |
| CN103757133B (en) | Gene chip kit that a kind of hydrocoles DNA virus detects and its preparation method and application | |
| CN104321443A (en) | HEV assay | |
| Xiang et al. | Multiplexed DNA detection with a composite molecular beacon based on guanine-quenching | |
| US20240229121A1 (en) | Micro-rna detection method and kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20151124 Address after: 510535 Luogang District Kaiyuan Road, Guangdong, Guangzhou No. 190 Patentee after: Zeng Lingwen Address before: 214400 hundred bridge biological garden A806, 85 Sha Shan Road, Wuxi, Jiangsu, Jiangyin Patentee before: JIANGYIN TIANRUI BIOTECH LLC |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20161125 Address after: 430074 Hubei Province, East Lake hi tech Zone, high tech Avenue, No. 888 high agricultural park headquarters A District, building No. 2, layer 203, No., No. 16 Patentee after: WUHAN ZHONGKE ZHIKANG BIOTECHNOLOGY CO.,LTD. Address before: 510535 Luogang District Kaiyuan Road, Guangdong, Guangzhou No. 190 Patentee before: Zeng Lingwen |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140903 |