CN1450172A - Method for detecting SARS virus and its reagent kit - Google Patents
Method for detecting SARS virus and its reagent kit Download PDFInfo
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- CN1450172A CN1450172A CN 03116659 CN03116659A CN1450172A CN 1450172 A CN1450172 A CN 1450172A CN 03116659 CN03116659 CN 03116659 CN 03116659 A CN03116659 A CN 03116659A CN 1450172 A CN1450172 A CN 1450172A
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- 241000315672 SARS coronavirus Species 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 21
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 6
- 241000700605 Viruses Species 0.000 claims abstract description 8
- 238000003752 polymerase chain reaction Methods 0.000 claims description 13
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 claims description 12
- 108020004414 DNA Proteins 0.000 claims description 9
- 238000001514 detection method Methods 0.000 claims description 9
- 150000007523 nucleic acids Chemical group 0.000 claims description 9
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- 238000013016 damping Methods 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 239000002299 complementary DNA Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 4
- 238000005516 engineering process Methods 0.000 claims description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 4
- 238000009666 routine test Methods 0.000 claims description 4
- GLXHFRGBLGCWIK-UHFFFAOYSA-N 2-[carbamimidoyl(methyl)amino]tetradecanoic acid Chemical compound C(CCCCCCCCCCC)C(C(=O)O)N(C)C(N)=N GLXHFRGBLGCWIK-UHFFFAOYSA-N 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- 239000012472 biological sample Substances 0.000 claims description 3
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 claims description 3
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical class N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 claims description 3
- 230000003248 secreting effect Effects 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- 239000001632 sodium acetate Substances 0.000 claims description 3
- 229960004249 sodium acetate Drugs 0.000 claims description 3
- 235000017281 sodium acetate Nutrition 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- 230000002103 transcriptional effect Effects 0.000 claims description 3
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 3
- 229940038773 trisodium citrate Drugs 0.000 claims description 3
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 3
- 238000003745 diagnosis Methods 0.000 abstract description 4
- 241001493065 dsRNA viruses Species 0.000 abstract description 2
- 230000009385 viral infection Effects 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 abstract 1
- 238000003757 reverse transcription PCR Methods 0.000 abstract 1
- 241000711573 Coronaviridae Species 0.000 description 8
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- 229920000936 Agarose Polymers 0.000 description 3
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 2
- 229960005542 ethidium bromide Drugs 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- -1 2.5 μ l Substances 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 241000726445 Viroids Species 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009351 contact transmission Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
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- 238000002474 experimental method Methods 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
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- 239000003550 marker Substances 0.000 description 1
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- 238000010369 molecular cloning Methods 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for clinically detecting SARS virus and its reagent box. According to the characteristic analysis of SARS virus genome sequence, selecting specific region, and according to the characteristics of said virus being RNA virus, utilizing specific primer sequence and adopting RT-PCR and PCR related reagents and techniques to establish a set of method for quickly detecting SARS virus. The invention has the advantages of solving the problem of early and rapid diagnosis of the SARS virus infected person in clinic, having higher accuracy and providing a rapid and accurate diagnosis method and a kit for clinically detecting the SARS virus infection.
Description
Technical field
The present invention relates to detect the method and the test kit thereof of severe acute respiratory syndrome virus.
Background technology
Severe acute respiratory syndrome virus, be that SARS virus is a kind of serious acute respiratory disease syndromes (Severe Acute Respiratory Syndrome that causes, SARS), the virus that claims severe acute respiratory syndrome again, belong to coronavirus (coronavirus) viroid, genome contains 29,737 Nucleotide altogether.This disease is mainly by the closely air spittle and contact transmission closely.Also there is not effective immunochemistry detection method before this virales, can only diagnose according to clinical symptom (high heat, dry cough, the detection of lung X-ray light have shade etc.), be subjected to great limitation, can only after sufferer's morbidity, just can make diagnosis, and be difficult for and other pneumonia and general influenza difference.
Summary of the invention
The purpose of this invention is to provide a kind of method and test kit thereof that detects severe acute respiratory syndrome virus.
The method of the detection severe acute respiratory syndrome virus that invention provides, it comprises following step:
1) gets suspicious patient's biological samples such as serum, secretory product or tissue, extract RNA;
The method of extracting RNA can adopt common molecular biology experiment handbook, and the method as " molecular cloning " introduced also can adopt the commercial reagents box, as Trizol
R, by specification carries out.
2) add reverse primer and reversed transcriptive enzyme, synthetic cDNA;
The reverse primer that adds is designed for requiring according to severe acute respiratory syndrome virus specific nucleic acid squences and PCR reaction, and " severe acute respiratory syndrome virus specific nucleic acid squences " has the nucleotide sequence shown in Seq.ID N6.1, Seq.ID No.2, Seq.ID No.3, Seq.ID No.4, Seq.ID No.5, the Seq.ID No.6;
The primer sequence that adopts in the invention is the nucleotide sequence shown in the table 1, and R is a reverse primer in the table 1; Adopt commercial kit, as Invitrogen
TMCorporation's Super Script test kit, the by specification step is synthesized cDNA.
3) add forward primer, utilize the genomic nucleic acid sequence of polymerase chain reaction (PCR) technology amplification severe acute respiratory syndrome virus;
The forward primer that adds is designed for requiring according to severe acute respiratory syndrome virus specific nucleic acid squences and PCR reaction, and " severe acute respiratory syndrome virus specific nucleic acid squences " is for having the nucleotide sequence shown in Seq.ID No.1, Seq.ID No.2, Seq.ID No.3, Seq.ID No.4, Seq.ID No.5, the Seq.ID No.6;
The primer sequence that adopts in the invention is the nucleotide sequence shown in the table 1, and L is a forward primer in the table 1;
Among the present invention, the general standard that consists of of PCR reaction system is formed, and comprising: PCR reaction buffer (Tris.Cl 125mM PH 8.1; (NH
4)
2SO
4125mM; MgCl
212.5mM; BSA 1mg/ml; Formamide 25%); Reaction conditions refers to: the template denaturation temperature is 92 ℃-98 ℃, and preferred: 95 ℃, the time is 10 seconds-10 minutes, and is preferred: 30 seconds; The template annealing temperature is 45 ℃--65 ℃, preferred: 52 ℃, the time is 10 seconds-10 minutes, and is preferred: 30 seconds; The template elongating temperature is 65 ℃--85 ℃, preferred: 70 ℃, the time is 10 seconds-10 minutes, and is preferred: 40 seconds; Circulation 20--40 time, preferred: 30 times.
4) adopt Routine Test Lab to detect the dna technique detected result;
Routine Test Lab detects dna technique and detects, as adopts 1% agarose electrophoresis to detect: use ethidium bromide staining, observe under UV-light, the positive of band arranged, no band negative; Also can adopt the fluorescent PCR method to detect to improve detection sensitivity, its method all can be found at the Routine Test Lab handbook.
The test kit that is used to detect severe acute respiratory syndrome virus of the present invention, it comprises following reagent: (solution D contains: 2.5 gram guanidinium isothiocyanates, the Trisodium Citrate (PH7.0) of 1.76 milliliters of 0.75M, 2.46 milliliter 10% dodecyl creatine sodium for PBS damping fluid, solution D, add 29.3 milliliters in water, add the mercaptoethanol of 7.2 μ l/ml before using), phenol, chloroform and 4.0M (PH=3.8) sodium-acetate, RT damping fluid dNTP, primer and working instructions.
The present invention is according to the genome sequence analytical results of severe acute respiratory syndrome virus, choose the distinctive nucleotide sequence of this virus district, the design primer, and be the characteristics of a kind of ribonucleic acid virus (RNA viruses) at this virus, for clinical detection of SARS virus provides diagnostic method and test kit fast and accurately.Adopt this method to carry out gene test to suspicious the infected apace, solved clinical early stage quick diagnosis problem severe acute respiratory syndrome virus infection person.
Description of drawings
Fig. 1 is the result that agarose electrophoresis detects, the positive result of 1-5 from left to right, and the negative contrast of 7-8,10 is molecular weight marker.
Embodiment
Embodiment
1, gets suspicious patient's biological sample, extract RNA;
1) gets suspicious patient's serum, secretory product or tissue, add PBS damping fluid 50 μ l, solution D 160 μ l, phenol 160 μ l, chloroform 32 μ l and 4.0M (PH=3.8) sodium-acetate 9 μ l.
Solution D contains: 2.5 gram guanidinium isothiocyanates, the Trisodium Citrate (PH7.0) of 1.76 milliliters of 0.75M, 2.46 milliliter 10% dodecyl creatine sodium, add 29.3 milliliters in water, and add the mercaptoethanol of 7.2 μ l/ml before using.
2) the jolting mixed solution is 15 seconds, places on ice 15 minutes.
3) 14000rpm, 4 ℃ centrifugal 20 minutes.
4) get supernatant, add isopyknic Virahol, placed on ice 25 minutes.
5) 4 ℃, the centrifugal 20-30 of 14000rpm minute.
6) precipitation washes twice with-20 ℃ of precooled ethanol, and vacuum is drained, and is dissolved in the DEPC water.
2, add reverse primer and reversed transcriptive enzyme, synthetic cDNA;
Adopt Invitrogen
TMCorporation's Super Script test kit
1) listed reverse primer (0.5ug/ml) 2.5 μ l in an amount of sample RNA, the adding table 1,95 ℃ are incubated 3-4 minute, cooled on ice.
2) add 5 * RT damping fluid, 4.0 μ l, dNTP (10mM) 1.0 μ l, 0.1M DTT 2.0 μ l, Superscript (200u/ μ l) 1.0 μ l, Rnasein (25u) 1.0 μ l, 40 ℃ are incubated 1 hour, and 70 ℃ are incubated 5 minutes, cooled on ice.
3, add forward primer, utilize the genomic nucleic acid sequence of polymerase chain reaction (PCR) technology amplification severe acute respiratory syndrome virus;
Get forward primer 1.0 μ l listed in step 2 synthetic cDNA template 1.0 μ l, the adding table 1,10 * PCR reaction buffer, 2.5 μ l, Mg
2+(25mM) 1.75 μ l, dNTP (2.5mM) 0.8 μ l, Taq (5u/ μ l) 0.25 μ l add water to 25 μ l; 95 ℃ are incubated 5 minutes.95 ℃, 30 seconds; 54 ℃, 45 seconds; 72,1 minutes, circulate 30 times, 72 ℃ are incubated 7 minutes.
4, the result detects
1% agarose electrophoresis detects, and uses ethidium bromide staining, observes under the UV-light, and the positive of band arranged, no band negative.
The primer sequence that table 1 is used to detect
| The primer title | Primer sequence | The genome position |
| p00001.1.L | ACGAACTTTAAAATCTGTGTAGC | ????51 |
| p00001.59.L | ATGCCTTAAGCACCAATCAC | ????472 |
| p00001.1.R | ATTTCTGCAACCAGCTCAAC | ????504 |
| p00001.70.L | CAAGTCAATGTGCACTCTTTC | ????875 |
| p00001.59.R | CTCTTCGACTCGATGTAATCA | ????905 |
| p00001.7.L | TGTGGCACTGAAAATTTAGTTA | ????1278 |
| p00001.70.R | TAGGTACCCACATGTAGTAGGTC | ????1306 |
| p00001.18.L | GGCATCTTTCTCTGCTTCTAC | ????1679 |
| p00001.7.R | GGTTTTGAAAGACTTGTAATCAA | ????1729 |
| p00001.29.L | CTCAGTGGTTGTCTAATCTTTTG | ????2077 |
| p00001.18.R | CTGAGTTTTTCAACAGTAGTGC | ????2101 |
| p00001.40.L | AGGTGATTCACATGACACAGTA | ????2477 |
| p00001.29.R | GAACAACCTCCTCAGAGGTAA | ????2500 |
| p00001.51.L | AGAGGCTGTTGTGAAGACTTT | ????2879 |
| p00001.40.R | AGGAGATCAGAAACTGGTTGT | ????2900 |
| p00001.57.L | TGGTTATTTAAAACTTACTGACAAT | ????3269 |
| p00001.51.R | CCTTAACGATGTCAACACATTT | ????3303 |
| p00001.58.L | AACCACTTCAGTCTTTACAAGTG | ????3652 |
| p00001.57.R | GCTTTGTCATTGACTGCAATA | ????3704 |
| p00001.60.L | TGTTGTAATACCCTCCAAAAAG | ????4070 |
| p00001.58.R | TCTTGAGAGCATCTCAGTAGT | ????4101 |
| p00001.61.L | TTATTACGAAGCTGAACTCTCTA | ????4477 |
| p00001.60.R | ATTGTGACAAGCGGCTCATT | ????4500 |
| p00001.62.L | GACTATAAAAGTGTTCACAACTG | ????4877 |
| p00001.61.R | GTGTGTGGAGATTAGTGTTGTC | ????4902 |
| p00001.63.L | TATTATAGAGCCCGTGCTGGT | ????5274 |
| p00001.62.R | AGTATGAGTGCACAAAAGTTAGC | ????5301 |
| p00001.64.L | TCAGTGTGGTCATTACACTCATA | ????5672 |
| p00001.63.R | AATACGATAGAGGGTCTCCTTAG | ????5701 |
| p00001.65.L | GCGATGTAGTGGCTATTGAC | ????6073 |
| p00001.64.R | TTCTTGAAACTCGCTGAATAGT | ????6100 |
| p00001.66.L | GGAAAACACAAGCATTACCAT | ????6479 |
| p00001.65.R | CCTAAGGCTAGTGAAAGCTCAT | ????6511 |
| p00001.67.L | TGGCTATTGTTGTTAAGTATTTG | ????6873 |
| p00001.66.R | GCAGTTACACAGATTAGAGAACC | ????6900 |
| p00001.68.L | GCTCATGTGGTTTATCATTAGTA | ????7268 |
| p00001.67.R | TACATCCTAACCATTGCAGAA | ????7310 |
| p00001.69.L | CGCTTCACCTCTACTTTGAC | ????7678 |
| p00001.68.R | CTCTCATAGGTCTTTTGACCAG | ????7702 |
| p00001.71.L | TCCTTTCTACATTCGTGTCAG | ????8065 |
| p00001.69.R | GTCAACATCGGTATCAACAAC | ????8100 |
| p00001.72.L | TGTTAGTACTTGTTTTAAACTTATGC | ????8474 |
| p00001.71.R | CGCACAATAATGTGGCCTTA | ????850 |
| p00001.73.L | CTACACACCTTCCAAACTCATT | ????8867 |
| p00001.72.R | AAGAACGCAAGCAGAGGTAG | ????8905 |
| p00001.74.L | TACTCCTCTTGTGCAACCTG | ????9272 |
| p00001.73.R | CACTACTGAAGCAGACACATCTA | ????930 |
| p00001.75.L | CATGTTTAATGGAGTTACATTTAGT | ????9674 |
| p00001.74.R | TTGTTGAGCAAAAAGGTACAC | ????9719 |
| p00001.2.L | TGTGGTTGGATGACACAGTAT | ????10057 |
| p00001.75.R | TAAGCATGTCTTCTGCTGTG | ????10100 |
| p00001.3.L | GAGTACACGCTGGTACTGACTTA | ????10477 |
| p00001.2.R | AAATGGACCATAGAATTTACCTT | ????1050 |
| p00001.4.L | ACCTTCCAAGGTAAGTTCAAG | ????10878 |
| p00001.3.R | GATGAGTGCCCTTAACAATTT | ????10900 |
| p00001.5.L | GTTTATGATGATGCTGCTAGAC | ????11277 |
| p00001.4.R | GACATTCATCAGTGTCCAAAC | ????1130 |
| p00001.6.L | CCTAAGAGTAGTATTGATGCTTTC | ????11670 |
| p00001.5.R | CTCCAATACCCAACAACTTAAT | ????11703 |
| p00001.8.L | GCTGTAGCTAATGGTGATCTG | ????12078 |
| p00001.6.R | CTTTAACTTTTTGAGAACGACTT | ????12100 |
| p00001.9.L | CAAGTTGTTGATGCGGATAG | ????12477 |
| p00001.8.R | TTAATTTCACTAAGTTGAACAATC | ????12500 |
| p00001.10.L | CAAAGGCTTAAACAACCTAAATA | ????12872 |
| p00001.9.R | CCTGAAGACGTACTGTAGCA | ????12920 |
| p00001.11.L | CACTTAGAAACACAGTCTGTACC | ????13270 |
| p00001.10.R | AGCCATAACCTTTCCACATT | ????13301 |
| p00001.12.L | GTGACATGGTACCACATATATCA | ????13677 |
| p00001.11.R | CAGCCATTGTGTATTTAGTTAGA | ??????13708 |
| p00001.13.L | GCCCATCCTCACTTTGACTA | ????14080 |
| p00001.12.R | AGCATCCATATGGGACTCAG | ????14112 |
| p00001.14.L | TATGCTGCTGATCCAGCTAT | ????14474 |
| p00001.13.R | AGCAATAAATTGCCAGAAG | ????14502 |
| p00001.15.L | TAATAAATGGGGTAAGGCTAGA | ????14872 |
| p00001.14.R | ATCTTGATCCTCATAACTCATTG | ????14907 |
| p00001.16.L | CAAACATAACACTTGCTGTAACTT | ????15274 |
| p00001.15.R | CTAACCTGTAGAAACGGTGTG | ????15300 |
| p00001.17.L | GTTTAGTAGCTAGCATTAAGAACT | ????15675 |
| p00001.16.R | CAGACATGAACACATTATTTTGA | ????15718 |
| p00001.19.L | TGCTAACTAATGATAACACCTCAC | ????1607 |
| p00001.17.R | ATAGCCTCATAAAACTCAGGTTC | ????16103 |
| p00001.20.L | ATAGCAACATGTGATTGGACTAA | ????16475 |
| p00001.19.R | ACAAGTGTTGGCAAGTATGTAAT | ????16506 |
| p00001.21.L | CAAGAGCACTATGTGAGAATTAC | ????16877 |
| p00001.20.R | ATGTTGAGTGTTGGGTACAAG | ????16903 |
| p00001.22.L | TTGATGAAATCTCTATGGCTACT | ????17268 |
| p00001.21.R | GTCTAGCATTGACAACACTCAA | ????17300 |
| p00001.23.L | AAAAGCTGTTTTTATCTCACCTT | ????1767 |
| p00001.22.R | TGAAGCTACAGCGTTCTGTG | ????17700 |
| p00001.24.L | ACATACCAGGCATACCAAAG | ????18075 |
| p00001.23.R | CATAGAGATGAGTCTACGGTAGGT | ????1810 |
| p00001.25.L | GGATTGTCAGACAGAGTCGT | ????18479 |
| p00001.24.R | TCTTGACAAAGTACTTCATTGAT | ????18532 |
| p00001.26.L | CTTGCAGAAAAGTACAACACAT | ????18876 |
| p00001.25.R | AAGCAATGCAGACTTCACAA | ????18900 |
| p00001.27.L | TTGCCTTTCTTTTACTATTCTGA | ????19277 |
| p00001.26.R | CCATGAGACTCACAAGGACTA | ????19300 |
| p00001.28.L | ACTTCCTGTTAATGTTGCATTT | ????19678 |
| p00001.27.R | CACTGGTTTAATGTTACGCTTAG | ????19710 |
| p00001.30.L | GTAGACGGCATTATTCAACAGT | ????20078 |
| p00001.28.R | GCTCTGAGTAAAGTAGGTTTCAG | ????20103 |
| p00001.31.L | CAAAAGTGGTCAAGGTTACAAT | ????20478 |
| p00001.30.R | CACCAAAGCATGAATGAAAT | ????20513 |
| p00001.32.L | TCAGATCTTAATGACTTCGTCTC | ????20864 |
| p00001.31.R | TGCACAGTCTCCAATTAAAGT | ????20900 |
| p00001.33.L | ATCCTATCCAGTTGTCTTCCTAT | ????21276 |
| p00001.32.R | ATTTGCTCATGTCAAAGAGTG | ????21300 |
| p00001.34.L | TTCTAATGTTACAGGGTTTCATA | ????21664 |
| p00001.33.R | CATCCTTAAAAGGTATGACAGG | ????21710 |
| p00001.35.L | GGCTATCAACCTATAGATGTAGTTC | ????22070 |
| p00001.34.R | CAAAGTGTTAAAACCAGAAGGTA | ????22101 |
| p00001.36.L | ACTAAATTCCCTTCTGTCTATGC | ????22469 |
| p00001.35.R | TAATCAGCAACACAATTAGAAAT | ????22508 |
| p00001.37.L | CCCACCTGCTCTTAATTGTTA | ????22879 |
| p00001.36.R | TGGTGTAAAAACCATAATCATTTA | ????22908 |
| p00001.38.L | TTAACTGCACTGATGTTTCTACA | ????23277 |
| p00001.37.R | GAGTTGATCTGCATGAATTG | ????23301 |
| p00001.39.L | CTTCTCCAATATGGTAGCTTTT | ????23678 |
| p00001.38.R | GAGTGCACGATTTAGTTGTGT | ????23702 |
| p00001.41.L | CTGGATGGACATTTGGTGCT | ????24072 |
| p00001.39.R | GCATAGCAAAAGGTATTTGAAG | ????24101 |
| p00001.42.L | ATCAGGGCTTCTGCTAATCT | ????24473 |
| p00001.41.R | CAAGAACACACTCAGACATTTTA | ????24502 |
| p00001.43.L | TGGACAAGTACTTCAAAAATCAT | ????24876 |
| p00001.42.R | GTCGCCAAGATCAACATCTG | ????24906 |
| p00001.44.L | TTTACTCTTGGATCAATTACTGC | ????25273 |
| p00001.43.R | AGAAGCATTGTCAATTTTTACTG | ????25301 |
| p00001.45.L | TTATGATGCCAACTACTTTGTTT | ????25671 |
| p00001.44.R | TGGTATACAGTAGTCATAGTTATGTG | ????25703 |
| p00001.46.L | AGCACAAGAAAGTGAGTACGA | ????26076 |
| p00001.45.R | TCCGAAACGAATGAGTACATA | ????26100 |
| p00001.47.L | ATGTTACTACAATTTGCCTATTC | ????26475 |
| p00001.46.R | TATTATGTACAAAAACCTGTTCC | ????26503 |
| p00001.48.L | AAGAGATCACTGTGGCTACATC | ????26875 |
| p00001.47.R | ACGCTCCTAATTTGTAATAAGA | ????26907 |
| p00001.49.L | TCTCTTCCTGACATTGATTGTAT | ????27268 |
| p00001.48.R | CTCTAACACACTCCTGATAGTGA | ????27310 |
| p00001.50.L | TTTCTGCTATTCCTTGTTTTAAT | ????27667 |
| p00001.49.R | ATTTCGAGTGAAAACCAAAATA | ????27701 |
| p00001.52.L | GTTGTTTTAAATAAACGAACAAA | ????28076 |
| p00001.50.R | GGGTCCATTATCAGACATTTTA | ????28100 |
| p00001.53.L | GCTAACAAAGAAGGCATCGTA | ????28479 |
| p00001.52.R | GTATTCAAGGCTCCCTCAGT | ????28509 |
| p00001.54.L | AGCCTCGCCAAAAACGTACT | ????28876 |
| p00001.53.R | TTGAGTGACGTTGTACTGTTT | ????28901 |
| p00001.55.L | AAAGGACAAAAAGAAAAAGACTG | ????29213 |
| p00001.56.L | AAAGGACAAAAAGAAAAAGACTG | ????29213 |
| p00001.54.R | CTGGAGAAATCATCCATGTC | ????29301 |
| p00001.55.R | AATTTTACACATTAGGGCTCTTC | ????29636 |
| p00001.56.R | AATTTTACACATTAGGGCTCTTC | ????29636 |
Nucleic acid sequence table:
<110> Genomics R & D center in Hangzhou *
<120> SARS virus gene detection method and kit
<160> 6
<170> PatentIn Version 3.1
<210> 1
<211> 1953
<212> DNA
<213> coronavirus SARS
<400> 1
ctacccagga aaagccaacc aacctcgatc tcttgtagat ctgttctcta aacgaacttt 60
aaaatctgtg tagctgtcgc tcggctgcat gcctagtgca cctacgcagt ataaacaata 120
ataaatttta ctgtcgttga caagaaacga gtaactcgtc cctcttctgc agactgctta 180
cggtttcgtc cgtgttgcag tcgatcatca gcatacctag gtttcgtccg ggtgtgaccg 240
aaaggtaaga tggagagcct tgttcttggt gtcaacgaga aaacacacgt ccaactcagt 300
ttgcctgtcc ttcaggttag agacgtgcta gtgcgtggct tcggggactc tgtggaagag 360
gccctatcgg aggcacgtga acacctcaaa aatggcactt gtggtctagt agagctggaa 420
aaaggcgtac tgccccagct tgaacagccc tatgtgttca ttaaacgttc tgatgcctta 480
agcaccaatc acggccacaa ggtcgttgag ctggttgcag aaatggacgg cattcagtac 540
ggtcgtagcg gtataacact gggagtactc gtgccacatg tgggcgaaac cccaattgca 600
taccgcaatg ttcttcttcg taagaacggt aataagggag ccggtggtca tagctatggc 660
atcgatctaa agtcttatga cttaggtgac gagcttggca ctgatcccat tgaagattat 720
gaacaaaact ggaacactaa gcatggcagt ggtgcactcc gtgaactcac tcgtgagctc 780
aatggaggtg cagtcactcg ctatgtcgac aacaatttct gtggcccaga tgggtaccct 840
cttgattgca tcaaagattt tctcgcacgc gcgggcaagt caatgtgcac tctttccgaa 900
caacttgatt acatcgagtc gaagagaggt gtctactgct gccgtgacca tgagcatgaa 960
attgcctggt tcactgagcg ctctgataag agctacgagc accagacacc cttcgaaatt 1020
aagagtgcca agaaatttga cactttcaaa ggggaatgcc caaagtttgt gtttcctctt 1080
aactcaaaag tcaaagtcat tcaaccacgt gttgaaaaga aaaagactga gggtttcatg 1140
gggcgtatac gctctgtgta ccctgttgca tctccacagg agtgtaacaa tatgcacttg 1200
tctaccttga tgaaatgtaa tcattgcgat gaagtttcat ggcagacgtg cgactttctg 1260
aaagccactt gtgaacattg tggcactgaa aatttagtta ttgaaggacc tactacatgt 1320
gggtacctac ctactaatgc tgtagtgaaa atgccatgtc ctgcctgtca agacccagag 1380
attggacctg agcatagtgt tgcagattat cacaaccact caaacattga aactcgactc 1440
cgcaagggag gtaggactag atgttttgga ggctgtgtgt ttgcctatgt tggctgctat 1500
aataagcgtg cctactgggt tcctcgtgct agtgctgata ttggctcagg ccatactggc 1560
attactggtg acaatgtgga gaccttgaat gaggatctcc ttgagatact gagtcgtgaa 1620
cgtgttaaca ttaacattgt tggcgatttt catttgaatg aagaggttgc catcattttg 1680
gcatctttct ctgcttctac aagtgccttt attgacacta taaagagtct tgattacaag 1740
tctttcaaaa ccattgttga gtcctgcggt aactataaag ttaccaaggg aaagcccgta 1800
aaaggtgctt ggaacattgg acaacagaga tcagttttaa caccactgtg tggttttccc 1860
tcacaggctg ctggtgttat cagatcaatt tttgcgcgca cacttgatgc agcaaaccac 1920
tcaattcctg atttgcaaag agcagctgtc acc 1953
<210> 2
<211> 249
<212> DNA
<213> coronavirus SARS
<400> 2
gatgaggaag aagaggacga tgcagagtgt gaggaagaag aaattgatga aacctgtgaa 60
catgagtacg gtacagagga tgattatcaa ggtctccctc tggaatttgg tgcctcagct 120
gaaacagttc gagttgagga agaagaagag gaagactggc tggatgatac tactgagcaa 180
tcagagattg agccagaacc agaacctaca cctgaagaac cagttaatca gtttactggt 240
tatttaaaa 249
<210> 3
<211> 732
<212> DNA
<213> coronavirus SARS
<400> 3
ccagttgatg agtatataac cacgtaccct ggacaaggat gtgctggtta tacacttgag 60
gaagctaaga ctgctcttaa gaaatgcaaa tctgcatttt atgtactacc ttcagaagca 120
cctaatgcta aggaagagat tctaggaact gtatcctgga atttgagaga aatgcttgct 180
catgctgaag agacaagaaa attaatgcct atatgcatgg atgttagagc cataatggca 240
accatccaac gtaagtataa aggaattaaa attcaagagg gcatcgttga ctatggtgtc 300
cgattcttct tttatactag taaagagcct gtagcttcta ttattacgaa gctgaactct 360
ctaaatgagc cgcttgtcac aatgccaatt ggttatgtga cacatggttt taatcttgaa 420
gaggctgcgc gctgtatgcg ttctcttaaa gctcctgccg tagtgtcagt atcatcacca 480
gatgctgtta ctacatataa tggatacctc acttcgtcat caaagacatc tgaggagcac 540
tttgtagaaa cagtttcttt ggctggctct tacagagatt ggtcctattc aggacagcgt 600
acagagttag gtgttgaatt tcttaagcgt ggtgacaaaa ttgtgtacca cactctggag 660
agccccgtcg agtttcatct tgacggtgag gttctttcac ttgacaaact aaagagtctc 720
ttatccctgc gg 732
<210> 4
<211> 1177
<212> DNA
<213> coronavirus SARS
<400> 4
ggtgtcaaat tacattacac ataaacgaac ttatggattt gtttatgaga ttttttactc 60
ttggatcaat tactgcacag ccagtaaaaa ttgacaatgc ttctcctgca agtactgttc 120
atgctacagc aacgataccg ctacaagcct cactcccttt cggatggctt gttattggcg 180
ttgcatttct tgctgttttt cagagcgcta ccaaaataat tgcgctcaat aaaagatggc 240
agctagccct ttataagggc ttccagttca tttgcaattt actgctgcta tttgttacca 300
tctattcaca tcttttgctt gtcgctgcag gtatggaggc gcaatttttg tacctctatg 360
ccttgatata ttttctacaa tgcatcaacg catgtagaat tattatgaga tgttggcttt 420
gttggaagtg caaatccaag aacccattac tttatgatgc caactacttt gtttgctggc 480
acacacataa ctatgactac tgtataccat ataacagtgt cacagataca attgtcgtta 540
ctgaaggtga cggcatttca acaccaaaac tcaaagaaga ctaccaaatt ggtggttatt 600
ctgaggatag gcactcaggt gttaaagact atgtcgttgt acatggctat ttcaccgaag 660
tttactacca gcttgagtct acacaaatta ctacagacac tggtattgaa aatgctacat 720
tcttcatctt taacaagctt gttaaagacc caccgaatgt gcaaatacac acaatcgacg 780
gctcttcagg agttgctaat ccagcaatgg atccaattta tgatgagccg acgacgacta 840
ctagcgtgcc tttgtaagca caagaaagtg agtacgaact tatgtactca ttcgtttcgg 900
aagaaacagg tacgttaata gttaatagcg tacttctttt tcttgctttc gtggtattct 960
tgctagtcac actagccatc cttactgcgc ttcgattgtg tgcgtactgc tgcaatattg 1020
ttaacgtgag tttagtaaaa ccaacggttt acgtctactc gcgtgttaaa aatctgaact 1080
cttctgaagg agttcctgat cttctggtct aaacgaacta actattatta ttattctgtt 1140
tggaacttta acattgctta tcatggcaga caacggt 1177
<210> 5
<211> 1059
<212> DNA
<213> coronavirus SARS
<400> 5
cagtaagtga caacagatgt ttcatcttgt tgacttccag gttacaatag cagagatatt 60
gattatcatt atgaggactt tcaggattgc tatttggaat cttgacgtta taataagttc 120
aatagtgaga caattattta agcctctaac taagaagaat tattcggagt tagatgatga 180
agaacctatg gagttagatt atccataaaa cgaacatgaa aattattctc ttcctgacat 240
tgattgtatt tacatcttgc gagctatatc actatcagga gtgtgttaga ggtacgactg 300
tactactaaa agaaccttgc ccatcaggaa catacgaggg caattcacca tttcaccctc 360
ttgctgacaa taaatttgca ctaacttgca ctagcacaca ctttgctttt gcttgtgctg 420
acggtactcg acatacctat cagctgcgtg caagatcagt ttcaccaaaa cttttcatca 480
gacaagagga ggttcaacaa gagctctact cgccactttt tctcattgtt gctgctctag 540
tatttttaat actttgcttc accattaaga gaaagacaga atgaatgagc tcactttaat 600
tgacttctat ttgtgctttt tagcctttct gctattcctt gttttaataa tgcttattat 660
attttggttt tcactcgaaa tccaggatct agaagaacct tgtaccaaag tctaaacgaa 720
catgaaactt ctcattgttt tgacttgtat ttctctatgc agttgcatat gcactgtagt 780
acagcgctgt gcatctaata aacctcatgt gcttgaagat ccttgtaagg tacaacacta 840
ggggtaatac ttatagcact gcttggcttt gtgctctagg aaaggtttta ccttttcata 900
gatggcacac tatggttcaa acatgcacac ctaatgttac tatcaactgt caagatccag 960
ctggtggtgc gcttatagct aggtgttggt accttcatga aggtcaccaa actgctgcat 1020
ttagagacgt acttgttgtt ttaaataaac gaacaaatt 1059
<210> 6
<211> 255
<212> DNA
<213> coronavirus SARS
<400> 6
agacaacttc aaaattccat gagtggagct tctgctgatt caactcaggc ataaacactc 60
atgatgacca cacaaggcag atgggctatg taaacgtttt cgcaattccg tttacgatac 120
atagtctact cttgtgcaga atgaattctc gtaactaaac agcacaagta ggtttagtta 180
actttaatct cacatagcaa tctttaatca atgtgtaaca ttagggagga cttgaaagag 240
ccaccacatt ttcat 255
...
Claims (4)
1. method that detects severe acute respiratory syndrome virus, it comprises following step:
1) gets suspicious patient's biological samples such as serum, secretory product or tissue, extract RNA.
2) add reverse primer and reversed transcriptive enzyme, synthetic cDNA.
3) add forward primer, utilize the genomic nucleic acid sequence of polymerase chain reaction (PCR) technology amplification severe acute respiratory syndrome virus.
4) adopt Routine Test Lab to detect the dna technique detected result.
2. by the described detection severe acute respiratory syndrome of claim 1 virus method, it is characterized in that the genomic nucleic acid sequence of the severe acute respiratory syndrome virus of said PCR reaction amplification has the nucleotide sequence shown in sequence Seq.ID No.1, Seq.IDNo.2, Seq.ID No.3, Seq.ID No.4, Seq.ID No.5, the Seq.ID No.6.
3. by the described detection severe acute respiratory syndrome of claim 1 virus method, it is characterized in that the forward and reverse primer that adds has the nucleotide sequence as table 1, L is a forward primer in the table, and R is a reverse primer.
4. press the test kit of the described detection severe acute respiratory syndrome of claim 1 virus method, it is characterized in that it comprises following reagent: (solution D contains: 2.5 gram guanidinium isothiocyanates, the Trisodium Citrate (PH7.0) of 1.76 milliliters of 0.75M, 2.46 milliliter 10% dodecyl creatine sodium for PBS damping fluid, solution D, add 29.3 milliliters in water, add the mercaptoethanol of 7.2 μ l/ml before using), phenol, chloroform and 4.0M (PH=3.8) sodium-acetate, RT damping fluid dNTP, primer and working instructions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03116659 CN1450172A (en) | 2003-04-26 | 2003-04-26 | Method for detecting SARS virus and its reagent kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03116659 CN1450172A (en) | 2003-04-26 | 2003-04-26 | Method for detecting SARS virus and its reagent kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1450172A true CN1450172A (en) | 2003-10-22 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 03116659 Pending CN1450172A (en) | 2003-04-26 | 2003-04-26 | Method for detecting SARS virus and its reagent kit |
Country Status (1)
| Country | Link |
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| CN (1) | CN1450172A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004094675A2 (en) * | 2003-04-17 | 2004-11-04 | Gen-Probe Incorporated | Compositions and methods for determining the presence of sars coronavirus in a sample |
| CN110511977A (en) * | 2019-09-05 | 2019-11-29 | 武汉华大基因技术服务有限公司 | Extracting method, sequencing approach and the kit of genomic DNA |
| CN111286558A (en) * | 2020-02-12 | 2020-06-16 | 谱尼测试集团北京检验认证科学研究院有限公司 | Novel coronavirus 2019-nCoV nucleic acid detection technology |
| CN111471800A (en) * | 2020-05-26 | 2020-07-31 | 海关总署(北京)国际旅行卫生保健中心 | Kit for detecting novel coronavirus and amplification primer composition thereof |
| CN111647685A (en) * | 2020-05-19 | 2020-09-11 | 广州中科抗体生物技术有限公司 | Primer group, probe group and kit for detecting COVID-19 virus and application thereof |
| CN113215231A (en) * | 2021-03-30 | 2021-08-06 | 成都里来生物科技有限公司 | Dual PCR detection method for SARS-CoV and COVID-19 virus |
| WO2021155638A1 (en) * | 2020-02-06 | 2021-08-12 | 广州达安基因股份有限公司 | Dual detection kit for 2019 novel corona virus |
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2003
- 2003-04-26 CN CN 03116659 patent/CN1450172A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004094675A2 (en) * | 2003-04-17 | 2004-11-04 | Gen-Probe Incorporated | Compositions and methods for determining the presence of sars coronavirus in a sample |
| WO2004094675A3 (en) * | 2003-04-17 | 2005-02-10 | Gen Probe Inc | Compositions and methods for determining the presence of sars coronavirus in a sample |
| CN110511977A (en) * | 2019-09-05 | 2019-11-29 | 武汉华大基因技术服务有限公司 | Extracting method, sequencing approach and the kit of genomic DNA |
| WO2021155638A1 (en) * | 2020-02-06 | 2021-08-12 | 广州达安基因股份有限公司 | Dual detection kit for 2019 novel corona virus |
| US11649511B2 (en) | 2020-02-06 | 2023-05-16 | Daan Gene Co., Ltd. | Multiplex PCR method for the detection of SARS-CoV-2 |
| CN111286558A (en) * | 2020-02-12 | 2020-06-16 | 谱尼测试集团北京检验认证科学研究院有限公司 | Novel coronavirus 2019-nCoV nucleic acid detection technology |
| CN111647685A (en) * | 2020-05-19 | 2020-09-11 | 广州中科抗体生物技术有限公司 | Primer group, probe group and kit for detecting COVID-19 virus and application thereof |
| CN111471800A (en) * | 2020-05-26 | 2020-07-31 | 海关总署(北京)国际旅行卫生保健中心 | Kit for detecting novel coronavirus and amplification primer composition thereof |
| CN113215231A (en) * | 2021-03-30 | 2021-08-06 | 成都里来生物科技有限公司 | Dual PCR detection method for SARS-CoV and COVID-19 virus |
| CN113215231B (en) * | 2021-03-30 | 2022-08-05 | 成都里来生物科技有限公司 | Dual PCR detection method for SARS-CoV and COVID-19 virus |
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