CN1054618A - 检测间日疟原虫的新方法 - Google Patents
检测间日疟原虫的新方法 Download PDFInfo
- Publication number
- CN1054618A CN1054618A CN90109920A CN90109920A CN1054618A CN 1054618 A CN1054618 A CN 1054618A CN 90109920 A CN90109920 A CN 90109920A CN 90109920 A CN90109920 A CN 90109920A CN 1054618 A CN1054618 A CN 1054618A
- Authority
- CN
- China
- Prior art keywords
- ttt
- att
- taa
- cat
- ttg
- 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.)
- Pending
Links
- 241000223810 Plasmodium vivax Species 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 24
- 239000000523 sample Substances 0.000 claims abstract description 87
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 11
- 241000251539 Vertebrata <Metazoa> Species 0.000 claims abstract description 4
- 108020004414 DNA Proteins 0.000 claims description 42
- 238000009396 hybridization Methods 0.000 claims description 27
- 238000001514 detection method Methods 0.000 claims description 10
- 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 claims description 8
- 239000013612 plasmid Substances 0.000 claims description 6
- 241000588724 Escherichia coli Species 0.000 claims description 5
- 241000607142 Salmonella Species 0.000 claims description 4
- 229930003756 Vitamin B7 Natural products 0.000 claims description 4
- 239000011735 vitamin B7 Substances 0.000 claims description 4
- 235000011912 vitamin B7 Nutrition 0.000 claims description 4
- 239000012472 biological sample Substances 0.000 claims description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- 238000013467 fragmentation Methods 0.000 claims 3
- 238000006062 fragmentation reaction Methods 0.000 claims 3
- 125000006853 reporter group Chemical group 0.000 claims 2
- 108020004635 Complementary DNA Proteins 0.000 claims 1
- 238000010804 cDNA synthesis Methods 0.000 claims 1
- 239000002299 complementary DNA Substances 0.000 claims 1
- 239000002245 particle Substances 0.000 claims 1
- 210000004369 blood Anatomy 0.000 abstract description 17
- 239000008280 blood Substances 0.000 abstract description 17
- 150000007523 nucleic acids Chemical group 0.000 abstract description 11
- 201000004792 malaria Diseases 0.000 abstract description 9
- 238000007899 nucleic acid hybridization Methods 0.000 abstract description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 14
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 12
- 239000012528 membrane Substances 0.000 description 12
- 239000011159 matrix material Substances 0.000 description 10
- 239000002773 nucleotide Substances 0.000 description 10
- 125000003729 nucleotide group Chemical group 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 239000000020 Nitrocellulose Substances 0.000 description 9
- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 0.000 description 9
- 229920001220 nitrocellulos Polymers 0.000 description 9
- 108020004707 nucleic acids Proteins 0.000 description 9
- 102000039446 nucleic acids Human genes 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 8
- 102000053602 DNA Human genes 0.000 description 7
- 244000045947 parasite Species 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 238000013519 translation Methods 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 5
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 5
- 241000224016 Plasmodium Species 0.000 description 5
- 241000223960 Plasmodium falciparum Species 0.000 description 5
- 230000003196 chaotropic effect Effects 0.000 description 5
- 238000002372 labelling Methods 0.000 description 5
- 150000003839 salts Chemical class 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- BQCIDUSAKPWEOX-UHFFFAOYSA-N 1,1-Difluoroethene Chemical compound FC(F)=C BQCIDUSAKPWEOX-UHFFFAOYSA-N 0.000 description 4
- 206010035503 Plasmodium vivax infection Diseases 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 4
- 230000000155 isotopic effect Effects 0.000 description 4
- -1 poly(vinylidene fluoride) Polymers 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 241001505293 Plasmodium ovale Species 0.000 description 3
- 108020004682 Single-Stranded DNA Proteins 0.000 description 3
- IYKNGWWPNRDDHK-ONEGZZNKSA-N [[5-[2,4-dioxo-5-[(e)-3-[5-(2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl)pentanoylamino]prop-1-enyl]pyrimidin-1-yl]-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound O1C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)CC1N1C(=O)NC(=O)C(\C=C\CNC(=O)CCCCC2C3NC(=O)NC3CS2)=C1 IYKNGWWPNRDDHK-ONEGZZNKSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 238000002405 diagnostic procedure Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 230000003071 parasitic effect Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 2
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000009434 installation Methods 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- ZHPDOBDGGAXVTC-UHFFFAOYSA-N 2-cyanoguanidine;sulfuric acid Chemical compound OS(O)(=O)=O.NC(N)=NC#N ZHPDOBDGGAXVTC-UHFFFAOYSA-N 0.000 description 1
- 241000256186 Anopheles <genus> Species 0.000 description 1
- 108020004394 Complementary RNA Proteins 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 101100173437 Fusarium pseudograminearum (strain CS3096) FCK2 gene Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 102000001675 Parvalbumin Human genes 0.000 description 1
- 108060005874 Parvalbumin Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 206010035500 Plasmodium falciparum infection Diseases 0.000 description 1
- 241000223821 Plasmodium malariae Species 0.000 description 1
- 206010035501 Plasmodium malariae infection Diseases 0.000 description 1
- 206010035502 Plasmodium ovale infection Diseases 0.000 description 1
- 201000009976 Plasmodium vivax malaria Diseases 0.000 description 1
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 1
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 1
- 108020004518 RNA Probes Proteins 0.000 description 1
- 108010065868 RNA polymerase SP6 Proteins 0.000 description 1
- 239000003391 RNA probe Substances 0.000 description 1
- 241001468001 Salmonella virus SP6 Species 0.000 description 1
- 208000005469 Vivax Malaria Diseases 0.000 description 1
- ZKHQWZAMYRWXGA-MVKANHKCSA-N [[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxy(32P)phosphoryl] phosphono hydrogen phosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO[32P](O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-MVKANHKCSA-N 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 230000002186 photoactivation Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000008521 reorganization Effects 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
- C07K14/445—Plasmodium
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
公开了特异于间日疟原虫的探针的DNA顺序
及获得这些探针的方法。通过核酸杂交测定证明这
些核酸顺序在检测由间日疟原虫引起的人疟疾病方
面是很有用的。这些测定的高灵敏度及操作方便,使
得它们可用来对脊椎动物和无脊椎动物的血样和其
它组织进行分析。
Description
疟疾是由属于疟原虫属的原生动物寄生虫引起的。寄生虫的生活周期为二个阶段,即在脊椎动物身上的无性阶段和在蚊虫身上(通常为按蚊属)的有性阶段。造成人疟疾病的四种疟原虫(Plasmodium)是恶性疟原虫(P.falciparum)、间日疟原虫(P.vivax)、三日疟原虫(P.malariae)和卵形疟原虫(P.ovale)。
在这些疟原虫中,前两种是最常见的。恶性疟原虫是造成疟疾最严重的一种疟原虫,在某些情况下可致死。而且,这种寄生虫还对通常使用的疟疾药物产生抗性。由间日疟原虫引起疾病的频率视不同国家而不同,一般约为50-80%。三日疟原虫和卵形疟原虫很少出现。
目前诊断疟疾的方法是利用血样涂片观察。这种方法很麻烦,并需要专家评价。而且,一个熟练的显微镜学家一天最多只能看六十张片子。也可以通过血清学诊断,但由于抗体的持久性,无法将现时的感染与过去的感染区分开。新的诊断测试的研究包括了检测寄生虫核酸作为寄生虫存在的指征。这种测试只需要很少量的血(5-50μl),可从刺破手指得到,这种测试方法灵敏迅速。10μl血中有50个寄生虫,可通过核酸杂交检测(1)。只要有一点初级训练,一天就可分析几百个样品。这种检测方法的灵敏性使得这种检测可用于筛选输血用的血库。
为了鉴别作为载体的载体种类,也可在昆虫组织的样品中进行核酸杂交。这一信息会有助于加强载体的对照测量以限制疟疾的地理分布。或者,可在这些区域采用化学预防,利用核酸杂交来完成对这一措施的评价。
双链的DNA是互补的。在某些温度和盐浓度条件下,DNA的互补双链会变性或解离并且能再生性地重新组合。这种重新组合还可能发生在DNA和互补的RNA之间。重组的DNA和RNA可用检测装置(同位素的或非同位素的)标记,也可采用适当的检测方法。在间日疟原虫中,还没有任何潜在的DNA探针顺序的报道。为了确定间日疟原虫在地理限定区域中的影响,发展检测间日疟原虫的特异性核酸顺序是很重要。术语“探针用于表示一组用生物学方法或合成方法得到的DNA顺序。
核酸杂交法可用于任何怀疑有寄生虫的生物样品。例如,血样可直接拿来溶于碱中,并利用标准方法(2)点在硝酸纤维素或类似的固体支承物上。DNA被固定在支承物上,并在适当的温度、离子强度等条件下与特定的探针接触(3),所述离子强度有利于探针和目标核酸特异性重退火。如果探针带有同位素标记(通常用32P),则利用放射自显影检测负样品和正样品。如果探针带有非同位素标记如生物素(4),则采用酶体系(例如抗生物素蛋白-碱性磷酸脂酶),经过一联串的反应后,样品中出现颜色的,就是测试的正反应。
在另一项技术中(5),可直接将待分析的血样或组织样品收集到离液序列高的试剂如4M硫氰酸胍中。杂交过程在溶液中进行,然后将混合物在只促进目标-探针结合而过量的探针不结合到基质上的条件下通过固体支承物过滤,放射性自显影或比色检测可在基质上进行。当用单链RNA探针时,这种杂交形式特别有用。
本发明描述了间日疟原虫特异性DNA顺序和用于间日疟原虫的探针的核苷酸顺序的鉴定。基于这些特性,发展了采用核酸杂交法特异性地检测间日疟原虫的诊断方法。这种测试快速、灵敏并且可大规模地用于流行病检测。
本发明涉及:
1.新的构建体或质粒pARC117、pARC145和pARC1153,以转化的大肠杆菌形式保藏在National Collection of Industrial Bacteria,Torry Research Station,Aberdeen(NCIB)保藏号如下:
pARC 117: NCIB 40114,E.Coli RR1 M15 pARC 117,Feb.15,1989
pARC 145: NCIB 40110,E.Coli RR1 M15 pARC 145,Feb.7,1989
pARC 1153: NCIB 40108 E.Coli RR1 M15 pARC 1153,Feb.7,1989
2.pARC117、pARC145和pARC1153的DNA顺序和其亚结构包括至少20个必要的核苷酸。
3.一种用于检测间日疟原虫的DNA探针,其核苷酸顺序的构成同后面的pARC117。
4.一种用于检测间日疟原虫的DNA探针,其核苷酸顺序的构成同后面的pARC145。
5.一种用于检测间日疟原虫的DNA探针,其核苷酸顺序的构成同后面的pARC1153。
6.一种检测脊椎动物和无脊椎动物生物样品中间日疟原虫的方法,该方法将含有放射性或非放射性标记形式的与pARC117、pARC145和pARC1153核苷酸顺序序相同的探针与它们各自在间日疟原虫基因组上的同源顺序杂交,然后检测结合到间日疟原虫基因组上的标记探针。
本发明以疾病的诊断为例子,但不限于此。流行病筛选、法医研究、测定食品污染、公共卫生调查、预防医学、兽医及农业应用,在侵染试剂诊断方面都可用本发明的方法。
特异于间日疟原虫的DNA顺序的鉴定
由于间日疟原虫还不能在体外培养,这些寄生虫的基因组DNA得自感染病人的血液。在除去尽可能多的淡黄色外壳后,由标准方法(6)制备基因组DNA,用Sau 3A酶消化,然后克隆到已知质粒载体pUC18的BamH1位点上。特异性DNA顺序的种类通过用缺口翻译的间日疟原虫DNA和缺口翻译的正常人DNA在差示筛选形式下筛选重复滤膜(dupli-cate filters)而鉴定。得到三种间日疟原虫的特异性探针,表示为pARC117、pARC145和pARC1153,将它们亚克隆到M13载体上,它们的DNA顺序由双脱氧链末端法(di-deoxy chain termination method)而测定(7)。它们在排好顺序的区域内都没有任何特征性的重复单元。一种基于同源寻找的电脑,利用诸如Genbank和EMBL这类最新数据库,揭示任何已公开的核苷酸顺序都没有明显的同源性。用pARC117和大鼠小清蛋白(parvalbumm)得到最高的同源性(约41%),但这一数据对实际应用没有多大意义。
上述用于间日疟原虫的探针是特异性的,这些探针不与恶性疟原虫的分离物交叉反应(图1)。
利用特异性的间日疟原虫探针的检测方法
核酸杂交有两种方法。一种方法是把核酸样品固定在固体支承物上,而探针顺序在溶液中(2)。另一种方法是核酸样品和探针顺序都在溶液中(5)。
因此,一种测试核酸杂交的方法是将血或组织样品点在固体支承物(如硝酸纤维)上,然后在适当的温度等条件下与(同位素/非同位素标记的)探针杂交(3)。这类方法在文献中均已有记载,并且在疟疾诊断中被许多研究者应用。但是,组织样品必须进行寄生虫DNA的提取,然后才能用于圆斑分析。这在设计用于大量筛选的诊断测试时是不实用的。根据发明者的经验,将感染的血液直接点在基质上是不行的。血液中的红血球膜蛋白和其它血浆组份(可能为蛋白)和其它组织在使用时会在空间上阻碍寄生虫DNA结合到基质上。结果,在杂交过程中,点样样品会脱离基质,导致特异性信号消失。这个问题可通过预先洗涤样品以除去引起测试麻烦的干扰血浆成份而克服,或通过点样非常小量的会影响检测灵敏度的血样而克服。
本发明者因此采取了无需样品处理的溶液杂交形式,比滤膜杂交形式进行的更快。测试使用了下列步骤。
a)将组织样品直接收集在离液序列高的盐溶液或变性试剂中,例如,将25μl血液收集在50μl 6M硫氰酸胍中。如果探针有同位素标记,可用4M的硫氰酸胍作为离液序列高的盐。如果探针是非同位素标记的(如生物素),必须用酶反应检测,则不能用硫氰酸胍,因为硫氰酸胍会结合到硝酸纤维素滤膜、聚偏二氟乙烯(PVDF)滤膜和其它类似的基质上,并使反应中随后使用的酶变性。但是,只要将基质预处理,则硫氰酸胍可与非同位素探针一起使用。
在本发明中,当用盐酸胍作溶解试剂时,发现它与硫氰酸胍一样有效。并且它还有另外的优点,即它不结合到固体基质上,因此可用于非同位素探针。
b)加入探针(lng)。所加入的探针最好是单链核酸,DNA或RNA,所述DNA或RNA可以从生物上得到或利用本领域记载的方法合成。RNA是优选的探针。
c)样品在85℃加热5分钟,然后在室温(约28-35℃)下冷却2小时。
d)将样品用含有核糖核酸酶A(RNA se A)的盐溶液稀释至少10倍,再保温15分钟。当用单链DNA作探针时,末杂交的探针可通过羟基磷灰石层析而除去。
e)将样品通过基质(如硝酸纤维或聚偏二氟乙烯滤膜)过滤,滤液在含有核糖核酸酶A的盐溶液中冲洗两次,每次15分钟。
f)然后用适当的检测方法检测。
制备杂交探针
杂交探针可通过在适当的载体上克隆基因单元而制备。这些载体可以是质粒(大肠杆菌质粒)、丝状噬菌体(M13)、类λ(lamboid)噬菌体。装配型质粒、沙门氏菌噬菌体和酵母。本领域记载的任何载体都可使用。
杂交探针通过利用P用DNA聚合酶和〔α-P〕dNTP进行缺口翻译而标记。对非放射性标记而言,可以用Bio-dUTP进行缺口翻译。短的寡核苷酸探针可以用〔γ-P〕或〔α-P〕dNTP分别在其5′或3′末端进行末端标记。
用P标记的单链DNA探针可利用M13载体体系(9)得到。单链DNA探针可用生物素标记,方法是在Bio-dUTP、其它脱氧核糖核酸和Taq DNA聚合酶的存在下进行聚合酶链反应(10)。
在利用RNA杂交反应的杂交测定中,须制备互补于DNA的标记RNA。构建这类体系的方法在本领域的文献中已有记载(11)。本发明也包括这类测定。因此,本发明包括用双链DNA、单链DNA和RNA作探针检测间日疟原虫基因组的应用。
实例1
本实例说明了获得间日疟原虫特异性探针的方法。
1.从间日疟原虫感染的血液中分离总DNA,用Sau 3A酶消化,并克隆于质粒载体pUC18的BamH1位点上。
2.将克隆拷贝到两张硝酸纤维素滤膜上。一张滤膜用缺口翻译的放射性人DNA筛选,另一张滤膜用缺口翻译的放射性间日疟原虫DNA筛选。
3.将杂交的拷贝进行放射自显影响以产生杂交信号。
4.与间日疟原虫DNA反应而不与人DNA反应的克隆认为是特异性克隆种,选作间日疟原虫的杂交探针。
5.用这种方法鉴定出三种克隆,这三种克隆分别表示为pARC117、pARC145和pARC1153。每个克隆的核酸顺序用标准方法测定,结果如下。
pARC117的核苷酸顺序:
5'-------AT GTA AGA GCA CAT
GAG ATT TTA TAA GGA TTT CAT TTT
ACT CAG GGT GAA ATG AAG AAG CAC
TAA AAG ATT TTG AGT AGA GTT TCA
T-----3'
pARC145的核苷酸顺序:
5'-----CC AAG TGA AGA AAG
GTG GAA GGG CCA GCA GGA GAG CTG
GTC ACT GCA TTG TCT CTC TGA GGT
CTG TAG GCC AGA AGC TCC CCA GGA
CTT AGA CCC TAC TAA ATG GGG TAG
AGA GTA AGG GGC AGC CAT CAC TTA
TCA CTG GCT GTC CTG AGG GTT TGG
TGT ACA GCA TGG CTT GTG GTC AGA
GGC CTG TCA GCT GGG CTC CAA GAG
TCC TAG TGA ATG TAA ACA GTG CAG
ACC TTT TCT GGG GGG AAG G-----3'
pARC1153的核苷酸顺序:
5'------TTT GTG TGA TTT TTG TGA TTT TTG ATG
GAA ACC TGA ATA TTT GGG GTA ATT ATG TGA TTA GAC
TCA GGA TTT TAT TTA AAT CTT CTG TTT TAG CTA GCC
TCC TCT GAC ACT AGC TTG GCA GGA ACG AGG GCA GGA
GAG CAT TGC TGA TGC ATT CCT GCC TCT TTT CTT CCT
TGT TAC TCC CAA GTG GGT GTA AAA ATC CAG GTT TCC
CAC TGT TTC CTC CTT TAA ATT AAT TAA TTA ATT TTT
AAT GTT GGC AAA TAA AAA TTA TAT ATT GTG TAT ATT
TAT GGG GTA CAA CAT GAT ATT TTG ATA TAT GTA TAC
ATT GCA GAA TGG CTA AAT TAA GCT AAT TAA CAT ACA
TAT TAC CTC ACA TAA TCA ATT TTT TTG TGG TGA GAG
CAC CTG CAA TCT ACT CTT TTA GCA ATT TTC AAG TAT
ATA AAA CAT TGT TAT TAA CTA TGG TCA CCT CAT TGT
ACA ATA TGT TTT TTG AAC TTA TTC CTC CTA AGT ATA
ATT TTG TAC TCT TTG ACC AAC ATC TCC CCA GAC CCC
TCA ATG CCC ACC CTC TGG TAA CCA ACA TTC TAC TCT
TTG CTT TTC AAC TTT TAT AGA TTC CAT ATG AAG TAG
GAT CAT GCT GTA TTT GTC TTT GTG CCT GGC TTA TTT
CCT TTA CAT ACT GTT CTC TAG GTG ------3'
实例2
这个实例说明特异性杂交探针的制备。
杂交探针的制备方法是:在大肠杆菌DNA聚合酶或T4 DNA聚合酶及〔α-32P〕dNRTs的存在下通过缺口翻译标记插入在pARC117、pARC145和pARC1153中的DNA,产生比活为107cpm~5×107cpmμg的DNA。单链或合成得到的DNA可利用聚核苷酸激酶和〔α-32P〕ATP在5′端进行末端标记。也可利用末端转多酶和〔α-32P〕dNTPs在3′端标记。生物素化的单链DNA可通过利用Taq DNA聚合酶和Bio-dUTP进行聚合酶链反应而合成。
单链或双链的核酸可利用可光活化的生物素而生物素化。也可进行核酸的化学生物素化作用。
单链RNA可通过将目标DNA融合到在适当载体中的沙门氏菌噬菌体SP6启动子的片段上而产生。载体可在大肠杆菌中繁殖。该载体的DAN可利用SP6 RNA聚合酶在体外转录。在这个加工期间,可利用〔α-32P〕UTP获得高比活的放射性RNA。该RNA的生物素化作用也是可能的。
实例3
本实例说明实例1所描述的三种间日疟原虫克隆(pARC117、pARC145和pARC1153)的特异性。
制备间日疟原虫9种不同分离物的基因组DNA,点在硝酸纤维素滤膜上。分别用几种有放射性的间日疟原虫特异性探针探测滤膜,探针只与间日疟原种样品反应,而不与恶性疟原虫及人DNA反应(图1)。
图1显示了间日疟原虫三种探针pARC117(A)、pARC145(B)和pARC1153(C)的特异性。以从这三种克隆中分离出的双链插入片段作为测试探针。Pv1到Pv9代表从9种间日疟原虫感染血样中制备的DNA。FCK2和T9代表恶性疟接虫DNA。NH代表正常人DNA。
实例4
本实例说明利用溶液杂交方式测定间日疟原虫。
将感染的血液(20μl)或组织直接放入50μl含有1ng经标记的pARC117 RNA探针(如实例2描述)的硫氰酸胍溶液中。
样品在85℃加热5分钟,然后在室温下放置2小时。
将样品在含有10ng RNA se A的2X SSC中稀释10倍,再在室温下保温15分钟。
将样品通过硝酸纤维素或PVDF滤膜基质过滤,滤膜在含有RNA se A的缓冲液2X SSC中冲洗两次,每次15分钟。
将滤膜干燥,对膜进行放射性自显影以显示杂交信号。
所描述的测定很容易进行,只需要很少的实验装置,例如在大量调查期间周围环境条件上所存在的那些实验设备。
实例5
本实例说明用盐酸胍代替实例4描述的硫氰酸胍的溶液杂交方法。
4M硫酸氰胍作为离液序列高的试剂与由蛋白结合物检测的非放射性探针不相容。离液序列高的盐会结合到硝酸纤维和PVDF滤膜上,并使得用于显色反应的蛋白结合物变性。
盐酸胍用作溶解试剂时,和硫氰酸胍一样有效。由于盐酸胍不会结合到基质(硝酸纤维或PVDF滤膜)上,它使得由蛋白结合物检测的非放射性探针可以使用。
硫氰酸胍只有在滤膜预处理过(如在硫氰酸胍溶液通过以前用3%BSA预处理)的条件下,才可以与非放射性探针结合使用。
参考文献
1. Pollack,Y.,Metzger,S.,Shemer,R.,Landau,D.,Spira,D.T.and Golenser,J.(1985).Am.J.Trop.Med.Hyg.34,663.
2. Kafatos,F.C.,Jones,C.W.and Efstratiadis,A.(1979)Nucl.Acids.Res 7,1541.
3. Maniatis,T.,Fritsch,E.F.and Sambrook,J.,Molecular Cloning,A Laboratory Manual,Cold Spring Harbour,N.Y.1982.
4. Langer,P.R.,Waldorp,A.A.and Ward,D.C.(1981)Proc.Natl.Acad Sci.U.S.A.78,6633.
5. Thompson,J.and Gillespie,D.(1987)Anal.Biochem.163,281.
6. Panyim,S.,Wilairat,P.and Yuthavong,Y.Application of Genetic Engineering to Research on Tropical Disease Patho-gens with special reference to Plasmodia-WHO publication.
7. Sanger,F.,Coulson,A.R.,Barrell,B.G.,Smith,A.G.H.and Roe,B.A.(1980).J.Mol.Biol.143,161.
8. Holmberg,M.,Bjorkmann,A.,Franzen.L.,Aslund,L.,Lebbad,M.,Pettersson U.and Wigzell,H.(1980),Bull,W.H.O.64,579.
9. Burke,J.F.,(1984)Gene 30,63.
10. Saiki,R.K.,et al (1988)Science 239,487
11. Green,M.R.,Maniatis,T.and Melton,D.A,(1983),Cell 32,681.
Claims (16)
1、构建体pARC117,保藏号NCIB40114。
2、构建体pARC145,保藏号NCIB40110。
3、构建体pARC1153,保藏号NCIB40108。
4、一种具有以下顺序的DNA片段
5'-------AT GTA AGA GCA CAT
GAG ATT TTA TAA GGA TTT CAT TTT
ACT CAG GGT GAA ATG AAG AAG CAC
TAA AAG ATT TTG AGT AGA GTT TCA
T-----3'
5、一种具有以下顺序的DNA片段
5'-----CC AAG TGA AGA AAG
GTG GAA GGG CCA GCA GGA GAG CTG
GTC ACT GCA TTG TCT CTC TGA GGT
CTG TAG GCC AGA AGC TCC CCA GGA
CTT AGA CCC TAC TAA ATG GGG TAG
AGA GTA AGG GGC AGC CAT CAC TTA
TCA CTG GCT GTC CTG AGG GTT TGG
TGT ACA GCA TGG CTT GTG GTC AGA
GGC CTG TCA GCT GGG CTC CAA GAG
TCC TAG TGA ATG TAA ACA GTG CAG
ACC TTT TCT GGG GGG AAG G-----3'
6、一种具有以下顺序的DNA片段
5'------TTT GTG TGA TTT TTG TGA TTT TTG ATG
GAA ACC TGA ATA TTT GGG GTA ATT ATG TGA TTA GAC
TCA GGA TTT TAT TTA AAT CTT CTG TTT TAG CTA GCC
TCC TCT GAC ACT AGC TTG GCA GGA ACG AGG GCA GGA
GAG CAT TGC TGA TGC ATT CCT GCC TCT TTT CTT CCT
TGT TAC TCC CAA GTG GGT GTA AAA ATC CAG GTT TCC
CAC TGT TTC CTC CTT TAA ATT AAT TAA TTA ATT TTT
AAT GTT GGC AAA TAA AAA TTA TAT ATT GTG TAT ATT
TAT GGG GTA CAA CAT GAT ATT TTG ATA TAT GTA TAC
ATT GCA GAA TGG CTA AAT TAA GCT AAT TAA CAT ACA
TAT TAC CTC ACA TAA TCA ATT TTT TTG TGG TGA GAG
CAC CTG CAA TCT ACT CTT TTA GCA ATT TTC AAG TAT
ATA AAA CAT TGT TAT TAA CTA TGG TCA CCT CAT TGT
ACA ATA TGT TTT TTG AAC TTA TTC CTC CTA AGT ATA
ATT TTG TAC TCT TTG ACC AAC ATC TCC CCA GAC CCC
TCA ATG CCC ACC CTC TGG TAA CCA ACA TTC TAC TCT
TTG CTT TTC AAC TTT TAT AGA TTC CAT ATG AAG TAG
GAT CAT GCT GTA TTT GTC TTT GTG CCT GGC TTA TTT
CCT TTA CAT ACT GTT CTC TAG GTG ------3'
7、一种含有权利要求4、5和6所定义的核苷酸顺序或其相似片段的杂交探针,它能与间日疟原虫基因组特异性地杂交。
8、一种在适当载体上的权利要求7的杂交探针。
9、一种权利要求8的杂交探针,其中所述载体选自大肠杆菌质粒、丝状噬菌体(M13)、类λ噬菌体、装配型质粒、沙门氏噬菌体和酵母。
10、权利要求9的杂交探针,其中所述载体是大肠杆菌质粒。
11、权利要求10的杂交探针,其中所述大肠杆菌质粒是pUC18或pUC19。
12、权利要求9-11的杂交探针,其中所述载体含有沙门氏菌SP6噬菌体启动子或T7噬菌体启动子。
13、权利要求7-12的杂交探针,其中的探针用P、I、发色团或信息基团标记。
14、权利要求13的杂交探针,其中所述信息基团为生物素。
15、一种检测脊椎动物和无脊椎动物生物样品中间日疟原虫的方法,该方法是将权利要求7-14的探针与间日疟原虫基因组中各自的同源顺序杂交,然后检测结合到间日疟原虫基因组上的探针。
16、权利要求15的方法,其中所述杂交探针包含一个含有所述DNA顺序,互补DNA(单链)或RNA的载体。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SE8904100 | 1989-12-05 | ||
SE8904100A SE8904100D0 (sv) | 1989-12-05 | 1989-12-05 | New analysis method |
Publications (1)
Publication Number | Publication Date |
---|---|
CN1054618A true CN1054618A (zh) | 1991-09-18 |
Family
ID=20377694
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN90109920A Pending CN1054618A (zh) | 1989-12-05 | 1990-12-05 | 检测间日疟原虫的新方法 |
Country Status (12)
Country | Link |
---|---|
US (1) | US5250411A (zh) |
EP (1) | EP0504253A1 (zh) |
JP (1) | JPH05502582A (zh) |
CN (1) | CN1054618A (zh) |
AU (1) | AU6955691A (zh) |
BR (1) | BR9007895A (zh) |
IL (1) | IL96431A0 (zh) |
JO (1) | JO1656B1 (zh) |
PT (1) | PT96081A (zh) |
SE (1) | SE8904100D0 (zh) |
WO (1) | WO1991008293A1 (zh) |
ZA (1) | ZA909109B (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013159293A1 (en) * | 2012-04-25 | 2013-10-31 | Institute Of Basic Medical Sciences Chinese Academy Of Medical Sciences | Method, composition and kit for high throughput detection of genus plasmodium |
CN103965320A (zh) * | 2013-01-29 | 2014-08-06 | 苏州偲聚生物材料有限公司 | 多肽、包含该多肽的检测器件和检测试剂盒 |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH06261758A (ja) * | 1993-03-12 | 1994-09-20 | Wakunaga Pharmaceut Co Ltd | マラリアの検出 |
US5770368A (en) * | 1996-05-09 | 1998-06-23 | Metropolitan Water District Of Southern California | Cryptosporidium detection method |
US6436638B1 (en) | 1996-05-09 | 2002-08-20 | Metropolitan Water District Of Southern California | Cryptosporidium detection method |
KR20020024958A (ko) * | 2000-09-27 | 2002-04-03 | 임채승 | 삼일열 말라리아 더피 혈액형 부착 표면 단백질 유전자 및그를 이용한 말라리아 검출방법 |
KR20020028385A (ko) * | 2000-10-09 | 2002-04-17 | 박제철 | 마커를 이용한 말라리아 원충에 감염된 모기 검출방법 |
Family Cites Families (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3878836A (en) * | 1973-08-23 | 1975-04-22 | Products Int Marketing | Disposable speculum for tympanic thermometer |
DE2953811A1 (de) * | 1979-09-12 | 1982-02-11 | M Jacobs | Hand-held digital temperature measuring instrument |
US4358535A (en) * | 1980-12-08 | 1982-11-09 | Board Of Regents Of The University Of Washington | Specific DNA probes in diagnostic microbiology |
EP0135108A3 (en) * | 1983-08-12 | 1988-07-13 | Rockefeller University | Nucleotide hybridization assay for protozoan parasites |
GB8404378D0 (en) * | 1984-02-20 | 1984-03-28 | Biogen Nv | Dna sequence recombinant dna molecules |
US4602642A (en) * | 1984-10-23 | 1986-07-29 | Intelligent Medical Systems, Inc. | Method and apparatus for measuring internal body temperature utilizing infrared emissions |
US4957869A (en) * | 1985-07-12 | 1990-09-18 | New York University | Immunogenic peptide corresponding to P. vivax CS protein |
WO1987000533A1 (en) * | 1985-07-12 | 1987-01-29 | New York University | Immunogenic peptide antigen corresponding to plasmodium vivax circumsporozoite protein |
US4693994A (en) * | 1985-11-19 | 1987-09-15 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Protective synthetic peptide against malaria and encoding gene |
IL85905A0 (en) * | 1987-03-30 | 1988-09-30 | Univ New York | Immunogenic recombinant yeast expression product and method for purifying it |
US5101017A (en) * | 1987-04-06 | 1992-03-31 | New York Blood Center, Inc. | Antibodies for providing protection against P. vivax malaria infection |
EP0309746A1 (de) * | 1987-09-08 | 1989-04-05 | F. Hoffmann-La Roche Ag | Antimalaria-Vakzine |
US5112749A (en) * | 1987-10-02 | 1992-05-12 | Praxis Biologics, Inc. | Vaccines for the malaria circumsporozoite protein |
-
1989
- 1989-12-05 SE SE8904100A patent/SE8904100D0/xx unknown
-
1990
- 1990-11-13 ZA ZA909109A patent/ZA909109B/xx unknown
- 1990-11-21 IL IL96431A patent/IL96431A0/xx unknown
- 1990-12-04 US US07/624,313 patent/US5250411A/en not_active Expired - Lifetime
- 1990-12-04 EP EP91900983A patent/EP0504253A1/en not_active Withdrawn
- 1990-12-04 JO JO19901656A patent/JO1656B1/en active
- 1990-12-04 PT PT96081A patent/PT96081A/pt not_active Application Discontinuation
- 1990-12-04 JP JP3501387A patent/JPH05502582A/ja active Pending
- 1990-12-04 AU AU69556/91A patent/AU6955691A/en not_active Abandoned
- 1990-12-04 WO PCT/SE1990/000801 patent/WO1991008293A1/en not_active Application Discontinuation
- 1990-12-04 BR BR909007895A patent/BR9007895A/pt not_active Application Discontinuation
- 1990-12-05 CN CN90109920A patent/CN1054618A/zh active Pending
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013159293A1 (en) * | 2012-04-25 | 2013-10-31 | Institute Of Basic Medical Sciences Chinese Academy Of Medical Sciences | Method, composition and kit for high throughput detection of genus plasmodium |
CN104284986A (zh) * | 2012-04-25 | 2015-01-14 | 中国医学科学院基础医学研究所 | 用于高通量检测疟原虫的方法、组合物和试剂盒 |
CN104284986B (zh) * | 2012-04-25 | 2016-10-12 | 中国医学科学院基础医学研究所 | 用于高通量检测疟原虫的方法、组合物和试剂盒 |
CN103965320A (zh) * | 2013-01-29 | 2014-08-06 | 苏州偲聚生物材料有限公司 | 多肽、包含该多肽的检测器件和检测试剂盒 |
Also Published As
Publication number | Publication date |
---|---|
BR9007895A (pt) | 1992-09-29 |
ZA909109B (en) | 1991-08-28 |
US5250411A (en) | 1993-10-05 |
JO1656B1 (en) | 1991-11-27 |
SE8904100D0 (sv) | 1989-12-05 |
JPH05502582A (ja) | 1993-05-13 |
IL96431A0 (en) | 1991-08-16 |
AU6955691A (en) | 1991-06-26 |
EP0504253A1 (en) | 1992-09-23 |
PT96081A (pt) | 1991-09-30 |
WO1991008293A1 (en) | 1991-06-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Guy et al. | Real-time PCR assay for detection and genotype differentiation of Giardia lamblia in stool specimens | |
Slapeta et al. | Coprodiagnosis of Hammondia heydorni in dogs by PCR based amplification of ITS 1 rRNA: differentiation from morphologically indistinguishable oocysts of Neospora caninum | |
Amann | Fluorescently labelled, rRNA‐targeted oligonucleotide probes in the study of microbial ecology | |
KR101642784B1 (ko) | 바이러스성 출혈성 패혈증 바이러스 판별용 유전자 마커, 및 이를 이용한 원인바이러스의 판별방법 | |
US6238866B1 (en) | Detector for nucleic acid typing and methods of using the same | |
Vadopalas et al. | Application of real-time PCR for simultaneous identification and quantification of larval abalone | |
Takeyama et al. | Mitochondrial DNA sequence variation within and between tuna Thunnus species and its application to species identification | |
CN1522304A (zh) | 脓毒症或脓毒相关综合症生物芯片在诊断上的应用 | |
CN1308685A (zh) | 核酸检测方法 | |
KR101642783B1 (ko) | 참돔 이리도바이러스병 원인바이러스의 검출용 유전자 마커, 및 이를 이용한 원인바이러스의 검출 방법 | |
CN1256714A (zh) | 使用三核苷酸重复序列诊断神经精神疾病的方法及试剂盒 | |
US20060141503A1 (en) | Detection of sequence variation of nucleic acid by shifted termination analysis | |
KR101782489B1 (ko) | 바이러스성 출혈성 패혈증 바이러스의 검출용 pna 프로브 및 그 용도 | |
Sanburn et al. | Rapid titer determination using quantitative real-time PCR | |
WO2017188631A1 (ko) | 고병원성 바이러스성 출혈성 패혈증 바이러스 검출용 바이오마커 및 진단방법 | |
CN1054618A (zh) | 检测间日疟原虫的新方法 | |
CN1810989A (zh) | 一种等温反应检测具序列特异性的dna和rna的方法 | |
Abal-Fabeiro et al. | High-throughput genotyping assay for the large-scale genetic characterization of Cryptosporidium parasites from human and bovine samples | |
JP5855335B2 (ja) | 海水性二枚貝検出用プライマーセットとこれを利用した海水性二枚貝幼生の検出・定量方法 | |
CN1059910A (zh) | 一种用dna探针检测病原体的新方法 | |
Fong et al. | Genetic characterisation of the erythrocyte-binding protein (Pk β II) of Plasmodium knowlesi isolates from Malaysia | |
WO2013065957A1 (ko) | 가루이류 분류를 위한 프로브 조성물, dna 칩 및 키트 그리고 이를 이용한 가루이류 분류방법 | |
Ramzy | Recent advances in molecular diagnostic techniques for human lymphatic filariasis and their use in epidemiological research | |
Uera et al. | Detection of enzootic bovine leukosis in cattle using nested polymerase chain reaction assay | |
WO2021060311A1 (ja) | 脳腫瘍を検査する方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C01 | Deemed withdrawal of patent application (patent law 1993) | ||
WD01 | Invention patent application deemed withdrawn after publication |