RU2003129269A - Способ быстрой детекции и идентификации биоагентов - Google Patents
Способ быстрой детекции и идентификации биоагентов Download PDFInfo
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- RU2003129269A RU2003129269A RU2003129269/13A RU2003129269A RU2003129269A RU 2003129269 A RU2003129269 A RU 2003129269A RU 2003129269/13 A RU2003129269/13 A RU 2003129269/13A RU 2003129269 A RU2003129269 A RU 2003129269A RU 2003129269 A RU2003129269 A RU 2003129269A
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- 238000000034 method Methods 0.000 title claims 52
- 238000001514 detection method Methods 0.000 title 1
- 230000003321 amplification Effects 0.000 claims 26
- 238000003199 nucleic acid amplification method Methods 0.000 claims 26
- 150000007523 nucleic acids Chemical class 0.000 claims 19
- 108020004707 nucleic acids Proteins 0.000 claims 14
- 102000039446 nucleic acids Human genes 0.000 claims 14
- 239000003155 DNA primer Substances 0.000 claims 9
- 239000013615 primer Substances 0.000 claims 9
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 claims 8
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 claims 8
- 239000000203 mixture Substances 0.000 claims 8
- 238000004252 FT/ICR mass spectrometry Methods 0.000 claims 6
- 238000004949 mass spectrometry Methods 0.000 claims 6
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 claims 4
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 claims 4
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 claims 4
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 claims 4
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 claims 4
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 claims 4
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 claims 4
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 claims 4
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 claims 4
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 claims 4
- 229940029575 guanosine Drugs 0.000 claims 4
- 239000002773 nucleotide Substances 0.000 claims 4
- 125000003729 nucleotide group Chemical group 0.000 claims 4
- MWWATHDPGQKSAR-UHFFFAOYSA-N propyne Chemical compound CC#C MWWATHDPGQKSAR-UHFFFAOYSA-N 0.000 claims 4
- 229940104230 thymidine Drugs 0.000 claims 4
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 3
- 238000003795 desorption Methods 0.000 claims 3
- 238000000132 electrospray ionisation Methods 0.000 claims 3
- 238000000534 ion trap mass spectrometry Methods 0.000 claims 3
- 238000007834 ligase chain reaction Methods 0.000 claims 3
- 239000011159 matrix material Substances 0.000 claims 3
- 238000003752 polymerase chain reaction Methods 0.000 claims 3
- JZTCJRMDKBFLOO-XLPZGREQSA-N 1-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]imidazole-4-carboxamide Chemical compound C1=NC(C(=O)N)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 JZTCJRMDKBFLOO-XLPZGREQSA-N 0.000 claims 2
- LOJNBPNACKZWAI-UHFFFAOYSA-N 3-nitro-1h-pyrrole Chemical compound [O-][N+](=O)C=1C=CNC=1 LOJNBPNACKZWAI-UHFFFAOYSA-N 0.000 claims 2
- OZFPSOBLQZPIAV-UHFFFAOYSA-N 5-nitro-1h-indole Chemical compound [O-][N+](=O)C1=CC=C2NC=CC2=C1 OZFPSOBLQZPIAV-UHFFFAOYSA-N 0.000 claims 2
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 claims 2
- 229930024421 Adenine Natural products 0.000 claims 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims 2
- 241000894006 Bacteria Species 0.000 claims 2
- 239000002126 C01EB10 - Adenosine Substances 0.000 claims 2
- 229930010555 Inosine Natural products 0.000 claims 2
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 claims 2
- 108060004795 Methyltransferase Proteins 0.000 claims 2
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 claims 2
- 108091034117 Oligonucleotide Proteins 0.000 claims 2
- 102000004167 Ribonuclease P Human genes 0.000 claims 2
- 108090000621 Ribonuclease P Proteins 0.000 claims 2
- 241000700605 Viruses Species 0.000 claims 2
- 229960000643 adenine Drugs 0.000 claims 2
- 229960005305 adenosine Drugs 0.000 claims 2
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 claims 2
- 210000004027 cell Anatomy 0.000 claims 2
- 230000000295 complement effect Effects 0.000 claims 2
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 claims 2
- 238000010265 fast atom bombardment Methods 0.000 claims 2
- 229960004198 guanidine Drugs 0.000 claims 2
- 229960003786 inosine Drugs 0.000 claims 2
- 150000002991 phenoxazines Chemical class 0.000 claims 2
- 108020004418 ribosomal RNA Proteins 0.000 claims 2
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 claims 2
- 229940045145 uridine Drugs 0.000 claims 2
- 239000000427 antigen Substances 0.000 claims 1
- 102000036639 antigens Human genes 0.000 claims 1
- 108091007433 antigens Proteins 0.000 claims 1
- 239000008280 blood Substances 0.000 claims 1
- 210000004369 blood Anatomy 0.000 claims 1
- 108091036078 conserved sequence Proteins 0.000 claims 1
- 201000010099 disease Diseases 0.000 claims 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims 1
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- 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
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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Claims (48)
1. Способ идентификации неизвестного биоагента, включающий:
(a) приведение в контакт нуклеиновой кислоты из указанного биоагента с по меньшей мере одной парой олигонуклеотидных праймеров, которые гибридизуются с последовательностями указанной нуклеиновой кислоты, где упомянутые последовательности фланкируют вариабельную последовательность нуклеиновой кислоты биоагента;
(b) амплификацию вариабельной последовательности нуклеиновой кислоты с получением продукта амплификации;
(c) определение молекулярной массы указанного продукта амплификации; и
(d) сравнение упомянутой молекулярной массы с одной или несколькими молекулярными массами продуктов амплификации, полученными посредством выполнения стадий (a)-(c) для ряда известных организмов, где совпадение позволяет идентифицировать неизвестный биоагент.
2. Способ по п.1, где указанные последовательности, с которыми гибридизуется упомянутая по меньшей мере одна пара олигонуклеотидных праймеров, являются высококонсервативными.
3. Способ по п.1, где указанная стадия амплификации включает полимеразную цепную реакцию.
4. Способ по п.1, где указанная стадия амплификации включает лигазную цепную реакцию или амплификацию с заменой цепи.
5. Способ по п.1, где указанный биоагент представляет собой бактерию, вирус, клетку или спору.
6. Способ по п.1, где указанная нуклеиновая кислота представляет собой рибосомальную РНК.
7. Способ по п.1, где указанная нуклеиновая кислота кодирует РНКазу Р или РНК-зависимую РНК-полимеразу.
8. Способ по п.1, где перед определением молекулярной массы указанный продукт амплификации ионизируют.
9. Способ по п.1, дополнительно включающий стадию выделения нуклеиновой кислоты из указанного биоагента перед приведением в контакт указанной нуклеиновой кислоты с упомянутой по меньшей мере одной парой олигонуклеотидных праймеров.
10. Способ по п.1, дополнительно включающий стадию, заключающуюся в выполнении стадий a)-d) с использованием другой пары олигонуклеотидных праймеров и сравнении результатов с одной или несколькими молекулярными массами продуктов амплификации, полученными посредством выполнения стадий a)-c) для ряда известных организмов, отличающихся от тех, которые использовались на стадии d).
11. Способ по п.1, где указанные одна или несколько молекулярных масс содержатся в базе данных молекулярных масс.
12. Способ по п.1, где указанный продукт амплификации ионизируют ионизацией электрораспылением, лазерной десорбцией с использованием матрицы или бомбардировкой быстрыми атомами.
13. Способ по п.1, где указанную молекулярную массу определяют методом масс-спектрометрии.
14. Способ по п.11, где указанный метод масс-спектрометрии выбирают из группы, состоящей из масс-спектрометрии ионного циклотронного резонанса с Фурье-преобразованием (FT-ICR-MS), МС с ионной ловушкой, квадрупольной МС, МС с магнитным сектором, время-пролетной (TOF) МС, Q-TOF и тройной квадрупольной МС.
15. Способ по п.1, дополнительно включающий выполнение стадии (b) в присутствии аналога аденина, тимидина, гуанозина или цитидина, имеющего молекулярную массу, отличающуюся от молекулярной массы аденозина, тимидина, гуанозина или цитидина.
16. Способ по п.1, где указанный олигонуклеотидный праймер включает аналог основания в положениях 1 и 2 каждого триплета в пределах упомянутого праймера, где указанный аналог основания связывается с комплементарным основанием с более высоким сродством, чем нативное основание.
17. Способ по п.16, где указанный праймер включает универсальное основание в положении 3 каждого триплета в пределах упомянутого праймера.
18. Способ по п.16, где указанный аналог основания выбирают из группы, состоящей из 2,6-диаминопурина, пропина Т, пропина G, феноксазинов и G-клэмпа.
19. Способ по п.16, где указанное универсальное основание выбирают из группы, состоящей из инозина, гуанидина, уридина, 5-нитроиндола, 3-нитропиррола, dP, dK и 1-(2-дезокси-β-D-рибофуранозил)имидазол-4-карбоксамида.
20. Способ идентификации неизвестного биоагента, включающий
(a) приведение в контакт нуклеиновой кислоты из указанного биоагента с по меньшей мере одной парой олигонуклеотидных праймеров, которые гибридизуются с последовательностями упомянутой нуклеиновой кислоты, где указанные последовательности фланкируют вариабельную последовательность нуклеиновой кислоты;
(b) амплификацию указанной вариабельной последовательности нуклеиновой кислоты с получением продукта амплификации;
(c) определение состава оснований упомянутого продукта амплификации; и
(d) сравнение указанного состава оснований с одним или несколькими составами оснований продуктов амплификации, полученными посредством выполнения стадий a)-c) для ряда известных организмов, где совпадение позволяет идентифицировать неизвестный биоагент.
21. Способ по п.20, где указанные последовательности, с которыми гибридизуется упомянутая по меньшей мере одна пара олигонуклеотидных праймеров, являются высоко консервативными.
22. Способ по п.20, где указанная стадия амплификации включает полимеразную цепную реакцию.
23. Способ по п.20, где указанная стадия амплификации включает лигазную цепную реакцию или амплификацию с заменой цепи.
24. Способ по п.20, где указанный биоагент представляет собой бактерию, вирус, клетку или спору.
25. Способ по п.20, где указанная нуклеиновая кислота представляет собой рибосомальную РНК.
26. Способ по п.20, где указанная нуклеиновая кислота кодирует РНКазу Р или РНК-зависимую РНК-полимеразу.
27. Способ по п.20, где перед определением состава оснований указанный продукт амплификации ионизируют.
28. Способ по п.20, дополнительно включающий стадию выделения нуклеиновой кислоты из указанного биоагента перед приведением в контакт указанной нуклеиновой кислоты с упомянутой по меньшей мере одной парой олигонуклеотидных праймеров.
29. Способ по п.20, дополнительно включающий стадию, заключающуюся в выполнении стадий a)-d) с использованием другой пары олигонуклеотидных праймеров и сравнении результатов с одним или несколькими составами оснований продуктов амплификации, полученными посредством выполнения стадий a)-c) для ряда известных организмов, отличающихся от тех, которые использовались на стадии d).
30. Способ по п.20, где указанные одна или несколько сигнатур нуклеотидных составов содержатся в базе данных сигнатур нуклеотидных составов.
31. Способ по п.20, где указанный продукт амплификации ионизируют ионизацией электрораспылением, лазерной десорбцией с использованием матрицы или бомбардировкой быстрыми атомами.
32. Способ по п.20, где указанную сигнатуру нуклеотидного состава определяют методом масс-спектрометрии.
33. Способ по п.32, где указанный метод масс-спектрометрии выбирают из группы, состоящей из масс-спектрометрии ионного циклотронного резонанса с Фурье-преобразованием (FT-ICR-MS), МС с ионной ловушкой, квадрупольной МС, МС с магнитным сектором, время-пролетной (TOF) МС, Q-TOF и тройной квадрупольной МС.
34. Способ по п.20, дополнительно включающий выполнение стадии (b) в присутствии аналога аденина, тимидина, гуанозина или цитидина, имеющего молекулярную массу, отличающуюся от молекулярной массы аденозина, тимидина, гуанозина или цитидина.
35. Способ по п.20, где указанный олигонуклеотидный праймер включает аналог основания в положениях 1 и 2 каждого триплета в пределах упомянутого праймера, где указанный аналог основания связывается с комплементарным основанием с более высоким сродством, чем нативное основание.
36. Способ по п.35, где указанный праймер включает универсальное основание в положении 3 каждого триплета в пределах упомянутого праймера.
37. Способ по п.35, где указанный аналог основания выбирают из группы, состоящей из 2,6-диаминопурина, пропина Т, пропина G, феноксазинов и G-клэмпа.
38. Способ по п.36, где указанное универсальное основание выбирают из группы, состоящей из инозина, гуанидина, уридина, 5-нитроиндола, 3-нитропиррола, dP, dK и 1-(2-дезокси-β-D-рибофуранозил)имидазол-4-карбоксамида.
39. Способ детекции единичного нуклеотидного полиморфизма у субъекта, включающий стадии:
(a) выделения нуклеиновой кислоты из указанного субъекта;
(b) приведения в контакт упомянутой нуклеиновой кислоты с олигонуклеотидными праймерами, которые гибридизуются с участками указанной нуклеиновой кислоты, фланкирующими участок, включающий упомянутый возможный полиморфизм;
(с) амплификации указанного участка с получением продукта амплификации;
(d) определения молекулярной массы упомянутого продукта амплификации; и
(е) сравнения указанной молекулярной массы с молекулярной массой упомянутого участка субъекта, который заведомо обладает полиморфизмом, где если молекулярные массы одинаковы, то субъект обладает указанным полиморфизмом.
40. Способ по п.39, где указанный полиморфизм связан с заболеванием.
41. Способ по п.39, где указанным полиморфизмом является антиген группы крови.
42. Способ по п.39, где указанная стадия амплификации представляет собой полимеразную цепную реакцию.
43. Способ по п.39, где указанная стадия амплификации представляет собой лигазную цепную реакцию или амплификацию с заменой цепи.
44. Способ по п.39, где перед определением массы указанный продукт амплификации ионизируют.
45. Способ по п.39, где указанный продукт амплификации ионизируют ионизацией электрораспылением, лазерной десорбцией с использованием матрицы или бомбардировкой быстрыми атомами.
46. Способ по п.39, где указанные праймеры гибридизуются с консервативными последовательностями.
47. Способ по п.39, где указанную молекулярную массу определяют методом масс-спектрометрии.
48. Способ по п.47, где указанный метод масс-спектрометрии выбирают из группы, состоящей из масс-спектрометрии ионного циклотронного резонанса с Фурье-преобразованием (FT-ICR-MS), МС с ионной ловушкой, квадрупольной МС, МС с магнитным сектором, время-пролетной (TOF) МС, Q-TOF и тройной квадрупольной МС.
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