RU2014141207A - Способ обнаружения внутриклеточных возбудителей инфекции в клетках спермы - Google Patents
Способ обнаружения внутриклеточных возбудителей инфекции в клетках спермы Download PDFInfo
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Abstract
1. Способ исследования на присутствие вирусов, хламидий, паразитов и других возбудителей инфекции внутриклеточно в сперматозоидах, при этом способ включает следующие этапы:- процедуру для- облегчения плотной структуры ДНК клеток спермы, в том числе сперматозоидов,- прямую и непрямую иммунофлуоресценцию внутри клеток спермы,- визуализацию и оценку результатов с помощью проточной цитометрии,и данный способ отличается тем, что этап облегчения плотной структуры ДНК обязательно должен происходить до иммунофлуоресценции.2. Способ по п. 1, где этап визуализации и оценки результатов с помощью проточной цитометрии включает инкубацию клеточных осадков с 7-аминоактиномицином D (7AAD) в WB для того, чтобы дать возможность различить клетки 1N и 2N.3. Способ по любому из пп. 1 или 2, который применяют для определения причин бесплодия, срыва беременности на ранних сроках, выкидыша или гибели плода и для профилактики врожденных инфекций или для профилактики вертикальной передачи, а также для исследования воспаления и инфекций мужской половой системы.4. Способ по п. 1, который применяют специально для исследования одного из следующих возбудителей инфекции: цитомегаловируса (CMV), вируса простого герпеса I (HSV I), вируса простого герпеса II (HSV II), вируса Эпштейна-Барра (EBV), HHV6, HHV7, HHV8, парвовируса 19, вируса гепатита В (HBV), вируса гепатита С (HCV), коксаки-вируса, вирусов иммунодефицита человека (HIV-1, HIV-2), аденоассоциированного вируса (AAV), вируса краснухи, HPV, хламидий, токсоплазмы и норовируса.5. Способ по п. 1, который применяют для исследования на присутствие хламидий в сперматозоидах в комбинации со спермограммой и посевами с использованием одного и того же образца, или на разных образцах.6. Способ по п. 1, где облегчение очен
Claims (13)
1. Способ исследования на присутствие вирусов, хламидий, паразитов и других возбудителей инфекции внутриклеточно в сперматозоидах, при этом способ включает следующие этапы:
- процедуру для
- облегчения плотной структуры ДНК клеток спермы, в том числе сперматозоидов,
- прямую и непрямую иммунофлуоресценцию внутри клеток спермы,
- визуализацию и оценку результатов с помощью проточной цитометрии,
и данный способ отличается тем, что этап облегчения плотной структуры ДНК обязательно должен происходить до иммунофлуоресценции.
2. Способ по п. 1, где этап визуализации и оценки результатов с помощью проточной цитометрии включает инкубацию клеточных осадков с 7-аминоактиномицином D (7AAD) в WB для того, чтобы дать возможность различить клетки 1N и 2N.
3. Способ по любому из пп. 1 или 2, который применяют для определения причин бесплодия, срыва беременности на ранних сроках, выкидыша или гибели плода и для профилактики врожденных инфекций или для профилактики вертикальной передачи, а также для исследования воспаления и инфекций мужской половой системы.
4. Способ по п. 1, который применяют специально для исследования одного из следующих возбудителей инфекции: цитомегаловируса (CMV), вируса простого герпеса I (HSV I), вируса простого герпеса II (HSV II), вируса Эпштейна-Барра (EBV), HHV6, HHV7, HHV8, парвовируса 19, вируса гепатита В (HBV), вируса гепатита С (HCV), коксаки-вируса, вирусов иммунодефицита человека (HIV-1, HIV-2), аденоассоциированного вируса (AAV), вируса краснухи, HPV, хламидий, токсоплазмы и норовируса.
5. Способ по п. 1, который применяют для исследования на присутствие хламидий в сперматозоидах в комбинации со спермограммой и посевами с использованием одного и того же образца, или на разных образцах.
6. Способ по п. 1, где облегчение очень плотной структуры ДНК сперматозоидов выполняют с расщеплением ДНК.
7. Способ по п. 6, где расщепление ДНК осуществляют с помощью фермента, который разрывает ДНК.
8. Способ по любому из пп. 6 или 7, где ферментом, который разрывает ДНК, является ДНКаза I.
9. Способ по п. 1, где для флуоресценции можно применять любое соответствующим образом меченое антитело к антителу.
10. Способ по п. 9, где для флуоресценции можно применять любой флуорохром, среди которых любой из следующего: флуоресцеин-5-изотиоцианат (FITC), аминометилкумарина ацетат (АМСА 350), производное 6,8-дифтор-7-гидроксикумарина (Marina Blue), Cascade Blue, Alexa fluor 405, производное 6,8-дифтор-7-гидроксикумарина (Pacific Blue), Alexa Fluor 430, Cascade Yellow, Alexa Fluor 488, фикоэритрин (РЕ), фикоэритрин-техасский красный (PE-Texas Red), фикоэритрин-цианин 5 (PE-Cy5), перидинин-хлорофилл протеин (РегСР), перидинин-хлорофилл протеин-цианин 5.5 (РегСР-Су5.5), фикоэритрин-цианин 7 (PE-Cy7), родамин TR, аллофикоцианин (АРС), ALexa Fluor 647, аллофикоцианин-цианин 7 (APC-Cy7), BD APC-H7 или Alexa Fluor 700.
11. Способ по п. 1, где включен дополнительный этап поверхностной иммунофлуоресценции.
12. Набор для исследования на присутствие вирусов, хламидий, паразитов и других возбудителей инфекции внутри сперматозоидов, который включает по меньшей мере следующее:
вещество, которое может облегчить ДНК сперматозоидных клеток, одно или несколько антител, подходящих для применения против представляющих интерес возбудителей инфекции.
13. Набор по п. 12, где вещество для расщепления ДНК является любым расщепляющим ДНК ферментом.
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| GR20120100185 | 2012-03-29 | ||
| PCT/GR2013/000016 WO2013144662A1 (en) | 2012-03-29 | 2013-03-29 | Method of intracellular infectious agent detection in sperm cells |
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| CN111474339A (zh) * | 2020-04-17 | 2020-07-31 | 浙江康佰裕生物科技有限公司 | 一种利用荧光标记痘病毒颗粒的方法及其应用 |
| RU2738798C1 (ru) * | 2020-08-31 | 2020-12-16 | Василий Васильевич Ашапкин | Способ количественного определения вирусного инфицирования сперматозоидов |
| CN115032391A (zh) * | 2022-06-21 | 2022-09-09 | 成都思瑞多医疗科技有限公司 | 精子唾液酸酶1/3检测试剂盒及其制备方法、检测精子唾液酸酶1/3表达水平的方法 |
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| GB0016920D0 (en) * | 2000-07-10 | 2000-08-30 | Univ Cambridge Tech | Decondensation of DNA |
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| GR1008033B (el) | 2013-11-18 |
| IN2014DN08201A (ru) | 2015-05-01 |
| US20150064691A1 (en) | 2015-03-05 |
| CN104303059B (zh) | 2016-08-17 |
| IL234844A0 (en) | 2014-12-31 |
| SG10201704802YA (en) | 2017-07-28 |
| SG11201406096QA (en) | 2014-10-30 |
| HK1201579A1 (en) | 2015-09-04 |
| EP2831588A1 (en) | 2015-02-04 |
| GR20120100185A (el) | 2013-10-15 |
| RU2664734C2 (ru) | 2018-08-22 |
| WO2013144662A1 (en) | 2013-10-03 |
| AU2013239416A1 (en) | 2014-10-16 |
| CA2868707A1 (en) | 2013-10-03 |
| KR20150042146A (ko) | 2015-04-20 |
| ZA201407818B (en) | 2016-05-25 |
| CN104303059A (zh) | 2015-01-21 |
| AU2018275005A1 (en) | 2019-01-03 |
| JP6301310B2 (ja) | 2018-03-28 |
| CL2014002584A1 (es) | 2015-06-19 |
| JP2015513103A (ja) | 2015-04-30 |
| AU2018274968A1 (en) | 2019-01-03 |
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