WO2013144662A1 - Method of intracellular infectious agent detection in sperm cells - Google Patents

Method of intracellular infectious agent detection in sperm cells Download PDF

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Publication number
WO2013144662A1
WO2013144662A1 PCT/GR2013/000016 GR2013000016W WO2013144662A1 WO 2013144662 A1 WO2013144662 A1 WO 2013144662A1 GR 2013000016 W GR2013000016 W GR 2013000016W WO 2013144662 A1 WO2013144662 A1 WO 2013144662A1
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Prior art keywords
dna
spermatozoa
virus
cells
chlamydia
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Ceased
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PCT/GR2013/000016
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English (en)
French (fr)
Inventor
Vassilios Tsilivakos
Aggelos GRITZAPIS
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Individual
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Priority to HK15101974.1A priority Critical patent/HK1201579A1/xx
Priority to JP2015502463A priority patent/JP6301310B2/ja
Priority to CA2868707A priority patent/CA2868707A1/en
Priority to AU2013239416A priority patent/AU2013239416A1/en
Priority to IN8201DEN2014 priority patent/IN2014DN08201A/en
Priority to EP13721796.4A priority patent/EP2831588A1/en
Priority to SG11201406096QA priority patent/SG11201406096QA/en
Priority to RU2014141207A priority patent/RU2664734C2/ru
Priority to US14/387,794 priority patent/US20150064691A1/en
Application filed by Individual filed Critical Individual
Priority to KR20147029210A priority patent/KR20150042146A/ko
Priority to CN201380023706.3A priority patent/CN104303059B/zh
Publication of WO2013144662A1 publication Critical patent/WO2013144662A1/en
Priority to IL234844A priority patent/IL234844A0/en
Anticipated expiration legal-status Critical
Priority to ZA2014/07818A priority patent/ZA201407818B/en
Priority to AU2018274968A priority patent/AU2018274968A1/en
Priority to AU2018275005A priority patent/AU2018275005A1/en
Ceased legal-status Critical Current

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    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56905—Protozoa
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56927—Chlamydia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction

Definitions

  • azoospermia and menopause are indisputable causes of subfertility.
  • Oligospermia and premenopause which are the earlier stages of azoosperimia and menopause are also of interest as they are progressively important factors leading to sub-fertility. Oligospermia in particular, should be a problem easily resolved through IVF, since a single spermatozoon should be enough for conception. In practice however, this is not the case.
  • the method described for the investigation of the presence of viruses, Chlamydia, parasites and other infectious pathogens intracellular ⁇ in spermatozoa comprises the following steps:
  • this enzyme could be DNase I.
  • any suitable fluorescent antibody or anti antibodies can be used.
  • any fluorochrome can be used including any one of the following that are known today: Fluorecein-5- isothiocyanate (FITC), aminomethylcoumarin Acetate (AMCA 350), 6,8-difluoro-7-hydroxycoumarin derivative (Marina Blue), Cascade Blue, Alexa fluor 405, 6,8-difluoro-7- hydroxycoumarin derivative (Pacific Blue), Alexa Fluor 430, Cascade Yellow, Alexa Fluor 488, phycoerythrin (PE), phycoerythrin Texas Red (PE-Texas Red), phycoerythrin-cyanin 5 (PE-Cy5) , peridinin chlorophyll protein (PerCP), peridinin chlorophyll protein -cyanin 5.5 (PerCP-Cy5.5), phycoerythrin-cyanin 7( PE-Cy7), Rhodamine TR, allophycocyanin (APC), ALex
  • FITC Fluor
  • the method of the present invention may include an additional step for detecting surface antigens.
  • PFA fixation preserves the physical characteristics of cells: i.e. following fixation the cells exhibit the same scatter characteristics shown, during analysis done with flow cytometry. In addition PFA fixation allows the subsequent application of either an extracellular or intracellular staining procedure.
  • an example of such a substance is a DNA digestion enzyme.
  • DNA digestion substance we have used the enzyme DNAse I.
  • Figure 3 illustrates the failure of pathogen detection without the step of DNase I digestion.
  • Indirect staining is used in our example because it is less costly than direct staining.
  • the present invention can also function if it alternatively utilizes directly conjugated antibodies as described below.
  • a fraction of cells is spun down, resuspended, and incubated for 30 min with 100-500 ⁇ Phosphate Buffer Saline (PBS) containing 4% PFA and 0.1% saponin (medium A).
  • PBS Phosphate Buffer Saline
  • the cells are then washed with 2ml PBS containing 0.1 % saponin and 2% Fetal calf serum (FCS) (wash buffer-WB).
  • FCS Fetal calf serum
  • the supernatant is discarded and the pellet incubated with 100-500 ⁇ PBS containing 10% Dimethyl sulfoxide (DMSO) and 0.1% saponin for 10 min.
  • DMSO Dimethyl sulfoxide
  • HSV I Herpes Simplex Virus I
  • HSV II Herpes Simplex Virus II
  • EBV Epstein Bar virus
  • the incubation of cells with antibodies takes place either in separate tubes for each one of the pathogens or in the same tube, which allows the simultaneous detection of pathogens, provided that there are directly conjugated antibodies with discrete fluorophores, that emit colors which contrast to each other. After 30 min incubation at 4° C, the cells are washed with WB and the supernatant is discarded.
  • Fluorecein-5- isothiocyanate (FITC), aminomethylcoumarin Acetate (AMCA 350), 6,8- difluoro-7-hydroxycoumarin derivative (Marina Blue), Cascade Blue, Alexa fluor 405, 6,8-difluoro-7-hydroxycoumarin derivative (Pacific Blue), Alexa Fluor 430, Cascade Yellow, Alexa Fluor 488, phycoerythrin (PE), phycoerythrin Texas Red (PE-Texas Red), phycoerythrin-cyanin 5 (PE-Cy5) , peridinin chlorophyll protein (PerCP), peridinin chlorophyll protein -cyanin 5.5 (PerCP-Cy5.5), phycoerythrin-cyanin 7( PE- Cy7), Rhodamine TR, allophycocyanin (APC), ALexa Fluor 647, allophycocyanin cyanin 7 (APC-Cy7), BD APC-H7, Alexa Fluor 700.
  • fluorophore can be used including, but not limited to the following known fluorophores: Fluorecein-5- isothiocyanate (FITC), aminomethylcoumarin Acetate (A CA 350), 6,8-difluoro-7-hydroxycoumarin derivative (Marina Blue), Cascade Blue, Alexa fluor 405, 6,8-difluoro-7-hydroxycoumarin derivative (Pacific Blue), Alexa Fluor 430, Cascade Yellow, Alexa Fluor 488, phycoerythrin (PE), phycoerythrin Texas Red (PE-Texas Red), phycoerythrin-cyanin 5 (PE-Cy5) , peridinin chlorophyll protein (PerCP), peridinin chlorophyll protein -cyanin 5.5 (PerCP-Cy5.5), phycoerythrin- cyanin 7( PE-Cy7), Rhodamine TR, allophycocyanin (APC), ALexa Fluor 647, allophycocyanin cyanin 7 (
  • the tubes are washed with PBS containing 2% FCS (PBS - 2%FCS), and the supernatant is discarded.
  • the cells are re-suspended and placed in a flow cytometry apparatus for acquisition and analysis.
  • the inventors of the present invention believe that these are two different kinds of approach, with different clinical interpretations. For example, the inventors attribute subclinical viremias and chlamydiaemias to progenitor sperm cell infection through penetration of the blood-testis barrier.
  • the membrane localization of microorganisms is mainly associated with infection of the sperm release pathway (epididymis, prostate, urethra).

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Virology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • AIDS & HIV (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
PCT/GR2013/000016 2012-03-29 2013-03-29 Method of intracellular infectious agent detection in sperm cells Ceased WO2013144662A1 (en)

Priority Applications (15)

Application Number Priority Date Filing Date Title
US14/387,794 US20150064691A1 (en) 2012-03-29 2013-03-29 Method of intracellular infectious agent detection in sperm cells
CA2868707A CA2868707A1 (en) 2012-03-29 2013-03-29 Method of intracellular infectious agent detection in sperm cells
KR20147029210A KR20150042146A (ko) 2012-03-29 2013-03-29 정자 세포에서 세포내 감염제의 검출 방법
IN8201DEN2014 IN2014DN08201A (enExample) 2012-03-29 2013-03-29
EP13721796.4A EP2831588A1 (en) 2012-03-29 2013-03-29 Method of intracellular infectious agent detection in sperm cells
SG11201406096QA SG11201406096QA (en) 2012-03-29 2013-03-29 Method of intracellular infectious agent detection in sperm cells
RU2014141207A RU2664734C2 (ru) 2012-03-29 2013-03-29 Способ обнаружения внутриклеточных возбудителей инфекции в клетках спермы
HK15101974.1A HK1201579A1 (en) 2012-03-29 2013-03-29 Method of intracellular infectious agent detection in sperm cells
AU2013239416A AU2013239416A1 (en) 2012-03-29 2013-03-29 Method of intracellular infectious agent detection in sperm cells
JP2015502463A JP6301310B2 (ja) 2012-03-29 2013-03-29 精細胞中の細胞内感染因子の検出方法
CN201380023706.3A CN104303059B (zh) 2012-03-29 2013-03-29 检测精细胞中细胞内感染原的方法
IL234844A IL234844A0 (en) 2012-03-29 2014-09-28 A method for identifying the cause of intercellular contamination in sperm cells
ZA2014/07818A ZA201407818B (en) 2012-03-29 2014-10-27 Method of intracellular infectious agent detection in sperm cells
AU2018274968A AU2018274968A1 (en) 2012-03-29 2018-12-06 Method of intracellular infectious agent detection in sperm cells
AU2018275005A AU2018275005A1 (en) 2012-03-29 2018-12-07 Method of intracellular infectious agent detection in sperm cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GR20120100185 2012-03-29
GR20120100185A GR1008033B (el) 2012-03-29 2012-03-29 Μεθοδος διερευνησης της παρουσιας ενδοκυτταριων μολυσματικων παραγοντων σε κυτταρα του σπερματος

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WO2013144662A1 true WO2013144662A1 (en) 2013-10-03

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PCT/GR2013/000016 Ceased WO2013144662A1 (en) 2012-03-29 2013-03-29 Method of intracellular infectious agent detection in sperm cells

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US (1) US20150064691A1 (enExample)
EP (1) EP2831588A1 (enExample)
JP (1) JP6301310B2 (enExample)
KR (1) KR20150042146A (enExample)
CN (1) CN104303059B (enExample)
AU (3) AU2013239416A1 (enExample)
CA (1) CA2868707A1 (enExample)
CL (1) CL2014002584A1 (enExample)
GR (1) GR1008033B (enExample)
HK (1) HK1201579A1 (enExample)
IL (1) IL234844A0 (enExample)
IN (1) IN2014DN08201A (enExample)
RU (1) RU2664734C2 (enExample)
SG (2) SG10201704802YA (enExample)
WO (1) WO2013144662A1 (enExample)
ZA (1) ZA201407818B (enExample)

Families Citing this family (5)

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Publication number Priority date Publication date Assignee Title
WO2016154286A1 (en) * 2015-03-23 2016-09-29 Virocyt, Inc. Evaluating biological material for unassociated virus-size particles of adenovirus or adeno-associated virus viral type
CN107121547A (zh) * 2016-01-21 2017-09-01 广东和信健康科技有限公司 直接免疫荧光检测肠道病毒71型和柯萨奇病毒a16型的试剂盒及其应用
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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WO2002004666A2 (en) * 2000-07-10 2002-01-17 Cambridge University Technical Services Limited Decondensation of dna
WO2003060520A2 (en) * 2001-12-24 2003-07-24 Helen Lee Sample preparation for the detection of infectious agents
US20060099661A1 (en) 2004-04-16 2006-05-11 Stuart Elizabeth S Detection and quantification of intracellular pathogens

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PT1673105E (pt) * 2003-07-25 2007-06-20 Serono Lab ''utilizaçao da hormona estimulante do folículo para a redução de aberrações cromossómicas em espermatozóides em indivíduos do sexo masculino''

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WO2002004666A2 (en) * 2000-07-10 2002-01-17 Cambridge University Technical Services Limited Decondensation of dna
WO2003060520A2 (en) * 2001-12-24 2003-07-24 Helen Lee Sample preparation for the detection of infectious agents
US20060099661A1 (en) 2004-04-16 2006-05-11 Stuart Elizabeth S Detection and quantification of intracellular pathogens

Non-Patent Citations (2)

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Title
AYNAUD 0 ET AL.: "Frequency of herpes simplex virus, cytomegalovirus and human papillomavirus DNA in semen", INT J STD AIDS, vol. 13, no. 8, August 2002 (2002-08-01), pages 547 - 50
L. A. MITCHELL ET AL: "The TUNEL assay consistently underestimates DNA damage in human spermatozoa and is influenced by DNA compaction and cell vitality: development of an improved methodology", INTERNATIONAL JOURNAL OF ANDROLOGY, vol. 34, no. 1, 11 February 2011 (2011-02-11), pages 2 - 13, XP055067917, ISSN: 0105-6263, DOI: 10.1111/j.1365-2605.2009.01042.x *

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CA2868707A1 (en) 2013-10-03
KR20150042146A (ko) 2015-04-20
CN104303059B (zh) 2016-08-17
HK1201579A1 (en) 2015-09-04
AU2018275005A1 (en) 2019-01-03
RU2014141207A (ru) 2016-05-27
GR20120100185A (el) 2013-10-15
SG10201704802YA (en) 2017-07-28
CL2014002584A1 (es) 2015-06-19
ZA201407818B (en) 2016-05-25
US20150064691A1 (en) 2015-03-05
IL234844A0 (en) 2014-12-31
IN2014DN08201A (enExample) 2015-05-01
JP6301310B2 (ja) 2018-03-28
SG11201406096QA (en) 2014-10-30
AU2013239416A1 (en) 2014-10-16
CN104303059A (zh) 2015-01-21
EP2831588A1 (en) 2015-02-04
RU2664734C2 (ru) 2018-08-22
JP2015513103A (ja) 2015-04-30
GR1008033B (el) 2013-11-18
AU2018274968A1 (en) 2019-01-03

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