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Sommaire du brevet 2888949 

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Disponibilité de l'Abrégé et des Revendications

L'apparition de différences dans le texte et l'image des Revendications et de l'Abrégé dépend du moment auquel le document est publié. Les textes des Revendications et de l'Abrégé sont affichés :

  • lorsque la demande peut être examinée par le public;
  • lorsque le brevet est émis (délivrance).
(12) Demande de brevet: (11) CA 2888949
(54) Titre français: METHODE ET COMPOSITIONS ASSOCIEES POUR DETECTER UN ACIDE NUCLEIQUE CIBLE DANS DES ECHANTILLONS BIOLOGIQUES ET DES LIQUIDES ORGANIQUES
(54) Titre anglais: METHOD AND ITS COMPOSITIONS FOR DETECTION OF NUCLEIC ACID TARGET FROM BIOLOGICAL SAMPLES AND BODY FLUIDS
Statut: Morte
Données bibliographiques
(51) Classification internationale des brevets (CIB):
  • C12Q 1/6806 (2018.01)
  • C12Q 1/6844 (2018.01)
  • C12N 1/06 (2006.01)
  • C12N 15/10 (2006.01)
  • C12Q 1/68 (2006.01)
(72) Inventeurs :
  • TULP, INDREK (Estonie)
  • KROLOV, KATRIN (Estonie)
  • LEHES, MARKO (Estonie)
  • LANGEL, ULO (Suède)
(73) Titulaires :
  • SELFDIAGNOSTICS OU (Estonie)
(71) Demandeurs :
  • SELFDIAGNOSTICS OU (Estonie)
(74) Agent: AIRD & MCBURNEY LP
(74) Co-agent:
(45) Délivré:
(86) Date de dépôt PCT: 2013-10-20
(87) Mise à la disponibilité du public: 2014-04-24
Licence disponible: S.O.
(25) Langue des documents déposés: Anglais

Traité de coopération en matière de brevets (PCT): Oui
(86) Numéro de la demande PCT: PCT/EP2013/071906
(87) Numéro de publication internationale PCT: WO2014/060604
(85) Entrée nationale: 2015-04-21

(30) Données de priorité de la demande:
Numéro de la demande Pays / territoire Date
61/716,495 Etats-Unis d'Amérique 2012-10-20

Abrégés

Abrégé français

Cette invention concerne une méthode rapide de prétraitement d'un échantillon qui permet de détecter avec une extrême sensibilité et une extrême spécificité un acide nucléique cible (par exemple l'ADN du génome humain, l'ADN du génome d'un pathogène de l'homme, l'ADN du génome d'un agent non pathogène de l'homme) par amplification directe de matériel génomique contenu dans des lysats d'échantillons biologiques non purifiés bruts prélevés chez l'homme (par exemple des échantillons d'urine, de salive, de sang, des prélèvements de l'urètre et du col de l'utérus et d'autres échantillons contenant du matériel biologique). En particulier, la méthode de prétraitement d'un échantillon biologique selon l'invention permet aux cellules de l'homme et du pathogène de libérer rapidement leur matériel génomique. L'invention concerne également des composants compatibles avec la méthode d'amplification de l'acide nucléique décrite ci-dessous. A titre d'exemple, l'invention décrit également des protocoles et des séquences d'amorces pour l'amplification isotherme de l'acide nucléique [amplification par recombinase-polymérase (ARP), technique d'amplification isotherme de l'ADN induite par boucle (LAMP)], qui permettent d'établir un diagnostic extrêmement spécifique et sensible du matériel génomique de l'Homo sapiens, de Chlamydia trachomatis et de Mycoplasma genitalium à partir de lysats d'échantillons biologiques bruts et/ou d'ADN total purifié. L'amplification utilisée peut être associée à la détection d'un produit immunochromatographique sur bandelettes à écoulement latéral et permet une amplification isotherme rapide de l'acide nucléique (moins de 20 minutes) pour le diagnostic de C. trachomatis et de M. genitalium dans des échantillons urinaires recueillis chez l'homme. La méthode selon l'invention ne nécessite pas d'équipement de laboratoire spécifique ni de personnel qualifié, et convient donc parfaitement aux applications effectuées au point d'intervention.

Abrégé anglais

Current invention is directed for rapid sample pretreatment method that allows highly sensitive and specific detection of target nucleic acid (e.g. human genomic DNA, human pathogen genomic DNA, human non-pathogen genomic DNA) by amplification directly from crude unpurified biological samples lysates (e.g. human 5 urine, saliva, blood, urethra and cervical swabs and other samples containing biological material). Invention is focused on the description of the biological sample pretreatment method that enables fast release of the genomic material from human and pathogen cells, components of what are compatible with the following nucleic acid amplification method. As an example of the application, 10 invention also discloses protocols and primer sequences for isothermal nucleic acid amplification (recombinase polymerase amplification RPA, loop-mediated isothermal amplification - LAMP), that enable highly specific and sensitive diagnostics of the genomic material from Homo sapiens, Chlamydia trachomatis and Mycoplasma genitalium from crude biological sample lysates and/or purified 15 total DNA. The example amplification can be combined with immunochromotographic product detection using lateral-flow strips and allows rapid (under 20 min) isothermal nucleic acid amplification based C. trachomatis and M. genitalium diagnostics from human urine samples, that does not require specific laboratory equipment nor qualified personnel, and is therefore well suited 20 for point-of-care settings applications.

Revendications

Note : Les revendications sont présentées dans la langue officielle dans laquelle elles ont été soumises.



19

CLAIMS

1. A method for detection of nucleic acid target from biological samples and
body
fluids comprises following steps:
a) sample pretreatment comprising cell lysis and release of nucleic acid
targets in biological samples and body fluids;
b) amplification of nucleic acid(s);
c) detection of amplification product(s),
wherein lytic peptides are used to release nucleic acid targets in biological
samples or body fluids.
2. The method according to claim 1, wherein detergents are used to release
nucleic acid targets in biological samples or body fluids.
3. The method according to claim 1, wherein combination of lytic peptides and
detergents are used to release nucleic acid targets in biological samples or
body fluids.
4. The method according to claim 1, wherein one or more specific target based
sequences are amplified.
5. The method according to claim 1, wherein sample solution obtained during
the
step (1) is directly subjected for further amplification procedure.
6. The method according to claim 1, wherein qualitative and quantitative
detection
is performed with crude sample solution.
7. The method according to claim 1, wherein the Chlamydia trachomatis nucleic
acid target(s) with the use of specific target region provided in Table 1 is
detected.
8. The method according to claim 1, wherein the Mycoplasma genitalium nucleic
acid target(s) with the use of specific target region provided in Table 1 is
detected.
9. The method according to claim 1, wherein the Chlamydia trachomatis nucleic
acid target(s) the use of specific primer(s) and/or its labeled derivative(s)
sequences provided in Table 2 and 3 is detected.


20

10. The method according to claim 1, wherein the Mycoplasma genitalium nucleic

acid target(s) with the use of specific primer(s) and/or its labeled
derivative(s)
sequences provided in Table 2 and 3 is detected.
11. The method according to claim 1, wherein the human genomic GAPDH target
is used for detection as an internal validation and platform assessing
technique.
12. The method according to claim 1, wherein the human genomic GAPDH target
is used for detection as an internal validation and platform assessing
technique
with specific primer(s) and/or its labeled derivative(s) sequences provided in

Tables 2, 3.
13. A molecular diagnostics method of Chlamydia trachomatis, wherein the TRPB
gene is used as molecular diagnostics target.

Description

Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.


CA 02888949 2015-04-21
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METHOD AND ITS COMPOSITIONS FOR DETECTION OF NUCLEIC ACID
TARGET FROM BIOLOGICAL SAMPLES AND BODY FLUIDS
FIELD OF THE INVENTION
The invention is directed to compositions and method for rapid biological
sample
pretreatment that allows following nucleic acid amplification based detection
of the
target nucleic acid from biological samples and body fluids.
BACKGROUND OF THE INVENTION
Current diagnostics relies majorly on the nucleic acid amplification
techniques
(NAAT). Most commonly known method for specific DNA amplification is PCR that
gives reasonable sensitivity on the laboratory level. Lately new emerging
techniques have been developed of isothermal amplification, such as
recombinase
polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP),

helicase dependent amplification (HDA). These isothermal NAATs do not require
thrermocycling of the reaction and have shown extremely high levels of
sensitivity,
resulting in detectable amplification product from as few as 1-2 template
copies.
Isothermal reaction makes them well suited for point-of-care (POC) settings
(eg
GP office, at home), bringing diagnostics test conveniently and immediately to
the
patient and decreasing time to result. In the field of sexually transmitted
diseases,
POC diagnostics also allows private and non-invasive testing, that has a
potential
to significantly reduce the spread of the pathogens, especially those that
exist in
asymptomatic form like C. trachomatis and M. genitaium.
Both M. genitalium and C. trachomatis infections are known as "silent"
diseases as
they often remain asymptomatic. Thus regular diagnostic screening of these
sexually transmitted pathogens is of high importance. Classically C.
trachomatis
infection has been diagnosed from urethral or cervical swab specimens by
tissue
culture method. Because culturing identifies only viable C. trachomatis cells,

sensitivity of the diagnostics is affected by the freshness of the specimen
depending on the time between collection and processing in the laboratory.
Thus
during 1980s antigen and nucleic acid detection technologies have been
developed for C. trachomatis diagnostics that have lesser demand of cost,
time,
expertise, preservation of infectivity during transport. Furthermore nucleic
acid
detection techniques have proved to have much higher sensitivity levels as
they

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2
can detect pathogen DNA from unviable cells or cell debris. Microbiological
detection of M. genitalium is also mostly performed by specific amplification
of the
pathogen DNA by PCR. M. genitalium culture is extremely difficult and is not
performed routinely. Serological detection methods of M. genitalium are weakly
sensitive and specific.
Although NAAT open up crucial opportunity for highly effective diagnostics, to
date
they are routinely used only on the laboratory level. NAATs are complicated to

perform, require trained personnel and expensive machinery. Thus NAAT based
diagnostics is centered to large hospitals and diagnostics centers. One of the
major limitations of the NAAT techniques is the requirement for pure DNA
sample.
The purity of the sample can affect significantly performance of the NAAT-s,
especially PCR. Novel isothermal NAAT-s like RPA, LAMP, HDA etc seem to be
less sensitive towards nucleic acid sample purity and are able to efficiency
amplify
DNA present in eg human urine samples.
Current invention discloses a method and its compounds for biological sample
pretreatment that allows efficient release of the genomic DNA from cellular
material. Described sample pretreatment method is compatible with the
following
nucleic acid amplification procedure allowing detection of the target DNA from

crude sample lysates. The invention allows skipping of the DNA purification
step
prior to NAAT analysis, having therefore an important impact on the complexity
and speed of the diagnostic technique. Current invention facilitates
significantly
implementation of the highly sensitive and specific NAAT diagnostics in the
POC
settings.
Because examples of the invention implementation is concentrated on human
sexually transmitted pathogen diagnostics, the overview of the Chlamydia
trachomatis and Mycoplasma genitalium will be given hereafter.
C. trachomatis and M. genitalium are sexually transmitted human pathogens.
Both
of them are associated with non-gonococcal (non-specific) urethritis in men
and
several inflammatory reproductive tract syndromes in women such as cervicitis
and pelvic inflammatory disease. Inflammatory diseases caused by acute
untreated infections of C. trachomatis and M. genitalium are one of the
leading
causes of female infertility worldwide.

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The prevalence of M. genitalium ranges globally from 1-4% in men and 1-6% in
women. Reported prevalence data within populations at higher risk (eg within
sexually transmitted disease (STD) testing centers) reach 38%. C. trachomatis
prevalence rates among sexually active young people vary from 5-10% depending
on the age, ethnic origin etc. C. trachomatis infection is almost always more
prevalent among women and has shown an increasing trend globally during past
decades.
M. genitalium is a small (0.2-0.3 pm) pleomorphic bacterium that lacks cell
wall
making it resistant to common antibiotics targeting cell wall (eg penicillin).
M.
genitalium cells are flask shaped and carry a specific adhesion organelle that
allows bacteria to adhere to various materials and cells including human
epithelial
cells. Adhesion is the main mechanism of M. genitalium pathogenesis that
involves at least seven adhesins including major adhesin MgPa (encoded by
MGPB gene).
C. trachomatis is a gram-negative, obligate intracellular pathogen that has a
unique biphasic developmental cycle during which they exist in two
developmental
forms: the EB (or elementary body) and RB (or reticulate body). EB is smaller
(0.2
pM), metabolically inactive, infectious extracellular form of the organism and
RB is
larger (0.8 pM) metabolically active intracellular form. Chlamydial infection
involves
attachment of the EB to a host cell and its subsequent internalization into a
membrane-bound vesicle. Inclusion differentiates into RB which uses host cell
ATP and metabolites to undergo 8-12 round of cell division. RB differentiates
and
matures into infectious EB that are released by host cell lysis. C.
trachomatis
strains are serologically classified into 15 serovars based on antigenic
variation of
the major outer membrane protein. A-C serovars are eye pathogens causing
ocular trachoma. Serovars D-K and L1-L2 are sexually transmitted pathogens
that
infect columnar epithelial cells of the genital tract.
Adaptive immunity against C. trachomatis involves INF-y mediated host cell
responce that deprives chlamydial RBs of tryptophan, which ultimately prevents
their growth and replicative capabilities. C. trachomatis genital serovars
have
retained some of the eubacterial tryptophan biosynthesis genes, TRPA and TRPB
encoding a and 13 subunits of the tryptophan synthase that catalyzes
conversion of
the indole into tryptophan. Thus genital C. trachomatis serovars have retained
the

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capacity to use exogenous indole secreted by genital trakt normal microflora
that
allows them to overcome INF-y mediated growth restriction and promotes long
term establishment of the infection.
M. genitalium has a small AT rich (68%) 0.58 Mb genome that encodes 485
genes. Despite its small size, 4% of the genome consists of repeated elements
(MgPa repeats) that present homology with the MGPB gene. C. trachomatis also
carries a small genome of approximately 1Mb chromosome and 7.5 kb cryptic
plasmid. Almost all C. trachomatis strains harbor four to ten plasmid copies
per
chromosome. Although some plasmid-free C. trachomatis isolates have been
described, their virulence is significantly reduced as compared to the plasmid
carrying strains. Chlamydia plasmid sequence is highly conserved (< 1%
variation)
and contains eight major coding sequences (CDSs) along with a replication
origin
formed by four 22 bp tandem repeats. In silico analysis has identified plasmid

encoded proteins to have a function in replication.
DESCRIPTION OF THE INVENTION
Current invention discloses a method and its compounds for biological sample
pretreatment that allows efficient release of the genomic DNA from cellular
material. Major advantage of the described sample pretreatment method is its
compatibility with downstream nucleic acid amplification procedures allowing
detection of the target DNA from crude sample lysates. Thus current invention
allows skipping of the DNA purification step prior to NAAT analysis, having
therefore an important impact on the complexity and speed of the diagnostic
technique.
The invention discloses cell lytic compounds that allow fast (within 5 min at
RT C)
and efficient release of the genomic material from mammalian cells, their
pathogen
and commensal microorganisms, bacterial and fungi cultures etc. Sample
pretreatment buffer consists of membrane active (cell-penetrating) peptides,
mild
detergents or a combination of the above two.
Membrane active peptides have antibacterial and antimicrobial effect acting
disruptively on bacterial membranes. They are also known as cell membrane
penetrating agents that can deliver different cargo molecules into mammalian
cells
(eg oligonucleotides, siRNA, plasmids, peptides). Current invention targets
novel

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usage of the cell-penetrating peptides for diagnostics purposes. At higher (pM
-
mM) concentrations cell-penetrating peptides disrupt cellular membranes, that
allows the release of the genomic DNA that can be used as a target in the
following nucleic acid amplification reaction. Cell membrane disruptive
peptides
5 have shown no or minimal inhibiting effect on nucleic acid amplification
even at
high concentrations, thus can be efficiently used as agents facilitating
genomic
material release.
Detergents are very good solubilizing agents, but they tend to denature
proteins by
destroying native three dimensional structures. Certain combination of the
mild
ionic or non-ionic detergents (eg Triton X-100, Triton X-114, NP-40, CHAPS,
Octy1-3-glucoside, Octy1-3-thioglucopyronoside) at low (eg 0.1-1%)
concentration
allow efficient cell wall disruption in order to release genomic material
enclosed
within cells. These mild detergents do not interfere significantly with
nucleic acid
amplification procedure, and are able to induce or facilitate the release of
the
sufficient amount of the target nucleic acid. The composition and
concentration of
the detergents is set to efficiently lyse cells within 5 min RT C incubation.
The ability of the membrane active peptide and/or detergent mediated sample
pretreatment to convert biological sample into material well usable for the
nucleic
acid amplification is the major focus of the invention and has been confirmed
by
establishing detection of the Chlamydia trachomatis, Mycoplasma genitalium and
Homo sapiens genomic DNA from crude human urine lysates.
For that a diagnostic method for highly specific and sensitive C. trachomatis
and
M. genitalium detection from human samples has been developed based on
isothermal nucleic acid amplification (RPA, LAMP) and including
immunochromotographic product detection using lateral-flow strips. For both
pathogens we have used double target system, where simultaneous detection of
two different genomic targets is performed. This reduces probability of the
false
negative diagnostics test result in case deletions or mutations are introduced
into
pathogen genomic DNA regions used as the amplification targets. All target
regions were selected based on their high homology among different pathogen
strains and lack of identity with similar species.

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For C. trachomatis detection we have used genomic sequence regions from a
well-established diagnostic target ¨ coding sequence 2 of the multicopy
cryptic
plasmid (CDS2). For the second target we have chosen 33 subunit of the
tryptophan synthase gene TRPB. For M. genitalium detection we have used
genomic sequence regions from gene encoding MgPa dominant adhesin (MGPB)
that is the main component of multiple repeats throughout its genome. For the
second target we used 16S rRNA gene that is also present in multiple copies
within M. genitalium genome. M. genitalium 16S rRNA gene however is highly
conserved between different Mycoplasma species (eg 98% identity with M.
pneumoniae, 91% with M. gallisepticum). Thus multiple mutations containing
regions were chosen for the isothermal amplification and additional
specificity
testing was performed for this particular target.
For each target, optimal primer pair combinations were established that enable

highest sensitivity levels for the assay. Optimized RPA reaction allowed well
detectable and stable product amplification with minimum of 20-50 target
sequence copies. Optimized LAMP reaction with loop primers allowed product
amplification with minimum of 5-10 target sequence copies. Each diagnostics
target was tested for specificity of the reaction with 50 000 copies (0.16 ng)
of H.
sapiens genomic DNA and in case of M. genitalium 16S rRNA target also with
100 000 copies of M. pneumoniae genomic DNA. Isothermal amplification
sensitivity and specificity was verified with total DNA extracted from human
urine
samples.
Major objective of the current invention was to develop a diagnostic assay
applicable under point-of-care conditions. Thus we have integrated
immunochromotographic amplification product detection into the diagnostics
system. For that purpose, forward primer sequences were 5' labeled with biotin

and reverse primers with fluorescein amidite (FAM). During amplification
reaction
a dually labeled products were produced, that were detected within minutes
using
lateral-flow strips. Integration of the innmunochromotographic product
detection
required additional primer optimization. Primers gaining template independent
lateral-flow strip detectable signal were eliminated from the selection.
RPA and LAMP isothermal amplification based diagnostics methods were also
showed to be suitable for simultaneous multiple target detection. Both assays

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were optimized for H. sapiens GAPDH gene target to be used as a positive
control
of the diagnostics test with human samples. PCR and isothermal amplification
(RPA/LAMP/HDA) protocols were adjusted for optimal sensitivity and high
specificity of the diagnostics test.
The present method for detection of nucleic acid target(s) from biological
crude
samples and body fluids comprises following steps:
a) sample pretreatment comprising cell lysis and release of nucleic acid
targets in
biological samples and body fluids such as tissue, urine, saliva, blood,
stool,
hair, etc. and their derivatives, but not limited to the examples list,
wherein the
lytic peptides are used to release nucleic acid targets in biological samples;
b) amplification of nucleic acid(s) comprising nucleic acid, such as DNA, RNA
and
their derivatives but not limited to the list, amplification initiated by
presence of
target and comprise amplification methods such as PCR (Polymerase Chain
Reaction), HCR (Hybridization Chain Reaction), RCA (Rolling Circle
Amplification), RPA (Recombinase Polymerase Amplification), LAMP (Loop
mediated isothermal AMPlification), HDA (Helicase Dependent Amplification),
etc. and their derivatives, but not limited to the examples list, wherein one
or
more specific target based sequences are amplified or sample solution
obtained during the step (1) is directly subjected for further amplification
procedure;
C) detection of amplification product(s) comprising the use of qualitative or
quantitative detection methods such as sandwich assays, ELISAs (Enzyme
Linked ImmunoSorbent Assay), LF (Lateral Flow) immunochromatographic
assays, wavelength changing (visible spectrum, chemiluminescence,
fluorescence, phosphorescence and etc.) dyes, denrimeres, etc. or
corresponding moiety conjugated detector molecules and ligands, with or
without optical apparatus, appropriate wavelength emitter or reader or their
combination, wherein qualitative and quantitative detection is performed with
crude sample solution.
The pretreatment method is specifically designed to detect nucleic acid
target(s):
- of Chlamydia trachomatis with the use of specific target region provided in
Table 1 or with the use of specific primer(s) and/or its labeled derivative(s)

sequences provided in Table 2, 3; and

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- Mycoplasma genitalium with the use of specific target region provided in
Table
1 or with the use of specific primer(s) and/or its labeled derivative(s)
sequences
provided in Table 2, 3.
The present method with human genomic GAPDH target is used for detection:
- as an internal validation and platform assessing technique;
- as an internal validation and platform assessing technique with specific
primer(s) and/or its labeled derivative(s) sequences provided in Tables 2, 3.
The pretreatment method that relates to molecular diagnostics of Chlamydia
trachomatis wherein TRPB gene is used as molecular diagnostics target.
EXAMPLES OF THE IMPLEMENTATION
Example 1. Fast diagnostics of the presence of Chlamydia trachomatis in a
urine
sample
Present protocol describes method and its components for highly sensitive
Chlamydia trachomatis diagnostics from human urine sample. The whole
procedure including sample pretreatment, target isothermal amplification and
product detection takes under 20 min and requires 10 min incubation at 37 C.
Described method detects two C. trachomatis targets TRPB sequence in the
genomic region and CDS2 sequence in the cryptic plasmid region (Table 1).
Table 1. Genomic regions of Chlamydia trachomatis, Mycoplasnna genitalium and
Homo sapiens used for isothermal amplification based detection
Target organism Sequence name Genebank accession nr
PL CDS2 FM865439.1
C. trachomatis -
sequence 756-1748
FN652779.2
TRPB
sequence 193461-194639
16S rRNA CP003773.1
sequence 169843-1 71 366
M. genitalium
CP003773.1
MGPA
sequence 221365-225744
H. sapiens GAPDH NG 007073.2
Both of the C. trachomatis targets are amplified using highly specific and
sensitive
primers that carry same labeling, forward primers are labeled with biotin and
reverse with FAM. Thus C. trachomatis specific products are not distinguished
during immunochromatographic detection on lateral-flow strips. Detection of
the

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two C. trachomatis regions is used to ensure positive test results in case one
of
the target regions is mutated or deleted. The reaction also contains primers
targeting H. sapiens GAPDH gene that produce DIG and FAM labeled product.
This product is recognized as a separate lane on the lateral-flow strip and
serves
as a positive control for the whole procedure (release of the genomic material
from
cells, amplification and detection). Analytical sensitivity of the described
method is
50 C. trachomatis cells and 50 H. sapiens cells per test. This allows
detection of
the C. trachomatis in the first void urine at pathogen concentration of 10 000
cells
per 1 ml of urine or higher.
Patient urine sample is mixed with equal volume of sample pretreatment buffer
containing 0.2% Triton X-114, 150 mM NaCl, 50 mM Tris pH 7.0, and incubated 5
min at RT C. 10 pl of the treated sample is used in the RPA reaction
containing
following components: C. trachomatis PL-CDS2 5' biotin labeled FW3 primer at
0.4 pM final concentration, C. trachomatis PL-CDS2 5' FAM labeled RV1 primer
at
0.4 pM final concentration, C. trachomatis TRPB 5' biotin labeled FW2 primer
at
0.4 pM final concentration, C. trachomatis TRPB 5' FAM labeled RV3 primer at
0.4
pM final concentration, H. sapiens GAPDH 5' DIG labeled FW3 primer at 0.4 pM
final concentration, H. sapiens GAPDH 5' FAM labeled RV2 primer at 0.4 pM
final
concentration (see Table 2 for primer sequences), 14 mM magnesium acetate,
TwistDX RPA enzyme pellet and 29,5 pl of the rehydration buffer. Reaction is
incubated at 37 C for 10 min. The products are diluted 1:10 ratio with
dilution
buffer and analyzed on lateral-flow strips detecting Biotin-FAM and DIG-FAM
labeled molecules.
Table 2. Specific primer sequences for recombinase polymerase amplification
(RPA) against targets provided in Table 1
Target Sequence (5' ¨ 3')
organism
and region
C. Forward FW1 5' ¨
trachomatis (FW) CTTCTTTGAAGCGTTGTCTTCTCGAGAAGATTT
PL-CDS2 primer FW2 5' -
sequences CTTCTCGAGAAGATTTATCGTACGCAAATATC
FW3 5' -
CCTTCATTATGTCGGAGTCTGAGCACCCTAGGC
FW4 5' -
AGGCGTTTGTACTCCGTCACAGCGGTTGCTCG

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Reverse RV1 5'-
(RV) CTCTCAAGCAGGACTACAAGCTGCAATCCCTT
primer RV2 5' -
sequences ATGGTGGGGTTAAGGCAAATCGCCCGCACGTT
RV3 5'- TCT TCG TAA CTC GCT CCG GM AAA
TGG TGG GG
RV4 5'- CTT TCT ACA AGA GTA CAT CGG TCA
ACG AAG AGG
C. Forward FW1 5'- ACT ATG CGG GGA GAC AM CTC CTC
trachomatis (FW) TGA CTG MG
TRP B primer FW2 5'- TCT TM ACG CGA AGA TCT UT GCA
sequences TAC AGG AGC
FW3 5'- CAT ACA GGA GCA CAT AM CTG MT
MT GCT CU GG
FW4 5'- CTC TTG GTC AGT GTT TGC TTG CTA
AAT ATC TTG
Reverse RV1 5'- TCC CCC ACC TGT TTC AGC TAC MC
(RV) ACG TGT TT
primer RV2 5'- CTG TTG CTG TTG CTA CTC CAT GTT
sequences GTC CCG CAC
RV3 5'- TCC CAT GTA TAC TAC ACA ATC TM
TCC TAG ATA
RV4 5'- TTC TGT CGT TCC ACA TCT TTT GCT
CCC ATG TAT
M. Forward FW1 5'- AGC GCA ACC CTT ATC GTT AGT TAC
genitalium (FW) ATT GTT TM
1 6S rRNA primer FW2 5'- CGT TAG TTA CAT TGT TTA ACG AGA
sequences CTG CTA ATG T
FW3 5'- ACG TGC TAC MT GGC CM TAC AM
GAG TAG CCA A
Reverse RV1 5'- TTG CAG CCC TCA ATC CGA ACT GAG
(RV) ACC AAC TTT T
primer RV2 5'- CAT AGC TGA TTC GCG ATT ACT AGT
sequences GAT TCC AGC
RV3 5'- TTC CM TAA AGG TTA GCA ACA OCT
TTT TAA ATA
M. Forward FW1 5' ¨
genital ium (FW) TTGGACTTGAAACAATAACAACTTCTCTTCACT
MGPA primer FW2 5' ¨
sequences AAGATTACTGGAGAGAACCCAGGATCATTTGGA
FW3 5'- GAG TGG GCA GAC TAT GTC TTA OCT
TTG ATT GTA
FW4 5'- TTA TCC TTA GTG TTA CU TGG GAT
TAA CGA TTG G
FW5 5'-
CAATGCACAGAAACAAAAAGGCATTACAAGCAGG
Reverse RV1 5'- TCT GAT TGC AAA GTT TTG CTG ACC
(RV) ATC AAG GTA

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primer RV2 5'- CTC TAC CGT TGT TAT CAT ACC TTC
sequences TGA TTG C
RV3 5'- TTC TGT TAA TGA TCT CTT TAA AGA
CAC TAC CAA
RV4 5'- CTT AGG AGC GTT AGA GAT CCC TGT
TCT GTT AAT G
RV5 5'- CTT GTT TTA ACT TCT TAG GAG CGT
TAG AGA TCC C
RV6 5'-
TTACTGGAGGTTTTGGTGGGGTTTTAGGAGTTGG
H. sapiens Forward FW1 5' -
GAPDH (FW) CTCCTCCGGGTGATGCTTTTCCTAGATTATTCTC
primer FW2 5' - CTA ACC CTG CGC TCC TGC CTC GAT
sequences GGG TGG AG
FW3 5'- AAG TCA GGT GGA GCG AGG CTA GCT
GGC CCG ATT
Reverse RV1 5'- TCC UT TCC MC TAC CCA TGA CTC
(RV) AGC TTC TCC C
primer RV2 5'- CAC CAT GCC ACA GCC ACC ACA CCT
sequences CTG CGG GGA
RV3 5'- CCA CCA CCA GAG GGG CCA TTT TGC
GGT GGA AAT
Chlamydia tests positive if the test gives 2 lines (Biotin-FAM and DIG-FAM),
negative if the test gives 1 line DIG-FAM. The results of the test are invalid
if none
of the lines are present or only iotin-FAM line is present.
Example 2. Fast diagnostics of the presence of Mycoplasma genitalium in a
urine
sample
Present protocol describes method and its components for highly sensitive
Mycoplasma genitalium diagnostics from human urine sample. The whole
procedure including sample pretreatment, target isothermal amplification and
product detection takes under 20 min and requires 10 min incubation at 37 C.
Described method detects two M. genitalium targets MGPA and 16S rRNA
sequences in the pathogen genome (Table 1). Both of the M. genitalium targets
are amplified using highly specific and sensitive primers that carry same
labeling,
forward primers are labeled with biotin and reverse with FAM. Thus M.
genitalium
specific products are not distinguished during immunochromatographic detection
on lateral-flow strips.
Detection of the two M. genitalium regions is used to ensure positive test
results in
case one of the target regions is mutated or deleted. The reaction also
contains

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primers targeting H. sapiens GAPDH gene that produce DIG and FAM labeled
product. This product is recognized as a separate lane on the lateral-flow
strip and
serves as a positive control for the whole procedure (release of the genomic
material from cells, amplification and detection). Analytical sensitivity of
the
described method is at least 50 M. genitalium cells and 50 H. sapiens cells
per
test. This allows detection of the M. genitalium in the first void urine at
pathogen
concentration of 10 000 cells per 1 ml of urine or higher.
Patient urine sample is mixed with equal volume of sample pretreatment buffer
containing 0.2% NP-40, 150 mM NaCl, 50 mM Tris pH 7.0, and incubated 5 min at
RT C. 10 pl of the treated sample is used in the RPA reaction containing
following
components: M. genitalium MGPA 5' biotin labeled FW4 primer at 0.4 pM final
concentration, M. genitalium MGPA 5' FAM labeled RV4 primer at 0.4 pM final
concentration, M. genitalium 16S rRNA 5' biotin labeled FW1 primer at 0.4 pM
final
concentration, M. genitalium 16S rRNA 5' FAM labeled RV1 primer at 0.4 pM
final
concentration, H. sapiens GAPDH 5' DIG labeled FW3 primer at 0.4 pM final
concentration, H. sapiens GAPDH 5' FAM labeled RV2 primer at 0.4 pM final
concentration (see Table 2 for primer sequences), 14 mM magnesium acetate,
TwistDX RPA enzyme pellet and 29,5 pl of the rehydration buffer. Reaction is
incubated at 37 C for 10 mind The products are diluted 1:10 ratio with
dilution
buffer and analyzed on lateral-flow strips detecting Biotin-FAM and DIG-FAM
labeled molecules.
M. genitalium tests positive if the test gives 2 lines (Biotin-FAM and DIG-
FAM),
negative if the test gives 1 line DIG-FAM. The results of the test are invalid
if none
of the lines are present or only Biotin-FAM line is present
Example 3. Highly sensitive diagnostics of the presence of Chlamydia
trachomatis
from a patient sample extracted total DNA.
Present method uses highly sensitive loop mediated isothermal amplification
(LAMP) for specific detection of C. trachomatis DNA. Analytical sensitivity of
the
described method is at least 5 C. trachomatis cells per test. LAMP reaction is
prepared as follows: C. trachomatis PL-CDS2 SET4 primers F3 and B3 at 0.2 pM
concentration each, C. trachomatis PL-CDS2 SET4 5' biotin labeled FIP and 5'
FAM labeled BIP primers at 1.6 4M each, C. trachomatis PL-CDS2 SET4 5' biotin

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labeled LF and 5' FAM labeled LB loop primers at 0.8 jiM each (see Table 3 for
primer sequences), 5.6 ILIM dNTP, 6 mM MgSO4, 0.8 M betain, 8 units of Bst
polymerase, 2.5 jil of 10x Bst polymerase buffer and 5 1_11 of total DNA
extracted
from patient sample per 25 41 reaction. Incubate reaction for 1 h at 63 C,
dilute
diluted 1:10 ratio with dilution buffer and analyzed on lateral-flow strips
detecting
Biotin-FAM labeled molecules.
In a parallel reaction C. trachomatis TRPB targeting LAMP can be performed
with
SET1 primers (Table 3) for additional positive control (with analytical
sensitivity of
at least 5 C. trachonnatis cells per test). Additionally H. sapiens GAPDH
targeting
LAMP with SET 1 primers (Table 3) could be used as a positive control of the
reaction.
Table 3. Specific primer sequences for loop mediated isothermal amplification
(LAMP) against targets provided in Table 1
Target Sequence (5' ¨ 3')
organism
and
region
C. SET 1 F3 GCTTGTTGGAAACAAATCTGA
trachoma B3 TCGAACATTTTTTAAAACCAGG
tis PL- FIP GATCGCCCAGACAATGCTCCTAATCTCCAAGCTTAAG
CDS2 ACTTCA
BIP AACCAATCCCGGGCATTGATAAAAACGGATGCGATGA
AC
SET 2 F3 AAAGTGCATAAACTTCTGAGG
B3 CTAAAAAAAATCAATGCCCGG
Fl P TGTTTCCAACAAGCTACCATTTCTTATAATCCTCTTTT
CTGTCTGACG
BIP AATCTCCAAGCTTAAGACTICAGAGATTGGTTGATCG
CCCAGA
SET 3 F3 TCTAAAGACAAAAAAGATCCTCG
B3 TGTGATGGGTAAAGGGATT
FIP GCATGAAAAGCTTCTCCTTATTCGAATGATCTACAAGT
ATGTTTGTTGAG
BIP CCAATAGGATTCTTGGCGAATTTTTTGCAGCAAGAAA
TGTCGTTA
SET 4 F3 CGACTATTTTCTTGTTTAGAAGGTT
B3 GAAAAGATTGGTCTATTGTCCT
Fl P AGCAGCAAGCTATATTTCCTTAACAGCTATAGCGACT
ATTCCTTGA

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BIP GTCTTGGCAGAGGAAACTTTTTTAATGGATATGAATCT
GCAAGAGTT
LF1 GATTC,CTAAACAGGATGAC
LB1 TCGCATCTAGGATTAGAT
LF3 AGATTCCTAAACAGGATGAC
LB2 CGCATCTAGGATTAGATTATG
SET 5 F3 AATATCATCTTTGCGGTTGC
B3 TCTACAAGAGTACATCGGTCA
FIP TCGAGCAACCGCTGTGACGACCTTCATTATGTCGGAG
TC
BIP GCAGCTTGTAGTCCTGCTTGAGTCTTCGTAACTCGCT
CC
LF TAC AAA CGC CTA GGG TGC
LB CGG GCG ATT TGC CTT AAC
C. SET 1 F3 GCA GTT GCA GGA AGA GAT C
trachomati B3 GTC ATC TTG AAG AAG ATA CGA A
s TRPB FIP GGA CTT TTG GAT TCG GGA TAA AAT GCT GAT AU
CTG ATT GCA TGT ATC G
BIP GGA GGA CTG GGC ATT TCT TCA TGG AAT ACT CCA
GGT CGC
[Fl AGCGTTGGAGCCACCTC
LB1 GAAAACATGCAGCACGTTTTGCA
LF2 CAATAGCGTTGGAGCCACCT
LB2 AACATGCAGCACGTTTTGCA
SET 2 F3 CAAGATGACGATGGACAAGT
B3 CCAGATAAGTTAACGATGACGA
FIP GGCTCGTCCTGACTCATGCTCCGCTGGATTAGATTAT
CCT
BIP CCGATGAAGAGGCGTTACGAGGAGCATGTGAAGA
CTCCAAT
LF CAT GAT CTG GCC CM CTG A
LB TCC TGC TTA CTA GM ATG AGG G
M. SET 1 F3 ATTGGTTAACTTACCTAGTGGC
genitalium B3 ACTTCTTAGGAGCGTTAGAGA
MGPA FIP GACATAGTCTGCCCACTGGTTGATCCTCAAACCCAAC
AGTT
BIP AGGCATTACAAGCAGGGTTTGAAAGACACTACCAACT
GCTT
LF AAAGGGTTGAAAGACAGTTTGG
LB AAGGTTGATGTCTTGACCA
SET 2 F3 CACCTTACCAGTAACTGAACT
B3 AACCCTGCTTGTAATGCC
FIP TTAAGCGGATTGAAGCTTGATCTGTCTATGACCAGTA
TGTACCA
BIP CCCAACAGTTTATCCCGGTACTAAGGTAAGACATAGT

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CTGCC
LF GCCACTAGGTAAGTTAACCAAT
LB AATGCATCAAGTACAGGTCC
SET 3 F3 CACCTTACCAGTAACTGAACT
B3 AACCCTGCTTGTAATGCC
El P TTAAGCGGATTGAAGCTTGATCTCTATGACCAGTATG
TACCACT
BIP CCCAACAGTTTATCCCGGTACTAAGGTAAGACATAGT
CTGCC
LF GCCACTAGGTAAGTTAACCAAT
LB AATGCATCAAGTACAGGTCC
SET 4 F3 CACCTTACCAGTAACTGAACT
B3 AACCCTGCTTGTAATGCC
Fl P TTAAGCGGATTGAAGCTTGATCGTCTATGACCAGTAT
GTACCAC
BIP CCCAACAGTTTATCCCGGTACTAAGGTAAGACATAGT
CTGCC
LF GCCACTAGGTAAGTTAACCAAT
LB AATGCATCAAGTACAGGTCC
SET 5 F3 GATCCTCAAACCCAACAGTT
B3 TTAGGAGTTGGTTTGGTTGG
F IP GACATAGTCTGCCCACTGGTTTGCATCAAGTACAGGT
CC
BIP AGGCATTACAAGCAGGGTTTGAACTTCTTAGGAGCGT
TAGAGA
LF AAAGGGTTGAAAGACAGTTTGG
LB AAGGTTGATGTCTTGACCAA
SET 6 F3 TGTCTATGACCAGTATGTACCA
B3 AACCCTGCTTGTAATGCC
Fl P ACTGTTGGGTTTGAGGATCTTTATTGGTTAACTTACCT
AGTGGC
BIP CCCGGTACTAAATGCATCAAGTAAGGTAAGACATAGT
CTGCC
LF TTAAGCGGATTGAAGCTTGATC
LB CCAAACTGTCTTTCAACCCTTT
SET 7 F3 CACCTTACCAGTAACTGAACT
B3 AACCCTGCTTGTAATGCC
F IP TTACCTTTAAGCGGATTGAAGCTGACCAGTATGTACC
ACTATTG
= BIP CCCAACAGTTTATCCCGGTACTAAGGTAAGACATAGT
CTGCC
LF GATCAAAGCCACTAGGTAAGTT
LB AATGCATCAAGTACAGGTCC
SET 8 F3 CTATGACCAGTATGTACCACTA
B3 AACCCTGCTTGTAATGCC
Fl P ACTGTTGGGTTTGAGGATCTITTTGGTTAACTTACCTA
GTGGC
B I P CCCGGTACTAAATGCATCAAGTAAGGTAAGACATAGT
CTGCC

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LF TTAAGCGGATTGAAGCTTGATC
LB CCAAACTGTCTTTCAACCCTTT
SET 9 F3 TGACCAGTATGTACCACTAT
B3 AACCCTGCTTGTAATGCC
F IP ACTGTTGGGTTTGAGGATCTTTTGGTTAACTTACCTAG
BIP CCCGGTACTAAATGCATCAAGTAAGGTAAGACATAGT
CTGCC
LF TTAAGCGGATTGAAGCTTGATC
LB CCAAACTGTCTTTCAACCCTTT
SET F3 TGACCAGTATGTACCACTATTG
B3 AACCCTGCTTGTAATGCC
FIP ACTGTTGGGTTTGAGGATCTTTGTTAACTTACCTAGTG
GCTT
BIP CCCGGTACTAAATGCATCAAGTAAGGTAAGACATAGT
CTGCC
LF TTAAGCGGATTGAAGCTTGATC
LB CCAAACTGTCTTTCAACCCTTT
SET E3 GACCAGTATGTACCACTATT
11 B3 AACCCTGCTTGTAATGCC
F IP ACTGTTGGGTTTGAGGATCTTTGGTTAACTTACCTAGT
GGCTT
BIP CCCGGTACTAAATGCATCAAGTAAGGTAAGACATAGT
CTGCC
LF TTAAGCGGATTGAAGCTTGATC
LB CCAAACTGTCTTTCAACCCTTT
M. SET 1 F3 CGTGAACGATGAAGGTCTT
genital urn B3 ACCACACTCTAGACTGATAGTT
16S rRNA FIP GCGACTGCTGGCACATAGTTAAGAATGACTCTAGCAG
GCA
BIP ACATAGGTCGCAAGCGTTATCCCTGCCTTTAACACCA
GACTT
LF GTACt GTCAAACTCCAGCCA
LB GGATTTATTGGGCGTAAAGCAA
SET 2 E3 CGTGAACGATGAAGGTCTT
B3 ACCACACTCTAGACTGATAGTT
FIP GCGACTGCTGGCACATAGTAATGACTCTAGCAGGCA
ATG
BIP ACATAGGTCGCAAGCGTTATCCCTGCCTTTAACACCA
GACTT
LF TGGTACAGTCAAACTCCAGC
LB _ GGATTTATTGGGCGTAAAGCAA
SET 3 F3 CGTGAACGATGAAGGTCTT
B3 ACCACACTCTAGACTGATAGTT
F IP GCTGGCACATAGTTAGTCGTCAGAAGAATGACTCTAG
CAGGC
BIP ACATAGGTCGCAAGCGTTATCCCTGCCTTTAACACCA
GACTT
LF GTACAGTCAAACTCCAGCCA

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LB GGATTTATTGGGCGTAAAGCAA
SET 4 F3 CGTGAACGATGAAGGTCTT
B3 ACCACACTCTAGACTGATAGTT
F IP GCGACTGCTGGCACATAGTTAGAATGACTCTAGCAG
GCAAT
BIP ACATAGGTCGCAAGCGTTATCCCTGCCTTTAACACCA
GACT7
LF TGGTACAGTCAAACTCCAGC
LB GGATTTATTGGGCGTAAAGCAA
SET 5 F3 CATTACTGACGCTTAGGCTT
B3 GCCAAGGATGTCAAGTCTAG
F IP CTTCACTACCGAAGGGATCGCCCTAGTAGTCCACACC
GTAA
BIP GCCTGGGTAGTACATTCGCAAAACATGCTCCACCACT
TG
LF TCCGACAGCTAGTATCTATCGT
LB TGAAACTCAAACGGAATTGACG
SET 6 F3 CGTGAACGATGAAGGTCTT
B3 ACCACACTCTAGACTGATAGTT
FIP GCGACTGCTGGCACATAGTGACTCTAGCAGGCAATG
BIP ACATAGGTCGCAAGCGTTATCCCTGCCTTTAACACCA
GACTT
LF AAAGTGGTACAGTCAAACTCCA
LB GGATTTATTGGGCGTAAAGCAA
SET 7 F3 CAAGTGGTGGAGCATGTT
B3 TCCCTTCCTTCCTCCAATT
FIP CGACAACCATGCACCACCTCTAGACTTGACATCCTTG
GC
BIP CAGCTCGTGTCGTGAGATGTTTAACTAACGATAAGGG
TTGCG
LF GTCACTCGGTTAACCTCCATT
LB GGTTAAGTCCCGCAACGA
SET 8 F3 AATGACTCTAGCAGGCAATG
B3 ACCACACTCTAGACTGATAGTT
F IP CGGATAACGCTTGCGACCTTAAGTGACGACTAACTAT
GTGC
BIP AAGCGCAGGCGGATTGAACCAATGCATACAACTGTTA
AGC
LF TGTATTACCGCGACTGCTG
LB AGTCTGGTGTTAAAGGCAGC
SET 9 F3 AATGACTCTAGCAGGCAATG
B3 ACCACACTCTAGACTGATAGTT
FIP CGGATAACGCTTGCGACCTAAGTGACGACTAACTATG
TGC
BIP AAGCGCAGGCGGATTGAACCAATGCATACAACTGTTA
AGC
LF TGTATTACCGCGACTGCTG
LB AGTCTGGTGTTAAAGGCAGC

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SET F3 CAAGTGGTGGAGCATGTT
B3 GTTTGCAGCCCTAGACATAA
FIP CGACACGAGCTGACGACAACCTTGGCAAAGTTATGG
AAAC
BIP TGGGTTAAGTCCCGCAACGCCAATTTACATTAGCAGT
CTCG
LF CATGCACCACCTGTCACT
LB CGCAACCCTTATCGTTAGTTAC
SET F3 CGCATAAGAACTTTAGTTCGC
11 B3 AAGACCTTCATCGTTCACG
FIP TAGCTACACGTCATTGCCTTGGAGGGTTCGTTATTTG
ATGAGG
BIP CACAATGGGACTGAGACACGGAGCTTTCGCTCATTGT
GAA
LF CCTACCAACTAGCTGATATGGC
LB TACTCCTACGGGAGGCAG
H. SET 1 F3 TGGGTGTGAACCATGAGA
sapiens
GAPDH B3 AGTCCTTCCACGATACCAA
FIP TCCATAGGGTGCCAGGCTGTATGACAACAGCCTCA
AGAT
13IP CTTTCTTTGCAGCAATGCCTCCAGTTGTCATGGATGA
CCTTG
LF CTG CCT TCC TCA CCT GAT G
LB TGC ACC ACC AAC TGC TTA
SET 2 F3 CCCCAAAGGCCAGGCT
B3 AGAAGGGATGGGAGAGAGC
FIP GGAATGGGGAGAAGGGCAGGTTAAATGTCACCGGGA
GGATTG
131P CGGAAACCAGATCTCCCACCGGCTACAGAAAGGTCA
GCAGC
SET 3 F3 ATCAAGTGGGGCGATGCT
B3 GGGCAGAGATGATGACCCT
FIP GCACTCACCCCAGCCTTCTCGCTGAGTACGTCGTGG
AGT
BIP AAGCTGACTCAGCCCTGCAAACCCTGCAAATGAGCCT
ACA
F3 GTT GAC CCG ACC CCA AAG
B3 AAG GGA TGG GAG AGA GCC
FIP CGG AAT GGG GAG AAG GGC AGA TGT CAC CGG
GAG GAT TGG
BIP CGG AAA CCA GAT CTC CCA CCG CCA GCT ACA
GAA AGG TCA GC

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États administratifs

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États administratifs

Titre Date
Date de délivrance prévu Non disponible
(86) Date de dépôt PCT 2013-10-20
(87) Date de publication PCT 2014-04-24
(85) Entrée nationale 2015-04-21
Demande morte 2019-10-22

Historique d'abandonnement

Date d'abandonnement Raison Reinstatement Date
2018-10-22 Absence de requête d'examen
2018-10-22 Taxe périodique sur la demande impayée

Historique des paiements

Type de taxes Anniversaire Échéance Montant payé Date payée
Rétablissement des droits 200,00 $ 2015-04-21
Le dépôt d'une demande de brevet 200,00 $ 2015-04-21
Taxe de maintien en état - Demande - nouvelle loi 2 2015-10-20 50,00 $ 2015-10-07
Taxe de maintien en état - Demande - nouvelle loi 3 2016-10-20 50,00 $ 2016-10-03
Taxe de maintien en état - Demande - nouvelle loi 4 2017-10-20 50,00 $ 2017-10-03
Titulaires au dossier

Les titulaires actuels et antérieures au dossier sont affichés en ordre alphabétique.

Titulaires actuels au dossier
SELFDIAGNOSTICS OU
Titulaires antérieures au dossier
S.O.
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Description du
Document 
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(yyyy-mm-dd) 
Nombre de pages   Taille de l'image (Ko) 
Abrégé 2015-04-21 1 78
Revendications 2015-04-21 2 55
Description 2015-04-21 18 884
Page couverture 2015-05-19 1 50
PCT 2015-04-21 16 502
Cession 2015-04-21 7 179
Taxes 2015-10-07 1 33
Changement de nomination d'agent 2016-01-19 2 58
Lettre du bureau 2016-01-29 1 25
Requête d'assignation d'un agent 2016-01-29 1 34
Changement de nomination d'agent 2016-02-05 4 113
Lettre du bureau 2016-02-26 1 26
Lettre du bureau 2016-02-26 1 28
Changement à la méthode de correspondance 2016-11-15 2 42