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  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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  • 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
• • C—CHEMISTRY; METALLURGY
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/16—Primer sets for multiplex assays

Definitions

• 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.
• NAAT nucleic acid amplification techniques
• 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).
• RPA recombinase polymerase amplification
• LAMP loop-mediated isothermal amplification
• HDA helicase dependent amplification
• POC point-of-care
• 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.
• 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.
• 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.
• nucleic acid detection techniques have proved to have much higher sensitivity levels as they 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.
• 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.
• 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.
• 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. 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 ⁇ ) 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).
• 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 ⁇ ), metabolically inactive, infectious extracellular form of the organism and RB is larger (0.8 ⁇ ) 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.
• 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.
• C. trachomatis genital serovars have retained some of the eubacterial tryptophan biosynthesis genes, TRPA and TRPB encoding a and ⁇ subunits of the tryptophan synthase that catalyzes conversion of the indole into tryptophan.
• TRPA and TRPB eubacterial tryptophan biosynthesis genes
• 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 1 Mb 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.
• 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 usage of the cell-penetrating peptides for diagnostics purposes. At higher ( ⁇ - 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 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-1 14, NP-40, CHAPS, Octyl- -glucoside, Octyl- -thioglucopyronoside
• 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.
• trachomatis detection we have used genomic sequence regions from a well-established diagnostic target - coding sequence 2 of the multicopy cryptic plasmid (CDS2).
• CDS2 multicopy cryptic plasmid
• For the second target we have chosen ⁇ subunit of the tryptophan synthase gene TRPB.
• 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.
• MGPB MgPa dominant adhesin
• 16S rRNA gene 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.
• 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.
• immunochromotographic amplification product detection into the diagnostics system.
• forward primer sequences were 5 ' labeled with biotin and reverse primers with fluorescein amidite (FAM).
• FAM fluorescein amidite
• a dually labeled products were produced, that were detected within minutes using lateral-flow strips.
• Integration of the immunochromotographic 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 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.
• 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 AMPIification), 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;
• PCR Polymerase Chain Reaction
• HCR Hybridization Chain Reaction
• RCA Rolling Circle Amplification
• RPA Recombinase Polymerase Amplification
• LAMP Loop mediated isothermal AMPIification
• HDA Helicase Dependent Amplification
• 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.
• 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
• 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 - 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;
• 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 ).
• 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.
• C. trachomatis specific products are not distinguished during immunochromatographic detection on lateral-flow strips. Detection of the 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-1 14, 150 mM NaCI, 50 mM Tris pH 7.0, and incubated 5 min at RT°C. 10 ⁇ 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 ⁇ final concentration, C. trachomatis PL-CDS2 5' FAM labeled RV1 primer at 0.4 ⁇ final concentration, C.
• H. sapiens GAPDH 5' FAM labeled RV2 primer at 0.4 ⁇ final concentration see Table 2 for primer sequences
• 14 mM magnesium acetate TwistDX RPA enzyme pellet and 29,5 ⁇ 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.
• RV1 CTCTCAAGCAGGACTACAAGCTGCAATCCCTT primer RV2 5' - sequences ATGGTGGGGTTAAGGCAAATCGCCCGCACGTT
• RV1 5'- TCT GAT TGC AAA GTT TTG CTG ACC (RV)
• RV2 5'- CTC TAC CGT TGT TAT CAT ACC TTC sequences TGA TTG C
• 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.
• M. genitalium specific products are not distinguished during immunochromatographic detection on lateral-flow strips.
• 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 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 NaCI, 50 mM Tris pH 7.0, and incubated 5 min at RT°C. 10 ⁇ of the treated sample is used in the RPA reaction containing following components: M. genitalium MGPA 5' biotin labeled FW4 primer at 0.4 ⁇ final concentration, M. genitalium MGPA 5' FAM labeled RV4 primer at 0.4 ⁇ final concentration, M. genitalium 16S rRNA 5' biotin labeled FW1 primer at 0.4 ⁇ final concentration, M. genitalium 16S rRNA 5' FAM labeled RV1 primer at 0.4 ⁇ final concentration, H.
• LAMP loop mediated isothermal amplification
• trachomatis PL-CDS2 SET4 5' biotin labeled LF and 5' FAM labeled LB loop primers at 0.8 ⁇ each (see Table 3 for primer sequences), 5.6 ⁇ dNTP, 6 mM MgSO , 0.8 M betain, 8 units of Bst polymerase, 2.5 ⁇ of 10x Bst polymerase buffer and 5 ⁇ of total DNA extracted from patient sample per 25 ⁇ 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.
• 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. trachomatis 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.

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