Abstract:
Current invention is directed for rapid sample pretreatment method that allows highly sensitive and specific detection of target nucleic acid (eg human genomic DNA, human pathogen genomic DNA, human non-pathogen genomic DNA) by amplification directly from crude unpurified biological samples lysates (eg human 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, 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 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 for point-of-care settings applications.

Description:
PRIORITY 
       [0001]    This application is a national entry of PCT/EP2013/071906 filed on Oct. 20, 2013 and claiming claims priority of U.S. 61/616,495 filed on Jun. 4, 2010, both of which are fully incorporated herein by reference. 
     
    
     SEQUENCE LISTING 
       [0002]    This application contains sequence data provided on a computer readable diskette and as a paper version. The paper version of the sequence data is identical to the data provided on the diskette. 
       FIELD OF THE INVENTION 
       [0003]    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 
       [0004]    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.    
         [0005]    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 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. 
         [0006]    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. 
         [0007]    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. 
         [0008]    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. 
         [0009]      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. 
         [0010]    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. 
         [0011]      M. genitalium  is a small (0.2-0.3 μm) 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). 
         [0012]      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 μM), metabolically inactive, infectious extracellular form of the organism and RB is larger (0.8 μM) 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. 
         [0013]    Adaptive immunity against  C. trachomatis  involves INF-γ 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 α and β subunits of the tryptophan synthase that catalyzes conversion of the indole into tryptophan. Thus genital  C. trachomatis  serovars have retained the capacity to use exogenous indole secreted by genital trakt normal microflora that allows them to overcome INF-γ mediated growth restriction and promotes long term establishment of the infection. 
         [0014]      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 (&lt;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 
       [0015]    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. 
         [0016]    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. 
         [0017]    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 (μM-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. 
         [0018]    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, Octyl-β-glucoside, Octyl-β-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. 
         [0019]    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. 
         [0020]    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. 
         [0021]    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 β 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. 
         [0022]    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. 
         [0023]    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 immunochromotographic product detection required additional primer optimization. Primers gaining template independent lateral-flow strip detectable signal were eliminated from the selection. 
         [0024]    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. 
         [0025]    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.   
 
         [0029]    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.       
 
         [0032]    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.       
 
         [0035]    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 
       [0036]    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). 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Genomic regions of  Chlamydia trachomatis , 
               
               
                   Mycoplasma genitalium  and  Homo sapiens  used 
               
               
                 for isothermal amplification based detection 
               
             
          
           
               
                   
                 Target organism 
                 Sequence name 
                 Genebank accession nr 
               
               
                   
                   
               
               
                   
                 
                   C. trachomatis 
                 
                 PL-CDS2 
                 FM865439.1 
               
               
                   
                   
                   
                 sequence 756-1748 
               
               
                   
                   
                 TRPB 
                 FN652779.2 
               
               
                   
                   
                   
                 sequence 193461-194639 
               
               
                   
                 
                   M. genitalium 
                 
                 16S rRNA 
                 CP003773.1 
               
               
                   
                   
                   
                 sequence 169843-171366 
               
               
                   
                   
                 MGPA 
                 CP003773.1 
               
               
                   
                   
                   
                 sequence 221365-225744 
               
               
                   
                 
                   H. sapiens 
                 
                 GAPDH 
                 NG_007073.2 
               
               
                   
                   
               
             
          
         
       
     
         [0037]    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 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. 
         [0038]    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 μl 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 μM final concentration,  C. trachomatis  PL-CDS2 5′ FAM labeled RV1 primer at 0.4 μM final concentration,  C. trachomatis  TRPB 5′ biotin labeled FW2 primer at 0.4 μM final concentration,  C. trachomatis  TRPB 5′ FAM labeled RV3 primer at 0.4 μM final concentration,  H. sapiens  GAPDH 5′ DIG labeled FW3 primer at 0.4 μM final concentration,  H. sapiens  GAPDH 5′ FAM labeled RV2 primer at 0.4 μM final concentration (see Table 2 for primer sequences), 14 mM magnesium acetate, TwistDX RPA enzyme pellet and 29.5 μl 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. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Specific primer sequences for recombinase polymerase amplification (RPA) 
               
               
                 against targets provided in Table 1 
               
             
          
           
               
                 Target 
                   
                   
                 SEQ 
               
               
                 organism 
                   
                   
                 ID 
               
               
                 and region 
                   
                 Sequence (5′-3′) 
                 NO 
               
               
                   
               
               
                   C .  trachomatis   
                 Forward 
                 FW1 5′- CTTCTTTGAAGCGTTGTCTTCTCGAGAAGATTT 
                  1 
               
               
                 PL-CDS2 
                 (FW) 
                 FW2 5′- CTTCTCGAGAAGATTTATCGTACGCAAATATC 
                  2 
               
               
                   
                 primer 
                 FW3 5′- 
                  3 
               
               
                   
                 sequences 
                 CCTTCATTATGTCGGAGTCTGAGCACCCTAGGC 
                   
               
               
                   
                   
                 FW4 5′- AGGCGTTTGTACTCCGTCACAGCGGTTGCTCG 
                  4 
               
               
                   
               
               
                   
                 Reverse 
                 RV1 5′- CTCTCAAGCAGGACTACAAGCTGCAATCCCTT 
                  5 
               
               
                   
                 (RV) 
                 RV2 5′- ATGGTGGGGTTAAGGCAAATCGCCCGCACGTT 
                  6 
               
               
                   
                 primer 
                 RV3 5′- TCT TCG TAA CTC GCT CCG GAA AAA TGG 
                  7 
               
               
                   
                 sequences 
                 TGG GG 
                   
               
               
                   
                   
                 RV4 5′- CTT TCT ACA AGA GTA CAT CGG TCA ACG AAG 
                  8 
               
               
                   
                   
                 AGG 
                   
               
               
                   
               
               
                   C .  trachomatis   
                 Forward 
                 FW1 5′- ACT ATG CGG GGA GAC AAA CTC CTC TGA 
                  9 
               
               
                 TRPB 
                 (FW) 
                 CTG AAG 
                   
               
               
                   
                 primer 
                 FW2 5′- TCT TAA ACG CGA AGA TCT TTT GCA TAC AGG 
                 10 
               
               
                   
                 sequences 
                 AGC 
                   
               
               
                   
                   
                 FW3 5′- CAT ACA GGA GCA CAT AAA CTG AAT AAT GCT 
                 11 
               
               
                   
                   
                 CTT GG 
                   
               
               
                   
                   
                 FW4 5′- CTC TTG GTC AGT GTT TGC TTG CTA AAT ATC 
                 12 
               
               
                   
                   
                 TTG 
                   
               
               
                   
               
               
                   
                 Reverse 
                 RV1 5′- TCC CGC ACC TGT TTC AGC TAC AAC ACG TGT 
                 13 
               
               
                   
                 (RV) 
                 TT 
                   
               
               
                   
                 primer 
                 RV2 5′- CTG TTG CTG TTG CTA CTC CAT GTT GTC CCG 
                 14 
               
               
                   
                 sequences 
                 CAC 
                   
               
               
                   
                   
                 RV3 5′- TCC CAT GTA TAC TAC ACA ATC TAA TCC TAG 
                 15 
               
               
                   
                   
                 ATA 
                   
               
               
                   
                   
                 RV4 5′- TTC TGT CGT TCC ACA TCT TTT GCT CCC ATG 
                 16 
               
               
                   
                   
                 TAT 
                   
               
               
                   
               
               
                   M .  genitalium   
                 Forward 
                 FW1 5′- AGC GCA ACC CTT ATC GTT AGT TAC ATT GTT 
                 17 
               
               
                 16S rRNA 
                 (FW) 
                 TAA 
                   
               
               
                   
                 primer 
                 FW2 5′- CGT TAG TTA CAT TGT TTA ACG AGA CTG CTA 
                 18 
               
               
                   
                 sequences 
                 ATG T 
                   
               
               
                   
                   
                 FW3 5′- ACG TGC TAC AAT GGC CAA TAC AAA CAG 
                 19 
               
               
                   
                   
                 TAG CCA A 
                   
               
               
                   
               
               
                   
                 Reverse 
                 RV1 5′- TTG CAG CCC TCA ATC CGA ACT GAG ACC 
                 20 
               
               
                   
                 (RV) 
                 AAC TTT T 
                   
               
               
                   
                 primer 
                 RV2 5′- CAT AGC TGA TTC GCG ATT ACT AGT GAT TCC 
                 21 
               
               
                   
                 sequences 
                 AGC 
                   
               
               
                   
                   
                 RV3 5′- TTC CAA TAA AGG TTA GCA ACA CGT TTT TAA 
                 22 
               
               
                   
                   
                 ATA 
                   
               
               
                   
               
               
                   M .  genitalium   
                 Forward 
                 FW1 5′- TTGGACTTGAAACAATAACAACTTCTCTTCACT 
                 23 
               
               
                 MGPA 
                 (FW) 
                 FW2 5′- 
                 24 
               
               
                   
                 primer 
                 AAGATTACTGGAGAGAACCCAGGATCATTTGGA 
                   
               
               
                   
                 sequences 
                 FW3 5′- CAG TGG GCA GAC TAT GTC TTA CCT TTG ATT 
                 25 
               
               
                   
                   
                 GTA 
                   
               
               
                   
                   
                 FW4 5′- TTA TCC TTA GTG TTA CTT TGG GAT TAA CGA 
                 26 
               
               
                   
                   
                 TTG G 
                   
               
               
                   
                   
                 FW5 5′- 
                 27 
               
               
                   
                   
                 CAATGCACAGAAACAAAAAGGCATTACAAGCAGGG 
                   
               
               
                   
               
               
                   
                 Reverse 
                 RV1 5′- TCT GAT TGC AAA GTT TTG CTG ACC ATC AAG 
                 28 
               
               
                   
                 (RV) 
                 GTA 
                   
               
               
                   
                 primer 
                 RV2 5′- CTC TAC CGT TGT TAT CAT ACC TTC TGA TTG 
                 29 
               
               
                   
                 sequences 
                 C 
                   
               
               
                   
                   
                 RV3 5′- TTC TGT TAA TGA TCT CTT TAA AGA CAC TAC 
                 30 
               
               
                   
                   
                 CAA 
                   
               
               
                   
                   
                 RV4 5′- CTT AGG AGC GTT AGA GAT CCC TGT TCT GTT 
                 31 
               
               
                   
                   
                 AAT G 
                   
               
               
                   
                   
                 RV5 5′- CTT GTT TTA ACT TCT TAG GAG CGT TAG AGA 
                 32 
               
               
                   
                   
                 TCC C 
                   
               
               
                   
                   
                 RV6 5′- 
                 33 
               
               
                   
                   
                 TTACTGGAGGTTTTGGTGGGGTTTTAGGAGTTGG 
                   
               
               
                   
               
               
                   H .  sapiens   
                 Forward 
                 FW1 5′- 
                 34 
               
               
                 GAPDH 
                 (FW) 
                 CTCCTCCGGGTGATGCTTTTCCTAGATTATTCTC 
                   
               
               
                   
                 primer 
                 FW2 5′- CTA ACC CTG CGC TCC TGC CTC GAT GGG 
                 35 
               
               
                   
                 sequences 
                 TGG AG 
                   
               
               
                   
                   
                 FW3 5′- AAG TCA GGT GGA GCG AGG CTA GCT GGC 
                 36 
               
               
                   
                   
                 CCG ATT 
                   
               
               
                   
               
               
                   
                 Reverse 
                 RV1 5′- TCC TTT TCC AAC TAC CCA TGA CTC AGC TTC 
                 37 
               
               
                   
                 (RV) 
                 TCC C 
                   
               
               
                   
                 primer 
                 RV2 5′- CAC CAT GCC ACA GCC ACC ACA CCT CTG 
                 38 
               
               
                   
                 sequences 
                 CGG GGA 
                   
               
               
                   
                   
                 RV3 5′- CCA CCA CCA GAG GGG CCA TTT TGC GGT 
                 39 
               
               
                   
                   
                 GGA AAT 
               
               
                   
               
             
          
         
       
     
         [0039]      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 Biotin-FAM line is present. 
       Example 2 
     Fast Diagnostics of the Presence of  Mycoplasma genitalium  in a Urine Sample 
       [0040]    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. 
         [0041]    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 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. 
         [0042]    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 μl of the treated sample is used in the RPA reaction containing following components:  M. genitalium  MGPA 5′ biotin labeled FW4 primer at 0.4 μM final concentration,  M. genitalium  MGPA 5′ FAM labeled RV4 primer at 0.4 μM final concentration,  M. genitalium  16S rRNA 5′ biotin labeled FW1 primer at 0.4 μM final concentration,  M. genitalium  16S rRNA 5′ FAM labeled RV1 primer at 0.4 μM final concentration,  H. sapiens  GAPDH 5′ DIG labeled FW3 primer at 0.4 μM final concentration,  H. sapiens  GAPDH 5′ FAM labeled RV2 primer at 0.4 μM final concentration (see Table 2 for primer sequences), 14 mM magnesium acetate, TwistDX RPA enzyme pellet and 29.5 μl 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. 
         [0043]      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 
       [0044]    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 μM concentration each,  C. trachomatis  PL-CDS2 SET4 5′ biotin labeled FIP and 5′ FAM labeled BIP primers at 1.6 μM each,  C. trachomatis  PL-CDS2 SET4 5′ biotin labeled LF and 5′ FAM labeled LB loop primers at 0.8 μM each (see Table 3 for primer sequences), 5.6 μM dNTP, 6 mM MgSO 4 , 0.8 M betain, 8 units of Bst polymerase, 2.5 μl of 10× Bst polymerase buffer and 5 μl of total DNA extracted from patient sample per 25 μl 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. 
         [0045]    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. 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. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Specific primer sequences for loop mediated isothermal amplification  
               
               
                 (LAMP) against targets provided in Table 1 
               
             
          
           
               
                 Target 
                   
                   
                 SEQ 
               
               
                 organism and 
                   
                   
                 ID 
               
               
                 region 
                   
                 Sequence (5′-3′) 
                 NO: 
               
               
                   
               
             
          
           
               
                   C .  trachomatis   
                 SET 
                 F3 
                 GCTTGTTGGAAACAAATCTGA 
                  40 
               
               
                 PL-CDS2 
                 1 
                 B3 
                 TCGAACATTTTTTAAAACCAGG 
                  41 
               
               
                   
                   
                 FIP 
                 GATCGCCCAGACAATGCTCCTAATCTCCAAGCTTAAGACTTCA 
                  42 
               
               
                   
                   
                 BIP 
                 AACCAATCCCGGGCATTGATAAAAACGGATGCGATGAAC 
                  43 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 AAAGTGCATAAACTTCTGAGG 
                  44 
               
               
                   
                 2 
                 B3 
                 CTAAAAAAAATCAATGCCCGG 
                  45 
               
               
                   
                   
                 FIP 
                 TGTTTCCAACAAGCTACCATTTCTTATAATCCTCTTTTCTGTCTGACG 
                  46 
               
               
                   
                   
                 BIP 
                 AATCTCCAAGCTTAAGACTTCAGAGATTGGTTGATCGCCCAGA 
                  47 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 TCTAAAGACAAAAAAGATCCTCG 
                  48 
               
               
                   
                 3 
                 B3 
                 TGTGATGGGTAAAGGGATT 
                  49 
               
               
                   
                   
                 FIP 
                 GCATGAAAAGCTTCTCCTTATTCGAATGATCTACAAGTATGTTTGTTGAG 
                  50 
               
               
                   
                   
                 BIP 
                 CCAATAGGATTCTTGGCGAATTTTTTGCAGCAAGAAATGTCGTTA 
                  51 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 CGACTATTTTCTTGTTTAGAAGGTT 
                  52 
               
               
                   
                 4 
                 B3 
                 GAAAAGATTGGTCTATTGTCCT 
                  53 
               
               
                   
                   
                 FIP 
                 AGCAGCAAGCTATATTTCCTTAACAGCTATAGCGACTATTCCTTGA 
                  54 
               
               
                   
                   
                 BIP 
                 GTCTTGGCAGAGGAAACTTTTTTAATGGATATGAATCTGCAAGAGTT 
                  55 
               
               
                   
                   
                 LF1 
                 GATTCCTAAACAGGATGAC 
                  56 
               
               
                   
                   
                 LB1 
                 TCGCATCTAGGATTAGAT 
                  57 
               
               
                   
                   
                 LF3 
                 AGATTCCTAAACAGGATGAC 
                  58 
               
               
                   
                   
                 LB2 
                 CGCATCTAGGATTAGATTATG 
                  59 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 AATATCATCTTTGCGGTTGC 
                  60 
               
               
                   
                 5 
                 B3 
                 TCTACAAGAGTACATCGGTCA 
                  61 
               
               
                   
                   
                 FIP 
                 TCGAGCAACCGCTGTGACGACCTTCATTATGTCGGAGTC 
                  62 
               
               
                   
                   
                 BIP 
                 GCAGCTTGTAGTCCTGCTTGAGTCTTCGTAACTCGCTCC 
                  63 
               
               
                   
                   
                 LF 
                 TAC AAA CGC CTA GGG TGC 
                  64 
               
               
                   
                   
                 LB 
                 CGG GCG ATT TGC CTT AAC 
                  65 
               
               
                   
               
               
                   C .  trachomatis   
                 SET 
                 F3 
                 GCA GTT GCA GGA AGA GAT C 
                  66 
               
               
                 TRPB 
                 1 
                 B3 
                 GTC ATC TTG AAG AAG ATA CGA A 
                  67 
               
               
                   
                   
                 FIP 
                 GGA CTT TTG GAT TCG GGA TAA AAT GCT GAT  
                  68 
               
               
                   
                   
                   
                 ATT CTG ATT GCA TGT ATC G 
                   
               
               
                   
                   
                 BIP 
                 GGA GGA CTG GGC ATT TCT TCA TGG AAT ACT 
                  69 
               
               
                   
                   
                   
                 CCA GGT CGC 
                   
               
               
                   
                   
                 LF1 
                 AGCGTTGGAGCCACCTC 
                  70 
               
               
                   
                   
                 LB1 
                 GAAAACATGCAGCACGTTTTGCA 
                  71 
               
               
                   
                   
                 LF2 
                 CAATAGCGTTGGAGCCACCT 
                  72 
               
               
                   
                   
                 LB2 
                 AACATGCAGCACGTTTTGCA 
                  73 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 CAAGATGACGATGGACAAGT 
                  74 
               
               
                   
                 2 
                 B3 
                 CCAGATAAGTTAACGATGACGA 
                  75 
               
               
                   
                   
                 FIP 
                 GGCTCGTCCTGACTCATGCTCCGCTGGATTAGATTATCCT 
                  76 
               
               
                   
                   
                 BIP 
                 CCGATGAAGAGGCGTTACGAGGAGCATGTGAAGA CTCCAAT 
                  77 
               
               
                   
                   
                 LF 
                 CAT GAT CTG GCC CAA CTG A 
                  78 
               
               
                   
                   
                 LB 
                 TCC TGC TTA CTA GAA ATG AGG G 
                  79 
               
               
                   
               
               
                   M .  genitalium   
                 SET 
                 F3 
                 ATTGGTTAACTTACCTAGTGGC 
                  80 
               
               
                 MGPA 
                 1 
                 B3 
                 ACTTCTTAGGAGCGTTAGAGA 
                  81 
               
               
                   
                   
                 FIP 
                 GACATAGTCTGCCCACTGGTTGATCCTCAAACCCAACAGTT 
                  82 
               
               
                   
                   
                 BIP 
                 AGGCATTACAAGCAGGGTTTGAAAGACACTACCAACTGCTT 
                  83 
               
               
                   
                   
                 LF 
                 AAAGGGTTGAAAGACAGTTTGG 
                  84 
               
               
                   
                   
                 LB 
                 AAGGTTGATGTCTTGACCA 
                  85 
               
               
                   
                   
                 F3 
                 CACCTTACCAGTAACTGAACT 
                  86 
               
               
                   
               
               
                   
                 SET 
                 B3 
                 AACCCTGCTTGTAATGCC 
                  87 
               
               
                   
                 2 
                 FIP 
                 TTAAGCGGATTGAAGCTTGATCTGTCTATGACCAGTATGTACCA 
                  88 
               
               
                   
                   
                 BIP 
                 CCCAACAGTTTATCCCGGTACTAAGGTAAGACATAGTCTGCC 
                  89 
               
               
                   
                   
                 LF 
                 GCCACTAGGTAAGTTAACCAAT 
                  90 
               
               
                   
                   
                 LB 
                 AATGCATCAAGTACAGGTCC 
                  91 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 CACCTTACCAGTAACTGAACT 
                  92 
               
               
                   
                 3 
                 B3 
                 AACCCTGCTTGTAATGCC 
                  93 
               
               
                   
                   
                 FIP 
                 TTAAGCGGATTGAAGCTTGATCTCTATGACCAGTATGTACCACT 
                  94 
               
               
                   
                   
                 BIP 
                 CCCAACAGTTTATCCCGGTACTAAGGTAAGACATAGTCTGCC 
                  95 
               
               
                   
                   
                 LF 
                 GCCACTAGGTAAGTTAACCAAT 
                  96 
               
               
                   
                   
                 LB 
                 AATGCATCAAGTACAGGTCC 
                  97 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 CACCTTACCAGTAACTGAACT 
                  98 
               
               
                   
                 4 
                 B3 
                 AACCCTGCTTGTAATGCC 
                  99 
               
               
                   
                   
                 FIP 
                 TTAAGCGGATTGAAGCTTGATCGTCTATGACCAGTATGTACCAC 
                 100 
               
               
                   
                   
                 BIP 
                 CCCAACAGTTTATCCCGGTACTAAGGTAAGACATAGTCTGCC 
                 101 
               
               
                   
                   
                 LF 
                 GCCACTAGGTAAGTTAACCAAT 
                 102 
               
               
                   
                   
                 LB 
                 AATGCATCAAGTACAGGTCC 
                 203 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 GATCCTCAAACCCAACAGTT 
                 104 
               
               
                   
                 5 
                 B3 
                 TTAGGAGTTGGTTTGGTTGG 
                 105 
               
               
                   
                   
                 FIP 
                 GACATAGTCTGCCCACTGGTTTGCATCAAGTACAGGTCC 
                 106 
               
               
                   
                   
                 BIP 
                 AGGCATTACAAGCAGGGTTTGAACTTCTTAGGAGCGTTAGAGA 
                 107 
               
               
                   
                   
                 LF 
                 AAAGGGTTGAAAGACAGTTTGG 
                 108 
               
               
                   
                   
                 LB 
                 AAGGTTGATGTCTTGACCAA 
                 109 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 TGTCTATGACCAGTATGTACCA 
                 110 
               
               
                   
                 6 
                 B3 
                 AACCCTGCTTGTAATGCC 
                 111 
               
               
                   
                   
                 FIP 
                 ACTGTTGGGTTTGAGGATCTTTATTGGTTAACTTACCTAGTGGC 
                 112 
               
               
                   
                   
                 BIP 
                 CCCGGTACTAAATGCATCAAGTAAGGTAAGACATAGTCTGCC 
                 113 
               
               
                   
                   
                 LF 
                 TTAAGCGGATTGAAGCTTGATC 
                 114 
               
               
                   
                   
                 LB 
                 CCAAACTGTCTTTCAACCCTTT 
                 115 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 CACCTTACCAGTAACTGAACT 
                 116 
               
               
                   
                 7 
                 B3 
                 AACCCTGCTTGTAATGCC 
                 117 
               
               
                   
                   
                 FIP 
                 TTACCTTTAAGCGGATTGAAGCTGACCAGTATGTACCACTATTG 
                 118 
               
               
                   
                   
                 BIP 
                 CCCAACAGTTTATCCCGGTACTAAGGTAAGACATAGTCTGCC 
                 119 
               
               
                   
                   
                 LF 
                 GATCAAAGCCACTAGGTAAGTT 
                 120 
               
               
                   
                   
                 LB 
                 AATGCATCAAGTACAGGTCC 
                 121 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 CTATGACCAGTATGTACCACTA 
                 122 
               
               
                   
                 8 
                 B3 
                 AACCCTGCTTGTAATGCC 
                 123 
               
               
                   
                   
                 FIP 
                 ACTGTTGGGTTTGAGGATCTTTTTGGTTAACTTACCTAGTGGC 
                 124 
               
               
                   
                   
                 BIP 
                 CCCGGTACTAAATGCATCAAGTAAGGTAAGACATAGTCTGCC 
                 125 
               
               
                   
                   
                 LF 
                 TTAAGCGGATTGAAGCTTGATC 
                 126 
               
               
                   
                   
                 LB 
                 CCAAACTGTCTTTCAACCCTTT 
                 127 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 TGACCAGTATGTACCACTAT 
                 128 
               
               
                   
                 9 
                 B3 
                 AACCCTGCTTGTAATGCC 
                 129 
               
               
                   
                   
                 FIP 
                 ACTGTTGGGTTTGAGGATCTTTTGGTTAACTTACCTAGTGGCT 
                 130 
               
               
                   
                   
                 BIP 
                 CCCGGTACTAAATGCATCAAGTAAGGTAAGACATAGTCTGCC 
                 131 
               
               
                   
                   
                 LF 
                 TTAAGCGGATTGAAGCTTGATC 
                 132 
               
               
                   
                   
                 LB 
                 CCAAACTGTCTTTCAACCCTTT 
                 133 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 TGACCAGTATGTACCACTATTG 
                 134 
               
               
                   
                 10 
                 B3 
                 AACCCTGCTTGTAATGCC 
                 135 
               
               
                   
                   
                 FIP 
                 ACTGTTGGGTTTGAGGATCTTTGTTAACTTACCTAGTGGCTT 
                 136 
               
               
                   
                   
                 BIP 
                 CCCGGTACTAAATGCATCAAGTAAGGTAAGACATAGTCTGCC 
                 137 
               
               
                   
                   
                 LF 
                 TTAAGCGGATTGAAGCTTGATC 
                 138 
               
               
                   
                   
                 LB 
                 CCAAACTGTCTTTCAACCCTTT 
                 139 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 GACCAGTATGTACCACTATT 
                 140 
               
               
                   
                 11 
                 B3 
                 AACCCTGCTTGTAATGCC 
                 141 
               
               
                   
                   
                 FIP 
                 ACTGTTGGGTTTGAGGATCTTTGGTTAACTTACCTAGTGGCTT 
                 142 
               
               
                   
                   
                 BIP 
                 CCCGGTACTAAATGCATCAAGTAAGGTAAGACATAGTCTGCC 
                 143 
               
               
                   
                   
                 LF 
                 TTAAGCGGATTGAAGCTTGATC 
                 144 
               
               
                   
                   
                 LB 
                 CCAAACTGTCTTTCAACCCTTT 
                 145 
               
               
                   
               
               
                   M .  genitalium   
                 SET 
                 F3 
                 CGTGAACGATGAAGGTCTT 
                 146 
               
               
                 16S rRNA 
                 1 
                 B3 
                 ACCACACTCTAGACTGATAGTT 
                 147 
               
               
                   
                   
                 FIP 
                 GCGACTGCTGGCACATAGTTAAGAATGACTCTAGCAGGCA 
                 148 
               
               
                   
                   
                 BIP 
                 ACATAGGTCGCAAGCGTTATCCCTGCCTTTAACACCAGACTT 
                 149 
               
               
                   
                   
                 LF 
                 GTACAGTCAAACTCCAGCCA 
                 150 
               
               
                   
                   
                 LB 
                 GGATTTATTGGGCGTAAAGCAA 
                 151 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 CGTGAACGATGAAGGTCTT 
                 152 
               
               
                   
                 2 
                 B3 
                 ACCACACTCTAGACTGATAGTT 
                 153 
               
               
                   
                   
                 FIP 
                 GCGACTGCTGGCACATAGTAATGACTCTAGCAGGCAATG 
                 154 
               
               
                   
                   
                 BIP 
                 ACATAGGTCGCAAGCGTTATCCCTGCCTTTAACACCAGACTT 
                 155 
               
               
                   
                   
                 LF 
                 TGGTACAGTCAAACTCCAGC 
                 156 
               
               
                   
                   
                 LB 
                 GGATTTATTGGGCGTAAAGCAA 
                 157 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 CGTGAACGATGAAGGTCTT 
                 158 
               
               
                   
                 3 
                 B3 
                 ACCACACTCTAGACTGATAGTT 
                 159 
               
               
                   
                   
                 FIP 
                 GCTGGCACATAGTTAGTCGTCAGAAGAATGACTCTAGCAGGC 
                 160 
               
               
                   
                   
                 BIP 
                 ACATAGGTCGCAAGCGTTATCCCTGCCTTTAACACCAGACTT 
                 161 
               
               
                   
                   
                 LF 
                 GTACAGTCAAACTCCAGCCA 
                 162 
               
               
                   
                   
                 LB 
                 GGATTTATTGGGCGTAAAGCAA 
                 163 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 CGTGAACGATGAAGGTCTT 
                 164 
               
               
                   
                 4 
                 B3 
                 ACCACACTCTAGACTGATAGTT 
                 165 
               
               
                   
                   
                 FIP 
                 GCGACTGCTGGCACATAGTTAGAATGACTCTAGCAGGCAAT 
                 166 
               
               
                   
                   
                 BIP 
                 ACATAGGTCGCAAGCGTTATCCCTGCCTTTAACACCAGACTT 
                 167 
               
               
                   
                   
                 LF 
                 TGGTACAGTCAAACTCCAGC 
                 168 
               
               
                   
                   
                 LB 
                 GGATTTATTGGGCGTAAAGCAA 
                 169 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 CATTACTGACGCTTAGGCTT 
                 170 
               
               
                   
                 5 
                 B3 
                 GCCAAGGATGTCAAGTCTAG 
                 171 
               
               
                   
                   
                 FIP 
                 CTTCACTACCGAAGGGATCGCCCTAGTAGTCCACACCGTAA 
                 172 
               
               
                   
                   
                 BIP 
                 GCCTGGGTAGTACATTCGCAAAACATGCTCCACCACTTG 
                 173 
               
               
                   
                   
                 LF 
                 TCCGACAGCTAGTATCTATCGT 
                 174 
               
               
                   
                   
                 LB 
                 TGAAACTCAAACGGAATTGACG 
                 175 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 CGTGAACGATGAAGGTCTT 
                 176 
               
               
                   
                 6 
                 B3 
                 ACCACACTCTAGACTGATAGTT 
                 177 
               
               
                   
                   
                 FIP 
                 GCGACTGCTGGCACATAGTGACTCTAGCAGGCAATGG 
                 178 
               
               
                   
                   
                 BIP 
                 ACATAGGTCGCAAGCGTTATCCCTGCCTTTAACACCAGACTT 
                 179 
               
               
                   
                   
                 LF 
                 AAAGTGGTACAGTCAAACTCCA 
                 180 
               
               
                   
                   
                 LB 
                 GGATTTATTGGGCGTAAAGCAA 
                 181 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 CAAGTGGTGGAGCATGTT 
                 182 
               
               
                   
                 7 
                 B3 
                 TCCCTTCCTTCCTCCAATT 
                 183 
               
               
                   
                   
                 FIP 
                 CGACAACCATGCACCACCTCTAGACTTGACATCCTTGGC 
                 184 
               
               
                   
                   
                 BIP 
                 CAGCTCGTGTCGTGAGATGTTTAACTAACGATAAGGGTTGCG 
                 185 
               
               
                   
                   
                 LF 
                 GTCACTCGGTTAACCTCCATT 
                 186 
               
               
                   
                   
                 LB 
                 GGTTAAGTCCCGCAACGA 
                 187 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 AATGACTCTAGCAGGCAATG 
                 188 
               
               
                   
                 8 
                 B3 
                 ACCACACTCTAGACTGATAGTT 
                 189 
               
               
                   
                   
                 FIP 
                 CGGATAACGCTTGCGACCTTAAGTGACGACTAACTATGTGC 
                 190 
               
               
                   
                   
                 BIP 
                 AAGCGCAGGCGGATTGAACCAATGCATACAACTGTTAAGC 
                 191 
               
               
                   
                   
                 LF 
                 TGTATTACCGCGACTGCTG 
                 192 
               
               
                   
                   
                 LB 
                 AGTCTGGTGTTAAAGGCAGC 
                 193 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 AATGACTCTAGCAGGCAATG 
                 194 
               
               
                   
                 9 
                 B3 
                 ACCACACTCTAGACTGATAGTT 
                 195 
               
               
                   
                   
                 FIP 
                 CGGATAACGCTTGCGACCTAAGTGACGACTAACTATGTGC 
                 196 
               
               
                   
                   
                 BIP 
                 AAGCGCAGGCGGATTGAACCAATGCATACAACTGTTAAGC 
                 197 
               
               
                   
                   
                 LF 
                 TGTATTACCGCGACTGCTG 
                 198 
               
               
                   
                   
                 LB 
                 AGTCTGGTGTTAAAGGCAGC 
                 199 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 CAAGTGGTGGAGCATGTT 
                 200 
               
               
                   
                 10 
                 B3 
                 GTTTGCAGCCCTAGACATAA 
                 201 
               
               
                   
                   
                 FIP 
                 CGACACGAGCTGACGACAACCTTGGCAAAGTTATGGAAAC 
                 202 
               
               
                   
                   
                 BIP 
                 TGGGTTAAGTCCCGCAACGCCAATTTACATTAGCAGTCTCG 
                 203 
               
               
                   
                   
                 LF 
                 CATGCACCACCTGTCACT 
                 204 
               
               
                   
                   
                 LB 
                 CGCAACCCTTATCGTTAGTTAC 
                 205 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 CGCATAAGAACTTTAGTTCGC 
                 206 
               
               
                   
                 11 
                 B3 
                 AAGACCTTCATCGTTCACG 
                 207 
               
               
                   
                   
                 FIP 
                 TAGCTACACGTCATTGCCTTGGAGGGTTCGTTATTTGATGAGG 
                 208 
               
               
                   
                   
                 BIP 
                 CACAATGGGACTGAGACACGGAGCTTTCGCTCATTGTGAA 
                 209 
               
               
                   
                   
                 LF 
                 CCTACCAACTAGCTGATATGGC 
                 210 
               
               
                   
                   
                 LB 
                 TACTCCTACGGGAGGCAG 
                 211 
               
               
                   
               
               
                   H .  sapiens   
                 SET 
                 F3 
                 TGGGTGTGAACCATGAGA 
                 212 
               
               
                 GAPDH 
                 1 
                 B3 
                 AGTCCTTCCACGATACCAA 
                 213 
               
               
                   
                   
                 FIP 
                 TCCATAGGGTGCCAGGCTGTATGACAACAGCCTCA AGAT 
                 214 
               
               
                   
                   
                 BIP 
                 CTTTCTTTGCAGCAATGCCTCCAGTTGTCATGGATGACCTTG 
                 215 
               
               
                   
                   
                 LF 
                 CTG CCT TCC TCA CCT GAT G 
                 216 
               
               
                   
                   
                 LB 
                 TGC ACC ACC AAC TGC TTA 
                 217 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 CCCCAAAGGCCAGGCT 
                 218 
               
               
                   
                 2 
                 B3 
                 AGAAGGGATGGGAGAGAGC 
                 219 
               
               
                   
                   
                 FIP 
                 GGAATGGGGAGAAGGGCAGGTTAAATGTCACCGGGAGGATTG 
                 220 
               
               
                   
                   
                 BIP 
                 CGGAAACCAGATCTCCCACCGGCTACAGAAAGGTCAGCAGC 
                 221 
               
               
                   
               
               
                   
                 SET 
                 F3 
                 ATCAAGTGGGGCGATGCT 
                 222 
               
               
                   
                 3 
                 B3 
                 GGGCAGAGATGATGACCCT 
                 223 
               
               
                   
                   
                 FIP 
                 GCACTCACCCCAGCCTTCTCGCTGAGTACGTCGTGGAGT 
                 224 
               
               
                   
                   
                 BIP 
                 AAGCTGACTCAGCCCTGCAAACCCTGCAAATGAGCCTACA 
                 225 
               
               
                   
                   
                 F3 
                 GTT GAC CCG ACC CCA AAG 
                 226 
               
               
                   
                   
                 B3 
                 AAG GGA TGG GAG AGA GCC 
                 227 
               
               
                   
                   
                 FIP 
                 CGG AAT GGG GAG AAG GGC AGA TGT CAC CGG  
                 228 
               
               
                   
                   
                   
                 GAG GAT TGG 
                   
               
               
                   
                   
                 BIP 
                 CGG AAA CCA GAT CTC CCA CCG CCA GCT ACA  
                 229 
               
               
                   
                   
                   
                 GAA AGG TCA GC