Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions

Definitions

• the present disclosure concerns diagnostic assays for detection of target nucleic acids, specifically for rapid detection of multiple pathogens in clinical samples.
• Isothermal amplification is one such technique that accumulates nucleic acid sequences (amplicons) at constant temperature thus overcoming the constraints associates with the classic polymerase chain reaction (PCR) which requires a sophisticated thermocycling machine for its performance. Isothermal amplification has been used for detecting DNA and RNA.
• RPA Recombinase polymerase amplification
• RCA Rolling circle amplification
• the RPA concept was originally described by Morrical et al., who showed that the replication-initiating activity of the uvsX protein greatly amplifies hairpin-primed DNA synthesis that is catalyzed by the T4 DNA polymerase holoenzyme on linear, single -stranded DNA templates.
• the key to RPA is the establishment of a dynamic reaction environment that balances the formation and disassembly of recombinase-primer filaments.
• uvsX 44kDa
• uvsY crowding agents shifts the equilibrium in favor of recombinase loading (Piepenburg et ai (2.006)).
• HR homologous recombination
• Bacteriophage T4 routinely generates ssDNA during replication of its linear chromosome ends.
• the production of ssDNA tails or gaps in otherwise duplex DNA allows the assembly of core recombination machinery including presynaptic filaments on ssDNA.
• Presynaptic filaments are helical nucleoprotein filaments consisting of a recombinase enzyme and its accessory proteins bound cooperatively to ssDNA.
• the presynapsis pathway in bacteriophage T4 homologous recombination generally comprises the following steps: a dsDNA end is resected to expose a 3’ ssDNA tail probably by the exonucleases Gp46 and Gp47 proteins.
• the exposed ssDNA is sequestered by the Gp32 ssDNA-binding protein, which denatures secondary structure in ssDNA and keeps it in an extended conformation.
• the UvsY recombination mediator protein forms a tripartite complex with Gp32 and ssDNA and “primes” the complex for recruitment of UvsX recombinase which is ATP dependent and binds in a sequence independent manner to both ssDNA and dsDNA.
• UvsY recruits ATP -bound UvsX protein and nucleates presynaptic filament formation.
• Gp32 is displaced in the process (Liu and Morrical (2010)).
• Zhang and Tanner describe isothermal amplification of DNA fragments facilitated by a single-stranded binding protein. They report the amplification of discrete target fragments from both double- and single-stranded circular template DNA. This amplification requires only the single-stranded DNA-binding protein gp32 from bacteriophage T4 and a strand-displacing DNA polymerase. Accordingly, T4 Gp32- primer complexes are generated. T4 Gp32 facilitates the primer strand invasion into double-stranded DNA allowing subsequent polymerase strand displacement activity.
• the duration of the protocol described by Zhang and Tanner is about 1 hour, it involves the identification of a single target pathogen (defined as “single-plex”) with a limit of detection of 3.5xlO 10 .
• RCA is an isothermal process where a short DNA or RNA primer is amplified to form a long single stranded DNA or RNA using a circular DNA probe and a DNA or RNA polymerase (All et al.).
• Murakami et al. describe a mode of RCA dubbed primer generation RCA (PG-RCA).
• the RCA product is a concatemer containing tens to hundreds of tandem repeats that are complementary to the circular probe . It has been used, among others, to develop diagnostic methods for detecting nucleic acids.
• the present invention provides a method for detection of a target nucleic acid in a sample, comprising: a. Converting said target nucleic acid into an amplicon; b. Further amplifying said amplicon by performing a rolling circle amplification (RCA) to obtain an RCA product; and c. Detecting said RCA product,
• a positive detection signal indicates the presence of said nucleic acid in the sample.
• the step of converting the target nucleic acid into an amplicon comprises isothermal amplification.
• the present invention provides a method for detection of a target nucleic acid in a sample, comprising: a. Obtaining nucleic acids, wherein said nucleic acids were extracted and eluted from said sample; b. Amplifying target specific nucleic acid sequences by isothermal amplification with target specific primers; c. Incubating the amplified target sequences obtained in step (b) with a sequence specific open circular probe and ligating the nick in the open circular probe; d. Performing a rolling circle amplification (RCA) to obtain RCA products; and e. Detecting said RCA product,
• a positive detection signal indicates the presence of the target nucleic acid in the sample.
• the method is for the detection of at least one pathogen in a sample.
• said nucleic acids are genomic DNA, or RNA.
• said nucleic acids were extracted by incubating the sample with a solution comprising a lysis reagent.
• the lysis reagent is Guanidinium Thiocyanate (GuTCN).
• said solution further comprises sodium N-lauroylsarcosinate and a protease.
• the nucleic acid elution is performed in a recombinase reaction buffer.
• said nucleic acid elution is performed by incubating the extracted nucleic acids in a buffer comprising at least one set of forward primer and reverse primer that hybridize to said target nucleic acid sequences, nucleoside triphosphates, magnesium acetate, optionally potassium acetate and optionally a hydrophilic polymer, e.g., polyethylene glycol (PEG).
• a buffer comprising at least one set of forward primer and reverse primer that hybridize to said target nucleic acid sequences, nucleoside triphosphates, magnesium acetate, optionally potassium acetate and optionally a hydrophilic polymer, e.g., polyethylene glycol (PEG).
• PEG polyethylene glycol
• said incubation is for 2 min at 56°C.
• the isothermal amplification is recombinase polymerase amplification (RPA).
• the RPA is performed by incubating the eluted nucleic acids with at least one recombinase, at least one polymerase, and optionally at least one reverse transcriptase.
• said incubation is for between about 5 minutes and about 40 minutes, e.g., 20 minutes at a temperature of between about 37°C and 42°C, e.g., 40°C.
• said at least one set of forward primer and reverse primer that hybridize to said target nucleic acid sequences are pathogen specific primers selected from a group consisting of SEQ ID Nos: 1-18, 47-52, 56 and 58.
• said isothermal amplification is performed by homologous recombination (HR).
• said homologous recombination is performed using a strand displacing DNA polymerase.
• said homologous recombination is performed by incubating the extracted nucleic acids with at least one strand displacing DNA polymerase, at least one set of forward primer and reverse primer that hybridize to said target nucleic acid sequences, nucleoside triphosphates, magnesium sulfate, optionally at least one set of outer forward primer and outer reverse primer, and optionally at least one reverse transcriptase.
• said strand displacing DNA polymerase is BST3 large fragment.
• said incubation is for between about 10 minutes and about 20 minutes, e.g., 15 minutes at a temperature of between about 60°C and 70°C, e.g., 65°C.
• said homologous recombination is performed using a strand displacing DNA polymerase together with bacteriophage T4 Gp32.
• said homologous recombination is performed by incubating the extracted nucleic acids with bacteriophage T4 Gp32, at least one DNA polymerase, at least one set of forward primer and reverse primer that hybridize to said target nucleic acid sequences, nucleoside triphosphates, magnesium acetate, optionally potassium acetate and optionally at least one reverse transcriptase.
• said incubation is for between about 5 minutes and about 15 minutes, e.g., 10 minutes at a temperature of between about 37°C and 65°C.
• said at least one set of forward primer and reverse primer that hybridize to said target nucleic acid sequences are pathogen specific primers selected from a group consisting of SEQ ID Nos: 29-46.
• said sequence specific open circular probe is a pathogen specific probe selected from a group consisting of SEQ ID Nos: 20-28, and 53-55.
• the method further comprises a step of exonuclease treatment after ligating the nick in the open circular probe and prior to performing the RCA.
• said RCA is performed using branching forward primers and branching reverse primers.
• the RCA is performed in the presence of nickase.
• said detecting the RCA product comprises: a. Cutting said RCA product; b. Addressing the cut RCA products onto a substrate comprising target specific capture probes; c. Adding a target specific detectably labelled reporter complex; and d. Visualizing the captured RCA products.
• said method is implemented in a cartridge.
• the method is for multiplex identification of a panel of pathogens.
• each of said panel of pathogens is responsible for a disease selected from a group consisting of a respiratory disease, a cerebrospinal fluid infection, a sexually transmitted disease, tuberculosis, gastrointestinal diseases, drug resistant pathogens, plant pathogens, animal pathogens, and sepsis.
• the present invention provides a cartridge for detecting at least one pathogen in a sample, comprising buffers, solutions, primers, probes and enzymes for performing: a. Pathogen lysis; b. Nucleic acid purification; c. Target amplification; d. Ligation of a circular probe with the amplified target; e. RCA; f. Digestion; and g. Detection.
• the present invention provides a kit for detecting at least one pathogen in a sample, comprising: a. A cartridge according to the invention; and b. Instructions for use.
• the present invention provides a system for detecting at least one pathogen in a sample, comprising: a. A benchtop analyzer; and b. At least one cartridge according to the invention.
• Fig. 1 shows RCA products detected using sensor imaging.
• the top panel shows the sensor design.
• the sensor comprises pathogen specific capture nucleic acids for: Chp (Chlamydia pneumonia), Bp (Bordetella pertussis), Mp (Mycoplasma pneumonia), RSV A (respiratory Syncytial virus A), RSV B (respiratory Syncytial virus B), Flu B (influenza B).
• Chp Chlamydia pneumonia
• Bp Bacilla pertussis
• Mp Mycoplasma pneumonia
• RSV A respiratory Syncytial virus A
• RSV B respiratory Syncytial virus B
• Flu B influenza B
• Fig. 2A and 2B show gel analysis of RCA products for detection of B. pertussis.
• Figure 2A Lanes 1 to 4 were loaded with RCA products obtained from a sample containing IxlO 3 copies of the pathogen, lanes 5 to 8 were loaded with RCA products obtained from a sample containing IxlO 2 copies of the pathogen.
• Lane 9 synthetic B. pertussis DNA.
• Lane 10 Non-specific sDNA (synthetic DNA).
• Lane M (molecular weight (Mw) ladder).
• Figure 2B Lanes 1 to 4 were loaded with RCA products obtained from a sample containing IxlO 1 copies of the pathogen.
• Lane 5 synthetic B. pertussis DNA.
• Lane 6 Non-specific sDNA.
• 2C-2D are graphs showing qPCR amplification curves (delta Rn) of the recombinase polymerase pre -amplification reactions as a function of reaction cycle.
• Fig. 2C and 2D are duplicates. Three time points were analyzed as indicated by the arrows in the graph: time 0 (no incubation, light grey lines), time 30 (30 minutes incubation, grey lines) and time 60 (60 minutes incubation, black lines).
• Fig. 2E is a graph showing a qPCR amplification curve (delta Rn) of RCA products prepared with the 60 minutes’ pre-amplified samples of Exp. a, and Exp. b (shown in Fig. 2C and Fig. 2D, respectively).
• delta Rn qPCR amplification curve
• 10 8 synthetic dsDNA target was used in the ligation step without pre-amplification step (No RPA).
• Fig. 3A is a gel analysis of RCA products for detection of mycoplasma pneumonia (Mp).
• Lane 1 was loaded with RCA products obtained from a sample containing 6.8xl0 2 copies of the pathogen
• Lane 2 was loaded with RCA products obtained from a sample containing 6.8X10 1 copies of the pathogen.
• Lane 3 Bordetella pertussis (Bp) gDNA IxlO 4 .
• Lane 4 Listeria monocytogenes (Lm) gDNA.
• Lane 5 double distilled water (DDW).
• Lane 6 synthetic Mp oligo.
• Lane 7 Uu (Ureaplasma urealyticum) oligo used as a positive control. The left lane is a Mw ladder.
• Fig. 3B is a gel analysis of RCA products for detection of Chlamydia trachomatis (Ct).
• Lane 1 was loaded with RCA products obtained from a sample containing 10 5 EBs of the pathogen
• Lane 2 was loaded with RCA products obtained from a sample containing 10 4 EBs of the pathogen.
• Lane 3 was loaded with RCA products obtained from a sample containing 10 3 EBs of the pathogen.
• Lane 4 was loaded with RCA products obtained from a sample containing 10 2 EBs of the pathogen.
• Lane 5 was loaded with RCA products obtained from a sample containing 10 EBs of the pathogen.
• Lane 7 Vircell genomic DNA HSV 10 2 copies.
• Lane 8 synthetic DNA 0. InM CT Tl.
• the left lane (M) is a Mw ladder.
• Fig. 3C is a gel analysis of RCA products for detection of Neisseria gonorrhea (NG).
• Lane 1 was loaded with RCA products obtained from a sample containing 10 7 cfu of the pathogen
• Lane 2 was loaded with RCA products obtained from a sample containing 10 6 cfu of the pathogen
• Lane 3 was loaded with RCA products obtained from a sample containing 10 5 cfu of the pathogen.
• Lane 4 was loaded with RCA products obtained from a sample containing 10 4 cfu of the pathogen.
• Lane 5 was loaded with RCA products obtained from a sample containing 10 3 cfu ofthe pathogen.
• Lane 7 Vircell genomic DNA NG 10 2 copies (T+RPA).
• Lane 8 synthetic DNA O.lnM NG T4.
• the left lane (M) is a Mw ladder.
• Fig. 3D is a gel analysis of RCA products for detection of HSV 1.
• Lane 1 was loaded with RCA products obtained from a sample containing 10 7 pfu of the pathogen
• Lane 2 was loaded with RCA products obtained from a sample containing 10 6 pfu of the pathogen.
• Lane 3 was loaded with RCA products obtained from a sample containing 10 5 pfu of the pathogen.
• Lane 4 was loaded with RCA products obtained from a sample containing 10 4 pfu of the pathogen.
• Lane 5 was loaded with RCA products obtained from a sample containing 10 3 pfu of the pathogen.
• Lane 7 Vircell genomic DNA NG 10 2 copies (T+RPA).
• Lane 8 synthetic DNA O. lnM NG T4 (T+RCA).
• the left lane (M) is a Mw ladder.
• Fig. 4 is a gel analysis of RCA products for detection of SARS-Cov-2 in clinical samples. Lanes 1 to 4 were loaded with RCA products obtained from clinical samples. Lane 5: non-specific gDNA. Lane 6: synthetic SARS-Cov-2. Lane 7: synthetic UU oligo. Lane M: Mw ladder.
• Fig. 5 shows RCA products of clinical samples detected using sensor imaging.
• the top panel shows the sensor design.
• the sensor comprises pathogen specific capture nucleic acids for: RSV B (respiratory Syncytial virus B), Flu B (influenza B), and CoV2 (SARS-CoV-2).
• RSV B respiratory Syncytial virus B
• Flu B influenza B
• CoV2 SARS-CoV-2
• Each of the lower panels represents results obtained with a clinical sample containing the indicated pathogen. Highlighted spots represent a positive result.
• the dots on the right and left sides represent positive control for the hydrogel integrity.
• Fig. 6 is a graphic representation of the method of the invention implemented on a cartridge.
• Fig. 7 is a gel analysis of Gp32 HR products and Gp32 HR+RCA products for detection of Influenza A (FluA).
• Lane M a Mw ladder.
• Lane 1 Gp32 HR products obtained from a sample containing 5,000 copies of the pathogen
• Lane 2 Gp32 HR+RCA products obtained from a sample containing 5,000 copies of the pathogen.
• Lane 3 Gp32 HR products obtained from a sample containing 1,000 copies of the pathogen.
• Lane 4 Gp32 HR+RCA products obtained from a sample containing 1,000 copies of the pathogen.
• Lane 5 Gp32 HR products obtained from a sample containing 5,000 copies of the pathogen
• Lane 6 Gp32 HR+RCA products obtained from a sample containing 5,000 copies of the pathogen.
• Lane 7 Gp32 HR products obtained from a sample containing 1,000 copies of the pathogen.
• Lane 8 Gp32 HR+RCA products obtained from a sample containing 1,000 copies of the pathogen.
• Lane 9 synthetic target FluA which underwent only RCA and served as the RCA control.
• Lane 10 double distilled water (DDW) (negative control).
• Fig. 8 is a gel analysis of Gp32 HR+RCA products for detection of Influenza A (FluA).
• Lane M a Mw ladder.
• Lanes 1 and 2 sample containing 1,000 copies of Chlamydophila Pneumonia.
• Lanes 3 and 4 sample containing 500 copies of FluA.
• Lanes 5 and 6 sample containing 100 copies of FluA.
• Lanes 7 and 8 sample containing 50 copies of FluA.
• Lane 9 synthetic target FluA (positive control).
• Lane 10 double distilled water (DDW) (negative control).
• Fig. 9 is a gel analysis of Gp32 HR+RCA products and RCA only (no prior amplification with Gp32 HR) products for detection of Mycoplasma Pneumoniae .
• Lane M a Mw ladder.
• Lanes 1 and 2 Gp32 HR+RCA products obtained from a sample containing 100 copies of the pathogen.
• Lanes 3 and 4 RCA products obtained from a sample containing 100 copies of the pathogen.
• Lanes 5 and 6 Gp32 HR+RCA products obtained from a sample containing 1,000 copies of the pathogen.
• Lanes 7 and 8 RCA products obtained from a sample containing 1,000 copies of the pathogen.
• Lane 9 synthetic target MP which underwent only RCA and served as the RCA control.
• Lane 10 double distilled water (DDW) (negative control).
• the present invention provides sensitive, specific, and multiplex identification/detection of target nucleic acid molecules in a sample. Specifically, the identification/detection of pathogens in clinical samples, with a significantly low limit of detection as compared to alternative methods.
• the system and methods of the invention offer rapid, easy (sample-to-answer), accurate, and comprehensive molecular diagnosis of various infectious agents in nearpatient settings.
• the methods of the invention can be performed using a fully automated system.
• the detection method of the invention comprises two isothermal amplification steps: (a) a first amplification step of converting a target nucleic acid (e.g., genomic DNA or RNA) into an amplicon, also referred to as the “preamplification step” and
• a second amplification step which employs rolling circle amplification (RCA) methodology performed using circular probes to further amplify a detection signal thereby achieving specific detection of low levels of the target nucleic acid.
• RCA rolling circle amplification
• the technology can be implemented in a variety of different formats, including microfluidic platforms and point-of care devices, for example on an electrophoretic chip, e.g., as described in WO 2018/122856 incorporated herein by reference.
• the first “preamplification” (PA) step which converts the genomic DNA or RNA into an amplicon, provides an enhanced starting point for the RCA thus significantly increasing its sensitivity and the outcome of the specific multiplex pathogens diagnostics method.
• This step may be performed using any method known in the art for nucleic acid amplification. Two such non limiting methods are exemplified herein, namely recombinase polymerase amplification (RPA) and homologous recombination, particularly strand displacing DNA polymerase mediated homologous recombination, with or without Gp32.
• RPA recombinase polymerase amplification
• homologous recombination particularly strand displacing DNA polymerase mediated homologous recombination, with or without Gp32.
• the term “detection” or “detecting” refers to discovering or evidencing the presence of a target nucleic acid in a biological/clinical sample. In one specific aspect of the invention, the term detecting refers to examining whether one or more pathogens are present in the biological/clinical sample.
• pathogen refers to an infectious agent that may be a bacterium or a virus.
• the infectious agent is a virus, such as an RNA virus (single stranded or double stranded) or a DNA virus (single stranded or double stranded).
• the virus is an RNA virus, e.g. an RNA virus of the Coronaviridae Family, particularly a virus of the species severe acute respiratory syndrome related coronavirus, more particularly SARS-Cov-2.
• RNA virus e.g. an RNA virus of the Coronaviridae Family, particularly a virus of the species severe acute respiratory syndrome related coronavirus, more particularly SARS-Cov-2.
• the virus is an RNA virus, e.g. an RNA virus of the Paramyxoviridae Family, particularly human respiratory virus (HSV) A or B.
• the virus is an RNA virus, e.g. an RNA virus of the Family, particularly influenza A or influenza B.
• the virus is a DNA virus, e.g. a DNA virus of the Herpesviridae family, particularly herpes simplex virus (HSV) 1 or 2.
• a DNA virus e.g. a DNA virus of the Herpesviridae family, particularly herpes simplex virus (HSV) 1 or 2.
• HSV herpes simplex virus
• the infectious agent is a bacterium.
• the bacterium is for example a bacterium responsible for respiratory infections such as Chlamydia pneumonia, Bordetella pertussis, Mycoplasma pneumonia, Mycobacterium tuberculosis, or klebsiella pneumonia, or a bacterium responsible for sexually transmitted infections such as Chlamydia trachomatis, or Neisseria gonorrhea.
• the bacterium is for instance E. Coli, Salmonella, Shigella, Pseudomonas aeruginosa, Borrelia burgdorferi, Vibrio Cholerae, Proteus mirabilis, or Enterococcus faecalis.
• a single or multiplexed assay may be conducted to detect simultaneously one or more infectious agents.
• the method generally comprises: a. extraction of nucleic acids from a sample, comprising lysis and elution steps.
• sample encompasses any sample that comprises biological material and may contain an infectious agent. It may be a clinical sample obtained from a human subject, or a biological sample obtained from an animal. The sample may be obtained from a bodily fluid or tissue such as blood, urine, feces, lung aspirate, cerebrospinal fluid, or semen. It also encompasses samples of water, soil or air, or samples that were taken from various surfaces that may contain biological material.
• the lysis step is performed in a lysis solution.
• the lysis solution comprises Guanidinium Thiocyanate, sodium N-lauroylsarcosinate, isopropanol (IP A), carrier RNA and a protease.
• the lysis solution may further comprise Dithiothreitol (DTT), e g., 80mM DTT.
• DTT Dithiothreitol
• the lysis solution is as described in the Examples below, namely a solution comprising 50% Solution A (2.5M Guanidinium Thiocyanate and 0.7% sodium N-lauroylsarcosinate in 0.1M Tris-HCl pH 7.4), 35%-50% Isopropanol, carrier RNA (1 mg/ml), DDW, and a protease (e.g., Proteinase K).
• Solution A 2.5M Guanidinium Thiocyanate and 0.7% sodium N-lauroylsarcosinate in 0.1M Tris-HCl pH 7.4
• Isopropanol carrier RNA (1 mg/ml)
• carrier RNA (1 mg/ml
• DDW DDW
• protease e.g., Proteinase K
• the extracted DNA is washed with a buffer, optionally comprising up to 70% ethanol.
• the wash may include one or more washing steps.
• the extracted DNA is subjected to two washing steps, the first with a buffer comprising 70% ethanol (e.g., Tris 66.8nM, pH-7 KC1 330.3mM + 70% ethanol) and the second with a buffer comprising 30% ethanol (e.g., Tris 66.8nM, pH-7 KC1 330.3mM + 30% ethanol).
• a buffer comprising 70% ethanol e.g., Tris 66.8nM, pH-7 KC1 330.3mM + 70% ethanol
• 30% ethanol e.g., Tris 66.8nM, pH-7 KC1 330.3mM + 30% ethanol.
• the isothermal amplification is performed using recombinase polymerase amplification (RPA) technique.
• RPA recombinase polymerase amplification
• the isothermal amplification is optionally performed in the presence of a reverse transcriptase enzyme, such that if RNA viruses are present in the sample their genomic RNA is reverse transcribed into cDNA.
• a reverse transcriptase enzyme such that if RNA viruses are present in the sample their genomic RNA is reverse transcribed into cDNA.
• the reverse transcriptase may be excluded from the reaction.
• the RPA reaction disclosed herein contains a recombinase, which may originate from prokaryotic, viral or eukaryotic origin.
• exemplary recombinases include RecA, and uvsX obtained from any species, e.g., the RecA/Rad51 ortholog of bacteriophage T4, and fragments or mutants thereof, and combinations thereof.
• the RPA reaction disclosed herein contains a DNA polymerase, which may be any polymerase suitable for isothermal amplification, for example Klenow exo, Bsu large fragment, phi29, or the large fragment of Bst DNA polymerase.
• a DNA polymerase which may be any polymerase suitable for isothermal amplification, for example Klenow exo, Bsu large fragment, phi29, or the large fragment of Bst DNA polymerase.
• the newly synthesized strand is displaced from the parental DNA and used as a template for an isothermal pre -amplification reaction.
• the homologous recombination is performed using a strand-displacing DNA polymerase with or without the presence of bacteriophage T4 Gp32.
• the extracted nucleic acids are contacted with suitable primers, bacteriophage T4 Gp32, and a strand-displacing DNA polymerase.
• the reaction is performed in a buffer comprising a buffering agent (e.g., Tris-HCl pH 8.0, potassium acetate and MgAc (or alternatively, NaCl and MgCl), DTT or TECP (Tris(2- carboxyethyl)phosphine hydrochloride), and dNTPs.
• a buffering agent e.g., Tris-HCl pH 8.0, potassium acetate and MgAc (or alternatively, NaCl and MgCl)
• DTT or TECP Tris(2- carboxyethyl)phosphine hydrochloride
• the extracted nucleic acids are contacted with suitable primers, and a strand-displacing DNA polymerase.
• the reaction is performed in a buffer comprising a buffering agent, magnesium sulfate, and dNTPs.
• the duration of the preamplification step (comprising a polymerization reaction and optionally reverse transcription) is very short, about 10 minutes, and it may even be shortened. It may be performed in a multiplex manner (i.e., 5 plex, 10 plex, or even 100 plex), and when combined with RCA (as will be described below) its limit of detection is lower than 50 copies (as shown for example for influenza A). It must be emphasized that this does not appear to be the lowest limit of detection, which is apparently lower than 50 copies, in the single digit range.
• the homologous recombination preamplification step may be performed using any isothermal strand-displacing DNA polymerase.
• Non-limiting examples include Bsu, BST (e.g., BST3 large fragment), Phi29, and Klenow exo.
• llie homologous recombination preamplification step may be performed using two or more primers (namely, a primer set comprising at least a forward and a reverse primer) or using a single primer for each target. Use of a single primer may increase the extent of multiplexing, and it may also facilitate a quantitative reaction.
• RNA viruses such as, but not limited to, SARS Cov2 influenza or HSV
• the extracted nucleic acids undergo reverse transcription prior to the amplification step.
• Idle reaction may therefore be performed in the presence of a reverse transcriptase (RT) (e g., RevertAid RT).
• RT reverse transcriptase
• the use of the HR reaction enables an ethanol tolerance of up to about 30% in the preamplification step. This is a highly advantageous feature since ethanol wash improves the efficiency of nucleic acid purification. However, its use within a cartridge is generally avoided since many enzymatic reactions have a low ethanol tolerance and the ethanol cannot be removed by drying when used within a cartridge. Therefore, high tolerance of the HR reaction to residual ethanol facilitates the use of an ethanol washing step in a cartridge even though no drying of the ethanol can take place. c. Open circular probe hybridization with the previously amplified target nucleic acid (the previously generated amplicon).
• Short circular probes also referred to as “padlock probes’’
• padlock probes short circular probes matching tire target nucleic acid sequences at both the 3' and 5' ends were designed.
• a perfect match at. the target region between the target nucleic acid and the padlock probe allows circularization of the padlock probe in the presence of thermostable ligase.
• the hybridization may be performed in solution, in a semi solid interface, in a bead assay, or on a solid support.
• the hybridization is performed using a bead assay whereby the open circular probe is attached to a magnetic bead.
• the attachment may be performed for example via biotin-streptavidin binding, namely streptavidin magnetic beads are associated with the open circular probe via a biotinylated capture probe which has a sequence that is complementary to a sequence within the open circular probe.
• d Ligation of positive circular probes which showed specific recognition.
• ligation enzymes include Hifi Taq ligase, ligase, taq ligase, 9°N DNA, ligase t4, and splintR ligase.
• ligation enzymes include Hifi Taq ligase, ligase, taq ligase, 9°N DNA, ligase t4, and splintR ligase.
• Elimination of non-ligated probes and nucleic acids For example by exonuclease treatment (e.g., using exonuclease I).
• RCA Rolling circle amplification
• the circularized padlock probe is then amplified with a DNA polymerase (e.g., BST, Klenow exo', Phi29, Bsu or Vent exo-DNA polymerase which are active in a temperature range of 37°C to 70°C) using primers derived from the central region (backbone) of the circular probe, in the presence of nucleotides.
• a DNA polymerase e.g., BST, Klenow exo', Phi29, Bsu or Vent exo-DNA polymerase which are active in a temperature range of 37°C to 70°C
• the reaction is performed with forward and reverse branching primers to exponentially amplify the produced signal, also referred to as hyper-branched RCA.
• linear RCA only a forward primer is used, and therefore a linear, long DNA strand is produced.
• a reverse primer is included (the forward and reverse primers are also referred to as sense and antisense primers) to increase the amount of synthesized DNA and decrease the incubation time. As a result, a reverse strand is produced, which, in turn, becomes a template for the forward primer. This process is called branching.
• ssDNA concatemenc single stranded DNA
• the RCA is conducted at a constant temperature, e.g., 65°C as shown in the Examples below .
• the RCA may be performed in the presence of the enzyme nickase which nicks only one strand of the nucleic acid and thereby generates additional priming sites for the polymerase amplification action which creates branching, thereby further amplifying the signal.
• Die suitable nickase is selected according to the unique target sequences, e.g., BSTNBI nicking endonuclease.
• Cutting RCA products to short oligos also referred to herein as “concatemer digestion”.
• the suitable cutting enzymes are selected according to the unique target sequences.
• Non-limiting examples for enzymes that can be used for cutting the RCA products include Haelll, Nhelll, and Aiul. h. Detection.
• Amplified DNA can be detected using any method known in the art. Nonlimiting example include hybridization and detection of amplified targets on screen printed carbon electrodes with fluorescent probes followed by sensor imaging, UV illumination after staining with Cybr-gold® (Molecular Probes), and molecular beacons. Compatible buffers are used for these reactions.
• fluorescent probe refers to any substance that emits electromagnetic energy at a certain wavelength (emission wavelength) when the substance is illuminated by radiation of a different wavelength (excitation wavelength) and is intended to encompass a chemical or biochemical molecule or fragment thereof that is capable of interacting or reacting specifically with an analyte of interest in a sample to provide one or more optical signals.
• fluorescent probes for use in the methods provided herein include for example, Cy5, Cy3, fluorescein amidites (FAM), and the like.
• the RCA products that were obtained by the amplification process of the invention were digested and electronically addressed to active sites/electrodes. Namely, since the digested RCA products or amplicons are negatively charged, they are electrically addressed (pulled) into a hydrogel, where capture-molecules bind complementary amplicons. In one embodiment, a power flow (current) of 200 pAmp is applied for 30 seconds to perform the addressing.
• addressing refers to the electrical pulling of the RCA products or amplicons into a hydrogel.
• the addressing is performed on an electrophoretic chip, for example as described in WO 2018/122856 incorporated herein by reference.
• the fluorescent report complex comprising the reporter oligo with 3 ’ and 5 ’ fluorophores (which are selected depending on the filters in the imaging system, for example but not limited to Cy5), which binds to the complementary sequence on the circular probe (CP) and to an additional oligo (e.g., 60 bp long) which complementarity binds the CP and additional reporter molecules (e.g., two or more). Resulting in multiple fluorophore molecules signaling for each CP captured on the array.
• the reporter fluorochrome is excited at the appropriate wavelength (e.g., 630 nm), and the sensor imaging system detects the emitted light (e.g., 670 nm) in an optical test chamber.
• the detection process is carried out in a single tube, microplate well, liquid handier or cartridge.
• the cartridge and systems for computerized automatic diagnosis disclosed in WO 2019/130,309, WO 2020/008463, WO 2018/12.2852, WO 2019/130290, , incorporated herein by reference.
• the invention thus encompasses assays integrated on microfluidics and one-step detection assays, in which a sample is introduced onto a cartridge and an assay report is produced showing the results of the multiplex detection (as exemplified in Fig. 6).
• the method provides primers and probes for detection of indication-specific pathogens, also referred to herein as “indication-specific anels”, or “panels”.
• the panels may include, but are not limited to, pathogens affecting the respiratory system, CNS-associated infections, sexually transmitted diseases, gastrointestinal diseases, drug resistant pathogens, plant, and animal pathogens (particularly plants/animals of agricultural relevance or pets), and sepsis.
• sexually transmitted diseases includes, but is not limited to Chlamydia trachomatis, Neisseria gonorrhea, and HSV1, as exemplified below.
• the method of the invention therefore provides a low limit of detection (LOD) of 10 to 100 copies in a reaction. Furthermore, the novel method of the invention allows the inclusion of this process in a single cartridge for sample-to-answer in vitro diagnostics.
• LOD low limit of detection
• the invention provides a system featuring a compact, easy- to-use, benchtop analyzer utilizing unique multiple sensors and a disposable fully integrated cartridge allowing the fully automatic sample-to-answer processing of clinical samples in 30 - 45 min. It features a high multiplexing capacity (up to 100 molecular targets per sample).
• the method of the invention is carried out within the cartridge.
• the system can be designed for any application or symptomatic condition and can be deployed as a point of care for a pandemic/endemic solution.
• nucleic acids in particular RNA into cells, specifically lipofection, for example as described below.
• the extracted DNA was washed with a Wash buffer I: Tris 66.8nM, pH-7 KC1 330.3mM + 70% ethanol, followed by a second wash with a Wash buffer II: Tris 66.8nM, pH-7 KC1 330.3mM + 30% ethanol.
• Nucleic acid elution in recombinase reaction buffer The nucleic acids were eluted by adding 55 pl elution buffer.
• the elution buffer contained: 32.4 pl recombinase reaction buffer (RRB): 50 mM Trizma hydrochloride solution (pH 8.0), 160mM Potassium acetate, 5% PEG (20000), 2mM DTT, 200pM dNTPs, 3mM ATP, 50mM Creatine phosphate, 14mM MgAc , 9.2 pl of a lOpM primer stock solution of each forward and reverse primer listed in Table 1, 2.75 pl magnesium acetate and 10.6 DDW.
• RRB recombinase reaction buffer
• RNA of RNA-based pathogens was thus reverse transcribed into cDNA to allow formation of the amplicon.
• This step results in the production of amplicons of the detected pathogens. These amplicons are next subjected to further amplification using a rolling circle amplification method.
• Cp+F primer (circular probe + forward primer) mix (Volume per sample: 3 pl of the 5’ biotinylated forward capture primer 5Bio_Fr_BSTNBI which is rich in TC repeats (at a final concentration of IpM in binding buffer (0.5M NaCl, 40 mM Tris-HCl pH 7.5), 0.6 pl per circular probe of CP_XXX_BSTNBI_HaeIII_TN (final concentration 0.02pM in binding buffer), and 26.4 pl binding buffer) were added to the beads, mixed, and incubated for 5 minutes at room temperature. The primer and the circular probe bind to each other through complementary sequences.
• the 5’-3’ sequence of 5Bio_Fr_BSTNBI is TCT CTC TCT CTC TCT CTC TCT CTC GAG TCG CTA CCT TGC CCT AAA CG (SEQ ID NO. 19). 5-Bio primer for CP+F mix preparation + TC repeats.
• a magnet was then applied to the side of the tube for 20 seconds and the supernatant was discarded.
• the beads were then washed twice with the Binding Buffer followed by washing with Wash buffer 2 (150mM NaCl, 40mM Tris-HCl pH 7.5), separated using a magnet and the supernatant was discarded.
• 5 pl ligation mix (0. 1 IM DL- Dithiothreitol solution, 1 lnMNAD + , Hifi Taq ligase XI 1, in ultrapure DNase/RNase free water) were added to the beads and pipetted to resuspend the beads in the solution.
• Denaturation step The eluent obtained after the preamplification step described above was incubated for 2 minutes at 95°C. Aliquots (5 pl) of beads (associated with the circular probe) in ligation mixture containing, 1 InM nicotinamide adenine dinucleotide (NAD +) , and Hifi Taq ligase XI 1, were added to 50pl of the denatured eluent and incubated for 10 minutes at 65°C.
• NAD + 1 InM nicotinamide adenine dinucleotide
• Hifi Taq ligase XI 1 Hifi Taq ligase XI
• Exonuclease treatment At the end of the ligation, 2pL of Exonuclease I were immediately added to the_reaction (for a final concentration of 0.7 units/pl), mixed and incubated for 5 minutes at 65 °C.
• the beads were separated using a magnet and the supernatant was discarded. 30pl RCA mix was added to the beads.
• the RCA mix was prepared as follows: isothermal buffer concentration XI, dNTPs 1.4mM, MgSO4 6mM, Fr (forward) -branching primer (3’PTO) 2pM, Rv (Reverse)-branching primer (3’PTO) 0.5pM, second reverse primer RV-TN (3’PTO) 0.5pM, BST polymerase 400 units/ml (total of 12 units/sample) in ultrapure Dnase/Rnase free water. The beads were suspended in the RCA mix and incubated for 15 minutes at 65°C.
• Example 1 visualizing the RCA products using sensor imaging
• the final stage of the protocol was tested, namely the digestion of the RCA product and its addressing/reporting on an array.
• the negatively charged DNA molecules namely, the digested/cut RCA products or amplicons
• the power flow (current) of 200 pAmp is applied for 30 seconds to perform the addressing.
• the fluorescent reporter complex comprising the reporter oligo with 3' and 5’ Cy5 fluorophores which binds to the complementary sequence on the circular probe (CP) and to an additional 60 bp long oligo which complementary binds the CP and two reported molecules is added. Therefore, six fluorophore molecules signal for each CP captured on the array.
• the reporter fluorochrome was excited at 630 nm, and the sensor imaging system caught the emitted light of 670 nm in an optical test chamber.
• the amplification process of the synthetic targets was sensitive and repetitive (as can be seen for example in the repetitive results obtained with Chlamydia pneumonia) resulting in a clear identification of the target pathogen with a high signal to noise ratio (as can be seen for example with the clear negative results obtained for RSV A, RSV B, and influenza).
• samples containing various amounts of the target pathogen were used.
• the samples contained 1000, 100, or 10 copies of the pathogen gDNA by Vircell.
• the RCA product was prepared as described in Example 1, using Bordetella pertussis specific primers as shown in Table 1 (SEQ ID Nos 15 and 16), and a circular probe as shown in Table 2 (SEQ ID No. 20).
• the RCA products were visualized on a gel (E-Gel® 2% Agarose, Thermo Fisher Scientific). As can be seen in Figures 2A and 2B, the method allowed the detection of as low as 10 copies of the pathogen per sample.
• Non-specific synthetic DNA namely an out of panel synthetic target, served as a negative control.
• An RCA reaction produces DNA products containing multiplications of circular probes; hence the products appear as a ladder of bands on the agarose gel.
• the Ct (cycle threshold) and the melting temperature (TM) values were measured for the RCA products.
• Ct indicates the number of amplicons received after the RCA reaction.
• the Ct is defined as the number of cycles required for the fluorescent signal to exceed background levels.
• Ct levels are inversely proportional to the amount of target nucleic acid in the sample (i.e., the lower the Ct level, the greater the amount of target nucleic acid in the sample).
• Ct was measured according to a threshold value of 1. This value was chosen as it falls in the linear stage of the amplification curve.
• Tm values are determined automatically by the QuantStudio Design (Applied Biosystmes by Thermo Fisher Scientific) and its Analysis software according to the peak in the melting curve.
• Table 3 shows the Ct and TM values for the Bordetella pertussis RCA products.
• the detection efficacy of the method of the invention which combines recombinase-based amplification (as a preamplification step) with RCA was compared to the detection efficacy of RCA alone.
• double strain synthetic oligonucleotide (Bordetella pertussis) with a well- known number of copies was used as a template and DDW as negative control.
• Reactions were performed at 40°C for 30 min or 60 min. Standard conditions were 50 mM Tris (Sigma, T2694, pH 8.0), 160 mM potassium acetate (Sigma, 95843F), 28 mM magnesium acetate (Sigma, 63052), 2 mM DTT (Sigma, 1719757), 5% PEG 20.000 (Sigma 8.18897), 1.4mM dNTPs (New England Biolabs: NEB, N0447L), 3 mM ATP (New England Biolabs: NEB, P0756S), 50 mM phosphocreatine (Sigma 19333-65-4), 100 ng/pL creatine kinase (Roche, 10127566001), and 0.33 U/ pL Bsu large fragment (New England Biolabs: NEB, M0330L).
• Figs. 2C and 2D show a qPCR amplification curve of the recombinase polymerase pre-amplification (RPA) reaction using specific primers to detect Bordetella Pertussis amplicon. Namely the experiments were performed without a further amplification step (no RCA).
• the RPA reactions were done in duplicate, referred to as Exp. la (Fig. 2C) and Exp. lb (Fig. 2D), in a single-plex mode using 10 8 synthetic dsDNA target. Three time points were analyzed: no incubation, 30 minutes incubation, and 60 minutes incubation. The reactions were diluted 1 : 100 in DDW prior to qPCR. A threshold value of 1 was used to calculate the Ct values.
• RPA recombinase polymerase pre-amplification
• Fig. 2E shows qPCR amplification curves of the RCA product amount.
• the RPA reactions after a 60-minute incubation (samples were taken from the experiments shown in Figs 2C and 2D) were used in the full RCA protocol. Namely the results present an amplification obtained after performing RPA + RCA.
• 10 8 synthetic dsDNA target was used in the ligation step without pre-amplification step.
• Mp Mycoplasma pneumonia
• the samples contained 680, or 68 copies of Mp gDNA by VirCell (a commercially available source of bacteria and viruses providing the number of copies per microliter, VirCell Microbiologists).
• the RCA product was prepared as described in Example 1, using Mycoplasma pneumonia specific primers as shown in Table 1 (SEQ ID Nos 5 and 6), and a circular probe as shown in Table 2 (SEQ ID No. 26).
• the RCA products were visualized on a gel. As can be seen in Figure 3A, the method allowed the detection of as low as about 60 copies of the pathogen per sample. To account for potential background non-relevant signal, the Ct, and the melting temperature (TM) values were measured for the RCA products.
• Table 4 shows the Ct and TM values for the Mycoplasma pneumonia RCA products, as compared with various controls.
• the limit of detection was also measured for Chlamydophila pneumonia, influenza B and SARS-Cov-2. For all these pathogens the limit of detection was in the range of 10 to 100 copies in a sample.
• the method of the invention was also employed for the detection of several exemplary sexually transmitted infections (STI), namely Chlamydia trachomatis, Neisseria gonorrhea, and HSV 1.
• STI sexually transmitted infections
• the samples contained 10 elementary bodies (EBs), 10 2 EBs, 10 3 EBs, 10 4 EBs or 10 5 EBs of Chlamydia trachomatis diluted in PBS.
• the RCA product was prepared as described in Example 1, using Chlamydia trachomatis .specific primers as shown in Table 1 (SEQ ID Nos 47 and 48), and a circular probe as shown in Table 2 (SEQ ID No. 53).
• the RCA products were visualized on a gel. As can be seen in Figure 3B, the method allowed the detection of as low as about 10 4 EBs of the pathogen per sample.
• NG Neisseria gonorrhoeae
• the samples contained 10 3 colony forming units (cfu), 10 4 cfu, 10 5 cfu, 10 6 cfu, or 10 7 cfu of Neisseria gonorrhoeae_diluted in PBS.
• the RCA product was prepared as described in Example 1, using Neisseria gonorrhoeae.specific primers as shown in Table 1 (SEQ ID Nos 49 and 50), and a circular probe as shown in Table 2 (SEQ ID No. 54).
• the RCA products were visualized on a gel. As can be seen in Figure 3C, the method allowed the detection of as low as about 10 5 cfu of the pathogen per sample.
• HSV1 Herpes simplex virus - 1
• the samples contained 10 3 plaque forming units (pfu), 10 4 pfu, 10 5 pfu, 10 6 pfu, or 10 7 pfu of HSV 1 diluted in PBS .
• the RCA product was prepared as described in Example 1, using HSV1 specific primers as shown in Table 1 (SEQ ID Nos 51 and 52), and a circular probe as shown in Table 2 (SEQ ID No. 55).
• the RCA products were visualized on a gel. As can be seen in Figure 3D, the method allowed the detection of as low as about 10 5 pfu of the pathogen per sample.
• Table 5 shows a non-limiting example of the extraction yield by quantifying HSV1 DNA immediately after extraction.
• the samples were analyzed for the detection of the following pathogens: SARS-Cov-2 (at least 19 samples were analyzed).
• Influenza B (at least 14 samples were analyzed).
• Influenza A (at least 4 samples were analyzed).
• Samples of patients with SARS-Cov-2 infection were used for the preparation of RCA products.
• the RCA products were prepared as described in Example 1, using SARS-Cov-2 specific primers as shown in Table 1 (SEQ ID Nos 17 and 18), and a circular probe as shown in Table 2 (SEQ ID No. 21).
• the RCA products were visualized on a gel. As can be seen in Figure 4, the method was sensitive and specific allowing the detection of SARS-Cov-2 in clinical samples obtained from patients.
• the Ct, and the melting temperature (TM) values were measured for the RCA products.
• Table 6 shows the Ct and TM values for SARS-Cov-2 RCA products obtained from different clinical samples (I-IV), as compared with various controls.
• Table 6 Ct and TM values Clinical samples were obtained from Shiba (Sars-Cov-2), Hadassah (RSV) and Ichilov (inffl). As can be seen in Figure 5, the amplification process performed on clinical samples was sensitive, resulting in a clear identification of the target pathogen with a high signal to noise ratio clearly indicating the presence of RSV-B, influenza A or SARS- CoV2 in separate clinical samples.
• Example 4 Performing the assay in a cartridge
• the amplification method of the invention can be performed in a specialized cartridge.
• the sample may be applied onto a cartridge in which all steps, including the isothermal amplification, are performed leading to an assay report providing the detection results.
• the steps that are performed in the cartridge comprise: nucleic acid (e.g., genomic DNA or RNA) extraction, pathogen selective nucleic acid isothermal amplification, circular probe hybridization with the target nucleic acid, ligation of positive circular probes, elimination of non-ligated probes and genomic DNA or RNA, RCA amplification of positive probes, cutting RCA products to short oligos, hybridization and detection of amplified targets with carbon array probes.
• nucleic acid e.g., genomic DNA or RNA
• Elution + preamplification The Elution step is performed in a solution comprising 30 pL H2O and 20 pL Gp32 pre-amplification buffer comprising: 50 mM Tris-Acetate (Sigma, T2694, pH 8.0), 7 mM Magnesium acetate (Sigma, 63052), 50 mM Potassium acetate (Sigma, 95843F), 2 mM DTT (Sigma, 1719757), 630pM dNTPs (New England Biolabs: NEB, N0447L), Forward and Reverse primers each IpM, 0.33 units/pL Bsu large fragment (New England Biolabs: NEB, M0330L) or 0.33 units/pL Klenow exo- DNA polymerases (New England Biolabs: NEB, M0212L), 200 units/pL RevertAid Reverse Transcriptase (Thermo Scientific, EP0441), and 500 ng/pL T4-gp32 (New England Biolabs:
• Unlabeled DNA oligonucleotides (primers) of the multi-plex targets were purchased from Integrated DNA Technologies (IDT) and their sequences are listed in Table 7.
• the samples contained 5,000, or 1,000 copies of Influenza A gRNA from Vircell.
• the Gp32 mediated homologous recombination products (referred to herein as the “Gp32 HR products”) and the products of the combined methods (referred to herein as the “Gp32 HR+RCA products”) were prepared as described above, using Influenza A specific primers as shown in Table 7 (SEQ ID Nos 39 and 40), and a circular probe as shown in Table 2 (SEQ ID No. 22).
• a gel E-Gel® 2% Agarose, Thermo Fisher Scientific.
• use of Gp32 mediated homologous recombination alone did not produce a detectable signal both for the sample containing 1000 copies and for the sample containing 5000 copies of FluA gRNA Vircell.
• the method combining Gp32 mediated homologous recombination with RCA resulted in a clear detection signal ⁇ allowing the detection of as low as about 1000 copies of the pathogen per sample.
• the Gp32 HR+RCA products were visualized on a gel (E-Gel® 2% Agarose, Thermo Fisher Scientific).
• E-Gel® 2% Agarose, Thermo Fisher Scientific As can be seen in Figure 8, the method combining Gp32 mediated homologous recombination with RCA resulted in a clear detection signal, allowing the detection of as low as about 50 copies of the pathogen gRNA (Vircell) per sample.
• the clarity of the results indicates that the assay did not reach yet its limit of detection, which appears to be, for influenza A, far lower than 50 copies. This advantage is especially emphasized by the fact that the duration of the preamplification step is only 10 minutes.
• Mp Mycoplasma pneumoniae
• the samples contained 100, or 1,000 copies of Mycoplasma pneumoniae gDNA (Vircell).
• the Gp32 HR+RCA products and the RCA only (without prior amplification with Gp32 HR) products were prepared as described above, using Mycoplasma pneumoniae specific primers as shown in Table 7 (SEQ ID Nos 33 and 34), and a circular probe as shown in Table 2 (SEQ ID No. 26).
• Tm values were measured for the qPCR of the RCA products.
• the Tm values were determined by the QuantStudio Design (Applied Biosystmes by Thermo Fisher Scientific) and its Analysis software according to the peak in the melting curve. Results revealed specific amplification for both 100 and 1,000 copies of Mp when the combined method of PA32 and RCA were performed. When only RCA was performed, no amplification was detected.
• Bp F3 forward outer primer 5'-CGACGCTACGGACCTTCG-3’ (SEQ ID NO: 57)
• Bp R3 reverse outer primer 5'-CGCTGGCCACCTACCA - 3’ (SEQ ID NO: 59)
• Pre-amplification step the DNA/primers solution obtained in the elution step was incubated for 15 minutes at 62°C in a solution comprising lx Isothermal Buffer Pack II (New England Biolabs: NEB, B0374S), 1.4 mM each dNTPs (New England Biolabs: NEB, N0447L), 8 mM Magnesium sulfate (New England Biolabs: NEB, B1003S), and 0.16 units/pL Bst3 large fragment (New England Biolabs: NEB, M0374L).

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