WO2018096496A1 - Procédé, kit et application associée - Google Patents

Procédé, kit et application associée Download PDF

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WO2018096496A1
WO2018096496A1 PCT/IB2017/057387 IB2017057387W WO2018096496A1 WO 2018096496 A1 WO2018096496 A1 WO 2018096496A1 IB 2017057387 W IB2017057387 W IB 2017057387W WO 2018096496 A1 WO2018096496 A1 WO 2018096496A1
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probe
amplification
nucleic acid
primer
group
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Sudeshna Adak
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Omix Research And Diagnostic Laboratories Private Ltd
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Omix Research And Diagnostic Laboratories Private Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • 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
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/30—Oligonucleotides characterised by their secondary structure
    • C12Q2525/301—Hairpin oligonucleotides
    • 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
    • C12Q2549/00—Reactions characterised by the features used to influence the efficiency or specificity
    • C12Q2549/10—Reactions characterised by the features used to influence the efficiency or specificity the purpose being that of reducing false positive or false negative signals
    • C12Q2549/119—Reactions characterised by the features used to influence the efficiency or specificity the purpose being that of reducing false positive or false negative signals using nested primers
    • 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
    • C12Q2561/00—Nucleic acid detection characterised by assay method
    • C12Q2561/101—Taqman

Definitions

  • the present disclosure describes a method for detecting nucleic acids, said method comprises combination of primary amplification and secondary amplification, wherein the primary amplification is nucleic acid amplification and secondary amplification is signal amplification.
  • the disclosure further describes a Kit comprising microplate comprising primers, enzymes, reagents, probes, buffering reagents and nucleoside triphosphate, buffer or water including but not limited to nuclease free water and double distilled water, or both.
  • nucleic acid amplification techniques cause non-specific amplification of the nucleic acid, thereby hampering the detection of nucleic acid, causing low specificity during detection of nucleic acid in a sample.
  • LAMP loop-mediated isothermal amplification
  • the present disclosure describes a method for detecting nucleic acid, said method comprises combination of primary amplification and secondary amplification, wherein the primary amplification is nucleic acid amplification and the secondary amplification is signal amplification.
  • the present disclosure further describes a kit comprising microplate having wells comprising primers, probes, enzymes, reagent and nucleoside triphosphate, and buffer or water or both.
  • FIGURE 1 illustrates a likely design of the microplate of the instant disclosure.
  • FIGURE 2 illustrates the outcome of the combination of primary amplification and secondary amplification of the present disclosure, wherein FIGURE 2a illustrates output of the primary amplification using negative control and lOcopies/ ⁇ and 100 copies/ ⁇ of pathogen DNA and FIGURE 2b illustrates the fold change overtime in the secondary amplification.
  • FIGURE 3 illustrates specificity of the method of the present disclosure, wherein FIGURE 3a illustrates output of the induced non-specific amplification in 4 of 8 negative controls during primary amplification, and FIGURE 3b illustrates the output of the secondary amplification, wherein the all 8 negative controls showed only basal signal, demonstrating absence of non-specific amplification detection.
  • FIGURE 4 illustrates the output of the secondary amplification of the present disclosure, detecting CTX-M-15 in about 10 copies/ ⁇ of DNA in a sample.
  • FIGURE 5 illustrates detection of Plasmodium falciparum in crude blood lysate.
  • FIGURE 6 illustrates molecular beacon probe and its mechanism of action during enzyme assisted target recycling.
  • FIGURE 7 illustrates the comparison of the polymerase chain reaction and the method of the present disclosure.
  • FIGURE 8 illustrates the output of the kit with DNA extracted from clinical samples of Plasmodium falciparum. DNA from 10 samples that were confirmed by microscopy and rapid diagnostics were used. All 10 samples showed positive amplification, above a threshold of 40, while the negative controls used did not show any amplification.
  • the present disclosure describes a method for detecting nucleic acid, said method comprises combination of primary amplification and secondary amplification, wherein the primary amplification is nucleic acid amplification and the secondary amplification is signal amplification.
  • the method detects target nucleic said, wherein the method combines primary amplification and secondary amplification.
  • the nucleic acid includes but is not limited to bacterial DNA, bacterial RNA, viral DNA, viral RNA and fungal DNA found in water, food, animal and human.
  • the method of present disclosure is a synergistic combination of nucleic acid amplification and signal amplification, which does not cause non-specific amplification of the nucleic acid, thereby aids in detecting as low as 1 copy/ ⁇ of DNA from the sample. In an embodiment, the method of present disclosure does not lead to false positives during detection of the nucleic acid.
  • the primary amplification described in the method of the present disclosure is target nucleic acid amplification, wherein the primary amplification includes but is not limited to polymerase chain reaction, ligase chain reaction, loop-mediated isothermal amplification, nucleic acid sequence based amplification, simple amplification based assay, helicase dependent amplification and rolling circle amplification.
  • the primary amplification described in the method of the present disclosure is loop mediated isothermal amplification (LAMP).
  • LAMP loop mediated isothermal amplification
  • the secondary amplification described in the method of the present disclosure includes but is not limited to enzyme assisted target recycling (EATR).
  • EATR enzyme assisted target recycling
  • the method for detecting nucleic acid comprises combination of loop mediated isothermal amplification and enzyme assisted target recycling.
  • the method for detecting target nucleic acid comprises combination of loop mediated isothermal amplification and enzyme assisted target recycling.
  • the primary amplification including but not limited to loop mediated isothermal amplification produces copies of target nucleic acid by amplification from a sample in a reaction mixture comprising oligonucleotide primers, enzymes, buffers, nucleoside triphosphate and any other component required for optimal primary amplification.
  • the primary amplification including but not limited to loop mediated isothermal amplification produces copies of target nucleic acid by amplification from a sample in a reaction mixture comprising oligonucleotide primers, enzymes, buffers, nucleoside triphosphate and any other component required for optimal primary amplification and oligonucleotide probes (EATR probes).
  • the enzymes employed during the primary amplification includes but is not limited to Taq polymerases, Bst polymerase-Bst 2.0, Bst polymerase-Bst 3.0, Bst large fragment polymerase, GspSSD LF DNA polymerase (from Optiigene) and OmniAmp (from Lucigen).
  • the buffers employed during the primary amplification includes but is not limited to mixtures containing some salts (chlorides, sulphates or acetates) of magnesium, potassium, or sodium, Tris HCL; DTT, BSA and volume displacement reagents such as PEG.
  • the reagent employed during the primary amplification includes but is not limited to dNTPs in a solution or buffer.
  • the primary implication is performed at temperature ranging from about 25°C to 70°C.
  • the enzymes employed during the loop mediated isothermal amplication includes but is not limited to Bst polymerase-Bst 2.0, Bst polymerase-Bst 3.0, Bst large fragment polymerase, GspSSD LF DNA polymerase (from Optiigene) and OmniAmp (from Lucigen).
  • the buffers employed during loop mediated isothermal amplification incudes but is not limited to salts comprising Mg salts comprising Mb and Tris, potassium salts.
  • the reagent employed during loop mediated isothermal amplification includes but is not limited to dNTPs, or a combination of dATP, DUTP, dGTP, dCTP, and Uracil DNA glycosylase.
  • the secondary amplification including but not limited to enzyme assisted target recycling comprises at least one labelled oligonucleotide probe, buffers, reagents and any other component required for optimal secondary amplification.
  • the enzyme employed during secondary amplification is required to have 3' to 5' exonuclease activity in cleaving double stranded DNA from 3' to 5' direction. And, such enzyme includes but is not limited to exonuclease III.
  • the oligonucleotide probe employed during the secondary amplification includes but is not limited to molecular beacon probe, strand displacement probes, hybridization probe or any probe that can be used in enzyme assisted target recycling.
  • amplicons from the primary amplification including but not limited to loop mediated isothermal amplification (LAMP) are hybridized to molecular beacon probe during secondary amplification including but not limited to enzyme assisted target recycling (EATR), wherein the molecular probe beacon ensure that only specific amplicon from the LAMP are hybridized leading to signal amplification in the secondary amplification.
  • LAMP loop mediated isothermal amplification
  • EATR enzyme assisted target recycling
  • EATR is amenable to multiplexing and overcomes any challenges that is associated with multiplexing with LAMP in the method of the present disclosure, thereby detection of the minimum DNA or low copy number nucleic acid, as low as 1 copy/ ⁇ DNA in the sample. And, the method of the present disclosure is accurate without any non-specific nucleic acid amplification and detection.
  • the primers for the primary amplification are designed in such a way that they always work in conjunction with the oligonucleotide probes of the secondary amplification
  • the amplicons of primary amplification in case of LAMP, are single stranded nucleic acid with several inverted repeats of target nucleic acid and cauliflower like structure formed by annealing between alternatively inverted repeats of the target nucleic acid.
  • the method of detecting the nucleic acid comprises steps of: contacting sample comprising nucleic acid with the nucleic acid amplification reaction mixture, followed by amplifying the nucleic acid to obtain amplicon;
  • the method of detecting the nucleic acid comprises steps of:
  • the cleaved probe produces signal; and hybridizing the released amplicon with another oligonucleotide probe, followed by cleaving the probe by the enzyme to release the amplicon, wherein the cleaved probe produces signal to detect the nucleic acid.
  • the step of hybridizing the amplicon and oligonucleotide probe is repeated plurality of times so that the enzyme can cleave the probes to amplify the signal to detect the nucleic acid.
  • the step of hybridizing the amplicon and oligonucleotide probe is repeated at least one time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times so that the enzyme can cleave the probes to amplify the signal to detect the nucleic acid.
  • the during the method of hybridizing the probe to amplicon cleaving the probe of the hybridized probe and amplicon produces amplification of the signal to will aid in detecting the nucleic acid as of lcopy/ ⁇ of DNA in the sample.
  • the during the method of hybridizing the probe to amplicon cleaving the probe of the hybridized probe and amplicon produces amplification of the signal to will aid in detecting the nucleic acid as of 5 copies/ ⁇ of DNA.
  • the nucleic acid amplification reaction mixture comprises- i. Primer selected from a group comprising outer primer, inner primer, middle primer and loop primer, or any combination thereof;
  • Enzyme selected from a group comprising Taq polymerase, Bst polymerase and other DNA polymerase that supports amplification of nucleic acid
  • Buffer selected from a group comprising magnesium chloride, magnesium sulphate, magnesium acetate, potassium chloride, potassium sulphate, potassium acetate, sodium chloride, sodium sulphate, sodium acetate, Tris HCL and DTT
  • Reagent selected from a group comprising Poly ethylene glycol, solution comprising dNTPs;
  • the primers in the nucleic acid amplification reaction mixture are outer primer, inner primer, middle primer and loop primer.
  • the inner primer is Pf-FIP and Pf-BIP, set forth as SEQ ID No. 1 and SEQ ID No. 2, respectively and the outer primer is Pf-F3 and Pf-B3, set forth as SEQ ID No. 7 and SEQ ID No. 8, respectively,
  • the inner primer is CTXM-FIP and CTXM, set forth as SEQ ID No. 3 and SEQ ID No. 4, respectively, and the outer primer is CTXM-F3 and CTXM-B3, set forth as SEQ ID No. 9 and SEQ ID No. 10, respectively
  • the inner primer is 16S-FIP and 16S-BIP, set forth as SEQ ID No. 5 and SEQ ID No. 6, respectively
  • the outer primer is 16S-F3 and 16S-B3, set forth as SEQ ID No. 11 and SEQ ID No. 12, respectively
  • the loop primer is 16S-LF1 and 16S-LB 1, set forth as SEQ ID No. 13 and SEQ ID No. 14, respectively
  • SEQ ID No.17 16s probe ACGTCATAGGTGGGGATGACGTCAAGTCA
  • the outer primer, middle primer and loop primer has melting temperature of about 55°C to 61°C, respectively.
  • the inner primer has melting temperature of about 62°C to 66°C.
  • the GC content of the outer primer, middle primer, loop primer and inner primer is about 40% to 60%.
  • the amplification of the nucleic acid is carried for a period of about 30 minutes to 90 minutes at temperature ranging from about 60°C to 70°C, and for a period of about 5 minutes to 10 minutes at temperature ranging from about 25°C to 40°C.
  • the amplicon is about 300bp to 400bp.
  • LAMP forward primers are designed in the flanking region within about lbp to 250bp upstream and LAMP backward primers are designed in about lbp to 250bp downstream (ideally should not exceed 150bp). The primers thus designed will produce primary amplicons that provide a template to which the probe will bind.
  • the oligonucleotide probe includes but is not limited to molecular beacon probe, strand displacement probes, hybridization probe or any probe that can be used in enzyme assisted target recycling.
  • the oligonucleotide probe is molecular beacon probe comprising single strand nucleotide in a step-loop hairpin structure having fluorophore and quencher at each end of the strand.
  • the fluorophore employed in the oligonucleotide probe have sufficient spectral overlap to ensure efficient quenching.
  • the fluorophore is selected from a group comprising FAM, HEX, TET, Cy3, Texas Red, ROX, TMR, LC Red 640, LC red 750 and Cy5.
  • the quencher is selected from a group comprising Dabcyl, BHQ1, BHQ2, BHQ3, Iowa Black RQ, Iowa Black FQ, QSY7 and QSY 21.
  • the molecular beacon probe comprises a fluorophore at the 5' end and quencher is at an internal position, at the end of the stem of the molecular beacon with an overhang of about 5 nucleotides to 10 nucleotides, preferably about 7 nucleotides that provides an open 3' end for action of the enzyme (illustrated in figure 6).
  • the molecular beacon is designed to target a specific region of about 22 bases in length of the amplicon, wherein the GC content of the beacon is about 40% to 60%, the melting temperature of stem and loop of the beacon is about 60°C to75°C with loop melting temperature higher than stem to favour amplicon-probe hybridization. Further, the molecular beacon comprises nucleotide T in specific position of 15 th base, nucleotide G not adjacent to fluorophore.
  • the molecular beacon employed in the method does have self-complementarity or low risk of secondary structure.
  • the oligo nucleotide probe is set forth as SEQ ID No. 15 (pf probe), SEQ ID No. 16 (CTXM probe), SEQ ID No. 17 (16s probe), SEQ ID No. 18 (16s probe_l), SEQ ID No. 19 (16s probe_2), SEQ ID No. 20 (16s probe_3) and SEQ ID No. 21 (16s probe_4).
  • the GC content of SEQ ID No. 17 (16s probe), SEQ ID No. 18 (16s probe_l), SEQ ID No. 19 (16s probe_2), SEQ ID No. 20 (16s probe_3) and SEQ ID No. 21 (16s probe_4) is about 51.72%, about 44.83%, about 58.62%, about 48.28% and about 44.83%, respectively.
  • the melting temperature of SEQ ID No. 17 (16s probe), SEQ ID No. 18 (16s probe_l), SEQ ID No. 19 (16s probe_2), SEQ ID No. 20 (16s probe_3) and SEQ ID No. 21 (16s probe_4) is about 71.9°C about 68.9°C, about 74.9°C, about 69.9°C and about 68.9°C, respectively.
  • the stem melting temperature of SEQ ID No. 17 (16s probe), SEQ ID No. 18 (16s probe_l), SEQ ID No. 19 (16s probe_2), SEQ ID No. 20 (16s probe_3) and SEQ ID No. 21 (16s probe_4) is about 20°C about 18°C, about 22°C, about 20°C and about 18°C, respectively.
  • the stem melting temperature of SEQ ID No. 17 (16s probe), SEQ ID No. 18 (16s probe_l), SEQ ID No. 19 (16s probe_2), SEQ ID No. 20 (16s probe_3) and SEQ ID No. 21 (16s probe_4) is about 28°C about 24°C, about 26°C, about 26°C and about 24°C, respectively.
  • hybridizing the amplicon and the probe, followed by cleaving the probe by the enzyme is carried out in buffer and reagent, wherein the buffer is selected from a group comprising magnesium chloride, magnesium sulphate, magnesium acetate, potassium chloride, potassium sulphate, potassium acetate, sodium chloride, sodium sulphate, sodium acetate, Tris HCL and DTT; and the reagent is selected from a group comprising BSA and PEG.
  • the hybridizing the amplicon and the probe, followed by cleaving the probe by the enzyme is carried out in buffer, wherein the buffer is selected from a group comprising magnesium chloride, magnesium sulphate, magnesium acetate, potassium chloride, potassium sulphate, potassium acetate, sodium chloride, sodium sulphate, sodium acetate, Tris HCL and DTT.
  • the buffer is selected from a group comprising magnesium chloride, magnesium sulphate, magnesium acetate, potassium chloride, potassium sulphate, potassium acetate, sodium chloride, sodium sulphate, sodium acetate, Tris HCL and DTT.
  • the hybridizing the amplicon to the probe is carried out at a temperature of about 25°C to 40°C for a period of about 5 minutes to lOminutes. Further, the hybridized amplicon and the probe is contacted with the enzyme and cleaving of the probe by the enzyme is carried out at a temperature of about 25 °C to 40°C, for a period of about 35 minutes to 90 minutes.
  • amplification of the nucleic acid to obtain the amplicon is primary amplification.
  • amplification of the signal by cleaving with the enzyme is secondary amplification.
  • the disclosure further relates to microplate having wells comprising primers, enzymes, reagents, probes and nucleoside triphosphate.
  • the microplate conducts the method of the present disclosure, wherein the microplate is capable of performing primary amplification and secondary amplification for detection of nucleic acid in the sample, wherein the primary amplification includes but is not limited to polymerase chain reaction, ligase chain reaction, loop-mediated isothermal amplification, nucleic acid sequence based amplification, simple amplification based assay, helicase dependent amplification and rolling circle amplification.
  • the primary amplification is loop-mediated isothermal amplification.
  • the secondary amplification includes but is not limited to enzyme assisted target recycling.
  • the microplate of the present disclosure has 6, 12, 24, 48. 96, 384, 1536, 3456 or 9600 wells, wherein the wells in the microplate are well differentiated to accommodate the contents of the primary amplification and secondary amplification to perform the method of the present disclosure to detect nucleic acid in the sample.
  • the wells dedicated for primary amplification comprises primers, enzymes, buffers and nucleoside triphosphates.
  • the primers are dried with or without stabilizing agents such as trehalose or lyophilized.
  • the enzymes are also dried in the presence of stabilizing agent such as trehalose and/or glycerol or alternatively the enzymes are crystallized as a micro crystal and maintained as dry powder.
  • the nucleoside triphosphates are also dried along with enzymes in the presence of stabilizing agent such as trehalose and glycerol.
  • the reagents are also dried in the presence of stabilizing agent such as trehalose.
  • each well in the microplate dedicated for primary amplification comprises primes, enzymes, reagents and nucleoside triphosphates, wherein the primers, enzymes, reagents and nucleoside triphosphate are dried in the presence of stabilizing agent such as trehalose or without any stabilizing agents.
  • the enzymes are crystallized as a microcrystal and added as dry powder.
  • the wells dedicated for secondary amplification comprises probes, enzymes and reagents.
  • the probes are dried in the presence of stabilizing agent such as trehalose or lyophilized or without any stabilizing agent.
  • the enzymes are dried in the presence of stabilizing agent such as trehalose or alternatively the enzymes are crystallized as a micro crystal and maintained as dry powder.
  • the reagents are also dried in the presence of stabilizing agent such as trehalose and glycerol.
  • each well in the microplate is dedicated for secondary amplification comprises probes, enzymes and reagents, wherein the probes, enzymes and reagent are dried in the presence of stabilizing agent such as trehalose or without any stabilizing agent.
  • the enzymes are crystallized as micro crystal and added as powder.
  • the microplate serves as storage of primers, probes, enzymes, buffers and nucleoside triphosphates.
  • predetermined amount of water including but not limited to nuclease free water and double distilled water or buffer or buffer and water is added to the wells and the contents are mixed.
  • the mixed contents are transferred to a tube/vial for the primary amplification, followed by secondary amplification.
  • the microplate is directly used for conducting the primary amplification and secondary amplification as described in the methods of the present disclosure, wherein predetermined amount of water including but not limited nuclease free water and double distilled water or buffer or buffer and water is added to the wells and the contents of the well are mixed.
  • the DNA sample is added and the microplate is placed in an appropriate device capable of supporting nucleic acid amplification, preferably in an isothermal condition.
  • the nucleic acid amplification (primary amplification) is completed, part or all the contents from the well dedicated to primary amplification is transferred manually by technique including but not limited to pipetting, to the wells dedicated to secondary amplification.
  • Predetermined amount of water including but not limited to nuclease free water and double distilled water or buffer and water including but not limited to nuclease free water and double distilled water is added to the wells, followed by mixing to initiate secondary amplification.
  • the present disclosure further relates to a kit comprising microplate having 6, 12, 24, 48, 96, 384, 536, 3456 or 9600 wells dedicated for primary amplification and secondary amplification, optionally along with buffer or water including but not limited to nuclease free water and double distilled water, or both.
  • the microplate in the kit having wells comprises primers, probes, enzymes, reagents and nucleoside triphosphate, capable of conducting primary amplification and secondary amplification, respectively as described in the method of the present disclosure to detect nucleic acid in a sample. Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon the description provided. The embodiments provide various features and advantageous details thereof in the description.
  • EXAMPLE 1 Detection of 16s rRNA gene from bacterial DNA using the methods of the present disclosure.
  • EATR probes (SEQ ID Nos. 17-21) are designed for a conserved region of 16s rRNA of Escherichia coli (E. coli).
  • LAMP primes (SEQ ID Nos. 5, 6, 11, 12, 13 and 14) are designed to flank the EATR probes.
  • a primary amplification is carried out on a mixture of DNA sample from a uropathogenic strain of E. coli and normal (uninfected) human DNA as mock sample representing DNA extracted from urine. Therefore, the amount of human DNA used is about 2500 copies/ ⁇ , representing the amount of DNA present in average normal urine sample.
  • varying amounts of E. coli DNA is spiked (about 10 copies/ ⁇ , about lOOcopies/ ⁇ and about lOOOcopies/ ⁇ ) representing low to high levels of infection.
  • Figure 2a illustrates the output from the primary amplification using negative control (no DNA-NTC), about 10 copies/ ⁇ and about 100 copies/ ⁇ .
  • EATR secondary amplification
  • LAMP primary amplification
  • EATR secondary amplification
  • lOnm to lOOnm probe about 4 units to 16 units of exonuclease III enzyme and buffer containing Tris-HCL, Bis-Propane, MgCl 2 , DTT is added, thereafter water is added to make up volume to lOOul.
  • Figure 2b illustrates the fold change overtime in the secondary amplification with EATR.
  • Inference Primary amplification for about 1 hour shows specific amplification in the samples containing DNA. In the negative controls, either no amplification or non-specific amplification is seen. However, during secondary amplification, only the specific amplicons from the primary amplification are amplified and this leads to no non-specific amplification. Moreover, in about 20 minutes of secondary amplification, peak fold change is achieved, thereby effectively detecting the DNA in the sample.
  • EXAMPLE 2 Illustrating the synergistic effect of combination of Primary amplification with Secondary amplification.
  • the mock urine sample is created as described in Example 1.
  • Non-specific amplification (primary amplification) is induced in 4 of the 8 negative controls by increasing the concentration of Mg in the buffer mixture, which is known to cause nonspecific amplification.
  • Non-specific amplification, which can occur at random is also observed in 1 of the 4 negative controls in which the Mg is at optimal concentration.
  • EXAMPLE 3 Detection of antimicrobial resistance genes by the method of the present disclosure.
  • EATR probe (SEQ ID No. 16) is designed for a conserved region of CTX-M-15 gene, which is responsible for resistance of beta lactam antibiotics such as cephalosporins. CTX-M-15 has been implicated in antibiotic resistance of E. coli and many other organisms. LAMP primers (SEQ ID Nos. 3, 4, 9 and 10) are designed to flank the EATR probe target region.
  • a primary amplification is carried out on DNA extracted from a uropathogenic strain of E. coli confirmed through PCR and sequencing to carry the CTX-M-15 gene.
  • the DNA from the E. colistrain and normal (uninfected) human DNA are mixed together to serve as mock samples representing DNA extracted from urine. Therefore, the amount of human DNA used is about 2500 copes/ ⁇ , representing the amount of DNA present in average normal urine samples.
  • varying amounts of E. coli DNA is spiked (about 10 copies/ ⁇ and about lOOOcopies/ ⁇ ) representing low to high levels of infection.
  • Figure 4 illustrates the result of the experiment, wherein the combination of primary amplification (LAMP) and secondary amplification (EATR) is clearly able to detect CTX-M- 15 in a sample having as low as about 10 copies/ ⁇ of DNA.
  • LAMP primary amplification
  • EATR secondary amplification
  • the detected 10 copies/ ⁇ would mean that the detected DNA is much lower than the copies of plasmid DNA carrying the CTX- M-15 gene, inferring that the method of the present disclosure is effectively sensitive and accurate.
  • EXAMPLE 4 Detection of Plasmodium falciparum in crude blood lysate by the method of the present disclosure.
  • EATR probe SEQ ID No. 15 and LAMP primers: SEQ ID No. 1, 2, 7 and 8, were designed and employed in this experiment.
  • About 180 ⁇ 1 of whole blood and 180 ⁇ 1 of extraction buffer are mixed together by vortexing for about 10 seconds and boiled at a temperature of about 95 °C for about 5 minutes.
  • Samples are centrifuged at room temperature for about 3 minutes at about 10,000g rpm and clear supernatant of about 200 ⁇ 1 is taken out.
  • To the supernatant various quantities of P.falciparum DNA (about Scopies/ ⁇ , 50 copies/ ⁇ and 100 copies/ ⁇ ) is added, respectively.
  • the sample with P.falciparum DNA is subject to primary amplification and secondary amplification.
  • Negative control (NTC), positive control and a comparison with pure DNA mix of about 5 copies/ ⁇ of P.falciparum and human DNA are included in the method.
  • Figure 5 illustrates that the method of the present disclosure is able to detect P '.falciparum in crude lysate having as low as 5 copies/ ⁇ P '.falciparum DNA.
  • EXAMPLE 5 Comparison of method of the present disclosure and polymerase chain reaction for detecting nucleic acid.
  • DNA was extracted using Qiagen miniAMP DNA kit and stored in Eppendorf tubes at room temperature. A quantity of 2 to 5 ⁇ 1 of DNA was used for PCR and similar quantity was used in the dual amplification of LAMP-EATR. PCR was carried out with the outer primers (Pf-F3 and Pf-B3) of LAMP and the limit of detection was 100 copies/ul. In the dual amplification, 10 copies/ul were detected, illustrated in figure 7. This clearly demonstrated the value of the dual amplification over PCR. Dual amplification with LAMP-EATR prevents the nonspecific amplification.
  • the DNA extracted from about 10 malarial samples was subjected to the combination of the primary amplification and secondary amplification described in the present disclosure, illustrated in figure 8
  • EXAMPLE 6 Illustration of pre-loading components for secondary Amplification.
  • Pre-loading of probes, reagents and enzymes for secondary amplification is done by drying under vacuum conditions in the presence of stabilizers such as trehalose.
  • Trehalose is known to impart stability to DNA probes such as Taqman probes and enzymes.
  • the EATR molecular beacon probe is dried under vacuum in the presence of varying amount of trehalose. With about 0.67mM trehalose, the probe is stable for about 25 days at 27 °C. However, with accelerated testing the probe is stable for about 6 days at 47°C. Table 1 illustrates the effect of varied concentration of trehalose on stability of the probes.
  • Table 1 Effect of varied concentration of Trehalose on stability of probe.
  • Microplate was prepared in a polymer material (comprising of one of acrylic or polypropylene). A block of the material measuring in thickness (2mm or 5mm or 10mm) and a width of 1cm. Wells were drilled and CNC machined for a smooth finish or laser cut. Well dimensions were determined to hold about ⁇ to 25 ⁇ 1 LAMP contents such as enzyme, buffer, primers and reagents and about ⁇ to 200 ⁇ 1 EATR contents such as oligonucleotide probe, enzyme, buffer. In each row, well for LAMP and EATR were made to ensure ease of transfer.
  • LAMP contents such as enzyme, buffer, primers and reagents
  • EATR contents such as oligonucleotide probe, enzyme, buffer. In each row, well for LAMP and EATR were made to ensure ease of transfer.
  • EATR master mix for LAMP was prepared as a solution or in dehydrated or lyophilized format and added to the LAMP well.
  • EATR master mix was prepared as a solution or in dehydrated or lyophilized format and added to the EATR well
  • Microplate was then covered with a detachable cover plated and stored in a sealed container with silica gel at room temperature or at 4°C, along with buffer or water or both.

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Abstract

La présente invention concerne un procédé de détection d'acides nucléiques dans un échantillon, ledit procédé comprenant une combinaison d'amplification primaire et d'amplification secondaire. L'invention concerne en outre un kit comprenant une microplaque et un tampon et/ou de l'eau. Ledit procédé et ledit kit de la présente invention détectent des acides nucléiques dans un échantillon sans aucune amplification non spécifique, permettant ainsi un résultat sans faux positifs. Ledit procédé et ledit kit de la présente invention permettent une détection très sensible et précise des acides nucléiques dans un échantillon.
PCT/IB2017/057387 2016-11-25 2017-11-24 Procédé, kit et application associée Ceased WO2018096496A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022033331A1 (fr) * 2020-08-11 2022-02-17 上海奕谱生物科技有限公司 Kit et procédé de détection d'acide nucléique et leurs utilisations
CN116694804A (zh) * 2023-06-16 2023-09-05 浙江大学 用于检测小麦禾谷镰孢菌的lamp引物探针组、试剂盒及其检测方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GERASIMOVA YV ET AL.: "Enzyme-assisted target recycling (EATR) for nucleic acid detection", CHEMICAL SOCIETY REVIEWS, vol. 43, no. 17, 2014, pages 6405 - 38, XP055324226, Retrieved from the Internet <URL:doi:10.1039/c4cs00083h> *
NAGDEV KJ ET AL.: "Loop-mediated isothermal amplification for rapid and reliable diagnosis of tuberculous meningitis", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 49, no. 5, 1 May 2011 (2011-05-01), pages 1861 - 5, XP055487910, Retrieved from the Internet <URL:doi:10.1128/JCM.00824-10> *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022033331A1 (fr) * 2020-08-11 2022-02-17 上海奕谱生物科技有限公司 Kit et procédé de détection d'acide nucléique et leurs utilisations
CN116694804A (zh) * 2023-06-16 2023-09-05 浙江大学 用于检测小麦禾谷镰孢菌的lamp引物探针组、试剂盒及其检测方法
CN116694804B (zh) * 2023-06-16 2024-02-02 浙江大学 用于检测小麦禾谷镰孢菌的lamp引物探针组、试剂盒及其检测方法

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