WO2011087782A2 - Compositions, trousses et procédés pour la détection de trypanosoma cruzi à l'aide d'une amplification isotherme médiée par boucle - Google Patents

Compositions, trousses et procédés pour la détection de trypanosoma cruzi à l'aide d'une amplification isotherme médiée par boucle Download PDF

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WO2011087782A2
WO2011087782A2 PCT/US2010/061500 US2010061500W WO2011087782A2 WO 2011087782 A2 WO2011087782 A2 WO 2011087782A2 US 2010061500 W US2010061500 W US 2010061500W WO 2011087782 A2 WO2011087782 A2 WO 2011087782A2
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Prior art keywords
primer
seq
target sequence
composition
fip
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PCT/US2010/061500
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WO2011087782A8 (fr
WO2011087782A3 (fr
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Yves Carlier
Eric Dumonteil
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The Administrators Of The Tulane Educational Fund
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Publication of WO2011087782A2 publication Critical patent/WO2011087782A2/fr
Publication of WO2011087782A3 publication Critical patent/WO2011087782A3/fr
Publication of WO2011087782A8 publication Critical patent/WO2011087782A8/fr

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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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • compositions, kits, and methods for detecting Trypanosoma cruzi using loop- mediated isothermal amplification (LAMP) are disclosed herein.
  • Chagas disease is a cause of morbidity and mortality in the Americas and is prevalent in Mexico, Central and South America where between eight and eleven million people are infected with the parasite, Trypanosoma cruzi.
  • the Pan American Health Organization estimates that 7.7 million people are currently infected in 21 endemic countries in Latin America, while, numerous infected people are also living in US, Europe, Japan and Australia, due to large scale migrations of Latin Americans over the last decades. Many of those infected are unaware of the infection due to the non-specific, usually mild symptoms with acute infection and the long latency period before the appearance of chronic Chagas disease.
  • Blood donations are routinely tested for T. cruzi in the United States, most Latin American countries, and some European countries to prevent transmission of the disease through diseased blood. Current testing in the clinical setting recommends the use of two tests and laboratory analysis. This means that a patient cannot be tested and receive results in the same visit to the doctor. This can hinder both diagnosis and treatment for those infected.
  • the current standard screening method for blood donation in the U.S. is the Ortho T. cruzi ELISA Test System, a serological screen that can detect human antibodies against the organism with an observed sensitivity of 99-100% and an estimated cost of $5-$9 per sample.
  • Serological assays are a screening method for blood donation in Central and South America and are also a common diagnostic technique. Serological tests can cost as little as $2.
  • Other screening procedures that test directly for the presence of T. cruzi DNA are generally polymerase chain reaction (PCR) based and are not widely used in clinical settings.
  • Some embodiments of the present invention can address one or more of these drawbacks.
  • some methods of the present invention can be specific and sensitive, so that a single test would be sufficient for diagnosis. Because, in some instances, the method requires only an hour or less to process, an accurate diagnosis may be obtained in a single visit to a health center.
  • blood samples from certain patient groups would no longer have to be analyzed by specialized labs, so the costs associated with diagnosis would decrease.
  • Embodiments of the method can be highly sensitive and thus could allow physicians to accurately monitor whether a patient is responding to a certain treatment; this would decrease the length of the process, resulting in more patients seeking treatment.
  • the method would also allow for more efficient research and development of new treatments for Chagas disease.
  • the method might be less expensive than PCR methods; and in some instances, there is no need for thermocycler machines.
  • the cost of LAMP diagnostics can be reduced, in some instances, to approximately $ 1 per test or cheaper.
  • Some embodiments of the present invention include a composition comprising an FIP primer from a target sequence, a BIP primer from the target sequence, an F3 primer from the target sequence, and a B3 primer from the target sequence, wherein the target sequence is from a sequence of T. cruzi.
  • Other embodiments of the composition further comprise an LB primer from the target sequence and an LF primer from the target sequence.
  • the target sequence is selected from the group consisting of a sequence unique to Trypanosoma species, a sequence unique to T. cruzi, SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3.
  • the target sequence length can be less than about 500 nucleotides.
  • the target sequence is SEQ ID NO: l
  • the F3 primer is SEQ ID NO:4, the B3 primer is SEQ ID NO:5, the FIP primer is SEQ ID NO:6, and the BIP primer is SEQ ID NO:7.
  • the target sequence is SEQ ID NO:2, the F3 primer is SEQ ID NO:4, the B3 primer is SEQ ID NO:5, the FIP primer is SEQ ID NO:6, and the BIP primer is SEQ ID NO:7.
  • the target sequence is SEQ ID NO:3, the F3 primer is SEQ ID NO: 10, the B3 primer is SEQ ID NO: 11, the FIP primer is SEQ ID NO: 12, and the BIP primer is SEQ ID NO: 13.
  • the target sequence is SEQ ID NO: l
  • the F3 primer is SEQ ID NO:4, the B3 primer is SEQ ID NO:5, the FIP primer is SEQ ID NO:6, the BIP primer is SEQ ID NO:7
  • the LF primer is SEQ ID NO:8, and the LB primer is SEQ ID NO:9.
  • the target sequence is SEQ ID NO:2, the F3 primer is SEQ ID NO:4, the B3 primer is SEQ ID NO:5, the FIP primer is SEQ ID NO:6, the BIP primer is SEQ ID NO:7, the LF primer is SEQ ID NO:8, and the LB primer is SEQ ID NO:9.
  • the target sequence is SEQ ID NO:3
  • the F3 primer is SEQ ID NO: 10
  • the B3 primer is SEQ ID NO: 11
  • the FIP primer is SEQ ID NO: 12
  • the BIP primer is SEQ ID NO: 13
  • the LF primer is SEQ ID NO: 14
  • the LB primer is SEQ ID NO: 15.
  • the concentration of the FIP primer and the concentration of the BIP primer are the same or different and are each from about 0.5 ⁇ to about 3.0 ⁇ , or the concentration of the F3 primer and the concentration of the B3 primer are the same or different and are each from about 0.05 ⁇ to about 1.0 ⁇ , or the concentration of the LF primer and the concentration of the LB primer are the same or different and are each from about 0.1 ⁇ to about 3.0 ⁇ .
  • the polymerase is a DNA polymerase at a concentration from about 1U to about 20U.
  • the composition comprises dNTPs, the concentration of each can be the same or different and are each at a concentration from about 100 ⁇ to about 2.5 mM.
  • Still other embodiments of the composition further comprise at least one helix destabilizer (e.g., from about 0.1 M betaine to about 2.0 M betaine).
  • kits comprising the ingredients of the composition as described above in the first paragraph of the SUMMARY section.
  • the kit is such that (a) the primers are each in separate containers, (b) the LIF primer and the BIF primer are in a single container, (c) the L3 primer and B3 primer are in a single container, or (d) the primers are together in a single container.
  • Still other embodiments of the invention include methods for determining the presence of T. cruzi in a sample comprising (a) maintaining an amplifying solution at a temperature to provide an amplified solution and (b) determining the presence of T. cruzi in the sample by detecting the presence or absence of amplification product in the amplified solution.
  • the amplifying solution comprises the sample, an FIP primer from a target sequence, a BIP primer from the target sequence, an F3 primer from the target sequence, a B3 primer from the target sequence, and a polymerase.
  • the target sequence is from a sequence of T. cruzi.
  • the amplifying solution further comprises an LB primer from the target sequence and an LF primer from the target sequence.
  • the amplifying solution comprises the composition as described above in the first paragraph of the SUMMARY section.
  • the sample is a specimen obtained from blood, serum, or blood plasma.
  • the sample comprises at least about 6 copies of the target sequence.
  • the temperature is from about 50°C to about 70°C.
  • Further embodiments of the invention include detecting that comprises adding DNA binding dye (e.g., SYBR green) to the amplified solution followed by visually observing the amplified solution.
  • DNA binding dye e.g., SYBR green
  • Figure 1 An illustrative depiction of an embodiment of region and primer design in the LAMP method with four primers.
  • (B) An illustrative depiction of an embodiment of region and primer design in the LAMP method with six primers.
  • Figure 2. Agarose gel electrophoresis of amplified solutions where the amplifying solution included (+) or did not include (-) T. cruzi DNA. Lane 1 shows molecular weight marker (size in base pair); lanes 2 and 3 show reactions run with primers F3, B3, FIP, and BIP; lanes 4 and 5 show reactions run with primers F3, B3, FIP, BIP, LF, and LB.
  • FIG. 3 Agarose gel electrophoresis of products from PCR (left) and LAMP (right) using dilutions of T. cruzi DNA in the PCR reaction solutions and amplifying solutions, respectively.
  • Figure 4 The visual observation of a positive (left - showing detection of amplification products) and negative (right - showing no detection of amplification product) amplification products after addition of SYBR to the amplified solution in the tube.
  • Figure 5. Agarose gel electrophoresis of amplified solutions where the amplifying solution included (+) or did not include (-) T. cruzi DNA. Lane 1 shows molecular weight marker (size in base pair); lanes 2 and 3 show reactions run with (+) and without (-) T. cruzi DNA, respectively.
  • FIG. Agarose gel electrophoresis of samples from LAMP (left) and PCR (right) using dilutions of T. cruzi DNA in the LAMP amplifying solutions and in the PCR reaction solutions, respectively.
  • Embodiments of the invention include a method to determine the presence of the human pathogen Trypanosoma cruzi using loop-mediated isothermal amplification.
  • the method uses loop-mediated isothermal amplification (LAMP) performed at constant temperature (e.g., 65° C), thus eliminating the need for the thermocycling required for conventional PCR.
  • LAMP loop-mediated isothermal amplification
  • the method relies on auto-cycling strand displacement DNA synthesis, performed with a DNA polymerase with strand displacement activity and a designed set of four primers which may displace one another; in other instances, an additional two primers can form hybridized stem-loops with various stem lengths and cauliflower-like structures, which can increase the amount of DNA synthesized.
  • the use of four to six primers can sometimes provide the method with a high specificity compared to PCR, and the large amount of DNA produced can sometimes increase sensitivity beyond that of PCR.
  • the primers are specific to T. cruzi, and thus there is no cross reactivity with other parasites in the Trypanosoma family or with human DNA.
  • the method in some embodiments, can allow for visual confirmation of the determination through the addition of DNA binding dye (e.g., SYBR Green) at the end of the reaction.
  • SYBR Green binds to the T. cruzi DNA and turns fluorescent green to the naked eye if sufficient T. cruzi DNA is present, minimizing the analysis required to determine the presence of T. cruzi in the sample.
  • Some embodiments of the invention include methods for determining the presence of T. cruzi in a sample comprising (a) maintaining an amplifying solution at a temperature to provide an amplified solution and (b) determining the presence of T. cruzi in the sample by detecting the presence of amplification product in the amplified solution.
  • the amplifying solution is heated (e.g., to denature DNA) and cooled (e.g., rapidly cooled on ice) prior to step (a).
  • the amplifying solution can comprise the sample, an FIP primer from a target sequence, a BIP primer from the target sequence, an F3 primer from the target sequence, a B3 primer from the target sequence, and a polymerase.
  • the amplified solution can change composition after step (a) and prior to step (b), if, for example, there are steps intermediate to step (a) and step (b) (e.g., if step (b) is not performed immediately after step (a)).
  • the amplified solution will comprise no detectable amplification product, such as but not limited to the circumstance where T. cruzi is not present in the sample.
  • the amplification product comprises at least one variety or form of the target sequence and portions thereof, such as, but not limited to, single-stranded DNA, double-stranded DNA, structures that have both single- stranded DNA and double-stranded DNA, stem-loop DNA with various stem lengths, and cauliflower-like structures with multiple loops.
  • the target sequence can be any suitable sequence, including but not limited to, a nucleotide sequence from T. cruzi, a nucleotide sequence unique to
  • Trypanosoma species a nucleotide sequence unique to T. cruzi, the DNA sequence of the Tc24 gene (GeneBank Accession Number AF 192980.2) (i.e., SEQ ID NO: 1) or a portion thereof (e.g., SEQ ID NO:2), or the DNA sequence of the 195 bp satellite repeat (GeneBank Accession Number AY520034) (i.e., SEQ ID NO:3) or a portion thereof.
  • the target sequence can be any suitable length, including but not limited to about 100 nucleotides, about 120 nucleotides, about 140 nucleotides, about 160 nucleotides, about 180 nucleotides, about 200 nucleotides, about 220 nucleotides, about 240 nucleotides, about 260 nucleotides, about 280 nucleotides, about 300 nucleotides, about 350 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, or about 800 nucleotides.
  • the target sequence can include one or more restriction sites.
  • the FIP primer, BIP primer, F3 primer, and B3 primer (and optionally both the LF primer and the LB primer) can be designed from the target sequence, as discussed herein.
  • the target sequence comprises (1) in the 5' to 3' direction, an F3 region, an F2 region, and an Fl region and (2) in the 3 ' to 5' direction, a B3c region, a B2c region, and a B lc region.
  • Figure 1A One embodiment of this design is disclosed in Figure 1A.
  • the regions can be separated from each another by additional nucleotides or not;
  • Figure 1 A shows an embodiment where all regions are separated by additional nucleotides.
  • the Fl region and the B lc region has the most separation between them.
  • four types of primers are used, as illustrated in Figure 1A.
  • the FIP primer comprises, in the 5' to 3 ' direction the F lc region (the complement of the Fl region) and the F2 region; in some embodiments the Flc region and the F2 region are separated by additional nucleotides.
  • the BIP primer comprises, in the 5' to 3 ' direction, the Blc region and the B2 region (the complement of B2c region); in some embodiments the Blc region and the B2 region are separated by additional nucleotides.
  • the other two primers are (1) the F3 primer which has the sequence of the F3 region and (2) the B3 primer which has the sequence complimentary to the B3c region.
  • One or more adjacent regions of the four primer design can be separated from each other by additional nucleotides or not.
  • Figure 1A shows an embodiment where all adjacent regions are separated by additional nucleotides.
  • two primers are used in addition to the inner primers and the outer primers discussed above.
  • the loop primers are referred to as the LF primer and the LB primer.
  • the LF primer comprises at least a portion of the complimentary nucleotide sequence between the F 1 region and the F2 region.
  • the LB primer comprises at least a portion of the nucleotide sequence between the Blc region and the B2c region.
  • One or more adjacent regions of the six primer design can be separated from each other by additional nucleotides or not.
  • Figure IB shows an embodiment where all adjacent regions are separated by additional nucleotides.
  • the length of the FIP primer, the BIP primer, the F3 primer, the B3 primer, the LF primer, and the LB primer can be the same or different and can be any suitable length including but not limited to, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, not more than 80 nucleotides, not more than 70 nucleotides, not more than 60 nucleotides, not more than 50 nucleotides, not more than 40 nucleotides, not more than 30 nucleotides, or not more than 20 nucleotides.
  • One or more primers can be chemically synthesized or naturally occurring. Each primer may be a single oligonucleotide or a mixture of a plurality of oligonucleotides.
  • the number of nucleotides between the F2 region and the F l region on the target sequence can be any suitable number, including but not limited to, at least about 10 nucleotides, at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, no more than about 90 nucleotides, no more than about 80 nucleotides, no more than about 70 nucleotides, no more than about 60 nucleotides, no more than about 40 nucleotides, no more than about 30, no more than about 20, or no more than about 10 nucleotides.
  • the number of nucleotides between the Blc region and the B2c region on the target sequence can be any suitable number, including but not limited to, at least about 10 nucleotides, at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, no more than about 90 nucleotides, no more than about 80 nucleotides, no more than about 70 nucleotides, no more than about 60 nucleotides, no more than about 40 nucleotides, no more than about 30, no more than about 20, or no more than about 10 nucleotides.
  • the target sequence is SEQ ID NO: l
  • the F3 primer is SEQ ID NO:4
  • the B3 primer is SEQ ID NO:5, the FIP primer is SEQ ID NO:6, and the BIP primer is SEQ ID NO:7.
  • the target sequence is SEQ ID NO:2, the F3 primer is SEQ ID NO:4, the B3 primer is SEQ ID NO:5, the FIP primer is SEQ ID NO:6, and the BIP primer is SEQ ID NO:7.
  • the target sequence is SEQ ID NO:3
  • the F3 primer is SEQ ID NO: 10
  • the B3 primer is SEQ ID NO: 11
  • the FIP primer is SEQ ID NO: 12
  • the BIP primer is SEQ ID NO: 13.
  • the target sequence is SEQ ID NO: l
  • the F3 primer is SEQ ID NO:4, the B3 primer is SEQ ID NO:5, the FIP primer is SEQ ID NO:6, the BIP primer is SEQ ID NO:7
  • the LF primer is SEQ ID NO:8, and the LB primer is SEQ ID NO:9.
  • the target sequence is SEQ ID NO:2, the F3 primer is SEQ ID NO:4, the B3 primer is SEQ ID NO:5, the FIP primer is SEQ ID NO:6, the BIP primer is SEQ ID NO:7, the LF primer is SEQ ID NO:8, and the LB primer is SEQ ID NO:9.
  • the target sequence is SEQ ID NO:3
  • the F3 primer is SEQ ID NO: 10
  • the B3 primer is SEQ ID NO: 11
  • the FIP primer is SEQ ID NO: 12
  • the BIP primer is SEQ ID NO: 13
  • the LF primer is SEQ ID NO: 14
  • the LB primer is SEQ ID NO: 15.
  • the concentration of the FIP primer, the BIP primer, the F3 primer, the B3 primer, the LB primer, and the LF primer can be the same or different.
  • the concentration of the FIP primer or the BIP primer can be the same or different and can be any suitable concentration including but not limited to about 0.01 ⁇ , about 0.05 ⁇ , about 0.1 ⁇ , about 0.15 ⁇ , about 0.2 ⁇ , about 0.25 ⁇ , about 0.3 ⁇ , about 0.35 ⁇ , about 0.4 ⁇ , about 0.5 ⁇ , about 0.6 ⁇ , about 0.7 ⁇ , about 0.8 ⁇ , about 0.9 ⁇ , about 1.0 ⁇ , about 1.2 ⁇ , about 1.4 ⁇ , about 1.6 ⁇ , about 1.8 ⁇ , about 2.0 ⁇ , about 2.5 ⁇ , about 3.0 ⁇ , about 3.5 ⁇ , about 4.0 ⁇ , about 5.0 ⁇ , about 6.0 ⁇ , about 7.0 ⁇ , about 8.0 ⁇ , about 9.0 ⁇ , or about 10.0
  • the concentration of the F3 primer or the B3 primer can be the same or different and can be any suitable concentration including but not limited to about 0.01 ⁇ , about 0.05 ⁇ , about 0.1 ⁇ , about 0.15 ⁇ , about 0.2 ⁇ , about 0.25 ⁇ , about 0.3 ⁇ , about 0.35 ⁇ , about 0.4 ⁇ , about 0.5 ⁇ , about 0.6 ⁇ , about 0.7 ⁇ , about 0.8 ⁇ , about 0.9 ⁇ , about 1.0 ⁇ , about 1.2 ⁇ , about 1.4 ⁇ , about 1.6 ⁇ , about 1.8 ⁇ , about 2.0 ⁇ , about 2.5 ⁇ , about 3.0 ⁇ , about 3.5 ⁇ , about 4.0 ⁇ , about 5.0 ⁇ , about 6.0 ⁇ , about 7.0 ⁇ , about 8.0 ⁇ , about 9.0 ⁇ , or about 10.0 ⁇ .
  • the concentration of the LF primer or the LB primer can be the same or different and can be any suitable concentration including but not limited to about 0.01 ⁇ , about 0.05 ⁇ , about 0.1 ⁇ , about 0.15 ⁇ , about 0.2 ⁇ , about 0.25 ⁇ , about 0.3 ⁇ , about 0.35 ⁇ , about 0.4 ⁇ , about 0.5 ⁇ , about 0.6 ⁇ , about 0.7 ⁇ , about 0.8 ⁇ , about 0.9 ⁇ , about 1.0 ⁇ , about 1.2 ⁇ , about 1.4 ⁇ , about 1.6 ⁇ , about 1.8 ⁇ , about 2.0 ⁇ , about 2.5 ⁇ , about 3.0 ⁇ , about 3.5 ⁇ , about 4.0 ⁇ , about 5.0 ⁇ , about 6.0 ⁇ , about 7.0 ⁇ , about 8.0 ⁇ , about 9.0 ⁇ , or about 10.0 ⁇ .
  • the ratio of the F3 primer concentration to the FIP primer concentration can be any suitable ratio, including but not limited to, about 0.01, about 0.05, about 0.10, about 0.15, about 0.20, about 0.25, about 0.3, about 0.4, or about 0.5.
  • the ratio of the B3 primer concentration to the BIP primer concentration can be any suitable ratio, including but not limited to, about 0.01, about 0.05, about 0.10, about 0.15, about 0.20, about 0.25, about 0.3, about 0.4, or about 0.5.
  • the F2 sequence and the B2 sequence are chosen so that their melting temperature (T m ) is within the optimal range of the polymerase used (e.g., T m can be about 60 °C to about 65 °C for Bst polymerase).
  • T m of the Flc sequence and of the B lc sequence can be set higher than the T m of the F2 sequence and of the B2 sequence; this can aid in more rapid stabilization of the loop structure.
  • the T m of the F3 sequence and of the B3 sequence can be set lower than that of the T m of the F2 sequence and of the B2 sequence, respectively, which can sometimes start the synthesis from the inner primers before the synthesis of the outer primers.
  • the DNA polymerase used in some embodiments of the present invention can be, but is not limited to, Bst polymerase, 5caBest DNA polymerase, Z-Taq DNA polymerase, Bsm DNA polymerase, or the Klenow fragment of E. Coli DNA polymerase I.
  • the concentration of the DNA polymerase can be any suitable concentration including but not limited to about 1U, about 2U, about 3U, about 4U, about 5U, about 6U, about 7U, about 8U, about 9U, about 10U, about 12U, about 14U, about 16U, about 18U, or about 20U.
  • the amplifying solution can include deoxyribonucleotide triphosphates (dNTPs).
  • each of the four dNTPs can be the same or different and can be any suitable concentration including but not limited to about 50 ⁇ , about 100 ⁇ , about 200 ⁇ , about 300 ⁇ , about 350 ⁇ , about 400 ⁇ , about 450 ⁇ , about 500 ⁇ , about 550 ⁇ , about 600 ⁇ , about 700 ⁇ , about 800 ⁇ , about 900 ⁇ , or about 1.0 mM.
  • the total concentration of the dNTPs can be any suitable concentration including but not limited to about 1.0 mM, about 1.2 mM, about 1.4 mM, about 1.6 mM, about 1.8 mM, about 2.0 mM, about 2.4 mM, about 2.8 mM, about 3.2 mM, about 3.6 mM, or about 4 mM.
  • at least one DNA helix destabilizer can be included in the amplifying solution to, for example, increase amplification efficiency. Any suitable DNA helix destabilizer can be used, including, but not limited to, betaine, L-proline, formamide, trehalose, or combinations thereof.
  • the concentration of one or more DNA helix destabilizers can be any suitable concentration, including but not limited to about 0.05 M, about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1.0 M, about 1.1 M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5 M, about 1.6 M, about 1.7 M, about 1.8 M, about 1.9 M, about 2.0 M, about 2.5 M, or about 3.0 M.
  • the sample can be any suitable specimen obtained from humans or other animals suspected of having Chagas disease.
  • the specimen can include, but is not limited to, blood, serum, blood plasma, spinal fluid, saliva, urine, stool, and tissue.
  • the sample in some embodiments can be a specimen or a pretreated specimen. Pretreatment of the specimen can include, but is not limited to, separation, extraction, concentration, purification, or combinations thereof.
  • the sample comprises a suitable number of copies of the target sequence, including but not limited to at least about 2 copies, at least about 4 copies, at least about 6 copies, at least about 10 copies, at least about 30 copies, at least about 60 copies, at least about 100 copies, at least about 200 copies, at least about 300 copies, at least about 600 copies, at least about 1000 copies, at least about 3000 copies, or at least about 6000 copies.
  • the amount of target sequence can be any suitable amount including but not limited to about 0.02 pg, about 0.1 pg, about 0.2 pg, about 1.0 pg, about 2.0 pg, about 20 pg, about 0.2 ng, about 2.0 ng, about 20 ng, about 0.2 mg, or at least about 2.0 mg.
  • the sample in some instances, can comprise nucleic acid (e.g., DNA) that is not associated with the target sequence.
  • the temperature of the amplifying solution can be maintained during the LAMP method.
  • maintaining the temperature can include any suitable maintenance method including keeping the temperature fixed or allowing the temperature to vary within a certain temperature range.
  • the temperature can be maintained at any suitable temperature including but not limited to about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, or about 75°C.
  • the temperature can be maintained within any suitable range including but not limited to about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about 10°C.
  • the amplifying time can be any suitable time including but not limited to about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, or about 120 minutes.
  • the reaction can be stopped or substantially slowed by any suitable method including but not limited to increasing the temperature to about 80°C for about 2 minutes, about 5 minutes, about 10 minutes, or about 15 minutes.
  • the methods used to detect the presence or absence of amplification product can be any suitable detection method, including but not limited to, visual inspection by eye, electrophoresis techniques, and UV absorption techniques. Visual inspection can be, for example, an observance of precipitation. In some instances, visual inspection can be done with the addition of a DNA binding dye (e.g., SYBR green I, SYBR Green II, SYBR Gold, Oxazole Yellow, Thiazole Orange, EvaGreen, or PicoGreen).
  • a DNA binding dye e.g., SYBR green I, SYBR Green II, SYBR Gold, Oxazole Yellow, Thiazole Orange, EvaGreen, or PicoGreen.
  • Electrophoresis techniques include, but are not limited to methods that use ID electrophoresis (e.g., agarose gel with a suitable dye such as ethidium bromide), methods that use 2D electrophoresis, molecular weight determination, restriction analysis, or Southern blots.
  • ID electrophoresis e.g., agarose gel with a suitable dye such as ethidium bromide
  • digestion of target sequence restriction sites in the amplification product can be performed to help detect amplification products or to determine the identity of some portions of the amplification product.
  • the digestion can be done in conjunction with the electrophoresis techniques to determine the identity of some portions of the amplification product.
  • Some embodiments of the present invention include compositions comprising an FIP primer, a BIP primer, an F3 primer, and a B3 primer.
  • compositions further comprise an LF primer and an LB primer.
  • compositions of the composition further comprise a DNA polymerase.
  • composition further comprises a DNA binding dye.
  • composition further comprise at least one DNA helix destabilizer.
  • composition further comprise deoxyribonucleotide triphosphates (dNTPs).
  • kits for detecting T. cruzi comprising an FIP primer, a BIP primer, an F3 primer, a B3 primer, and a DNA polymerase.
  • the kit further comprises a DNA binding dye (e.g., in a separate container).
  • Other embodiments of the kit further comprise an LF primer and an LB primer.
  • Still other embodiments of the kit further comprise at least one DNA helix destabilizer (e.g., in a separate container).
  • kits further comprise deoxyribonucleotide triphosphates (dNTPs)(e.g., in a separate container).
  • dNTPs deoxyribonucleotide triphosphates
  • kits include any suitable apparatus to heat the amplifying solution, including but not limited to an oven, a heating block, or an activatable container to produce heat (e.g., by mixing two or more chemicals within the activatable container).
  • the ingredients of the kit can be pre- mixed, in separate containers, or combinations thereof.
  • the primers are each in separate containers, (b) the LIF primer and the BIF primer are in a single container, (c) the L3 primer and B3 primer are in a single container, (d) all primers are together in a single container, or (e) applicable combinations thereof.
  • Example 1 - T. cruzi Tc24 gene [0048]
  • the target sequence in this example was a portion of T. cruzi sequence Tc24 gene (GeneBank Accession Number AF 192980.2) (SEQ ID NO: 1), which encodes for a characterized parasite antigen.
  • the Tc24 gene is also known as the 1F8 flagelar calcium binding protein 3 gene.
  • the corresponding recombinant protein has been used for the detection of specific antibodies in serologic tests.
  • a PCR diagnostic based on the specific PCR amplification of a 600 base pair (bp) Tc24 sequence has also been described. Because the Tc24 gene is not present in other Trypanosoma species (e.g., T.
  • the Tc24 gene or other sequences within the Tc24 gene could be used to design primers.
  • the primers designed for this example use the nomenclature according to Figure 1.
  • Tc24-F3 and Tc24-B3 were:
  • Tc24-FIP The two internal primers (Tc24-FIP and Tc24-BIP) were: [0054] 5 ' -AGCGCCGCGGGATCCTCGAACGAGGAGGAGCTCAAGCGC-3 [0055] Tc24-FIP (SEQ ID NO: 6)
  • Tc24-BIP (SEQ ID NO: 7)
  • the amplifying solutions included 2 ⁇ of Tc24-FIP, 2 ⁇ of Tc24-BIP, 0.2 ⁇ of Tc24-F3, 0.2 ⁇ of Tc24-B3, 0.8 ⁇ of Tc24-LF, 0.8 ⁇ of Tc24-LB, 400 ⁇ of each dNTP, 10 mM KCl, 10 mM (NH 4 ) 2 S0 4 , 4 mM MgS0 4 , 20 mM Triton X-100, 0.2 M betaine, and 8 U of Bst polymerase (New England Biolabs #M0275S) in a total volume of 25 ⁇ per reaction.
  • Some amplifying solutions comprise samples with no T. cruzi DNA.
  • Other amplifying solutions included samples that provide 20 ng of purified T. cruzi DNA in the amplifying solution.
  • the purified T. cruzi DNA included the entire T. cruzi genome and was extracted using a GeneMole® apparatus.
  • the amplifying solutions were maintained at 65°C for 1 hour. The amplified solutions were then held for 10 minutes at 80°C to stop the reaction. The amplified solutions were analyzed on 2% agarose gels and DNA was visualized with ethidium bromide ( Figure
  • AY520034.1 (SEQ ID NO:3) was chosen as the target sequence.
  • This DNA target is used for PCR detection of T. cruzi DNA as it one of the most repeated and abundant sequences in T. cruzi, but it is absent from other kinetoplastid parasite species (e.g., African trypanosomes or Leishmania), which can provide specificity.
  • Specific PCR primers TcZl and TcZ2 have been designed for diagnostic tests by PCR amplification of a 188 bp fragment of the repeated sequence (Moser et al, "Detection of Trypanosoma cruzi by DNA amplification using the polymerase chain reaction," J. Clin. Microbiol, 1989, Vol. 27, pp. 1477-1482).
  • TcZl and TcZ2 primers provide one possible primer set based upon sensitivity and specificity of the PCR assays for T. cruzi detection; the TcZl and TcZ2 primers could detect all major lineages of the parasite (Virreira et al, "Comparison of polymerase chain reaction methods for reliable and easy detection of congenital Trypanosoma cruzi infection," Am. J. Trop. Med. Hyg., 2003, Vol. 68, pp. 574-582). [0067] In this example, four additional primers were designed to use in combination with the primers TcZl and TcZ2 to detect the 195 bp satellite repeat of T. cruzi.
  • the TcZl and TcZ2 PCR primers were used as the external primers (F3 and B3, respectively) in the method.
  • the TcZl and TcZ2 PCR primers were produced according to Moser et al, "Detection of Trypanosoma cruzi by DNA amplification using the polymerase chain reaction," J. Clin. Microbiol, 1989, Vol. 27, pp. 1477-1482:
  • TcZ-FIP (SEQ ID NO: 12) [0075] 5 ' -TGAATGGTGGGAGTCAGAGGCACTGTTTGGTGTCCAG
  • TGTGTGAAC-3' TcZ-BIP (SEQ ID NO: 13)
  • the amplifying solutions included 2 ⁇ of TcZ-FIP, 2 ⁇ of TcZ-BIP, 0.2 ⁇ of TcZ-F3, 0.2 ⁇ of TcZ-B3, 0.8 ⁇ of TcZ-LF, 0.8 ⁇ of TcZ-LB, 400 ⁇ of each dNTP, 10 mM KC1, 10 mM (NH 4 ) 2 S0 4 , 4 mM MgS0 4 , 20 mM Triton X-100, 0.2 M betaine, and 8 U of Bst polymerase (New England Biolabs #M0275S) in a total volume of 25 ⁇ per reaction.
  • Some amplifying solutions included samples that had no T. cruzi DNA. Other amplifying solutions included samples that provided 20 ng of purified T. cruzi DNA in the amplifying solution.
  • the purified T. cruzi DNA included the entire T. cruzi genome and was extracted using a GeneMole® apparatus.
  • the amplifying solutions were maintained at 65°C for 1 hour. The amplified solutions were then held for 10 minutes at 80°C to stop the reaction. The amplified solutions were analyzed on 2% agarose gels and DNA was visualized with ethidium bromide ( Figure

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Abstract

La présente invention concerne des compositions, des trousses et des procédés pour la détection de Trypanosoma Cruzi à l'aide d'une amplification isotherme médiée par boucle (LAMP). Dans certains modes de réalisation de la présente invention, les trousses et les compositions comprennent des amorces internes, des amorces externes et facultativement des amorces en boucle, toutes les amorces étant conçues pour une séquence T. cruzi cible. Dans d'autres modes de réalisation, les procédés de détermination de la présence de T. cruzi dans un échantillon consistent à maintenir une solution d'amplification (qui comprend les amorces) à une température donnée, puis à détecter la présence ou l'absence du produit d'amplification.
PCT/US2010/061500 2009-12-22 2010-12-21 Compositions, trousses et procédés pour la détection de trypanosoma cruzi à l'aide d'une amplification isotherme médiée par boucle WO2011087782A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017009347A1 (fr) * 2015-07-13 2017-01-19 Kann Simone Oligonucléotides et leur utilisation
WO2021012063A1 (fr) * 2019-07-23 2021-01-28 MELOSSI JIMÉNEZ, Andrés Kit de diagnostic de fondement moléculaire pour la détection de séquences nucléotidiques et procédés pour détecter des agents infectieux à l'aidue dudit kit
WO2022261308A1 (fr) * 2021-06-10 2022-12-15 New England Biolabs, Inc. Test de diagnostic isotherme utilisant une protéine cas et une polymérase

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2009017736A1 (fr) * 2007-07-30 2009-02-05 University Of Georgia Research Foundation Inc. Test de diagnostic d'une infection à trypanosoma cruzi

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WO2009017736A1 (fr) * 2007-07-30 2009-02-05 University Of Georgia Research Foundation Inc. Test de diagnostic d'une infection à trypanosoma cruzi

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DATABASE GENBANK 02 August 2002 Database accession no. AF192980 *
DATABASE GENBANK 21 March 2005 Database accession no. AY520034 *
KUBOKI ET AL.: 'Loop-mediated isothermal amplification for detection of African trypanosomes' J CLIN MICROBIOL. vol. 41, no. 12, December 2003, pages 5517 - 5524 *
THEKISOE ET AL.: 'Species-specific loop-mediated isothermal amplification (LAMP) for diagnosis of trypanosomosis' ACTA TROP. vol. 102, no. 3, 18 May 2007, pages 182 - 189 *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017009347A1 (fr) * 2015-07-13 2017-01-19 Kann Simone Oligonucléotides et leur utilisation
WO2021012063A1 (fr) * 2019-07-23 2021-01-28 MELOSSI JIMÉNEZ, Andrés Kit de diagnostic de fondement moléculaire pour la détection de séquences nucléotidiques et procédés pour détecter des agents infectieux à l'aidue dudit kit
EP4008791A4 (fr) * 2019-07-23 2024-01-17 Laboratorio Pablo Cassara S.r.L. Kit de diagnostic de fondement moléculaire pour la détection de séquences nucléotidiques et procédés pour détecter des agents infectieux à l'aidue dudit kit
WO2022261308A1 (fr) * 2021-06-10 2022-12-15 New England Biolabs, Inc. Test de diagnostic isotherme utilisant une protéine cas et une polymérase

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