WO2010029520A1 - Amorce et procédé d’amplification pour former des amplicons d’adn monocaténaire - Google Patents

Amorce et procédé d’amplification pour former des amplicons d’adn monocaténaire Download PDF

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Publication number
WO2010029520A1
WO2010029520A1 PCT/IB2009/054027 IB2009054027W WO2010029520A1 WO 2010029520 A1 WO2010029520 A1 WO 2010029520A1 IB 2009054027 W IB2009054027 W IB 2009054027W WO 2010029520 A1 WO2010029520 A1 WO 2010029520A1
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Prior art keywords
nucleic acid
acid molecule
primer
lda
strand
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PCT/IB2009/054027
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English (en)
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Natela Rekhviashvili
Natasha Gous
Lesley Scott
Alexio Capovilla
Wendy Stevens
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University Of The Witwatersrand, Johannesburg
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Priority to AP2011005669A priority Critical patent/AP2011005669A0/xx
Publication of WO2010029520A1 publication Critical patent/WO2010029520A1/fr
Priority to ZA2011/02807A priority patent/ZA201102807B/en

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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6846—Common amplification features
    • 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
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates

Definitions

  • NAT Nucleic acid testing
  • POC point of care
  • NALF Nucleic Acid Lateral Flow
  • Protocols have been developed that describe time consuming and cumbersome post-amplification treatment of dsDNA amplicons in order to convert them into ssDNA amplicons.
  • isothermal In “isothermal” methods, the entire amplification process is performed under one constant temperature. Even though many isothermal amplifications use an initial denaturation at high temperature or an initial reverse transcription (RT), compared to PCR amplification there is no temperature cycling, as the amplification process is performed at one uniform temperature. Therefore, an isothermal amplification does not require a PCR instrument and it can be performed in a simple heating block. This feature makes isothermal amplification easily adaptable to the POC testing format.
  • RT initial reverse transcription
  • b-DNA branched DNA
  • RCA rolling circle amplification
  • target amplification methods are nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), strand displacement hybridization (SDA), loop-mediated isothermal amplification (LAMP) and helicase-dependant isothermal DNA amplification (HDA).
  • NASBA nucleic acid sequence-based amplification
  • TMA transcription mediated amplification
  • SDA strand displacement hybridization
  • LAMP loop-mediated isothermal amplification
  • HDA helicase-dependant isothermal DNA amplification
  • a front end isothermal amplification technique has to meet certain requirements. Firstly, it has to provide great power of amplification so that even low concentrations of target can be amplified sufficiently in a short period of time (1 hour, or ideally even less). In other words, the method has to show good sensitivity when using a short amplification time. Secondly, an amplification method has to convert the RNA or DNA target into short (maximum 200 bases), single stranded DNA (ssDNA) or RNA amplicons in order to be readily compatible with the lateral flow detection format.
  • ssDNA single stranded DNA
  • SDA and LAMP methods have good sensitivity, but in their existing format these methods cannot be employed for POC HIV-1 RNA tests that rely on lateral flow, since the final amplification products are double stranded DNA (dsDNA) fragments and long stretches of dsDNA, respectively.
  • dsDNA double stranded DNA
  • nucleic acid molecule for amplifying a target region of a target nucleic acid molecule, the nucleic acid molecule comprising:
  • a primer which is capable of binding to a strand of the target molecule, wherein the first nucleotide sequence prevents an amplicon, formed by amplifying the target region with the primer, from self-priming and the second nucleotide sequence results in the amplicon having two complementary regions which hybridize to form a partial loop, thereby forming a single stranded (ss) DNA amplicon which remains in this single stranded state.
  • the strand to which the primer binds may be the sense strand or the antisense strand of the target molecule.
  • a reverse primer may bind to the sense strand and a forward primer may bind to the antisense strand.
  • the portion of the target region to which the second nucleotide sequence is complementary may be sufficiently close to the 5' end of the target region to cause the complementary sequences to hybridise to form the partial loop.
  • the nucleic acid molecule may further include a third nucleotide sequence at the end of the second nucleotide sequence, the third nucleotide sequence comprising at least two nucleotides which are complementary to at least the last two nucleotides at the 3' end of the primer.
  • a fourth nucleotide sequence may link the second nucleotide sequence and the primer or the third nucleotide sequence and the primer.
  • the nucleic acid molecule may further comprise a restriction enzyme recognition site, such as a BsoB1 recognition site.
  • the restriction enzyme recognition site may be located within the primer or between the first and second nucleotide sequences.
  • the target molecule may be from a pathogen, such as a virus or microbe.
  • the target molecule may be HIV-1 or a region thereof.
  • the target region may be SEQ ID NO: 1 or a sequence which has at least 90% identity thereto.
  • the first nucleotide sequence may be from about 3 to about 5 nucleotides in length or from about 15 to about 18 nucleotides in length.
  • the second nucleotide sequence may be from about 7 to 12 nucleotides in length, and may have a GC content of about 50%.
  • the nucleic acid molecule may have at least 90% identity to any one of SEQ ID NOs: 3 to 10.
  • a primer set for amplifying a target region of a nucleic acid molecule including a forward primer comprising a nucleic acid molecule substantially as described above and a reverse primer.
  • the reverse primer may include:
  • kits for carrying out a point of care test comprising:
  • reagents for amplification of the nucleic acid (c) reagents for amplification of the nucleic acid; and/or (d) means for detecting the presence of a target region in the nucleic acid.
  • the detecting means may be a dipstick or microfluidic device.
  • a method of amplifying a target region of a target nucleic acid molecule including the steps of:
  • step (e) displacing the two strands of the double stranded DNA amplicon; and (f) allowing the complementary regions of the strand in step (d) to hybridize to each other and form a partially looped structure at its 3' end, thereby maintaining the strand in a single-stranded state.
  • the method may further include an initial reverse transcription step (RT) if the target molecule is RNA, so as to convert RNA to cDNA.
  • RT initial reverse transcription step
  • the strand of the target molecule to which the nucleic acid molecule binds may be a sense or antisense strand.
  • the method may further include the step of cleaving the strands of the DNA amplicon at a recognition site with a restriction enzyme after step (c).
  • Steps (c) and (e) may be initiated using Bst DNA polymerase and BsoBI restriction enzymes.
  • FIG. 1 Strand Displacement Amplification (SDA) (Adapted from Schweitzer and S.
  • Figure 2 Primer design according to a first embodiment of the invention.
  • Figure 3 Linear amplification method according to the invention.
  • Figure 4 Semi-cycling amplification method according to the invention.
  • Figure 5 MetaPhor gel (2%) showing PCR products digested with ⁇ soSI restriction enzyme.
  • Sequences obtained from four amplification reactions carried out according to the invention performed using four different combinations of primers are aligned with the ES DNA sequence (i.e. DNA sequence equivalent of RNA sequence of the ES RNA transcript that was used as a template in the RT- LDA reactions).
  • the sequence of ES DNA (128 bp; SEQ ID NO: 1) is highlighted in bold; sequences of primers/primer binding sites are underlined.
  • RT-LDA1 product (-105 bp; SEQ ID NO: 15) was amplified and sequenced using primers Fw#2/Rev#1 ; RT-LDA 2 product - Fw#3A/Rev#1 (-149 bp; SEQ ID NO: 16); RT-LDA 3 product - Fw#1C/Rev#1 (-170 bp; SEQ ID NO: 17); RT- LDA 4 product was PCR amplified and sequenced using the primer set Fw#3A/Rev#1 (-120 bp; SEQ ID NO: 18). (Note: for RT-LDA 4 product Fw#3A was used instead of Fw#4a since it has a much shorter overhang for more optimal PCR and sequencing reactions).
  • Figure 7 MetaPhor ® gel (1%) showing PCR product amplified from the linear RT-LDA reactions.
  • Figure 8 Melting curve analysis using FRET probes for detection of RT-LDA product.
  • Top graph Melting curve profiles of both RT-LDA samples (bold lines) show the presence of the specific amplification product with a typical melting peak at -69 0 C.
  • Bottom graph No melting peak is observed in the RT-LDA blank sample (grey line).
  • Figure 10 Melting curve analysis using FRET probes for verification of PCR product.
  • Figure 11 MetaPhor ® gel (1%) showing PCR product amplified from the partially cycling RT-LDA reactions.
  • Figure 12 Melting curve analysis for repeated RT-LDA experiments.
  • RT-LDA reproducibility experiments (2 out of 5) are illustrated in figures A and B 1 respectively. Specific RT-LDA amplification product is detected using
  • Figure 13 Detection of RT-LDA amplicons (arrow) using NALF dipsticks. From left to right: RT-LDA blank sample (lane 1 ); two positive controls containing an artificially synthesized looped amplicon (ampi29) (lanes 2 and 3); the next 6 dipsticks show the detection of diluted RT-LDA amplification products obtained from the different experiments (lanes 4-9).
  • Figure 14 Autoradiograph showing extracted PCR product following semi-cycling RT- LDA reaction.
  • This invention describes a novel primer design and amplification method utilising this primer.
  • the amplification method generates a ssDNA product from either RNA or DNA and can be directly combined with detection systems such as NALF, flow cytometry, microarrays and the like.
  • Primers according to the invention can be included in a kit for carrying out the amplification method and combined with a detection system for use in an assay, such as a POC test.
  • the primers include a short non-target specific sequence for preventing a displaced amplicon from self-priming; a sequence complementary to a strand extended from the primer ('overhang" sequence); a short artificial sequence that forms a link; and a sequence specific forward primer.
  • the forward primer binds to the anti-sense cDNA strand and produces a dsDNA target.
  • the overhang sequence is also extended and the anti-sense strand gets displaced from a nickable site, forming an amplicon with two complementary sequences. These hybridize, forming a partially looped ssDNA amplicon which is unable to bind to another forward primer and cannot be converted into a dsDNA target.
  • amplicon is intended to refer to any amplified product, and not only to products which have been amplified by PCR.
  • Primers designed according to the invention are referred to as “loop mediating primers” (or “LMPs”) and the amplification method of the invention is referred to as “reverse transcription loop dependent amplification” (or “RT- LDA”).
  • Strand displacement amplification provides great power of amplification. It consists of two phases (target generation and exponential target amplification) and employs four primers (a pair of bumping primers and a pair of primers containing a nickable site). During the second cycling phase of SDA, dsDNA amplicons accumulate exponentially. The typical target doubling time is 20-30 seconds, and a 10 billion-fold amplification of specific targets can be achieved in less than 15 minutes. SDA also makes use of two commercially available enzymes - BsoB ⁇ restriction enzyme and Bst DNA polymerase that possesses the strand displacement ability. In the case when RNA is used as a template, the reverse transcription (RT) step is performed (RT-SDA) by a third enzyme, Avian Myeloblastosis Virus (AMV) reverse transcriptase.
  • RT reverse transcription
  • AMV Avian Myeloblastosis Virus
  • a double-stranded DNA target (1) is denatured and hybridized with two primers (2).
  • One primer (B 1 ) is designated as a 'bumper' primer, and the other primer (S-O contains a BsoBl restriction enzyme sequence 5' to the target binding region.
  • the B 1 (3) and S 1 (4) primers are simultaneously extended by the thermostable enzyme Bst DNA polymerase in the presence of thiolated dCTP. Extension from the bumper primer displaces the S 1 extended product, which can then hybridize to the opposite strand primers, B 2 and S 2 (5).
  • the present invention therefore describes a novel primer and its use in a new isothermal amplification technique, based on SDA, which converts RNA or DNA into short ssDNA molecules (generally less than 200bp, although this will depend on each primer design) with high efficiency.
  • FIG. 2 An overview of a primer design according to the invention is shown in Figure 2: 1) Starting from the 5' end of the primer: a short artificial (i.e. non-target specific) sequence that serves to prevent a displaced amplicon from self-priming is shown by black line; a sequence complementary to a strand extended from this primer (so called "loop" overhang) is indicated in hashed line; this sequence (hashed line) also possesses at least two nucleotides that are complementary to the last two nucleotides at the 3'-end of the forward primer (i.e. a gene specific part of this forward LMP); a short artificial sequence that forms a link between the "loop" overhang and a forward primer is shown by grey line; a sequence specific forward primer is shown by black circles.
  • a short artificial (i.e. non-target specific) sequence that serves to prevent a displaced amplicon from self-priming is shown by black line; a sequence complementary to a strand extended from this primer (so called "
  • the forward primer with an overhang (parts indicated in black line, hashed line and grey line) binds to the anti-sense cDNA strand and gets extended. Extension of the forward primer produces a dsDNA target. An overhang sequence also gets extended by, for example, Bst polymerase from the 3' end of the cDNA strand.
  • the anti-sense strand gets displaced from a nickable site (similarly to SDA, see Figurei).
  • the displaced anti-sense strand which now can be termed an anti-sense ssDNA amplicon, contains a sequence complementary to an overhang of the forward primer.
  • 3B The two complementary sequences within one amplicon are in close proximity and the 3' end of the anti-sense amplicon folds forward and hybridizes to its complementary sequence (indicated by hashed line). As a result, a partially “looped" ssDNA amplicon is formed.
  • the "loop" structure at the 3' end of the amplicon prevents it from binding to another forward primer and from getting converted into a dsDNA target. Thus, most of the displaced anti-sense amplicons remain single stranded.
  • the "loop" structure must be sufficient to preserve the single stranded nature of the amplicons and at the same time it must not interfere with extension of the primer.
  • This technique was designed as a front amplification suitable for NAT based POC diagnostics assays, e.g. detection of genes, pathogens and diseases, including viruses and microbes, such as HIV-1 , tuberculosis or hepatitis.
  • the sense strand is thiolated with dCTP and is therefore not nicked and does not form a loop.
  • Reverse transcription is performed using only one, for example HIV-1, sequence specific primer and a type of AMV enzyme that has both reverse transcriptase and RNase H activity (H + ).
  • the method of the present invention does not use bumping primers.
  • cDNA anti-sense sequence
  • cDNA is released from the initial RNA/cDNA complex due to RNase H activity of AMV reverse transcriptase, which also makes the method different from SDA ( Figures 3 and 4).
  • ⁇ Amplification is achieved mainly due to the new primer (LMP) design.
  • the primer in this study, a forward primer
  • the primer has a 5' overhang containing a sequence complementary to a sense sequence of a DNA strand extended from this primer ( Figure 2).
  • dsDNA targets T1, T2 and T3
  • Figure 4 dsDNA targets
  • T1, T2 and T3 dsDNA targets
  • Figure 4 dsDNA targets
  • these strands/amplicons copy an overhang from the forward primer.
  • the displaced anti-sense amplicons have two regions at their 3 !
  • the primer design of the present invention preserves the single stranded structure of the DNA amplicons produced during RT-LDA.
  • RT-LDA reverse transcription bop dependant amplification
  • the new primer design can be used in combination with enzymes other than those described herein (these are the enzymes used in a commercial SDA technique as described above), provided that a DNA polymerase used has strand displacement ability. If a different restriction enzyme (not SsoBI as in SDA and RT-LDA) is used, than the nickable site in the primer will have a different sequence, which would correspond to the restriction enzyme used. Different RT (reverse transcriptase) enzymes can be used instead of AMV, in combination with a separate RNase H+ enzyme.
  • sample preparation which would include nucleic acid extraction from blood/plasma
  • amplification of the nucleic acid target amplification of the nucleic acid target
  • detection of the nucleic acid amplicon amplification of the nucleic acid amplicon
  • the invention also extends to a kit for use in performing the POC test.
  • the kit would include reagents for the extraction method, amplification reagents (RT-LDA reagents as described on pg 15) and dipsticks and/or a microfluidic device for detection.
  • HIV-1 has been used as an example of a target sequence to be amplified.
  • a person skilled in the art will understand that the primer and amplification method of the invention can be readily applied to any other target molecule, for example, other viral and microbial pathogens or genes.
  • RNA containing 128 bases of HIV-1 gag gene was used in the examples. Detection of the amplification product was performed using FRET hybridization probes and the LightCycler ® platform. This detection format was selected because it is cost effective, sensitive and sequence specific. Visual detection of amplicons using dipsticks was performed to confirm compatibility of the amplification technique of the invention with the NALF detection format.
  • a linear amplification method performed according to the invention is shown in Figure 3, with HIV-1 RNA as an initial template for amplification.
  • a reverse primer consisting of three regions. Starting from the 5' end they are as follows: an artificial (random) sequence, a recognition site sequence (so called nickable site) for the restriction enzyme BsoB ⁇ (indicated as a white circle), and an HIV-1 sequence specific (reverse) primer.
  • the reverse primer (R) binds viral RNA and gets extended by the RT activity of AMV reverse transcriptase (RT). A double stranded RNA/cDNA hybrid is formed.
  • the forward primer (F) converts cDNA into dsDNA target (LDA 1).
  • the forward primer (F) includes a forward primer with a 5' overhang. From the 5' end, the primer overhang contains "an artificial" sequence (black line), a sequence complementary to an amplicon ("loop" sequence) (hashed line) and a very short linking sequence (grey line). Due to the addition of chemically modified dCTPs (dCTP- ⁇ S) in the reaction mix, the double stranded SsoBI recognition site is hemithiolated and only one strand gets nicked by the restriction enzyme (see also Figure 4).
  • Bst DNA polymerase which also has strand displacement ability, extends the 3'-end of the newly formed nick and at the same time displaces the existing, anti- sense DNA strand (LDA 2).
  • LDA 2 anti- sense DNA strand
  • the SsoSI nickable site gets regenerated and can be nicked over and over again, thus producing a large number of "looped" anti-sense ssDNA amplicons.
  • the displaced amplicons assume a partially "looped” structure due to a carefully designed overhang of a forward primer, as described in Figure 4. Formation of partially “looped” amplicons prevents binding of the forward primer to the anti-sense amplicon and thus prevents the entire amplification process from cycling.
  • the RT-LDA design illustrated in this figure provides accumulation of partially "looped" ssDNA anti-sense amplicons in a linear fashion.
  • a primer and its use in a semi-cycling amplification method according to the invention are illustrated in Figure 4. Conversion of the initially designed linear amplification technique into semi-cycling was made possible due to another novel design applied to the forward primer.
  • the second design includes an HIV-1 specific forward primer (black circles) with a 5' overhang, but the structure of the overhang is different as it incorporates a nickable site (in Figure 3, only the reverse primer included the nickable site). From the 5' end the primer overhang contains "an artificial" sequence (black line), a BsoBl recognition site (white circle) and a sequence complementary to an amplicon ("loop" sequence) (hashed line) and a very short linking sequence (grey line).
  • the RT step for the partially cycling amplification is identical to the linear amplification ( Figure 3).
  • a modified forward primer that binds to cDNA is extended by Bst polymerase, and as a result a dsDNA target is formed (T1).
  • the strands complementary to the BsoBl restriction site are generated during the primer/strand extension and thus contain modified dCTP ⁇ S (thiolated), which prevents BsoBl from cutting dsDNA. Instead, only the unmodified nickable sites (in all targets - T1 , T2 and T3) from the primers get nicked (single stranded nick) by the enzyme.
  • T1 dsDNA has two nickable sites - one on a sense strand (forward primer; white circle) and another one on the anti-sense strand (reverse primer; white circle). Both nickable sites are recognized and nicked (cleaved) by BsoB). Next, the 3'- end of each nicked strand (sense and anti-sense) is extended by Bst DNA polymerase and the existing strands are displaced. The displaced sense strand forms a partially "looped" amplicon that has a sequence complementary to a reverse primer. The reverse primer binds this sense amplicon and converts it into a dsDNA target (T2).
  • T2 has only one ⁇ so ⁇ l restriction site and a process of nicking and strand displacement/polymerization occurs on the T2 target in a similar way as described above.
  • a cascade of processes between formations of T1 and T2 represents a linear phase of a RT-LDA.
  • the displaced anti-sense amplicons have a sequence at their 3'- end which is complementary to the "loop" overhang of a forward primer. Thus, once displaced, these amplicons assume a partially “looped" structure.
  • the anti-sense strand which is displaced from T1 , possesses a 3' sequence complementary to the entire forward primer and thus a forward primer binds to the anti-sense amplicon.
  • T3 dsDNA target
  • T3 produces "looped" sense amplicons similarly to T1.
  • These amplicons bind to the reverse primers which lead to the formation of dsDNA targets identical to T2.
  • a cascade of processes from the formation of T1 to T3 and then to T2 represents a partially cycling phase of this amplification technique.
  • T2 generated during a partially cycling phase also keeps producing "looped" ssDNA anti-sense amplicons.
  • both linear and semi-cycling phases of RT-LDA occur simultaneously and generate large amounts of a final amplification product - partially "looped" ssDNA anti-sense amplicons.
  • the partially cycling design provides at least a 3-fold increase in magnitude of amplification.
  • ES RNA in vitro transcribed RNA that represents an external quantitation standard (ES RNA) used for a LightCycter ® viral load assay (LUX assay), described in PCT Publication No. WO 2006/082496, the entire contents of which are incorporated by reference herein.
  • the ES RNA molecule includes a 128 base pair region of the HIV-1 gag gene:
  • the primer design of the invention was applied only to forward primers used in the amplification method. Seven types of forward primers having different lengths and base compositions of the loop forming overhang were evaluated during this study. These seven primers were individually combined with a reverse primer (Rev#1 ) in separate amplification reactions.
  • Forward #4A (a version of#3A with a nickable site for semi-cycling RT-LDA) 5'- AAC CTA TCC GGA CAA CGA TAA CCC GGG CC TCC TCA T ACT TGT TAA AAG ATACAATCAAT- 3' (59 mer) (SEQ ID NO: 10)
  • RT-LDA amplification reactions were performed in a total volume of 50 ⁇ l consisting of 20 ⁇ l of template RNA and 30 ⁇ l of reaction mix.
  • the reaction mix was assembled as follows: 35 mM potassium phosphate buffer (K 1 PO 4 ), 5% v/v dimethyl sulfoxide (DMSO; Sigma-Aldrich, USA), 4% v/v glycerol (Promega, Madison, Wl, USA), 5 ⁇ g acetylated bovine serum albumin (AcBSA; Promega, Madison, Wl), 0.8 mM 2'-deoxycytidine 5'-O-(1- thiotriphosphate) (dCTP ⁇ S; Amersham Biosciences, Piscataway, NJ, USA), 0.6 mM dUTP, 0.2 mM each dATP and dGTP (Promega, Madison, Wl, USA), 7.5 mM magnesium acetate (MgOAc 2 ; Sigma- Al
  • Synthetic in vitro RNA was used as a template for RT-LDA reactions.
  • the sequence was based on a reference gag sequence from the HIV-1 subtype B HXB2 isolate (http://hiv-web.lanl.gov).
  • Probes were manufactured by Metabion International AG (Germany). Reconstituted probes, each at 100 ⁇ M concentration, were kept at -20 0 C until use.
  • Post amplification detection using FRET probes was performed in 30 ⁇ l volume using 27 ⁇ l of amplification reaction and 0.5 ⁇ M of each probe.
  • Profile of the melting curve analysis was as follows: 95 0 C for 5 s, 40 0 C for 1 min and gradual heating up to 85-9O 0 C with 01°C/s ramping rate and continuous fluorescent signal acquisition. Melting curves were viewed using a combination of channels -640/Back530.
  • Reagents for nucleic acid lateral flow including buffers, magnetic conjugate, nitrocellulose DNP striped half dipsticks, capture and detection probes were provided by British Biocell International (Cardiff, UK). Sequences of the LF probes:
  • the positive control for NALF detection was also supplied by BBI.
  • Positive control (ampi29) was made to imitate RT-LDA amplicons and represents a synthetic ssDNA molecule that has a partially looped "structure" and the sequence similar to the real amplification product.
  • NALF detection was performed according to the manufacturer's instructions.
  • PCR was performed in 50 ⁇ l volume using 25 ⁇ l of ReadyMixTM Taq PCR reaction with MgCI 2 (Sigma, St. Louis, Missouri, USA), 0.5 ⁇ M forward and reverse primer, 15 ⁇ l of template (diluted RT-LDA product) and PCR grade water from the kit (Sigma).
  • PCR was performed using MyCyclerTM thermal cycler (BIO-RAD Laboratories, Inc., USA) according to the following profile: initial denaturation at 95 0 C for 3 min and then 40 cycles of amplification with denaturation at 95 0 C for 30 s, annealing at 52 0 C for 30 s, extension at 72 0 C for 30 s and final extension at 72 0 C for 7 minutes.
  • Detection of PCR product was performed using high resolution MetaPhor ® gels and conventional gel electrophoresis.
  • PCR product amplified using the RT-LDA reaction of the invention was used as a template for sequencing. Prior to sequencing, the PCR product was excised from the MetaPhor ® gel and purified using the MinEluteTM Gel Extraction kit (Qiagen, GmbH Hilden, Germany) according to the manufacturer's instructions. DNA was eluted in 10 ⁇ l of the elution buffer supplied in the kit.
  • the sequencing reaction was performed with the Big Dye ® Terminator version 3.1 cycle sequencing kit from Applied Biosystems (Foster City CA, USA) according to the manufacturer's instructions, on the ABI Prism 3100- ⁇ vanf Genetic Analyzer (Applied Biosystems (Foster City CA, USA) according to the manufacturer's instructions, on the ABI Prism 3100- ⁇ vanf Genetic Analyzer (Applied Biosystems (Foster City CA, USA) according to the manufacturer's instructions, on the ABI Prism 3100- ⁇ vanf Genetic Analyzer (Applied Biosystems).
  • a plasmid containing the applicants 128 bp sequence of interest was manufactured by Geneart (Regensburg, Germany) and run-off transcripts of 450 bp were synthesised by in vitro transcription off the plasmid. These larger RNA transcripts were then used as templates for further RT-LDA reactions and detection with autoradiography.
  • Template nucleic acid i.e. ES RNA
  • ES RNA was used at this stage of optimization only at high concentrations: ⁇ 4x10 7 and 4x10 s copies/ml.
  • Five forward primers (Fw#1 ,
  • Fw#1 A, Fw#1 B, Fw#2 and Fw#3) combined individually with the reverse primer (Rev#1 ) were first evaluated for the linear type of RT-LDA.
  • Forward primer Fw#2 is positioned more internally to Fw#1 and has the shortest "loop" overhang" with
  • Primer Fw#3 is positioned between Fw#1 and Fw#2 and partially overlaps with primer Fw#2. Each of these primers was combined individually with the reverse primer Rev#1 in separate RT-LDA reactions.
  • Initial RT-LDA experiments revealed no amplification product for all five primer combinations using 1% MetaPhor ® gel and FRET probes for detection.
  • ES RNA was reverse transcribed using SKT145 forward primer (a forward primer specific for HIV-1 gag gene and used in the LUX assay for HIV-1 viral load) and Rev#1 primer.
  • SKT145 forward primer a forward primer specific for HIV-1 gag gene and used in the LUX assay for HIV-1 viral load
  • Rev#1 primer Two different reaction mixes (RT-LDA reaction mix excluding ⁇ soSI and Bst enzymes and the reaction buffer supplied with AMV enzyme) and different concentrations of AMV enzyme were used.
  • RT was performed for 1 hour at 42 0 C and 20 ⁇ l of undiluted RT reaction (cDNA) was than amplified by conventional PCR using primers SKT145 and Rev#1.
  • PCR product was confirmed using 1% MetaPhor ® gel. Troubleshooting of the RT step revealed that the 2.5 units of AMV enzyme used initially was insufficient and an optimal concentration of 7.5 units of AMV per reaction was established.
  • cDNA reverse transcribed with primer Rev#1 under optimised RT conditions was PCR amplified using the set of primers SKT145/Rev#1.
  • PCR product was incubated with BsoB ⁇ restriction enzyme at 53 0 C for 3 hours. Digested and undigested PCR products were verified using 2% MetaPhor ® gel ( Figure 5). The presence of the digested product confirmed functional activity of BsoB ⁇ at 53 0 C and the presence of a corresponding restriction site in the reverse primer Rev#1.
  • RT-LDA reactions were performed using the combination of five forward primers described above and the Rev#1 primer. As with the first round of experiments, no RT-LDA amplification product was detected for all primer combinations using MetaPhor ® gel and FRET hybridization probes. Next, PCR was employed to confirm the presence/absence of RT-LDA product and to exclude insufficient sensitivity of the detection formats used. RT-LDA reactions were diluted 1 :50 and 1 :100 in molecular grade water and 15 ⁇ l of the diluted product was used for PCR. PCR was performed with the same primer combinations that were originally used for each RT-LDA reaction (e.g.
  • RNA i.e. purified dsDNA amplification product obtained using ES RNA and a conventional RT-PCR
  • NTC no template control
  • PCR revealed the presence of RT-LDA product only in the reaction performed with a set of primers Fw#2 and Rev#1. Together with a specific amplification product, the RT-LDA reaction and the blank sample revealed the presence of a non-specific product (data not shown).
  • RT-LDA reactions were performed using a two step temperature profile and a long amplification time as described above, and two combinations of primers: Fw#1C/Rev#1 and Fw#3A/Rev#1.
  • FRET hybridization probes revealed RT-LDA product amplified using both newly designed primers (Fw#1C and Fw#3A), but the fluorescent signals were weak (data not shown).
  • RT-LDA samples amplified with primers Fw#1C/Rev#1and Fw#3A/Rev#1 and their corresponding blank samples were diluted 1 :50 and 15 ⁇ l of each diluted sample was further PCR amplified with the corresponding RT-LDA primers.
  • ES DNA was used as a positive PCR control for both combinations of primers.
  • a PCR product was detected in the RT-LDA samples and in two positive control samples, but not in the RT-LDA blank samples ( Figure 7). Non-specific product observed using initially designed primers was not present in any of these PCR samples.
  • RT-LDA forward primers LMPs
  • the forward primer Fw#3A appeared to have an optimal design.
  • the LMP design for a partially cycling RT-LDA incorporated the features of primer Fw#3A.
  • primer Fw#4A for a partially cycling RT-LDA includes the same HIV-1 specific sequence and the loop overhang sequence as the primer Fw#3A and in addition it has a nickable site for SsoSI and the random primer sequence ( Figures 3 and 4).
  • New forward primer Fw#4A designed for partially cycling RT-LDA was evaluated in combination with primer Rev#1 using high concentrations of ES RNA ⁇ 4x10 7 and 4x10 8 copies/ml.
  • Detection with FRET probes demonstrated the presence of an amplification product in RT-LDA reactions performed with primer set Fw#4A/Rev#1 and no product in the blank sample ( Figure 8).
  • the characteristic melting temperature (T m ) observed for RT-LDA amplicons using these FRET probes was ⁇ 68.8-71°C. Unambiguous detection of RT-LDA amplicons with FRET probes was achieved and allowed further optimisation of the amplification method.
  • RT-LDA reaction components such as DMSO and phosphate buffer
  • concentration of DMSO was titrated from 4 to 10% v/v and the concentration of phosphate buffer from 35 mM to 50 mM. It was found that an increased concentration of DMSO requires higher concentrations of phosphate buffer to maintain sufficient amplification.
  • Preliminary data obtained from a NALF/MARTM (magnetic assay reader) feasibility study conducted by BBI demonstrated that higher concentrations of phosphate buffer decrease the intensity of magnetic signal detected with MARTM.
  • high (8-10%) concentrations of DMSO that could provide greater specificity of primer annealing required higher concentrations of phosphate buffer, which interferes with the MARTM detection.
  • RT-LDA RNA deoxyribonucleic acid
  • FRET fluorescence resonance energy transfer
  • a further reduction in amplification time was attempted using only one constant temperature for both the RT step and amplification steps.
  • Two amplification profiles were tested for the RT-LDA reactions: one at 53 0 C with a total reaction time of 30 minutes and at 53 0 C for 1 hour. Both amplification profiles were performed using a range of ES RNA concentrations: of 4x10 3 , 4x10 4 , 4x10 5 , 4x10 6 , 4x10 7 and 4x10 8 copies/ml.
  • the RT-LDA product was detected using FRET probes in all samples (excluding a blank sample) amplified for 30 minutes and 1 hour, respectively.
  • RT-LDA samples were used for PCR to provide an additional confirmation of successful RT-LDA using new time and temperature profiles.
  • the RT-LDA samples containing ES RNA at a starting concentration of 4x10 7 and 4x10 6 copies/ml that were amplified for 1 hour and a sample containing 4x10 3 copies/ml of ES RNA amplified for 30 minutes, were diluted 1 :10 and further amplified by PCR using primers Fw4A/Rev#1.
  • a PCR product for the RT-LDA sample of 4x10 7 copies/ml was clearly detected as a bright band on the 1% MetaPhor ® gel; the other two samples revealed faint bands (data not shown).
  • Primer Fw#3A has the same HIV-1 sequence specific region as the primer Fw#4A and was used due to the limited amounts of the primer Fw#4A available.
  • PCR product (-150 bp) was gel purified and used for sequencing ( Figure 11 ).
  • the sequence obtained using primers Fw#4A and Rev#1 was aligned with the ES DNA ( Figure 6).
  • RT-LDA Since successful RT-LDA was demonstrated using 1 hour and even 30 minutes amplification time at 53 0 C, initial evaluation of the method was performed to assess reproducibility of partially cycling RT-LDA under the new conditions. Reproducibility of RT-LDA was studied in five different experiments performed on five different days. Each experiment was performed using serial dilutions of a template ES RNA covering a range of concentrations: 4x10 3 , 4x10 4 , 4x10 5 , 4x10 6 , 4x10 7 and 4x10 8 copies/ml. RT-LDA reactions were performed at 53 0 C for 1 hour. Post-amplification detection of amplicons was performed using melting curve analysis and FRET hybridization probes.
  • FIG. 12 shows the melting curve analysis for two out of five repeated RT-LDA experiments.
  • RT-LDA Preliminary evaluation of partially cycling RT-LDA also involved detection of the amplicons using anti-DNP (dinitrophenyl) striped dipsticks and NALF.
  • RT-LDA reactions were performed using the short amplification profile described above and ES RNA template at concentrations of 4x10 8 and 4x10 7 copies/ml.
  • Different dilutions from 1 :10 to 1:30
  • input concentrations from 1 ⁇ l to 30 ⁇ l
  • the samples that gave positive detection in a visual detection range of NALF are shown in figure 13. The most optimal detection was found in samples diluted 1 :10 and 10-30 ⁇ l of that dilution. Reproducibility of NALF detection of the RT-LDA amplicons was confirmed in a number of lateral flow experiments.
  • the amplicon of the correct size (-173 bp: 128bp gag sequence plus the primer overhang) could be visualised in the extracted sample but was not detectable in the non-extracted sample control due to smear formation (Figure 14). Bands of various sizes were also visible in the water template control sample. However, the correct sized amplicon was absent (figure 14).
  • RT-LDA reverse transcription loop dependant amplification
  • the RT-LDA reverse primer similarly to primers used for strand displacement amplification, possesses a BsoB ⁇ recognition site that gets nicked, and this ssDNA nick initiates a series of strand displacement/polymerisation steps driven by Bst DNA polymerase.
  • a novel primer design is applied to the RT-LDA forward primer - it possesses a 5' overhang sequence, which is complementary to the sequence of a sense strand extended from this primer.
  • dsDNA targets T1 , T2 and T3 are formed ( Figure 4), and from each template, displacement of multiple anti-sense strands/amplicons begins. During strand polymerisation these strands/amplicons copy an overhang from the forward primer.
  • the displaced anti-sense amplicons have two regions at their 3' end with complementary sequences, which hybridize to each other causing a partially "looped" structure of the ssDNA amplicons ( Figures 2, 3 and 4).
  • Accumulation of partially “looped" ssDNA amplicons represents a key feature of RT-LDA.
  • the "loop" structure closes the region complementary to the forward primer and thus, prevents conversion of anti-sense ssDNA amplicons into dsDNA amplicons.
  • Another novel aspect of the RT-LDA design is the absence of bumping primers that are normally used in the SDA technique. This is done to avoid the production of multiple dsDNA species in SDA ( Figure 1) and to make RT-LDA less complex.
  • RT-LDA makes use of AMV reverse transcriptase that has combined RNase H activity, which digests viral RNA in the RNA/cDNA hybrid, leaving cDNA in a single stranded form. Functionality of the novel modifications applied to the RT-LDA design has been proven experimentally.
  • the length of a complementary "loop" sequence has to be long enough to preserve the single stranded structure of the DNA amplicons, but not too long to interfere with the extension of the forward primer.
  • the exact dimensions of the "loop" overhang in terms of the number of nucleotides and GC content were established experimentally. This was achieved by performing RT-LDA reactions using 7 forward primers with 5'- "loop” overhangs of different lengths and sequence composition. According to the experimental data obtained for linear RT-LDA reactions, the most optimal design for RT-LDA forward primer suggests short sequence (9 bases) of a "loop" forming overhang with a high ( ⁇ 50%) GC% content.
  • RT-LDA product from linear amplification could only be seen by further amplification using conventional PCR and was confirmed additionally using DNA sequencing. This issue was attributed to the insufficient power of amplification provided by the linear RT-LDA.
  • linear RT-LDA can still be used in combination with more sensitive detection formats, and for applications where the amplification methods have a long incubation time.
  • An advanced RT-LDA design is achieved by the addition of the second BsoB ⁇ nickable site into the sequence of the forward primer ( Figure 4).
  • This modification of a forward primer does not interfere with displacement of ssDNA partially "looped" anti-sense amplicons.
  • the modified forward primer enhances amplification due to formation of three different types of dsDNA targets (T1 , T2 and T3) as apposed to only one such target in a linear type of RT-LDA design ( Figures 2 and 3).
  • another novel primer design converted linear RT-LDA into a partially cycling amplification technique.
  • modification was applied to a forward primer Fw#3A, which was shown previously to produce the most optimal linear amplification.
  • Fw#4A a modified Fw#3A containing a BsoB ⁇ restriction site and a random sequence for "an anchoring artificial" primer was termed Fw#4A.
  • RT-LDA experiments performed with new primer Fw#4A and Rev#1 immediately showed an improved detection using melting curve analysis with the FRET hybridization probes.
  • Optimisation of reaction conditions allowed further improvement of RT-LDA.
  • Reduction in amplification time of RT-LDA that was performed using one constant temperature (53 0 C) revealed good sensitivity of this method. In particular, 4000 copies/ml of ES RNA template could be detected using only 30 minutes amplification.
  • the PCR product amplified from RT-LDA using the primer set Fw#4A/Rev#1 confirmed the presence of a specific product.
  • the PCR product was sequenced and the sequence of the amplicons aligned with HIV-1 subtype C sequence ( Figure 6).
  • Preliminary evaluation of semi-cycling RT-LDA was performed using a wide range of ES RNA concentrations from 4x10 3 to 4x10 8 copies/ml, which were tested in five replicate RT-LDA experiments. Each RT-LDA run was performed at 53 0 C for 1 hour. Repeated experiments showed that the newly designed RT-LDA technique is reproducible over six orders of magnitude.
  • Preliminarily sensitivity of a one hour RT-LDA reaction combined with the FRET detection format can be set at 4000 copies/ml.
  • Preliminary evaluation of RT-LDA also included NALF experiments in order to demonstrate compatibility of a new amplification technique with NALF detection using nitrocellulose dipsticks as one of the most widely used in POC tests detection formats. It was important to exclude possible interference between NALF reaction components and RT-LDA reactions mix.
  • the detection of RT-LDA product using NALF dipsticks without a prior denaturation step, provides experimental proof of the single stranded structure of the partially looped RT-LDA amplicons.
  • RT-LDA Positive detection observed for RT-LDA reactions ( Figure 13) showed that the newly developed amplification technique is fully compatible with NALF detection using anti-DNP striped dipsticks and anti-superparamagnetic conjugates.
  • LMP loop mediating primer

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Abstract

La présente invention concerne des amorces et un procédé d’amplification qui produisent des amplicons d’ADN monocaténaire à partir d’ARN ou d’ADN. Les amplicons d’ADNsb peuvent être directement combinés à des systèmes de détection tels que NALF, la cytométrie en flux, des micropuces et similaire. L’invention est particulièrement adaptée à une utilisation dans des tests en centre de soins. Les amorces comprennent une séquence spécifique courte non-cible pour empêcher un amplicon déplacé de s’auto-amorcer ; une séquence complémentaire d’un brin allongé à partir de l’amorce (séquence « saillante ») ; une séquence artificielle courte qui forme une liaison ; et une amorce sens séquence-spécifique. L’amorce directe se lie au brin d’ADNc antisens et produit un ADNdb cible. La séquence saillante est également allongée et le brin antisens est déplacé depuis un site de coupure simple brin, formant un amplicon avec deux séquences complémentaires. Celles-ci s’hybrident, formant un amplicon d’ADNsb partiellement bouclé qui est incapable de se lier à une autre amorce sens et ne peut pas être converti en ADNdb cible.
PCT/IB2009/054027 2008-09-15 2009-09-15 Amorce et procédé d’amplification pour former des amplicons d’adn monocaténaire WO2010029520A1 (fr)

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WO2017060316A1 (fr) * 2015-10-05 2017-04-13 Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Nouveau procédé de préparation de jeux d'amorces à code-barres
US10822645B1 (en) 2014-12-23 2020-11-03 Global Life Sciences Solutions Operations UK Ltd Methods and reagents for reverse-transcription polymerase chain reaction
US10858694B2 (en) 2014-12-23 2020-12-08 Global Life Sciences Solutions Operations UK Ltd Methods and reagents for reverse-transcription polymerase chain reaction
US10968478B2 (en) 2014-12-23 2021-04-06 Global Life Sciences Solutions Operations UK Ltd Methods and reagents for reverse-transcription polymerase chain reaction

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10822645B1 (en) 2014-12-23 2020-11-03 Global Life Sciences Solutions Operations UK Ltd Methods and reagents for reverse-transcription polymerase chain reaction
US10858694B2 (en) 2014-12-23 2020-12-08 Global Life Sciences Solutions Operations UK Ltd Methods and reagents for reverse-transcription polymerase chain reaction
US10968478B2 (en) 2014-12-23 2021-04-06 Global Life Sciences Solutions Operations UK Ltd Methods and reagents for reverse-transcription polymerase chain reaction
WO2017060316A1 (fr) * 2015-10-05 2017-04-13 Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Nouveau procédé de préparation de jeux d'amorces à code-barres
US10870879B2 (en) 2015-10-05 2020-12-22 Helmholtz Zentrum Münchendeutsches Forschungszentrum Für Gesundheit Und Umwelt Method for the preparation of bar-coded primer sets

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