WO2022201104A1 - Compositions, methods and kits comprising polyinosinic acid (poly(i)) for polymerase chain reaction (pcr) - Google Patents
Compositions, methods and kits comprising polyinosinic acid (poly(i)) for polymerase chain reaction (pcr) Download PDFInfo
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- WO2022201104A1 WO2022201104A1 PCT/IB2022/052729 IB2022052729W WO2022201104A1 WO 2022201104 A1 WO2022201104 A1 WO 2022201104A1 IB 2022052729 W IB2022052729 W IB 2022052729W WO 2022201104 A1 WO2022201104 A1 WO 2022201104A1
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- pcr
- poly
- virus
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Definitions
- the invention relates to the field of nucleic acids amplification and detection and more particularly to reagents for improved quantitative reverse transcription polymerase chain reaction (RT-PCR).
- RT-PCR quantitative reverse transcription polymerase chain reaction
- PCR Polymerase chain reaction
- RT-PCR Reverse transcription polymerase chain reaction
- SARS-CoV2 responsible of the COVID-19 pandemic.
- the method has its limitations, and it may result in a “false negative” when a test sample comprises a low quantity of virus. There is thus an urgent need for improving sensitivity of the method, particularly in efforts to combat the COVID-19 pandemic.
- the invention relates to a composition for polymerase chain reaction (PCR) comprising Polyinosinic acid (Poly(l)).
- the invention relates to a composition for reverse transcription polymerase chain reaction (RT-PCR) comprising Polyinosinic acid (Poly(l)).
- RT-PCR reverse transcription polymerase chain reaction
- the invention relates to ready-to-use reagent mixture for polymerase chain reaction (PCR), the mixture comprising: DNA polymerase, deoxyribonucleotide triphosphates, salt, buffer, magnesium, and Polyinosinic acid (Poly(l)).
- PCR polymerase chain reaction
- the invention relates to a ready-to-use reagent mixture for reverse transcription polymerase chain reaction (RT-PCR), the mixture comprising: reverse transcriptase, DNA polymerase, deoxyribonucleotide triphosphates, salt, buffer, magnesium, and Polyinosinic acid (Poly(l)).
- RT-PCR reverse transcription polymerase chain reaction
- the invention relates to the use of Polyinosinic acid (Poly(l)) in polymerase chain reaction (PCR) and/or in reverse transcription polymerase chain reaction (RT-PCR).
- PCR polymerase chain reaction
- RT-PCR reverse transcription polymerase chain reaction
- the invention relates to the use of Polyinosinic acid (Poly(l)) for increasing sensitivity of polymerase chain reaction (PCR) and/or for increasing sensitivity of reverse transcription polymerase chain reaction (RT-PCR).
- the invention relates to a method for increasing sensitivity of polymerase chain reaction (PCR), comprising: amplifying a DNA sequence of interest in a PCR amplification composition comprising Polyinosinic acid (Poly(l)).
- PCR polymerase chain reaction
- the invention relates to a method for increasing sensitivity of reverse transcription polymerase chain reaction (RT-PCR), the method comprising amplifying a RNA sequence of interest in a RT-PCR amplification composition comprising Polyinosinic acid (Poly(l)).
- RT-PCR reverse transcription polymerase chain reaction
- Additional aspects concerns kits, including a kit for carrying out any of the methods described herein, a kit for detection of viral RNA and a kit for reverse transcription polymerase chain reaction (RT-PCR).
- kits comprise the composition as defined herein and/or the ready-to-use reagent mixture as defined herein, and one or more of the followings: a sample collecting tube(s), reaction tube(s), microplate(s), buffer for the homogenization of the sample(s), incubation buffer(s), assay buffer(s), fluorescent and/or luminogenic detection materials, desalting column(s), purified control purified RNA or cDNA and a user manual or instructions.
- Figure 1 is a line graph showing amplification curves of TaqPathTM vs MM (MasterMix) with added hpRNA vs MM no hpRNA, using ATCC 1 :50,000 as a template for qPCR detection of SARS-CoV2 E gene on BioRadTM CFX96.
- Figure 2 is a line graph showing amplification curves of TaqPathTM vs MM with hpRNA vs MM no hpRNA, using ATCC 1 :50 as a template for qPCR detection of SARS- CoV2 E gene on BioRadTM CFX96.
- Figure 3 is a line graph showing amplification curves of TaqPathTM vs MM no Poly(l) vs MM with Poly(l) at 0.1 and 0.5 pg/mI, using ATCC 1 :50,000 as a template for qPCR detection of SARS-CoV2 E gene on BioRadTM CFX96.
- Figure 4 is a line graph showing amplification curves of MM lacking Poly(l) vs MM with Poly(l) from Sigma at 0.01 , 0.1 , and 0.25 pg/mI, using ATCC 1 :50,000 as a template for qPCR detection of SARS-CoV2 E gene on BioRadTM CFX96.
- Figure 5 is a line graph showing amplification curves of MM lacking Poly(l) vs
- Figure 6 is a line graph showing amplification curves of MM lacking Poly(l) vs MM with Poly(l) from Sigma at 0.1 pg/mI, using ATCC 1 :100 and 1 :50,000 as templates for qPCR detection of SARS-CoV2 E gene on BioRadTM CFX96.
- Figure 7 is a line graph showing amplification curves of MM lacking Poly(l) vs MM with Poly(l) from Sigma at 0.025, 0.1 , and 0.5 pg/mI, using ATCC 1 :50,000 as a template for qPCR detection of SARS-CoV2 E gene on BioRadTM CFX96.
- Figure 8 is a line graph showing amplification curves of TaqPathTM vs MM with Sigma Poly(l)(0.1 pg/mI), using ATCC 1 :100 and 1 :50,000 as a template for qPCR detection of SARS-CoV2 E gene on BioRadTM CFX96.
- Figure 9 is a line graph showing amplification curves of TaqPathTM vs MM with Sigma Poly(l) (0.1 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher QuantstudioTM 5.
- Figure 10 is a line graph showing amplification curves of TaqPathTM vs MM with Sigma Poly(l) (0.2 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher QuantstudioTM 5.
- Figure 11 is a line graph showing amplification curves of MM with Sigma Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.2 pg/mI), using ATCC 1 :100 and NIST 1 :1.75M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher
- Figure 12 is a line graph showing amplification curves of MM with Sigma Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.2 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher QuantstudioTM 5.
- Figure 13 is a line graph showing amplification curves of MM with TRC Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.1 pg/mI), using ATCC 1 :100 and NIST 1 :1 .75M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher QuantstudioTM 5.
- Figure 14 is a line graph showing amplification curves of MM with TRC Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.1 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher QuantstudioTM 5.
- Figure 15 is a line graph showing amplification curves of MM with TRC Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.2 pg/mI), using ATCC 1 :100 and NIST 1 :1 .75M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher QuantstudioTM 5.
- Figure 16 is a line graph showing amplification curves of MM with TRC Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.2 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher QuantstudioTM 5.
- Figure 17 is a line graph showing amplification curves of TaqPathTM vs MM with TRC Poly(l) (0.1 pg/mI), using ATCC 1 :100 and NIST 1 :1 .75M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher QuantstudioTM 5.
- Figure 18 is a line graph showing amplification curves of TaqPathTM vs MM with TRC Poly(l) (0.1 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher QuantstudioTM 5.
- Poly(l) greatly increases sensitivity of the polymerase chain reaction (PCR), and more particularly reverse transcription polymerase chain reaction (RT-PCR).
- PCR polymerase chain reaction
- RT-PCR reverse transcription polymerase chain reaction
- the present invention thus provides means of improvement of existing amplification compositions, ready-to-use reagent mixtures, and amplification methods.
- the present invention also addresses an urgent medical need in increasing detection sensitivity and reliability of detection of pathogens, and particularly viruses such as SARS-CoV2.
- the present description focuses primarily on the uses of Poly(l) for RT-PCR, the present invention encompasses the uses of Poly(l) in other nucleic acid amplification compositions, reagents and methods, including but not limited quantitative PCR (qPCR), real-time PCR, real-time RT-PCR, droplet digital PCR (ddPCR), isothermal amplification and loop-mediated isothermal amplification.
- qPCR quantitative PCR
- ddPCR droplet digital PCR
- isothermal amplification isothermal amplification
- loop-mediated isothermal amplification loop-mediated isothermal amplification.
- compositions and mixtures for amplification of nucleic acids molecules [00047]
- One aspect of the invention concerns a composition for amplification of nucleic acid molecules, the composition comprising Polyinosinic acid (Poly(l)).
- the composition comprises the compound Polyinosinic acid (Poly(l)).
- the compound may also be known by other names such as: 5'-lnosinic acid, polymers (8CI); NSC 120952; Poly(5'-inosinic acid); Poly(IMP) and Poly(rl).
- Polyinosinic acid may be used in different forms and may be obtained from different sources.
- the Poly(l) being used is Polyinosinic acid potassium salt (5'-lnosinic acid, homopolymer, potassium salt; Molecular Formula (CioHi 3 N 4 0gP) x . x K ; CAS Number 26936-41 -4).
- Such Poly(l) salt may be obtained for instance from Sigma-Aldrich (product #P4154).
- the Poly(l) being used is Polyinosinic acid homopolymer (5'-lnosinic acid, homopolymer; Molecular Formula (C IO H 13 N 4 0 8 P) X ; CAS Number 30918-54-8). Such Poly(l) may be obtained for instance from Toronto Research Chemicals (product #P698975).
- the Poly(l) being used is Polyinosinic acid homopolymer sodium salt (5'-lnosinic acid, homopolymer, sodium salt CAS Number 33378-44-8). Other Poly(l) salts are also envisionable.
- Polyinosinic acid is a homopolymer of inosine that may form with polycytidylic acid (poly(C)) the double-stranded homopolymer (Poly(l) ⁇ Poly(C)). Accordingly, it may be envisioned in accordance with the present invention to use Poly(l) in a double-stranded homopolymer form (i.e. Poly(l) ⁇ Poly(C)). Particular examples include, but are not limited to, (C10 H13 N4 08 P)x . (C9 H14 N3 08 P)x (CAS Registry Number 24939-03-5), the sodium salt C10 H13 N4 08 P)x .
- Polyinosinic acid may be used at any suitable concentration, for instance a concentration allowing to improve at least one of sensitivity fluorescent signal, of amplification methods and techniques.
- Poly(l) is used in the amplification tube at a final concentration of about 0.0001 pg/mI to about 100 pg/mI, or about 0.001 mg/ml to about 10 pg/mI, or about 0.01 mg/ml to about 0.5 pg/pl.
- a composition for amplification in accordance with the present invention e.g.
- MasterMix for PCR or RT-PCR as defined hereinafter comprises about 0.00011 pg/mI to about 400 pg/mI, or about 0.0011 pg/mI to about 40 pg/mI, or about 0.01 pg/mI to to about 10 pg/mI, or about 0.01 pg/mI to about 4 pg/mI, or about 0.1 pg/mI to about 1 pg/mI.
- the composition for amplification of nucleic acid molecules in accordance with the present invention minimally comprises components that are found in a minimal formulation for RT-PCR, including deoxyribonucleotide triphosphates, salt, buffer and magnesium.
- the composition further comprises additional components, including one or more of glycerol, BSA, a polar aprotic solvent (e.g. Dimethyl sulfoxide (DMSO)), a nonionic surfactant (e.g. Triton X-100TM) and a nonionic, non-denaturing detergent (e.g. IGEPAL CA-630).
- a polar aprotic solvent e.g. Dimethyl sulfoxide (DMSO)
- a nonionic surfactant e.g. Triton X-100TM
- a nonionic, non-denaturing detergent e.g. IGEPAL CA-630.
- the composition further comprises at least one of tetrapropylammonium hydroxide (CAS Number: 4499-86-9) and Hotstart DNA oligomer called HS2 or TQH6.
- HS2 a single stranded DNA sequence identified as TQH6 (SEQ ID NO:78) in US patent No. 6,183,967 (incorporated herein by reference in its entirety).
- TQH6 SEQ ID NO:78
- Such single stranded DNA sequence may be synthesized and, for instance its synthesis may commissioned from many suppliers (e.g. Integrated DNA Technologies, Inc, Genewiz, OligoFactory, etc.)
- the composition further comprises a reverse transcriptase / RNA-directed DNA polymerase (e.g. Moloney murine leukemia virus reverse transcriptase; CAS Number: 9068-38-6, Enzyme Commission Number: 2.7.7.49) and a DNA-directed DNA polymerase (e.g. Thermus aquaticus DNA polymerase; CAS Number: 9012-90-2, Enzyme Commission Number: 2.7.7.7)), and optionally at least one of a RNAse inhibitor (e.g. porcine RNAse inhibitor protein; CAS Number: 39369-21 -6) and a uracil DNA glycosylase (e.g.
- a reverse transcriptase / RNA-directed DNA polymerase e.g. Moloney murine leukemia virus reverse transcriptase; CAS Number: 9068-38-6, Enzyme Commission Number: 2.7.7.49
- a DNA-directed DNA polymerase e.g. Thermus aquaticus DNA polymerase
- the composition comprises the four (4) enzymes. It is also conceivable to use variations on these proteins and/or equivalent proteins from many other sources.
- composition or MM may be adapted in accordance with various factors such as the origin of the biological sample, the RNA or DNA sequence of interest, the PCR amplification method to be used, the isothermal amplification method or apparatus to be used, the amplification conditions, etc.
- the composition is a ready-to-use mixture is a MasterMix (MM) comprising the components of the mixtures defined in Table 1.
- the MM is as defined in the broader range of Table 1.
- the MM is as defined in the narrow range of Table 1.
- the MM is as defined in the first column of Table 1.
- the ready-to-use mixture advantageously further comprises Polyinosinic acid (Poly(l)) and, therefore, the ready-to-use mixture is a MasterMix (MM) as defined in any of the column of Table 1 + Poly(l) (e.g. about 0.01 pg/mI to about 1 pg/mI Poly(l), or about 0.1 pg/mI, or about 0.2 mg/ml, or about 0.5 pg/pl).
- the definite list of components and their relative concentrations in the composition may be adapted in accordance with factors such as the origin of the biological sample, the RNA or DNA sequence of interest, the PCR amplification method or apparatus to be used, the isothermal amplification method or apparatus to be used, the amplification conditions, etc.
- compositions in accordance with the present invention including ready-to- use mixtures and MasterMix (MM) may be formulated as a ready to use solution (i.e. 1X) or as a stock solution (e.g. 1 .1 X, 2X, 3X, 4X, 5X, 10X, etc.) for further dilution.
- a given quantity of a stock solution may be mixed within a reaction tube to obtain a final 1X concentration or, if appropriate, be previously diluted with a predetermined volume of filtered or distilled water.
- the pH of a resulting diluted solution may be adjusted to a desired pH by addition of suitable acidifying or alkaline agents.
- the present invention provides means for improving existing PCR-based amplification and detection methods.
- another aspect of the invention concerns methods for increasing sensitivity of polymerase chain reaction (PCR) and/or for increasing sensitivity of reverse transcription polymerase chain reaction (RT-PCR).
- the method comprises amplifying a nucleic acid molecule of interest (e.g. a DNA sequence in the case of PCR or a RNA sequence in the case of RT-PCR) in an amplification compositions comprising Polyinosinic acid (Poly(l)).
- a nucleic acid molecule of interest e.g. a DNA sequence in the case of PCR or a RNA sequence in the case of RT-PCR
- Poly(l) Polyinosinic acid
- the Poly(l) and amplification compositions are as defined hereinbefore.
- the methods of the invention are use for amplification of a nucleic acid of interest obtained from a biological sample, including but not limited to blood, urine, saliva, cerebrospinal fluid, nasal swab, nasopharyngeal swab, throat swab, etc.
- a biological sample including but not limited to blood, urine, saliva, cerebrospinal fluid, nasal swab, nasopharyngeal swab, throat swab, etc.
- Other sources of nucleic acid of interest obtained include animal, human and plant tissues, biopsies, cultures of cells, microbes such as viruses and bacteria, fungi, algae, or environmental samples of earth, water, air etc.
- the amplification is a real-time polymerase chain reaction (real-time PCR), also known as quantitative Polymerase Chain Reaction (qPCR) and is carried out in a thermocycler in accordance with parameters and conditions known in the art for real time PCR.
- real-time PCR also known as quantitative Polymerase Chain Reaction (qPCR)
- qPCR quantitative Polymerase Chain Reaction
- the amplification is a multiplex RT-qPCR reaction.
- amplification is a RT-qPCR reaction carried out in a 20 mI_ final reaction volume consisting of:
- amplified nucleic acid molecule copies or PCR products are detected by measuring light emission of a fluorescent chemical compound.
- the detection methods of PCR products in real-time PCR are: (1 ) non specific fluorescent dyes that intercalate with any double-stranded DNA; and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter, which permits detection only after hybridization and subsequent degradation of the probe from its complementary sequence. Both detection methods are suitable in accordance with the present invention.
- the methods in accordance with the present invention further comprise detecting a fluorescence signal associated with amplification of the nucleic acid sequence, wherein the fluorescence signal is increased compared to an amplification (e.g. PCR or RT-PCR) carried out in absence of the Poly(l).
- an amplification e.g. PCR or RT-PCR
- Having an increased fluorescence signal may provide many benefits to existing PCR amplification methods, including but not limited to increasing sensitivity, lowering the threshold detection level and reducing likelihood of false negatives.
- using Poly(l) in amplification methods in accordance with the present invention provides an increase sensitivity of at least 2 fold, or at least 2.5 fold, or at least 3 fold, or at least 4 fold, or at least 5 fold, or at least 6 fold, or at least 7 fold, or at least 10 fold or more, when compared an amplification (e.g. PCR or RT-PCR) carried out in absence of the Poly(l).
- an amplification e.g. PCR or RT-PCR
- using Poly(l) in amplification methods in accordance with the present invention provides an increase in the fluorescent signal of at least 10%, or at least 25%, or at least 50%, or at least 75%, or at least 100%, or at least 150 %, or at least 200%, or at least 300%, or at least 500%, or at least 1000%, at least 2000% or more, when compared an amplification (e.g. PCR or RT-PCR) carried out in absence of the Poly(l).
- an amplification e.g. PCR or RT-PCR
- the amplification methods of the present invention allow for detection of a lower copy number of a nucleic acid sequence of interest, compared to amplification carried out in absence of the Poly(l).
- the present invention may allow for detection of a reduced number of copies of a target nucleic acid, for instance detection of as low as 2 500 copies, or as low as 1 000 copies, or as low as 75 copies, or as low as 500 copies, or as low as 250 copies, or as low as 100 copies, or as low as 50 copies, or as low as 25 copies, or low as 15 copies, or low as 10 copies, or as low as 5 copies, or as low as 1 copy, in a sample.
- cycle threshold (Ct) value e.g. by at least 0.5, 1 , 2, 3, 4, 5, 10, 15, 20 or more cycles
- cycle threshold (Ct) value e.g. by at least 0.5, 1 , 2, 3, 4, 5, 10, 15, 20 or more cycles
- the starting quantity of the nucleic acid materials to be amplified is low (e.g. less than 10 copies, or less than 15 copies, or less than 25 copies, or less than 50 copies, or less than 75 or less than 100 copies, or less than 250 copies, or less than 500 copies or less than 1000 copies).
- the nucleic acid sequence of interest is viral RNA and the amplification methods in accordance with the present invention are used for the detection of viruses. Detection of virus may be particularly useful for diagnostic purposes, for treatment purposes, for safety measures (e.g. quarantine or isolation), etc.
- the methods and compositions may be used for the detection of various types of viruses, including those of Group I (double-stranded DNA viruses), Group II (single-stranded DNA viruses), Group III (double-stranded RNA viruses), Group IV (positive sense single-stranded RNA viruses), Group V (negative sense single-stranded RNA viruses), Group VI (single-stranded RNA viruses with a DNA intermediate in their life cycle) and Group VII (double-stranded DNA viruses with an RNA intermediate in their life cycle).
- Group I double-stranded DNA viruses
- Group II single-stranded DNA viruses
- Group III double-stranded RNA viruses
- Group IV positive sense single-stranded RNA viruses
- Group V negative sense single-stranded RNA viruses
- Group VI single-stranded RNA viruses with a DNA intermediate in their life cycle
- Group VII double-stranded DNA viruses with an RNA intermediate in their life cycle
- the methods and compositions of the present invention are for amplification and detection of human pathogenic viruses, including, but not limited to, adenovirus, cytomegalovirus, coronavirus, herpes virus, HIV, human papillomavirus, influenza virus, measles virus, Middle East respiratory syndrome-related coronavirus (MERS-CoV), mumps virus, parainfluenza virus, poliovirus, rabies virus, respiratory syncytial virus, and rubella virus.
- the pathogenic virus is SARS-CoV2.
- the methods and compositions of the present invention are for RT-PCR and are for amplification and detection of RNA viruses (e.g. Baltimore groups III, IV and V), including but not limited to rotavirus, orbivirus, coltivirus, banna virus, human astrovirus, Norwalk virus, human coronaviruses (e.g.
- Middle East respiratory syndrome-related coronavirus severe acute respiratory syndrome coronaviruses, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, TBE virus, Zika virus, Hepatitis E virus, Rubella virus, coxsackievirus, hepatitis A virus, poliovirus and rhinovirus.
- the amplification methods of the present invention may allow for the detection of a lower copy number of viral nucleic materials, and thus detection of a lower viral load or lower viral titer in a sample. This may thus reduce a number of “false negative” tests compared to existing amplification methods (e.g. PCR or RT-PCR amplification) carried out in absence of Poly(l).
- existing amplification methods e.g. PCR or RT-PCR amplification
- the methods of the invention allow for detection of a reduced number of viral particles or viral load in a sample.
- using Poly(l) may allow reliable detection of as low as 2 500 viral particles, or as low as 1000 viral particles, or as low as 75 viral particles, or as low as 500 viral particles, or as low as 250 viral particles, or as low as 100 viral particles, or as low as 50 viral particles, or as low as 25 viral particles, or low as 15 viral particles, or low as 10 viral particles, or as low as 5 viral particles, or as low as 1 viral particles, in a sample.
- Poly(l) could reduce of a minimal detection threshold (i.e. minimal number of virus particles) associated with positive detection of a virus in a sample.
- the minimal detection threshold associated with Poly(l) is reduced by at least 10 viral particles, or by at least 25 viral particles, or by at least 50 viral particles, by at least 75 viral particles, or by at least 100 viral particles, or by at least 250 viral particles, or by at least 500 viral particles, or by at least 1000 viral particles, or by at least 2500 viral particles, or by at least 5000 viral particles, or by at least 10000 viral particles or more, in a sample, when compared to amplification carried out in absence of the Poly(l).
- kits e.g. kits for polymerase chain reaction (e.g. PCR or RT-PCR) or diagnostic kits.
- the kits of the invention may be useful for the practice of the methods of the invention (e.g. for carrying out any of the amplification methods described hereinbefore) and/or for applications in humans (e.g. diagnostic or preventive applications for detection of pathogenic viruses).
- kits in accordance with the present invention comprises a composition for PCR or for RT-PCR as defined hereinbefore, or a ready-to-use reagent mixture or MasterMix as defined hereinbefore.
- a kit of the invention may further comprise one or more of the following elements: sample collection tubes (e.g. sterile tubes for collecting blood, urine, saliva, swabs, tissue samples, cell culture, environmental samples, etc.), a buffer for the homogenization of the blood, saliva, swabs sample(s), tissue samples, cell culture or environmental sample, etc., beads or other materials for isolating nucleic acid from samples, purified RNA or cDNA to be used as controls in PCR and/or RT-PCR amplification methods, incubation buffer(s), substrate and assay buffer(s), modulator buffer(s) and modulators (e.g. enhancers, inhibitors), standards, detection materials (e.g.
- sample collection tubes e.g. sterile tubes for collecting blood, urine, saliva, swabs, tissue samples, cell culture, environmental samples, etc.
- fluorochromes fluorochromes, luminogenic substrates, detection solutions, scintillation counting fluid, etc.
- laboratory supplies e.g. desalting columns, reaction tubes or microplates (e.g. 96- or 384-well plates), a user manual or instructions, etc.
- the kit and methods of the invention are configured such as to permit a quantitative detection or measurement of amplicons, DNA, RNA and/or nucleic acid sequence of interest.
- the kit may be optimized for various amplification methods and techniques, including but not limited quantitative PCR (qPCR), real-time PCR, real-time RT-PCR, droplet digital PCR (ddPCR), isothermal amplification.
- qPCR quantitative PCR
- ddPCR droplet digital PCR
- a kit for ddPCR may further comprise components for droplet generation, amplification and visualization, and digital droplet classification.
- the kit is optimized for qPCR detection of RNA viruses.
- kits of the invention may comprise at least one primer or probe (preferably at least two primers) which specifically hybridizes with nucleic acid molecules for a RNA virus such as SARS-CoV2. reaction buffers and instructional material.
- the primer or probe may contain a detectable tag.
- Certain kits may contain two or more of such primers or probes.
- a kit of the invention comprises components of the amplification system, including PCR reaction materials such as buffers, a thermostable polymerase and a reverse transcriptase.
- the kit of the present invention can be used in conjunction with commercially available amplification kits, such as those that may be obtained from GIBCO BRL (Gaithersburg, Md.), Stratagene (La Jolla, Calif.), Invitrogen (San Diego, Calif.).
- the kit may optionally include instructional materials, positive or negative control reactions, templates, or markers.
- the present invention may provide many benefits.
- the invention provides one or more of the following benefits:
- RNA and/or DNA targets in a single multiplex RT-qPCR reaction; robust performance across many sample types and instruments; - stock solutions (e.g. 1 .1 X, or 2X or 4X) which guarantees a simple reaction setup;
- hpRNA human placental RNA
- MM MasterMix; see Table 1
- Poly(l) poly-inosinic acid
- TaqPathTM TaqPathTM 1-Step Multiplex Master Mix (No ROX)
- ATCC ATCC-VR3276SD - Quantitative Synthetic SARS-CoV-2 RNA: ORF, E, N (ATCC® VR-3276SDTM). Listed as between 10 5 and 10 6 copies per mI.
- NIST NIST -10169 - SARS-CoV-2 Research Grade Test Material
- Cooling 1 cycle 40 S C - hold 30 sec (ramp rate 2.2)
- Figure 1 compares TaqPathTM vs MM with added hpRNA vs MM no hpRNA, using ATCC 1 :50,000 as a template for qPCR detection of SARS-CoV2 E gene on BioRadTM CFX96. These representative results indicate that a component of hpRNA can reduce the cycle threshold (Ct) value when the concentration of RNA template is low. After this observation, we sought to replicate and improve the observed effect of hpRNA with a different chemical.
- Ct cycle threshold
- Figure 2 compares TaqPathTM vs MM with hpRNA vs MM no hpRNA, using ATCC 1 :50 as a template for qPCR detection of SARS-CoV2 E gene on BioRadTM CFX96. These representative results indicate the effect of hpRNA is not observed when the concentration of RNA template is high. After this observation, we sought to replicate and improve the observed effect of hpRNA in Figure 1 with a different chemical.
- Figure 3 compares TaqPathTM vs MM no Poly(l) vs MM with Poly(l) at 0.1 and 0.5 pg/mI, using ATCC 1 :50,000 as a template for qPCR detection of SARS-CoV2 E gene on BioRadTM CFX96. These representative results indicate that Poly(l) can enhance peak fluorescence by amplifying more qPCR product when the concentration of RNA template is low.
- Figure 4 compares MM lacking Poly(l) vs MM with Poly(l) from Sigma at 0.01 , 0.1 , and 0.25 pg/mI, using ATCC 1 :50,000 as a template for qPCR detection of SARS- CoV2 E gene on BioRadTM CFX96. These representative results indicate that the effect of Poly(l) is dependent on its concentration in the MasterMix.
- Figure 5 compares MM lacking Poly(l) vs MM with Poly(l) from Sigma at 0.025,
- Figure 6 compares MM lacking Poly(l) vs MM with Poly(l) from Sigma at 0.1 pg/mI, using ATCC 1 :100 and 1 :50,000 as templates for qPCR detection of SARS-CoV2 E gene on BioRadTM CFX96.
- Figure 8 compares TaqPathTM vs MM with Poly(l) ( 0.1 pg/mI), using ATCC 1 :100 and 1 :50,000 as a template for qPCR detection of SARS-CoV2 E gene on BioRadTM CFX96. These representative results confirm the Mastermix containing Poly(l) has comparable performance to TaqPathTM when the concentration of RNA template is high (-28,000 copies) or low (-55 copies).
- Figure 9 compares TaqPathTM vs MM with Sigma Poly(l) (0.1 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher QuantstudioTM 5. These representative results confirm improved performance compared to TaqPathTM when 0.1 pg/mI Poly(l) (Sigma) is added to the MasterMix, especially at the limit of detection ( ⁇ 10 copies). The results also demonstrate that the effect can be seen when experiments are performed in another laboratory, with different personnel using another brand of thermal cycler.
- Figure 10 compares TaqPathTM vs MM with Sigma Poly(l) (0.2 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher QuantstudioTM 5. These representative results confirm improved performance compared to TaqPathTM when 0.2 pg/mI Poly(l) (Sigma) is added to the MasterMix. The improvement is seen when the concentration of RNA template is low ( ⁇ 55 copies), or near the estimated limit of detection ( ⁇ 10 copies) for that particular experiment.
- Figure 11 compares MM with Sigma Poly(l) (0.1 pg/mI) vs MM with Sigma
- Poly(l) (0.2 pg/mI), using ATCC 1 :100 and NIST 1 :1 .75M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher QuantstudioTM 5. These representative results confirm concentration-dependence of the effect of adding Poly(l) (Sigma) to the MasterMix, especially when the concentration of RNA template is low ( ⁇ 55 copies), or near the estimated limit of detection ( ⁇ 15 copies) for that particular experiment.
- Figure 12 compares MM with Sigma Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.2 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher QuantstudioTM 5. These representative results confirm concentration-dependence of the effect of adding Poly(l) (Sigma) to the MasterMix, especially when the concentration of RNA template is low ( ⁇ 55 copies), or near the estimated limit of detection ( ⁇ 10 copies) for that particular experiment.
- Figure 13 compares MM with TRC Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.1 pg/mI), using ATCC 1 :100 and NIST 1 :1.75M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher QuantstudioTM 5. These representative results confirm that adding Poly(l) (TRC) at 0.1 pg/mI gives improved performance compared to Poly(l) (Sigma) at 0.1 pg/mI, especially near the estimated limit of detection ( ⁇ 15 copies) for that particular experiment.
- Figure 14 compares MM with TRC Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.1 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher QuantstudioTM 5. These representative results confirm that adding Poly(l) (TRC) at O.l pg/mI gives improved performance compared to Poly(l) (Sigma) at 0.1 pg/mI, especially when the concentration of RNA template is low (-55 copies), or near the estimated limit of detection ( ⁇ 10 copies) for that particular experiment.
- Figure 15 compares MM with TRC Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.2 mg/ml), using ATCC 1 :100 and NIST 1 :1.75M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher QuantstudioTM 5. These representative results confirm comparable performance between MasterMixes containing either 0.1 pg/mI Poly(l) (TRC) or 0.2 pg/mI Poly(l) (Sigma).
- Figure 16 compares MM with TRC Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.2 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher QuantstudioTM 5. These representative results confirm comparable performance between MasterMixes containing either 0.1 pg/mI Poly(l) (TRC) or 0.2 pg/mI Poly(l) (Sigma) when the concentration of RNA template is low (-55 copies). Improved performance is seen with 0.1 pg/mI Poly(l) (TRC) near the estimated limit of detection ( ⁇ 10 copies) for that particular experiment.
- Figure 17 compares TaqPathTM vs MM with TRC Poly(l) (0.1 pg/mI), using ATCC 1 :100 and NIST 1 :1 .75M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher QuantstudioTM 5. These representative results confirm comparable performance compared to TaqPathTM when 0.1 pg/mI Poly(l) (TRC) is added to the MasterMix when the concentration of RNA template is high (-28,000 copies). Improved performance is seen with 0.1 pg/mI Poly(l) (TRC) near the estimated limit of detection (-15 copies) for that particular experiment.
- Figure 18 compares TaqPathTM vs MM with TRC Poly(l) (0.1 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher QuantstudioTM 5. These representative results confirm improved performance compared to TaqPathTM when 0.1 pg/mI Poly(l) (TRC) is added to the MasterMix, especially when when the concentration of RNA template is low (-55 copies), and near the estimated limit of detection (-10 copies) for that particular experiment.
- Headings are included herein for reference and to aid in locating certain sections.
Abstract
The invention provides reagents, methods and kits for improving nucleic acids amplification and detection. Described herein are compositions and ready-to-use reagent mixtures comprising, among other things, Polyinosinic acid (Poly(I)). Also described are kits and methods for increasing sensitivity of polymerase chain reaction (PCR) and/or for increasing sensitivity of reverse transcription polymerase chain reaction (RT-PCR).
Description
COMPOSITIONS, METHODS AND KITS COMPRISING POLYINOSINIC ACID (POLY(I)) FOR POLYMERASE CHAIN REACTION (PCR)
CROSS REFERENCE TO RELATED APPLICATION [0001] The present application claims priority to United States patent application number US 63/166,438 filed on March 21 , 2022, the content of which is incorporated herewith by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to the field of nucleic acids amplification and detection and more particularly to reagents for improved quantitative reverse transcription polymerase chain reaction (RT-PCR).
BACKGROUND OF THE INVENTION
[0003] Rapid diagnosis of viruses is crucial for efficient forecast and control of viral diseases. Polymerase chain reaction (PCR) is one of the most widely used diagnostic tests for detecting pathogens, including viruses, that cause diseases such as Ebola, African swine fever and foot-and-mouth disease.
[0004] Reverse transcription polymerase chain reaction (RT-PCR) is a rapid, sensitive and reliable method for virus detection. RT-PCR is a variation of PCR and it is used to detect viruses that only contains RNA, such as SARS-CoV2 responsible of the COVID-19 pandemic. However, the method has its limitations, and it may result in a “false negative” when a test sample comprises a low quantity of virus. There is thus an urgent need for improving sensitivity of the method, particularly in efforts to combat the COVID-19 pandemic.
[0005] Numerous reagent mixtures have been described for a rapid and efficient amplification of nucleic acid molecules and the detection and quantitation of RNA molecules. Such mixtures are described for instance in: US9353409; US20020119465; US20160097086; and US10900074. However, none of these patents or patent applications use Polyinosinic acid.
[0006] Polyinosinic acid or Poly(l) is a homopolymer of inosine. In the literature, Poly(l) has been suggested to be useful in the preparation and purification of RNAs from a biological sample (see US9057673; US20020137076; Steven G.Winslow* and Pierre A. Henkart, Nucleic Acids Research (1991), Vol. 19, No. 12 3251 ; D. Svec et. al., Frontiers in Oncology (2013), Vol. 3, Article 274, p. 1 -11) and on deoxyribonucleic acid template activity of isolated nuclei and soluble chromatin from rat liver (D. G. Brown and D.S Coffey, JBC (1972), Vol. 247, No. 23, pp. 7674-7683). However, these documents do not suggest using Polyinosinic acid in PCR, let alone to increase sensitivity of RT-PCR following extraction of RNA from samples. [0007] Accordingly, there is a need for improved PCR and RT-PCR methods with increased detection sensitivity and reliability.
[0008] There is also a need for reagents for PCR and RT-PCR that can improve the detection and quantitation of nucleic acid molecules, particularly viral nucleic acid molecules. [0009] There is particularly an urgent need for improved RT-PCR related methods, reagents and kits with increased sensitivity in the detection SARS-CoV2 in the global fight against the COVID-19 pandemic.
[00010] The present invention addresses these needs and other needs as it will be apparent from the review of the disclosure and description of the features of the invention hereinafter.
BRIEF SUMMARY OF THE INVENTION
[00011 ] According to one aspect, the invention relates to a composition for polymerase chain reaction (PCR) comprising Polyinosinic acid (Poly(l)).
[00012] According to another aspect, the invention relates to a composition for reverse transcription polymerase chain reaction (RT-PCR) comprising Polyinosinic acid (Poly(l)).
[00013] According to another aspect, the invention relates to ready-to-use reagent mixture for polymerase chain reaction (PCR), the mixture comprising: DNA polymerase,
deoxyribonucleotide triphosphates, salt, buffer, magnesium, and Polyinosinic acid (Poly(l)).
[00014] According to another aspect, the invention relates to a ready-to-use reagent mixture for reverse transcription polymerase chain reaction (RT-PCR), the mixture comprising: reverse transcriptase, DNA polymerase, deoxyribonucleotide triphosphates, salt, buffer, magnesium, and Polyinosinic acid (Poly(l)).
[00015] According to another aspect, the invention relates to the use of Polyinosinic acid (Poly(l)) in polymerase chain reaction (PCR) and/or in reverse transcription polymerase chain reaction (RT-PCR). [00016] According to another aspect, the invention relates to the use of Polyinosinic acid (Poly(l)) for increasing sensitivity of polymerase chain reaction (PCR) and/or for increasing sensitivity of reverse transcription polymerase chain reaction (RT-PCR).
[00017] According to another aspect, the invention relates to a method for increasing sensitivity of polymerase chain reaction (PCR), comprising: amplifying a DNA sequence of interest in a PCR amplification composition comprising Polyinosinic acid (Poly(l)).
[00018] According to another aspect, the invention relates to a method for increasing sensitivity of reverse transcription polymerase chain reaction (RT-PCR), the method comprising amplifying a RNA sequence of interest in a RT-PCR amplification composition comprising Polyinosinic acid (Poly(l)). [00019] Additional aspects concerns kits, including a kit for carrying out any of the methods described herein, a kit for detection of viral RNA and a kit for reverse transcription polymerase chain reaction (RT-PCR). In embodiments these kits comprise the composition as defined herein and/or the ready-to-use reagent mixture as defined herein, and one or more of the followings: a sample collecting tube(s), reaction tube(s), microplate(s), buffer for the homogenization of the sample(s), incubation buffer(s), assay buffer(s), fluorescent and/or luminogenic detection materials, desalting column(s), purified control purified RNA or cDNA and a user manual or instructions.
[00020] Additional aspects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments which are exemplary and should not be interpreted as limiting the scope of the invention. BRIEF DESCRIPTION OF THE FIGURES
[00021] In order for the invention to be readily understood, embodiments of the invention are illustrated by way of example in the accompanying figures.
[00022] Figure 1 is a line graph showing amplification curves of TaqPath™ vs MM (MasterMix) with added hpRNA vs MM no hpRNA, using ATCC 1 :50,000 as a template for qPCR detection of SARS-CoV2 E gene on BioRad™ CFX96.
[00023] Figure 2 is a line graph showing amplification curves of TaqPath™ vs MM with hpRNA vs MM no hpRNA, using ATCC 1 :50 as a template for qPCR detection of SARS- CoV2 E gene on BioRad™ CFX96.
[00024] Figure 3 is a line graph showing amplification curves of TaqPath™ vs MM no Poly(l) vs MM with Poly(l) at 0.1 and 0.5 pg/mI, using ATCC 1 :50,000 as a template for qPCR detection of SARS-CoV2 E gene on BioRad™ CFX96.
[00025] Figure 4 is a line graph showing amplification curves of MM lacking Poly(l) vs MM with Poly(l) from Sigma at 0.01 , 0.1 , and 0.25 pg/mI, using ATCC 1 :50,000 as a template for qPCR detection of SARS-CoV2 E gene on BioRad™ CFX96. [00026] Figure 5 is a line graph showing amplification curves of MM lacking Poly(l) vs
MM with Poly(l) from Sigma at 0.025, 0.1 , and 0.25 pg/mI, using ATCC 1 :50,000 as a template for qPCR detection of SARS-CoV2 E gene on BioRad™ CFX96.
[00027] Figure 6 is a line graph showing amplification curves of MM lacking Poly(l) vs MM with Poly(l) from Sigma at 0.1 pg/mI, using ATCC 1 :100 and 1 :50,000 as templates for qPCR detection of SARS-CoV2 E gene on BioRad™ CFX96.
[00028] Figure 7 is a line graph showing amplification curves of MM lacking Poly(l) vs MM with Poly(l) from Sigma at 0.025, 0.1 , and 0.5 pg/mI, using ATCC 1 :50,000 as a template for qPCR detection of SARS-CoV2 E gene on BioRad™ CFX96.
[00029] Figure 8 is a line graph showing amplification curves of TaqPath™ vs MM with Sigma Poly(l)(0.1 pg/mI), using ATCC 1 :100 and 1 :50,000 as a template for qPCR detection of SARS-CoV2 E gene on BioRad™ CFX96.
[00030] Figure 9 is a line graph showing amplification curves of TaqPath™ vs MM with Sigma Poly(l) (0.1 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher Quantstudio™ 5. [00031] Figure 10 is a line graph showing amplification curves of TaqPath™ vs MM with Sigma Poly(l) (0.2 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher Quantstudio™ 5.
[00032] Figure 11 is a line graph showing amplification curves of MM with Sigma Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.2 pg/mI), using ATCC 1 :100 and NIST 1 :1.75M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher
Quantstudio™ 5.
[00033] Figure 12 is a line graph showing amplification curves of MM with Sigma Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.2 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher Quantstudio™ 5.
[00034] Figure 13 is a line graph showing amplification curves of MM with TRC Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.1 pg/mI), using ATCC 1 :100 and NIST 1 :1 .75M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher Quantstudio™ 5.
[00035] Figure 14 is a line graph showing amplification curves of MM with TRC Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.1 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher Quantstudio™ 5.
[00036] Figure 15 is a line graph showing amplification curves of MM with TRC Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.2 pg/mI), using ATCC 1 :100 and NIST 1 :1 .75M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher Quantstudio™ 5.
[00037] Figure 16 is a line graph showing amplification curves of MM with TRC Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.2 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher Quantstudio™ 5.
[00038] Figure 17 is a line graph showing amplification curves of TaqPath™ vs MM with TRC Poly(l) (0.1 pg/mI), using ATCC 1 :100 and NIST 1 :1 .75M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher Quantstudio™ 5.
[00039] Figure 18 is a line graph showing amplification curves of TaqPath™ vs MM with TRC Poly(l) (0.1 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher Quantstudio™ 5.
[00040] Further details of the invention and its advantages will be apparent from the detailed description included below.
DETAILED DESCRIPTION OF EMBODIMENTS
[00041] In the following description of the embodiments, references to the accompanying figures are illustrations of an example by which the invention may be practiced. It will be understood that other embodiments may be made without departing from the scope of the invention disclosed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.
General overview
[00042] The present inventors have unexpectedly found that Polyinosinic acid (Poly(l)) greatly increases sensitivity of the polymerase chain reaction (PCR), and more particularly reverse transcription polymerase chain reaction (RT-PCR).
[00043] Indeed, as demonstrated in the following Example(s), addition of Poly(l) to a RT-PCR amplification mixture increases a resulting fluorescence signal compared to a RT-PCR amplification carried out in absence of Poly(l).
[00044] The present invention thus provides means of improvement of existing amplification compositions, ready-to-use reagent mixtures, and amplification methods.
[00045] The present invention also addresses an urgent medical need in increasing detection sensitivity and reliability of detection of pathogens, and particularly viruses such as SARS-CoV2.
[00046] Although the present description focuses primarily on the uses of Poly(l) for RT-PCR, the present invention encompasses the uses of Poly(l) in other nucleic acid amplification compositions, reagents and methods, including but not limited quantitative PCR (qPCR), real-time PCR, real-time RT-PCR, droplet digital PCR (ddPCR), isothermal amplification and loop-mediated isothermal amplification.
Compositions and mixtures for amplification of nucleic acids molecules [00047] One aspect of the invention concerns a composition for amplification of nucleic acid molecules, the composition comprising Polyinosinic acid (Poly(l)).
[00048] According to one aspect, the composition comprises the compound Polyinosinic acid (Poly(l)). The compound may also be known by other names such as: 5'-lnosinic acid, polymers (8CI); NSC 120952; Poly(5'-inosinic acid); Poly(IMP) and Poly(rl).
[00049] In accordance with the present invention, Polyinosinic acid may be used in different forms and may be obtained from different sources. In one embodiment, the Poly(l) being used is Polyinosinic acid potassium salt (5'-lnosinic acid, homopolymer, potassium salt; Molecular Formula (CioHi3N40gP)x . x K ; CAS Number 26936-41 -4). Such Poly(l) salt may be obtained for instance from Sigma-Aldrich (product #P4154). In another embodiment, the Poly(l) being used is Polyinosinic acid homopolymer (5'-lnosinic acid, homopolymer; Molecular Formula (CIOH13N408P)X; CAS Number 30918-54-8). Such
Poly(l) may be obtained for instance from Toronto Research Chemicals (product #P698975). In another embodiment, the Poly(l) being used is Polyinosinic acid homopolymer sodium salt (5'-lnosinic acid, homopolymer, sodium salt CAS Number 33378-44-8). Other Poly(l) salts are also envisionable. [00050] As is known, Polyinosinic acid (Poly(l)) is a homopolymer of inosine that may form with polycytidylic acid (poly(C)) the double-stranded homopolymer (Poly(l) · Poly(C)). Accordingly, it may be envisioned in accordance with the present invention to use Poly(l) in a double-stranded homopolymer form (i.e. Poly(l) · Poly(C)). Particular examples include, but are not limited to, (C10 H13 N4 08 P)x . (C9 H14 N3 08 P)x (CAS Registry Number 24939-03-5), the sodium salt C10 H13 N4 08 P)x . (C9 H14 N3 08 P)x . x Na (CAS 42424-50-0), the potassium salt (C10 H13 N4 08 P)x . (C9 H14 N3 08 P)x . x K (CAS 31852-29-6) and the lithium salt (C10 H13 N4 08 P)x . (C9 H14 N3 08 P)x . x Li (CAS 131212-70-9). Other Poly(l) · Poly(C) salts are also envisionable.
[00051] It is within the skills of those in the art to identify proper Poly(l) compound(s) (e.g. monomer, polymer or homopolymer) in accordance with the present invention.
[00052] In embodiments, Polyinosinic acid may be used at any suitable concentration, for instance a concentration allowing to improve at least one of sensitivity fluorescent signal, of amplification methods and techniques. In embodiments, Poly(l) is used in the amplification tube at a final concentration of about 0.0001 pg/mI to about 100 pg/mI, or about 0.001 mg/ml to about 10 pg/mI, or about 0.01 mg/ml to about 0.5 pg/pl. In embodiments, a composition for amplification in accordance with the present invention (e.g. MasterMix for PCR or RT-PCR as defined hereinafter) comprises about 0.00011 pg/mI to about 400 pg/mI, or about 0.0011 pg/mI to about 40 pg/mI, or about 0.01 pg/mI to to about 10 pg/mI, or about 0.01 pg/mI to about 4 pg/mI, or about 0.1 pg/mI to about 1 pg/mI. [00053] In embodiments, the composition for amplification of nucleic acid molecules in accordance with the present invention minimally comprises components that are found in a minimal formulation for RT-PCR, including deoxyribonucleotide triphosphates, salt, buffer and magnesium.
[00054] In embodiments, the composition further comprises additional components, including one or more of glycerol, BSA, a polar aprotic solvent (e.g. Dimethyl sulfoxide (DMSO)), a nonionic surfactant (e.g. Triton X-100™) and a nonionic, non-denaturing detergent (e.g. IGEPAL CA-630).
[00055] In embodiments, the composition further comprises at least one of tetrapropylammonium hydroxide (CAS Number: 4499-86-9) and Hotstart DNA oligomer called HS2 or TQH6. Particularly, HS2 a single stranded DNA sequence identified as TQH6 (SEQ ID NO:78) in US patent No. 6,183,967 (incorporated herein by reference in its entirety). Such single stranded DNA sequence may be synthesized and, for instance its synthesis may commissioned from many suppliers (e.g. Integrated DNA Technologies, Inc, Genewiz, OligoFactory, etc.)
[00056] In embodiments, the composition further comprises a reverse transcriptase / RNA-directed DNA polymerase (e.g. Moloney murine leukemia virus reverse transcriptase; CAS Number: 9068-38-6, Enzyme Commission Number: 2.7.7.49) and a DNA-directed DNA polymerase (e.g. Thermus aquaticus DNA polymerase; CAS Number: 9012-90-2, Enzyme Commission Number: 2.7.7.7)), and optionally at least one of a RNAse inhibitor (e.g. porcine RNAse inhibitor protein; CAS Number: 39369-21 -6) and a uracil DNA glycosylase (e.g. Atlantic cod uracil DNA glycosylase; CAS Number: 59088- 21 -0, Enzyme Commission Number: 3.2.2.27). In preferred embodiment, the composition comprises the four (4) enzymes. It is also conceivable to use variations on these proteins and/or equivalent proteins from many other sources.
[00057] Those skilled in art will understand that the definite list of components and their relative concentrations in the composition or MM may be adapted in accordance with various factors such as the origin of the biological sample, the RNA or DNA sequence of interest, the PCR amplification method to be used, the isothermal amplification method or apparatus to be used, the amplification conditions, etc.
[00058] In embodiments, the composition is a ready-to-use mixture is a MasterMix (MM) comprising the components of the mixtures defined in Table 1. In one particular embodiment, the MM is as defined in the broader range of Table 1. In one particular embodiment, the MM is as defined in the narrow range of Table 1. In one particular
embodiment, the MM is as defined in the first column of Table 1. In preferred embodiments the ready-to-use mixture advantageously further comprises Polyinosinic acid (Poly(l)) and, therefore, the ready-to-use mixture is a MasterMix (MM) as defined in any of the column of Table 1 + Poly(l) (e.g. about 0.01 pg/mI to about 1 pg/mI Poly(l), or about 0.1 pg/mI, or about 0.2 mg/ml, or about 0.5 pg/pl).
[00059] Those skilled in art understand that the definite list of components and their relative concentrations in the composition may be adapted in accordance with factors such as the origin of the biological sample, the RNA or DNA sequence of interest, the PCR amplification method or apparatus to be used, the isothermal amplification method or apparatus to be used, the amplification conditions, etc.
[00060] The compositions in accordance with the present invention, including ready-to- use mixtures and MasterMix (MM) may be formulated as a ready to use solution (i.e. 1X) or as a stock solution (e.g. 1 .1 X, 2X, 3X, 4X, 5X, 10X, etc.) for further dilution. A given quantity of a stock solution may be mixed within a reaction tube to obtain a final 1X concentration or, if appropriate, be previously diluted with a predetermined volume of filtered or distilled water. Whenever necessary, the pH of a resulting diluted solution may be adjusted to a desired pH by addition of suitable acidifying or alkaline agents.
Table 1 : Exemplary embodiments of MasterMix (MM) compositions
Component
Glycerol Tris-HCI pH 8 pH BSA (NH4)2S04 MgS04 DTT KCI
Tetrapropylammonium hydroxide
Improved amplification and detection methods
[00061] As indicated herein, the present invention provides means for improving existing PCR-based amplification and detection methods.
[00062] Accordingly, another aspect of the invention concerns methods for increasing sensitivity of polymerase chain reaction (PCR) and/or for increasing sensitivity of reverse transcription polymerase chain reaction (RT-PCR). In one embodiment the method comprises amplifying a nucleic acid molecule of interest (e.g. a DNA sequence in the case of PCR or a RNA sequence in the case of RT-PCR) in an amplification compositions comprising Polyinosinic acid (Poly(l)). [00063] In embodiments, the Poly(l) and amplification compositions are as defined hereinbefore.
[00064] In embodiments, the methods of the invention are use for amplification of a nucleic acid of interest obtained from a biological sample, including but not limited to blood, urine, saliva, cerebrospinal fluid, nasal swab, nasopharyngeal swab, throat swab, etc. Other sources of nucleic acid of interest obtained include animal, human and plant tissues, biopsies, cultures of cells, microbes such as viruses and bacteria, fungi, algae, or environmental samples of earth, water, air etc.
[00065] Preferably, the amplification is a real-time polymerase chain reaction (real-time PCR), also known as quantitative Polymerase Chain Reaction (qPCR) and is carried out in a thermocycler in accordance with parameters and conditions known in the art for real time PCR. In embodiments, the amplification is a multiplex RT-qPCR reaction.
[00066] In one particular embodiment, amplification is a RT-qPCR reaction carried out in a 20 mI_ final reaction volume consisting of:
4 mI_
20X Primers/Probe Set 1 mI_ 2X formulated MasterMix 10 mI_ RNA sample or control 5 mI_
[00067] In preferred embodiments, amplified nucleic acid molecule copies or PCR products are detected by measuring light emission of a fluorescent chemical compound. For instance, the detection methods of PCR products in real-time PCR are: (1 ) non specific fluorescent dyes that intercalate with any double-stranded DNA; and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter, which permits detection only after hybridization and subsequent degradation of the probe from its complementary sequence. Both detection methods are suitable in accordance with the present invention.
[00068] Therefore, in embodiments, the methods in accordance with the present invention further comprise detecting a fluorescence signal associated with amplification of the nucleic acid sequence, wherein the fluorescence signal is increased compared to an amplification (e.g. PCR or RT-PCR) carried out in absence of the Poly(l).
[00069] Having an increased fluorescence signal may provide many benefits to existing PCR amplification methods, including but not limited to increasing sensitivity, lowering the threshold detection level and reducing likelihood of false negatives.
[00070] In embodiments, using Poly(l) in amplification methods in accordance with the present invention provides an increase sensitivity of at least 2 fold, or at least 2.5 fold, or at least 3 fold, or at least 4 fold, or at least 5 fold, or at least 6 fold, or at least 7 fold, or at least 10 fold or more, when compared an amplification (e.g. PCR or RT-PCR) carried out in absence of the Poly(l).
[00071] In embodiments, using Poly(l) in amplification methods in accordance with the present invention provides an increase in the fluorescent signal of at least 10%, or at least 25%, or at least 50%, or at least 75%, or at least 100%, or at least 150 %, or at least 200%, or at least 300%, or at least 500%, or at least 1000%, at least 2000% or more, when compared an amplification (e.g. PCR or RT-PCR) carried out in absence of the Poly(l).
[00072] In embodiments, the amplification methods of the present invention allow for detection of a lower copy number of a nucleic acid sequence of interest, compared to amplification carried out in absence of the Poly(l). For instance, the present invention may
allow for detection of a reduced number of copies of a target nucleic acid, for instance detection of as low as 2 500 copies, or as low as 1 000 copies, or as low as 75 copies, or as low as 500 copies, or as low as 250 copies, or as low as 100 copies, or as low as 50 copies, or as low as 25 copies, or low as 15 copies, or low as 10 copies, or as low as 5 copies, or as low as 1 copy, in a sample.
[00073] It may also be envisioned that use of Poly(l) could reduce cycle threshold (Ct) value (e.g. by at least 0.5, 1 , 2, 3, 4, 5, 10, 15, 20 or more cycles) compared to an amplification carried out in absence of Poly(l). This may particularly be true if the starting quantity of the nucleic acid materials to be amplified is low (e.g. less than 10 copies, or less than 15 copies, or less than 25 copies, or less than 50 copies, or less than 75 or less than 100 copies, or less than 250 copies, or less than 500 copies or less than 1000 copies).
Improved virus detection methods
[00074] In particular embodiments, the nucleic acid sequence of interest is viral RNA and the amplification methods in accordance with the present invention are used for the detection of viruses. Detection of virus may be particularly useful for diagnostic purposes, for treatment purposes, for safety measures (e.g. quarantine or isolation), etc.
[00075] For instance, the methods and compositions may be used for the detection of various types of viruses, including those of Group I (double-stranded DNA viruses), Group II (single-stranded DNA viruses), Group III (double-stranded RNA viruses), Group IV (positive sense single-stranded RNA viruses), Group V (negative sense single-stranded RNA viruses), Group VI (single-stranded RNA viruses with a DNA intermediate in their life cycle) and Group VII (double-stranded DNA viruses with an RNA intermediate in their life cycle). [00076] In embodiments, the methods and compositions of the present invention are for amplification and detection of human pathogenic viruses, including, but not limited to, adenovirus, cytomegalovirus, coronavirus, herpes virus, HIV, human papillomavirus, influenza virus, measles virus, Middle East respiratory syndrome-related coronavirus (MERS-CoV), mumps virus, parainfluenza virus, poliovirus, rabies virus, respiratory
syncytial virus, and rubella virus. In one particular embodiment, the pathogenic virus is SARS-CoV2..
[00077] In embodiments, the methods and compositions of the present invention are for RT-PCR and are for amplification and detection of RNA viruses (e.g. Baltimore groups III, IV and V), including but not limited to rotavirus, orbivirus, coltivirus, banna virus, human astrovirus, Norwalk virus, human coronaviruses (e.g. 229E, NL63, OC43, HKU1 ,) Middle East respiratory syndrome-related coronavirus, severe acute respiratory syndrome coronaviruses, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, TBE virus, Zika virus, Hepatitis E virus, Rubella virus, coxsackievirus, hepatitis A virus, poliovirus and rhinovirus.
[00078] Because of their increased sensitivity, the amplification methods of the present invention may allow for the detection of a lower copy number of viral nucleic materials, and thus detection of a lower viral load or lower viral titer in a sample. This may thus reduce a number of “false negative” tests compared to existing amplification methods (e.g. PCR or RT-PCR amplification) carried out in absence of Poly(l).
[00079] In embodiments, the methods of the invention allow for detection of a reduced number of viral particles or viral load in a sample. In embodiments, using Poly(l) may allow reliable detection of as low as 2 500 viral particles, or as low as 1000 viral particles, or as low as 75 viral particles, or as low as 500 viral particles, or as low as 250 viral particles, or as low as 100 viral particles, or as low as 50 viral particles, or as low as 25 viral particles, or low as 15 viral particles, or low as 10 viral particles, or as low as 5 viral particles, or as low as 1 viral particles, in a sample.
Poly(l) could reduce of a minimal detection threshold (i.e. minimal number of virus particles) associated with positive detection of a virus in a sample. In particular embodiments, the minimal detection threshold associated with Poly(l) is reduced by at least 10 viral particles, or by at least 25 viral particles, or by at least 50 viral particles, by at least 75 viral particles, or by at least 100 viral particles, or by at least 250 viral particles, or by at least 500 viral particles, or by at least 1000 viral particles, or by at least 2500 viral
particles, or by at least 5000 viral particles, or by at least 10000 viral particles or more, in a sample, when compared to amplification carried out in absence of the Poly(l).
Kits
[00080] A further aspect of the invention relates to kits, e.g. kits for polymerase chain reaction (e.g. PCR or RT-PCR) or diagnostic kits. The kits of the invention may be useful for the practice of the methods of the invention (e.g. for carrying out any of the amplification methods described hereinbefore) and/or for applications in humans (e.g. diagnostic or preventive applications for detection of pathogenic viruses).
[00081] In embodiments, kits in accordance with the present invention comprises a composition for PCR or for RT-PCR as defined hereinbefore, or a ready-to-use reagent mixture or MasterMix as defined hereinbefore.
[00082] A kit of the invention may further comprise one or more of the following elements: sample collection tubes (e.g. sterile tubes for collecting blood, urine, saliva, swabs, tissue samples, cell culture, environmental samples, etc.), a buffer for the homogenization of the blood, saliva, swabs sample(s), tissue samples, cell culture or environmental sample, etc., beads or other materials for isolating nucleic acid from samples, purified RNA or cDNA to be used as controls in PCR and/or RT-PCR amplification methods, incubation buffer(s), substrate and assay buffer(s), modulator buffer(s) and modulators (e.g. enhancers, inhibitors), standards, detection materials (e.g. fluorochromes, luminogenic substrates, detection solutions, scintillation counting fluid, etc.), laboratory supplies (e.g. desalting columns, reaction tubes or microplates (e.g. 96- or 384-well plates), a user manual or instructions, etc.
[00083] Preferably, the kit and methods of the invention are configured such as to permit a quantitative detection or measurement of amplicons, DNA, RNA and/or nucleic acid sequence of interest. The kit may be optimized for various amplification methods and techniques, including but not limited quantitative PCR (qPCR), real-time PCR, real-time RT-PCR, droplet digital PCR (ddPCR), isothermal amplification. For instance, a kit for ddPCR may further comprise components for droplet generation, amplification and visualization, and digital droplet classification.
[00084] In one embodiment, the kit is optimized for qPCR detection of RNA viruses. For instance, a kit of the invention may comprise at least one primer or probe (preferably at least two primers) which specifically hybridizes with nucleic acid molecules for a RNA virus such as SARS-CoV2. reaction buffers and instructional material. Optionally, the primer or probe may contain a detectable tag. Certain kits may contain two or more of such primers or probes. In particular embodiments, a kit of the invention comprises components of the amplification system, including PCR reaction materials such as buffers, a thermostable polymerase and a reverse transcriptase. In other embodiments, the kit of the present invention can be used in conjunction with commercially available amplification kits, such as those that may be obtained from GIBCO BRL (Gaithersburg, Md.), Stratagene (La Jolla, Calif.), Invitrogen (San Diego, Calif.). The kit may optionally include instructional materials, positive or negative control reactions, templates, or markers.
[00085] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of this invention, and covered by the claims appended hereto. The invention is further illustrated by the following example, which should not be construed as further or specifically limiting.
Advantages and benefits [00086] As can be appreciated, the present invention may provide many benefits. In embodiments, the invention provides one or more of the following benefits:
- offers sensitive and reproducible detection of RNA and/or DNA targets in a single multiplex RT-qPCR reaction; robust performance across many sample types and instruments; - stock solutions (e.g. 1 .1 X, or 2X or 4X) which guarantees a simple reaction setup;
- ability to run multiplexed reactions;
- enhanced sensitivity for low-copy targets;
- compatible across a dynamic range of RT-qPCR equipment and real-time PCR systems such as Applied Biosystems, BioRad CFX™, ThermoFisher
Quantstudio™, Roche Lightcycler™ PCR platforms and Analytik Jena QTower 3™;and built in tolerance to common clinical sample inhibitors.
EXAMPLES Example 1 : RT-qPCR amplifications using MasterMix (MM) formulations comprising Poly(l)
[00087] A series of experiments were carried out to compare amplification and quantitative detection of RNAs using: (i) a commercial TaqPath™ master mix; or (ii) MasterMix formulations (MM) with or without Poly(l), in accordance with the present invention. Results are detailed hereinafter and shown in Figures 1 to 18.
[00088] Materials and methods
[00089] For the results of Figures 1 to 8, the supplier for Poly(l) was Sigma. For the results of Figures 9 to 18, the supplier for Poly(l) was either Sigma (denoted with an “S” in the legends) or Toronto Research Chemicals (TRC) (denoted with a “T” in the legends). [00090] A number in brackets after Poly(l) denotes the concentration used in pg/pl.
For example, if the legend says, “MM + Poly(l) (0.1)”, it means Poly(l) concentration in the MM was 0.1 pg/pl.
[00091] The experiments for Figures 1 to 8 were run on a BioRad™ CFX96 at the main campus of McGill University, and the experiments for Figures 9 to 18 were run on a ThermoFisher Quantstudio™ 5 at the Research Institute of the McGill University Health Centre by different operators. The data provided by these two machines differs in two ways that are noticeable in the Y-axis of these graphs. First, the label is RFU on the BioRad™ CFX96, and the label is ARxn on the ThermoFisher Quantstudio™ 5. Second, the scale differs by a thousand-fold. [00092] _The experiments for Figures 1 to 8 were run on a BioRad™ CFX96 at the main campus of McGill University, and the experiments for Figures 9 to 18 were run on a ThermoFisher Quantstudio™ 5 at the Research Institute of the McGill University Health
Centre by different laboratory personnel. They further demonstrate that the effect does not depend on the source of RNA template (ATCC or NIST).
[00093] Abbreviations and product numbers a. hpRNA (human placental RNA) (obtained from Thermofisher, catalog No AM7950) b. MM (MasterMix; see Table 1) c. Poly(l) (poly-inosinic acid) (obtained from Sigma or TRC as detailed above) d. TaqPath™ (TaqPath™ 1-Step Multiplex Master Mix (No ROX)) e. ATCC (ATCC-VR3276SD - Quantitative Synthetic SARS-CoV-2 RNA: ORF, E, N (ATCC® VR-3276SD™). Listed as between 105 and 106 copies per mI. f. NIST (NIST -10169 - SARS-CoV-2 Research Grade Test Material). Listed as 5.4 X 106 copies per mI for the E gene.
[00094] Preparation Protocol
• Prepare formulation of MasterMix at 2X concentration
• Prepare a 20X IDT Corman E gene Primer/Probe Set (12mM F1 , 12mM R2, 4mM P1 ) (Primer and probes may be purchased from Integrated DNA Technologies, Inc): o E_Sarbeco_F Forward Primer - Catalog number 10006889, DNA sequence* o E_Sarbeco_R Reverse Primer - Catalog number 10006891 , DNA sequence* o E_Sarbeco_P1 Probe - Catalog number 10006893 DNA Sequence * * DNA sequences for the two primers and probe are listed in Table 1 of the scientific publication: Corman Victor M et. at, Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020;25(3):pii=2000045; doi.org/10.2807/1560- 7917. ES.2020.25.3.2000045)
[00095] Reaction Protocol
• For 1 reaction of a 20 pL final reaction volume: o RNase-free H20 (or human placental RNA) 4 pL
o 20X E gene Primers/Probe Set 1 mI_ o 2X McGill Master Mix 10 mί. o RNA sample or control 5 uL
Total 20 mI_
[00096] Cycling protocol
• Reverse transcription 1 cycle: 53 SC - hold 10 mins (ramp rate 4.4)
• Denaturation 1 cycle: 95 SC - hold 2 mins (ramp rate 4.4)
• Amplification 45 cycles of: 95 SC - hold 15 sec (ramp rate 4.4)
60 SC - hold 30 sec (ramp rate 2.2)
• Cooling 1 cycle: 40 SC - hold 30 sec (ramp rate 2.2)
[00097] Results
[00098] Results of the comparative experiments are shown in Figures 1 to 18.
[00099] Figure 1 compares TaqPath™ vs MM with added hpRNA vs MM no hpRNA, using ATCC 1 :50,000 as a template for qPCR detection of SARS-CoV2 E gene on BioRad™ CFX96. These representative results indicate that a component of hpRNA can reduce the cycle threshold (Ct) value when the concentration of RNA template is low. After this observation, we sought to replicate and improve the observed effect of hpRNA with a different chemical.
[000100] Figure 2 compares TaqPath™ vs MM with hpRNA vs MM no hpRNA, using ATCC 1 :50 as a template for qPCR detection of SARS-CoV2 E gene on BioRad™ CFX96. These representative results indicate the effect of hpRNA is not observed when the concentration of RNA template is high. After this observation, we sought to replicate and improve the observed effect of hpRNA in Figure 1 with a different chemical.
[000101] Figure 3 compares TaqPath™ vs MM no Poly(l) vs MM with Poly(l) at 0.1 and 0.5 pg/mI, using ATCC 1 :50,000 as a template for qPCR detection of SARS-CoV2 E gene on BioRad™ CFX96. These representative results indicate that Poly(l) can enhance peak fluorescence by amplifying more qPCR product when the concentration of RNA template is low.
[000102] Figure 4 compares MM lacking Poly(l) vs MM with Poly(l) from Sigma at 0.01 , 0.1 , and 0.25 pg/mI, using ATCC 1 :50,000 as a template for qPCR detection of SARS- CoV2 E gene on BioRad™ CFX96. These representative results indicate that the effect of Poly(l) is dependent on its concentration in the MasterMix. [000103] Figure 5 compares MM lacking Poly(l) vs MM with Poly(l) from Sigma at 0.025,
0.1 , and 0.25 pg/mI, using ATCC 1 :50,000 as a template for qPCR detection of SARS- CoV2 E gene on BioRad™ CFX96. These representative results confirm that the effect of Poly(l) is dependent on its concentration in the MasterMix, and demonstrate that the experiment is repeatable. [000104] Figure 6 compares MM lacking Poly(l) vs MM with Poly(l) from Sigma at 0.1 pg/mI, using ATCC 1 :100 and 1 :50,000 as templates for qPCR detection of SARS-CoV2 E gene on BioRad™ CFX96. These representative results indicate that the effect of Poly(l) is modest when the concentration of RNA template is high, but it is robust when the concentration of RNA template is low. [000105] Figure 7 compares MM lacking Poly(l) vs MM with Poly(l) from Sigma at 0.025,
0.1 , and 0.5 pg/mI, using ATCC 1 :50,000 as a template for qPCR detection of SARS-CoV2 E gene on BioRad™ CFX96. These representative results confirm concentration- dependence of the effect of adding Poly(l) to the MasterMix when the concentration of RNA template is low. They also suggest a ceiling beyond which increased concentrations of Poly(l) have limited added effect.
[000106] Figure 8 compares TaqPath™ vs MM with Poly(l) ( 0.1 pg/mI), using ATCC 1 :100 and 1 :50,000 as a template for qPCR detection of SARS-CoV2 E gene on BioRad™ CFX96. These representative results confirm the Mastermix containing Poly(l) has comparable performance to TaqPath™ when the concentration of RNA template is high (-28,000 copies) or low (-55 copies)..
[000107] Figure 9 compares TaqPath™ vs MM with Sigma Poly(l) (0.1 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher Quantstudio™ 5. These representative results confirm improved performance compared to TaqPath™ when 0.1 pg/mI Poly(l) (Sigma) is added to the
MasterMix, especially at the limit of detection (~10 copies). The results also demonstrate that the effect can be seen when experiments are performed in another laboratory, with different personnel using another brand of thermal cycler.
[000108] Figure 10 compares TaqPath™ vs MM with Sigma Poly(l) (0.2 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher Quantstudio™ 5. These representative results confirm improved performance compared to TaqPath™ when 0.2 pg/mI Poly(l) (Sigma) is added to the MasterMix. The improvement is seen when the concentration of RNA template is low (~55 copies), or near the estimated limit of detection (~10 copies) for that particular experiment. [000109] Figure 11 compares MM with Sigma Poly(l) (0.1 pg/mI) vs MM with Sigma
Poly(l) (0.2 pg/mI), using ATCC 1 :100 and NIST 1 :1 .75M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher Quantstudio™ 5. These representative results confirm concentration-dependence of the effect of adding Poly(l) (Sigma) to the MasterMix, especially when the concentration of RNA template is low (~55 copies), or near the estimated limit of detection (~15 copies) for that particular experiment.
[000110] Figure 12 compares MM with Sigma Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.2 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher Quantstudio™ 5. These representative results confirm concentration-dependence of the effect of adding Poly(l) (Sigma) to the MasterMix, especially when the concentration of RNA template is low (~55 copies), or near the estimated limit of detection (~10 copies) for that particular experiment.
[000111] Figure 13 compares MM with TRC Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.1 pg/mI), using ATCC 1 :100 and NIST 1 :1.75M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher Quantstudio™ 5. These representative results confirm that adding Poly(l) (TRC) at 0.1 pg/mI gives improved performance compared to Poly(l) (Sigma) at 0.1 pg/mI, especially near the estimated limit of detection (~15 copies) for that particular experiment.
[000112] Figure 14 compares MM with TRC Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.1 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of
SARS-CoV2 E gene on ThermoFisher Quantstudio™ 5. These representative results confirm that adding Poly(l) (TRC) at O.l pg/mI gives improved performance compared to Poly(l) (Sigma) at 0.1 pg/mI, especially when the concentration of RNA template is low (-55 copies), or near the estimated limit of detection (~10 copies) for that particular experiment.
[000113] Figure 15 compares MM with TRC Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.2 mg/ml), using ATCC 1 :100 and NIST 1 :1.75M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher Quantstudio™ 5. These representative results confirm comparable performance between MasterMixes containing either 0.1 pg/mI Poly(l) (TRC) or 0.2 pg/mI Poly(l) (Sigma).
[000114] Figure 16 compares MM with TRC Poly(l) (0.1 pg/mI) vs MM with Sigma Poly(l) (0.2 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher Quantstudio™ 5. These representative results confirm comparable performance between MasterMixes containing either 0.1 pg/mI Poly(l) (TRC) or 0.2 pg/mI Poly(l) (Sigma) when the concentration of RNA template is low (-55 copies). Improved performance is seen with 0.1 pg/mI Poly(l) (TRC) near the estimated limit of detection (~10 copies) for that particular experiment.
[000115] Figure 17 compares TaqPath™ vs MM with TRC Poly(l) (0.1 pg/mI), using ATCC 1 :100 and NIST 1 :1 .75M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher Quantstudio™ 5. These representative results confirm comparable performance compared to TaqPath™ when 0.1 pg/mI Poly(l) (TRC) is added to the MasterMix when the concentration of RNA template is high (-28,000 copies). Improved performance is seen with 0.1 pg/mI Poly(l) (TRC) near the estimated limit of detection (-15 copies) for that particular experiment.
[000116] Figure 18 compares TaqPath™ vs MM with TRC Poly(l) (0.1 pg/mI), using ATCC 1 :50,000 and NIST 1 :2.5M as templates for qPCR detection of SARS-CoV2 E gene on ThermoFisher Quantstudio™ 5. These representative results confirm improved performance compared to TaqPath™ when 0.1 pg/mI Poly(l) (TRC) is added to the MasterMix, especially when when the concentration of RNA template is low (-55 copies), and near the estimated limit of detection (-10 copies) for that particular experiment.
[000117] Headings are included herein for reference and to aid in locating certain sections. These headings are not intended to limit the scope of the concepts described therein, and these concepts may have applicability in other sections throughout the entire specification. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
[000118] The singular forms “a”, “an” and “the” include corresponding plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes one or more of such compounds and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.
[000119] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, concentrations, properties, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present specification and attached claims are approximations that may vary depending upon the properties sought to be obtained. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the embodiments are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors resulting from variations in experiments, testing measurements, statistical analyses and such. [000120] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the present invention and scope of the appended claims.
Claims
1 . A composition for polymerase chain reaction (PCR), comprising Polyinosinic acid (Poly(l)).
2. The composition according to claim 1 , wherein the PCR is reverse transcription polymerase chain reaction (RT-PCR).
3. A composition for reverse transcription polymerase chain reaction (RT-PCR) comprising Polyinosinic acid (Poly(l)).
4. The composition according to any one of claims 1 to 3, wherein the composition comprises about 0.01 pg/mI to about 10 pg/mI of Polyinosinic acid.
5. The composition according to any one of claims 1 to 4, wherein the composition comprises Polyinosinic acid potassium salt.
6. The composition according to claim 5, wherein the composition comprises about 0.1 pg/mI of Polyinosinic acid potassium salt.
7. The composition according to any one of claims 1 to 6, wherein the composition comprises minimal components for PCR, said minimal components comprising deoxyribonucleotide triphosphates, salt, buffer and magnesium.
8. The composition according to any one of claims 1 to 7, wherein the composition further comprises at least one of tetrapropylammonium hydroxide and Hotstart DNA oligomer HS2.
9. The composition according to any one of claims 1 to 8, wherein the composition further comprises at least one of glycerol, BSA, a polar aprotic solvent, a nonionic surfactant and a nonionic, non-denaturing detergent.
10. The composition according to any one of claims 1 to 9, wherein the composition further comprises at least one of glycerol, Tris-HCI, BSA, (NH4)2S04, MgS04, DTT, KCI, DMSO, dNTPs, Reverse Transcriptase, Uracil DNA glycosylase, RNAse inhibitor protein, DNA Polymerase, Triton X-100™ and IGEPAL CA-630.
11 . The composition according to any one of claims 1 to 10, wherein the composition further comprises at least one of reverse transcriptase, DNA polymerase, RNAse inhibitor and uracil DNA glycosylase.
12. The composition according to any one of claims 1 to 11 , wherein said mixture is selected from the mixtures defined in Table 1.
13. A ready-to-use reagent mixture for polymerase chain reaction (PCR), said mixture comprising: DNA polymerase, deoxyribonucleotide triphosphates, salt, buffer, magnesium, and Polyinosinic acid (Poly(l)).
14. A ready-to-use reagent mixture for reverse transcription polymerase chain reaction (RT-PCR), said mixture comprising: reverse transcriptase, DNA polymerase, deoxyribonucleotide triphosphates, salt, buffer, magnesium, and Polyinosinic acid (Poly(l)).
15. The ready-to-use reagent mixture according to claim 14, comprising about 0.01 pg/mI to about 10 pg/mI of Polyinosinic acid.
16. The ready-to-use reagent mixture according to claim 14 or 15, comprising Polyinosinic acid potassium salt.
17. The ready-to-use reagent mixture according to any one of claims 14 to 16, comprising about 0.1 pg/mI of Polyinosinic acid potassium salt.
18. The ready-to-use reagent mixture according to any one of claims 14 to 17, wherein said reagent mixture further comprises at least one of a RNAse inhibitor and uracil DNA glycosylase.
19. The ready-to-use reagent mixture according to any one of claims 14 to 18, said reagent mixture further comprising at least one of glycerol, BSA, a polar aprotic solvent, a nonionic surfactant and a nonionic, and non-denaturing detergent.
20. The ready-to-use reagent mixture according to any one of claims 14 to 19, said reagent mixture further comprising at least one of tetrapropylammonium hydroxide and Hotstart DNA oligomer HS2.
21 . The ready-to-use reagent mixture according to any one of claims 14 to 20, wherein said ready-to-use reagent mixture is a MasterMix as defined in any one of the mixtures defined of Table 1.
22. The ready-to-use reagent mixture according to any one of claims 14 to 21 , wherein said ready-to-use reagent mixture is formulated as a 2X stock solution.
23. Use of Polyinosinic acid (Poly(l)) in polymerase chain reaction (PCR) and/or in reverse transcription polymerase chain reaction (RT-PCR).
24. Use of Polyinosinic acid (Poly(l)) for increasing sensitivity of polymerase chain reaction (PCR) and/or for increasing sensitivity of reverse transcription polymerase chain reaction (RT-PCR).
25. A method for increasing sensitivity of polymerase chain reaction (PCR), comprising: amplifying a DNA sequence of interest in a PCR amplification composition comprising Polyinosinic acid (Poly(l)).
26. A method for increasing sensitivity of reverse transcription polymerase chain reaction (RT-PCR), the method comprising amplifying a RNA sequence of interest in a RT-PCR amplification composition comprising Polyinosinic acid (Poly(l)).
27. The method according to claim 25 or 26, wherein the composition comprises about 0.01 pg/mI to about 10 pg/mI of Polyinosinic acid.
28. The method according to any one of claims 25 to 27, wherein the composition comprises Polyinosinic acid potassium salt.
29. The method according to claim 28, wherein the composition comprises about 0.1 pg/mI of Polyinosinic acid potassium salt.
30. The method according to any one of claims 25 to 29, wherein the composition comprises minimal components for PCR, said minimal components comprising deoxyribonucleotide triphosphates, salt, buffer and magnesium.
31. The method according to any one of claims 25 to 30, wherein the composition further comprises at least one of tetrapropylammonium hydroxide and Hotstart DNA oligomer HS2.
32. The method according to any one of claims 25 to 31 , wherein the composition further comprises at least one of glycerol, BSA, a polar aprotic solvent, a nonionic surfactant and a nonionic, non-denaturing detergent.
33. The method according to any one of claims 25 to 32, wherein the composition further comprises at least one of glycerol, Tris-HCI, BSA, (NH4)2S04, MgS04, DTT, KCI, DMSO, dNTPs, reverse transcriptase, uracil DNA glycosylase, RNAse inhibitor protein, DNA Polymerase, Triton X-100™ and IGEPAL CA-630.
34. The method according to any one of claims 25 to 33, wherein the composition further comprises at least one of reverse transcriptase, DNA polymerase, RNAse inhibitor and uracil DNA glycosylase.
35. The method according to any one of claims 25 to 34, wherein said mixture is selected from the mixtures defined in Table 1.
36. The method according to any one of claims 25 to 35, further comprising detecting a fluorescence signal associated with amplification of said nucleic acid sequence, wherein said fluorescence signal is increased compared to a RT-PCR amplification carried out in absence of said Poly(l).
37. The method according to any one of claims 25 to 36, wherein said PCR or RT- PCR amplification allows for detection of a lower copy number of said nucleic acid sequence compared to a corresponding PCR or RT-PCR amplification carried out in absence of said Poly(l).
38. The method according to any one of claims 25 to 37, wherein said PCR or RT- PCR amplification provides an increase sensitivity of at least 2 fold, or at least 2.5 fold, or at least 3 fold, or at least 4 fold, or at least 5 fold, or at least 6 fold, or at least 7 fold, or at least 10 fold or more, when compared to a corresponding PCR or RT-PCR amplification carried out in absence of the Poly(l).
39. The method according to any one of claims 25 to 38, wherein said PCR or RT- PCR amplification provides an increase in the fluorescent signal of at least 10%, or at least 25%, or at least 50%, or at least 75%, or at least 100%, or at least 150 %, or at least 200%, or at least 300% or more, when compared to a corresponding PCR or RT-PCR amplification carried out in absence of the Poly(l).
40. The method according to any one of claims 25 to 39, wherein said PCR or RT- PCR amplification allows for detection of as low as 2500 copies, or as low as 1 000 copies, or as low as 75 copies, or as low as 500 copies, or as low as 250 copies, or as low as 100 copies, or as low as 50 copies, or as low as 25 copies, or low as 10 copies, or as low as 5 copies, or as low as 1 copy, in a sample.
41. The method according to any one of claims 25 to 40, wherein amplification is a RT-PCR amplification, and wherein said nucleic acid sequence of interest is viral RNA.
42. The method according to claim 41 , wherein said RT-PCR amplification allows for detection of as low as 2 500 viral particles, or as low as 1 000 viral particles, or as low as 75 viral particles, or as low as 500 viral particles, or as low as 250 viral particles, or as low as 100 viral particles, or as low as 50 viral particles, or as low as 25 viral particles, or low as 10 viral particles, or as low as 5 viral particles, or as low as 1 viral particle, in a sample.
43. The method according to claim 41 or 42, wherein said method provides a reduction of a minimal detection threshold associated with positive detection of a virus in a sample, wherein said minimal detection threshold is reduced by at least 10 viral particles, or by at least 25 viral particles, or by at least 50 viral particles, by at least 75 viral particles, or by at least 100 viral particles, or by at least 250 viral particles, or by at least 500 viral particles, or by at least 1000 viral particles, or by at least 2500 viral particles, or by at least 5000
viral particles, in a sample, when compared to amplification carried out in absence of the Poly(l).
44. The method according to any one of claims 41 to 43, wherein said nucleic acid sequence of interest is a RNA sequence from a human pathogenic virus selected from the group consisting of adenovirus, cytomegalovirus, coronavirus, herpes virus, HIV, human papillomavirus, influenza virus, measles virus, Middle East respiratory syndrome-related coronavirus (MERS-CoV), mumps virus, parainfluenza virus, poliovirus, rabies virus, respiratory syncytial virus, and rubella virus.
45. The method according to any one of claims 41 to 43, wherein said nucleic acid sequence of interest is a RNA sequence from a RNA virus selected from the group consisting of rotavirus, orbivirus, coltivirus, banna virus, human astrovirus, Norwalk virus, human coronavirus, Middle East respiratory syndrome-related coronavirus, severe acute respiratory syndrome co ran a viruses, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, TBE virus, Zika virus, Hepatitis E virus, Rubella virus, coxsackievirus, hepatitis A virus, poliovirus and rhinovirus.
46. The method according to any one of claims 41 to 43, wherein said nucleic sequence of interest is a SARS-CoV2 RNA.
47. The method according to any one of claims 41 to 46, wherein said method provides a reduction of false negative, compared to a PCR or RT-PCR amplification carried out in absence of said Poly(l).
48. A kit for carrying out any of the methods of any one of claims 41 to 47 comprising:
(a) the composition as defined in any one of claims 1 to 12, or the ready-to- use reagent mixture as defined in any one of claims 13 to 22, and
(b) one or more of the following elements: a sample collecting tube(s), reaction tube(s), microplate(s), buffer for the homogenization of the sample(s), incubation buffer(s), assay buffer(s), fluorescent and/or luminogenic detection materials, desalting column(s), purified control purified RNA or cDNA and a user manual or instructions.
49. A kit for detection of viral RNA, comprising:
(a) the composition as defined in any one of claims 1 to 12, or the ready-to- use reagent mixture as defined in any one of claims 13 to 22, and
(b) one or more of the following elements: a sample collecting tube(s), reaction tube(s), microplate(s), buffer for the homogenization of the sample(s), incubation buffer(s), assay buffer(s), fluorescent and/or luminogenic detection materials, desalting column(s), purified control purified RNA or cDNA and a user manual or instructions.
50. A kit for reverse transcription polymerase chain reaction (RT-PCR), comprising:
(a) the composition as defined in any one of claims 1 to 12, or the ready-to- use reagent mixture as defined in any one of claims 13 to 22, and
(b) one or more of the following elements: a sample collecting tube(s), reaction tube(s), microplate(s), buffer for the homogenization of the sample(s), incubation buffer(s), assay buffer(s), fluorescent and/or luminogenic detection materials, desalting column(s), purified control purified RNA or cDNA and a user manual or instructions.
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US20020137076A1 (en) * | 2000-11-28 | 2002-09-26 | Shultz John W. | RNA polymers and uses thereof |
US10815539B1 (en) * | 2020-03-31 | 2020-10-27 | Diasorin S.P.A. | Assays for the detection of SARS-CoV-2 |
WO2022009116A1 (en) * | 2020-07-10 | 2022-01-13 | The Royal Institution For The Advancement Of Learning/Mcgill University | Protein constructs of moloney murine leukemia virus reverse transcriptase (mmlv-rt) |
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US20020137076A1 (en) * | 2000-11-28 | 2002-09-26 | Shultz John W. | RNA polymers and uses thereof |
US10815539B1 (en) * | 2020-03-31 | 2020-10-27 | Diasorin S.P.A. | Assays for the detection of SARS-CoV-2 |
WO2022009116A1 (en) * | 2020-07-10 | 2022-01-13 | The Royal Institution For The Advancement Of Learning/Mcgill University | Protein constructs of moloney murine leukemia virus reverse transcriptase (mmlv-rt) |
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