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WO2004018998A2 - Procede permettant d'utiliser l'amplification en chaine par polymerase en temps reel - Google Patents

Procede permettant d'utiliser l'amplification en chaine par polymerase en temps reel Download PDF

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Publication number
WO2004018998A2
WO2004018998A2 PCT/US2003/026177 US0326177W WO2004018998A2 WO 2004018998 A2 WO2004018998 A2 WO 2004018998A2 US 0326177 W US0326177 W US 0326177W WO 2004018998 A2 WO2004018998 A2 WO 2004018998A2
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Prior art keywords
nucleotide sequence
target nucleotide
genome
virus
sample
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WO2004018998A3 (fr
Inventor
Daniel C. Edelman
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University of Maryland Baltimore
University of Maryland College Park
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University of Maryland Baltimore
University of Maryland College Park
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Priority to AU2003272230A priority Critical patent/AU2003272230A1/en
Publication of WO2004018998A2 publication Critical patent/WO2004018998A2/fr
Publication of WO2004018998A3 publication Critical patent/WO2004018998A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • C12Q1/6851—Quantitative amplification

Definitions

  • aspects of the present invention generally relate to nucleic acid analysis, and more particularly, to a system and method for detecting a target or determining the quantity of a target within a sample.
  • the invention also relates to methods for assessing the presence of a target nucleotide sequence in a crude sample utilizing Quantitative Real-Time Polymerase Chain Reaction (QPCR).
  • QPCR Quantitative Real-Time Polymerase Chain Reaction
  • Vidaud et al. U.S. Patent No. 6,541,246 B2 is directed to a method of detecting or quantifying adenoviruses.
  • the genomic material containing the target nucleotide sequences are extensively extracted or washed, significantly affecting any quantitative measure in the sample.
  • Lunde et al. (15) used purified ⁇ DNA as the analyte in competitive PCR but targeted specific cut-and-join sites in the phage genome. These sites and not the number of phage were quantified using a fluorescently tagged primer.
  • the conventional process calls for a plaque assay.
  • This is an assay for the presence and concentration of infectious virus in a sample, by counting the number of clear plaques (i.e., a clear area of cell lysis caused by viral replication) on a continuous sheet of cultured susceptible cells in a cell monolayer that are infected with the virus sample. Theoretically each plaque is due to a single virus and its progeny destroying a group of contiguous cells.
  • the plaque assay is the traditional method for the quantification of virus and bacteriophage, including bacteriophage for ⁇ cloning vectors.
  • This technique takes a substantial investment in time and materials, and the quality of the data obtained may be inaccurate due to a subjective interpretation of the results.
  • Significant limitations of this method include: (i) the requirement for extensive hands-on time ( ⁇ 5 h) for preparation of the assay, at least 1.5 days to produce results, and the need for a significant quantity of reagents and supplies; (ii) a limited dynamic range of one log (30-300 plaques/plate); (Hi) media that are susceptible to environmental conditions (e.g., drying and aging), resulting in suboptimal bacterial growth with decreased phage infectivity; (iv) the dependence of accurate titers upon the viability of the host bacteria, with viability less than 100%, causing a reduction in the true titer; (v) the inherent subjectivity and potential error when visually counting plaques; (vi) a lack of reproducibihty in titer due to procedural errors from multiple dilution and pipetting steps; (vii) the detection of only functional phage rather than total phage DNA content in the preparation; and (viii) a procedure with a low throughput.
  • PCR amplification of the target sequence of DNA by PCR reaction proceeds through a series of temperature regulated cycles using the activity of a thermostable enzyme and a sequence specific primer set.
  • the primers hybridize to portions of the target DNA strand and the enzyme successively adds a plurality of nucleotide bases to elongate the primer which results in the production of progeny (daughter) strands.
  • Each progeny strand possesses a complementary composition relative to the target template strand from which it was derived, and can serve as a template in subsequent reaction cycles.
  • a fluorescent probe or other detectable reporter construct is incorporated into the reaction to provide a means for determining the progress of the template amplification.
  • the reaction fluoresces in relative proportion to the quantity of DNA product produced.
  • TaqManTM procedure Applied Biosystems, CA
  • Another probe according to the invention is SYBR Green ITM, a DNA-binding dye.
  • this system integrates the use of a detectable reporter construct, or probe, which can comprise, for instance, both a fluorescent label molecule and a quencher molecule.
  • a detectable reporter construct or probe
  • the quencher nullifies the majority of fluorescence which may be emitted by the probe.
  • both molecules are released from the probe allowing the fluorescent label to be detected.
  • the quantity or intensity of fluorescence may then be correlated with the amount of product formed in the reaction. Using this information, calculations can be made to determine the initial quantity of the starting template that was present. Quantitation in this manner is useful in applications including: determination of levels/concentrations of specific DNA and RNA sequences in tissue samples, identification of viral loads, gene expression, and numerous other applications.
  • a PCR-based method known as quantitative real-time PCR
  • QPCR quantitative polymerase chain reaction
  • This method utilizes a labeling dye which fluoresces in proportion to the amount of target DNA species that are produced by the PCR reaction.
  • Two principal determinants in the accurate and reproducible quantification of the initial target concentration are a background noise assessment and a threshold value determination.
  • the background noise assessment reflects the changing reaction conditions and environment during the early cycles of PCR amplification and is used to determine when target amplification is sufficiently above a background signal to enable accurate measurement of the fluorescence of the amplified target.
  • the threshold value reflects the level of fluorescence that is significantly above a background noise baseline which was determined previously.
  • the threshold value is important as it reflects the portion of the PCR reaction, the cross over threshold (C,), where a sufficient level of amplification has been achieved to allow for calculation of the initial target concentration.
  • C cross over threshold
  • the C,s of unknown samples are plotted against the C r s of the quantitative standards.
  • QPCR has only been demonstrated previously in the context of analyzing samples wherein the genomic material has been extracted or purified resulting in an uncontrolled loss of sample, and hence, an inaccurate final result.
  • melt curve data generated in QPCR can be used to detect the presence of a target in a crude sample.
  • melt curve data can be used to detect the presence of a target in a crude sample.
  • melt curve data is through assessing the melt-curve data for any crude sample against a positive control wherein the melt curve for a particular target and one or more probes has already been ascertained. If the melting curves match, then the presence of the target can be positively identified.
  • QPCR quantitative polymerase chain reaction
  • the potential advantages for using QPCR to supplant the traditional method of plaque assay include: (i) phage titer results within a few hours; (ii) a significant increase in data generation and through-put using 96-well microtiter PCR plates with real-time data processing; (Hi) cost savings by the reduction of labor and materials; (iv) a more accurate method of phage quantification, with a greater dynamic range and sensitivity; and (v) the possibility of direct and sensitive detection of phage in preparations, other than dilution, requiring no prior or separate sample preparation.
  • the present invention provides a method for quantifying and/or detecting a target nucleotide sequence in a crude sample, comprising producing an amplification product by amplifying the target nucleotide sequence in a real-time polymerase chain reaction within the crude sample using at least one primer directed to a nucleotide sequence containing the targeted nucleotide sequence, wherein said primer(s) is(are) an ohgonucleotide that hybridizes under conditions suitable for a polymerase chain reaction with a nucleotide sequence along the nucleotide sequence containing the target nucleotide sequence; and detecting the amplification product by using a probe comprising a structure, such as an ohgonucleotide, that hybridizes, under conditions suitable for a polymerase chain reaction, with a nucleotide sequence containing the targeted ohgonucleotide sequence, wherein the crude sample is derived from an organism, structure or composition containing the
  • Still another object of the invention is a method wherein the target nucleotide sequence is exposed for PCR purposes prior to performing PCR amplification by using heat to denature the organism or any other structure surrounding the target nucleotide sequence in the sample, or the target nucleotide sequence is exposed for PCR prior to performing PCR amplification using a denaturing agent to denature the organism or structure(s) surrounding target nucleotide sequence in the sample.
  • Yet still other objects of the invention are a method comprising a step for assessing the effect of a substance in the sample that inhibits the PCR reaction or interferes with the data collection, or a step for assessing the effect upon the PCR reaction or the real-time PCR data generation therefrom from a probe signal, wherein the effect is caused by a substance in the crude sample other than the target nucleotide sequence, or a step for assessing the effect of diluting the sample prior to performing any PCR reaction.
  • target nucleotide sequence is from the genome of a multicellular organism, a human gene, an animal gene, a plant gene, a single cell organism, a bacteria, a eubacteria, a parasite or a virus.
  • Another object of the invention are methods wherein the target nucleotide sequence is from the genome of a virus and the method further comprises a step for assessing the presence of free genomic material containing the target nucleotide sequence and not contained within a viral capsid structure.
  • Still other objects of the invention are methods in which the target nucleotide sequence is from the genome of a DNA virus, a Bacteriophage, a double stranded DNA virus, a Poxviridae virus, an Asfariviridae virus, an Iridoviridae virus, a Herpesviridae virus, a Baculoviridae virus, an Adenoviridae virus, a Polyomaviridae virus, a Papillomaviridae virus, a single stranded DNA virus, a
  • Parvoviridae virus an RNA virus, a double stranded RNA virus, a Reoviridae virus, a Birnaviridae virus, a single stranded (+) RNA virus, a Coronaviridae virus, a SARS virus, an Arteriviridae virus, a Togaviridae virus, a Flaviviridae virus, a Picornaviridae virus, an Astroviridae virus, a Caliciviridae virus, a single stranded (-) RNA virus, a Paramyxoviridae virus, a Filoviridae virus, a Rhabdoviridae virus, a Bornaviridae virus, an Orthomyxoviridae virus, a Bunyaviridae virus, an Arenaviridae virus, a single stranded RNA reverse transcriptase virus, a Retroviridae virus, a double stranded RNA reverse transcriptase virus, a Hep
  • Real-Time PCR is a relatively new analytical tool and the various applications are only beginning to be appreciated.
  • the use of Real- Time PCR has been limited to analyzing genomic material in samples wherein the genomic material was first extracted and/or washed prior to Real-Time PCR analysis, thus leading to increased costs and lower accuracy in obtaining quantitative data.
  • the invention relates to the discovery that Real-Time Quantitative PCR (QPCR) can be used to quantitatively analyze the genomic count in a crude sample with both high accuracy and high precision.
  • QPCR Real-Time Quantitative PCR
  • the method of the invention provides a substantial increase in the accuracy and precision from other methods of quantitative analysis, such as plaque assay techniques, while at a substantially reduced investment of time and cost.
  • the method of the invention can be practiced with a crude sample containing genomic material, and in particular, can be used in quantifying the genomic count from a crude sample containing the target nucleotide sequence of a single cell organism or a virus.
  • the method of the invention may comprise additional steps for assessing the effect of PCR inhibitory substances in the crude sample that inhibit the PCR reaction thereby affecting the evaluation of the quantitative results.
  • the invention may comprise other controls or standards for calibrating the accuracy or precision of the QPCR results.
  • FIG. 1 is a graphical representation of standard curve generated with primers having 100% identity with the hybridized sequence.
  • FIG. 2 is a graphical representation of standard curve generated with primers modified to have closely matching melting points to reduce the probability of unwanted secondary product formation, such as dimers, and to optimize the PCR reaction.
  • FIG. 3 A is a graphical representation of an amplification cycle graph for various titrations of a crude sample.
  • FIG. 3B is a graphical representation of a melt curve graph generated from the titrations of the crude sample in Figure 3 A showing melt curve peaks approximating 93 °C for all the titrations and with no significant secondary product formation.
  • real-time PCR applies to any technique which allows relative monitoring of the evolution of an ongoing polymerase chain reaction amplification.
  • crude sample is any sample that may contain a target nucleotide sequence that has not undergone substantial steps directed to the extraction of the genomic material containing the target nucleotide sequence. These types of substantial steps directed to the extraction of genomic material are well known by those of ordinary skill in the art as, for instance, genomic DNA purification kits, high purity RNA isolation kits, or tissue extraction kits.
  • the term “crude sample” may refer to a composition that is neat, diluted in a controlled manner, or otherwise substantially unchanged by the addition of inconsequential matter.
  • the term “crude sample” does not exclude a sample that has been lysed or treated to denature the components contained within to allow for access of the PCR components to the target nucleotide sequence. Furthermore, the term “crude sample” does not exclude a sample that has undergone separation that would remove a known or knowable quantity of the target nucleotide sequence, such as with measured dilution(s).
  • the crude samples of the invention can be drawn from, for instance, body fluid, tissue, plasma, feces, or any other biological or environmental sample source material. The crude sample may also be drawn from culture or any other artificially developed source of biological material.
  • probe is any species that binds a DNA product to communicate its presence in a reaction.
  • a probe can include, but is not limited to DNA-binding dyes, oligonucleotides and proteins.
  • a pair of primers are chosen for the PCR reaction to develop a well defined exponential growth phase.
  • two primers are chosen or modified to have closely matching melting points under the PCR conditions in any particular reaction conditions.
  • the two primers may comprise homology of 75-100% to the sequence identity to which they are to hybridize in order to allow amplification of a DNA fragment containing the target nucleotide sequence.
  • Primers can be chosen from convenient sources, either through design by the researcher in practicing the invention or from convenient commercial sources.
  • Probe is chosen to hybridize with the DNA fragment resulting from the amplification and to report successful hybridization through a signal.
  • Probes such as fluorescent probes, can be designed by one of ordinary skill in the art, or acquired through commercial sources.
  • probes may be used in any particular application of the invention.
  • Useful probes include, for instance, according to the invention, TaqmanTM and SYBR Green ITM.
  • recombinant phage (Lambda FIX II®) containing human herpes virus 8 sequences were obtained from the National Institutes of Health AIDS Research & Reference Reagent Program (McKesson-
  • the plate lysates were concentrated in Apollo® high-performance centrifugal concentrators, 70,000 and 100,000 MWCO (Orbital Biosciences®, Topsfield, MA, USA), according to the manufacturer's instructions. Before filtration, the lysates were treated with NaCl (1 M final concentration) to disassociate the phage from the bacterial debris (2).
  • DNase I endonuclease digestion (5) was used to quantify the contribution of uncoated phage DNA in the lysates that might add to the QPCR quantitative signal.
  • the lysates were treated with DNase I (Roche Applied Science, Indianapolis, IN, USA) by combining 25 ⁇ L lysate, 20 U DNase I, and LB broth for a final reaction volume of 30 ⁇ L.
  • DNase I Roche Applied Science, Indianapolis, IN, USA
  • LB broth a final reaction volume of 30 ⁇ L.
  • the samples were digested at 25°C for 30 min, and then at 99°C for 15 min to inactivate the enzyme, followed by cooling to room temperature.
  • the digests were stored at -20°C for later evaluation, at which time the samples were diluted 1 :200 in SM buffer and analyzed by QPCR.
  • Residual protein contamination was removed using the PUREGENE® DNA Isolation Kit (Gentra Systems, MN, USA), with the following modifications from the manufacturer's protocol. After the addition of two parts Protein Precipitation Solution, the samples were iced for 5 min, and, after the addition of a glycogen carrier (Invitrogen, Carlsbad, CA, USA) and inversion, the samples were stored at -
  • spectrophotometric OD 260/28 o measurements of the purified DNA were performed on a DU® 640C spectrophotometer (Beckman Coulter, Fullerton, CA, USA).
  • QPCR components consisted of lx Platinum® buffer (Invitrogen). 2.5 mM MgCl 2 (Invitrogen), 0.2 ⁇ M dNTP (Invitrogen), 0.25 ⁇ M each primer (The
  • the amplification and detection of ⁇ PCR products were performed using the iCycler® iQ Detection System (Bio-Rad Laboratories).
  • This System includes the iCycler Thermal Cycler, with real-time detection capabilities, and the iCycler iQ Real Time Detection System Software, which allows for the immediate determination of the cycle threshold ( ), melting curves, and quantification of unknown samples.
  • the PCR cycling parameters were an initial 1.5 min at 96°C and 1.5 min at 95°C, followed by 40 cycles of 15 s at 94°C and 1 min at 68°C (See
  • FIG 3A The protocol concluded with a melting curve program using 0.5°C increasing increments (10 s each; 68°-99°C) (See Fig. 3B). Standard curves were established from both purified and quantified (OD 260 28 o) phage DNA and from crude phage preparations (lysates) serially diluted over a six-log range (See Fig. 2). Precision and Reproducibihty Studies
  • the improved primer set detected an additional replicate of a 10- 8 dilution ofthe phage standard, which suggested an increase in PCR sensitivity.
  • the 1:100 sample ofthe phage stock had a mean C, of 16.88 (16.25-17.73) with a CV of 2.64%.
  • the mean ofthe 1:1000,000 standard was 30.61 (30.00-31.17) with a CV of 1.31%.
  • the precision was less.
  • the mean observed titers were 192, 76, and 39, with CNs of 7.6%, 14.9%, and 7.9%, respectively.
  • D ⁇ ase I- treated phage samples were compared to untreated phage samples via QPCR.
  • XL-1 Blue host bacteria 100 ⁇ L were inoculated with four different amounts of phage plug eluate
  • the precision ofthe assay is excellent, having CVs below 2%, which are much lower than those ofthe traditional plaque assay (7%-14%).
  • the QPCR is a more accurate method for quantification of phage titers. Excluding human error, samples can be assayed with confidence without resorting to duplicate reactions, thereby saving considerable cost and labor. Additionally, reproducibihty ofthe test and long-term stability ( ⁇ 3.6 months) ofthe phage target were established, demonstrating the high quality ofthe method.
  • the modified primer set could improve the efficiency ofthe QPCR by approximately 5.3%, with the complete absence of primer dimers.
  • specific ⁇ clones were used, this was only a convenience sample, and this method can be practiced with any phage possessing the same primer sites.
  • development of specific or consensus primers to other species can be designed or located as practiced above with ⁇ clones, perhaps in a multiplex QPCR format for use in such diverse applications as detecting phages in wastewater, recycling, and environmental samples. Phages have been reported as surrogate markers for the presence of human pathogens in these environmental sites (9-11).
  • the QPCR assay above is able to detect ⁇ phage as low as 0.014 pfu.
  • plaque assay titer shows a correlation with lysate quantified by QPCR if the standard and sample are treated similarly (i.e., DNase I digestion is not necessarily required for accurate titers of functional phages).
  • phage quantification by QPCR there are other advantages to phage quantification by QPCR. Often phage are grown for isolation of their cloned DNA insert and, because plaque assays only detect functional phage, they are not an adequate method for estimating the total amount of phage DNA in a preparation before DNA purification.
  • the data show that the QPCR can quantify the amount of phage DNA in crude lysates and in filter concentrate fractions and is useful for determining the loss and recovery of phage DNA from filtered stock preparations.
  • filter concentration is a relatively quick method to concentrate phage, it is not particularly efficient, most likely due to excessive binding ofthe virus to the filter membrane. Pre-blocking could prove to be efficacious for this application.
  • the assay could also be applied to diagnostics in the dairy industry, where there is interest in protecting milk from phage contamination before beginning fermentation or curding procedures (16). Financial losses occur to dairy farmers when the bacterial starter cultures do not grow due to phage infection (17).
  • Field- deployable QPCR units are already in existence (18) and can be used to compliment the inventive method for the survey of phages at field sites.
  • the use of the QPCR platform can also be used to accurately determine the amount of phage in a therapeutic dose and to monitor pharmacometrics in, among other things, the growing field of bacteriophage therapy.
  • the assay excels in the generation of accurate quantitative data, more efficiently and sensitively than previous methods used for the determination of phage titers.
  • the QPCR has multiple applications to other industries, for which it can provide a highly sensitive and rapid detection platform for phage from many diverse sample sources. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope ofthe invention encompassed by the appended claims.

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Abstract

La présente invention concerne un procédé permettant de réaliser une analyse quantitative ou qualitative d'un échantillon brut biologique ou prélevé dans l'environnement, au moyen d'une amplification en chaîne par polymérase quantitative en temps réel (QPCR).
PCT/US2003/026177 2002-08-20 2003-08-20 Procede permettant d'utiliser l'amplification en chaine par polymerase en temps reel Ceased WO2004018998A2 (fr)

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AU2003272230A AU2003272230A1 (en) 2002-08-20 2003-08-20 Method using real-time pcr

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US40462202P 2002-08-20 2002-08-20
US60/404,622 2002-08-20
US42765002P 2002-11-19 2002-11-19
US60/427,650 2002-11-19

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013006793A3 (fr) * 2011-07-06 2013-05-10 Quest Diagnostics Investments Incorporated Amplification directe et détection d'agents pathogènes viraux et bactériens
CN114369683A (zh) * 2021-09-16 2022-04-19 中山大学 马来西亚大虾病毒的检测试剂盒

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6033854A (en) * 1991-12-16 2000-03-07 Biotronics Corporation Quantitative PCR using blocking oligonucleotides
US6447994B1 (en) * 2000-06-20 2002-09-10 The General Hospital Corporation Production of replicative hepatitis C virus

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013006793A3 (fr) * 2011-07-06 2013-05-10 Quest Diagnostics Investments Incorporated Amplification directe et détection d'agents pathogènes viraux et bactériens
US9194006B2 (en) 2011-07-06 2015-11-24 Quest Diagnostics Investments Incorporated Direct amplification and detection of viral and bacterial pathogens
US9464331B2 (en) 2011-07-06 2016-10-11 Quest Diagnostics Investments Incorporated Direct amplification and detection of viral and bacterial pathogens
CN108866169A (zh) * 2011-07-06 2018-11-23 探索诊断投资公司 病毒和细菌病原体的直接扩增和检测
US10619220B2 (en) 2011-07-06 2020-04-14 Quest Diagnostics Investments Incorporated Direct amplification and detection of viral and bacterial pathogens
US11851720B2 (en) 2011-07-06 2023-12-26 Quest Diagnostics Investments Llc Direct amplification and detection of viral and bacterial pathogens
CN114369683A (zh) * 2021-09-16 2022-04-19 中山大学 马来西亚大虾病毒的检测试剂盒

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