WO2001066799A2 - Real time quantitative pcr with intercalating dye for single and multiplex target dna - Google Patents
Real time quantitative pcr with intercalating dye for single and multiplex target dna Download PDFInfo
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- WO2001066799A2 WO2001066799A2 PCT/US2001/007101 US0107101W WO0166799A2 WO 2001066799 A2 WO2001066799 A2 WO 2001066799A2 US 0107101 W US0107101 W US 0107101W WO 0166799 A2 WO0166799 A2 WO 0166799A2
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- This invention relates to the field of molecular biology and more particularly to DNA-based diagnostic protocols.
- BACKGROUND OF THE INVENTION it is desirable to quantify the level of a target nucleic acid in a sample. For example, it may be important to determine the amount of a pathogenic organism in a food or water sample, or the amount of a genetically modified organism (GMO) in a crop.
- GMO genetically modified organism
- PCR polymerase chain reaction
- the fluorescence so measured represents the total fluorescence of the mixture of specifically amplified target PCR products (target amplicon), as well as non-specific amplicons, which include single primer products, primer-dimers, and other aberrant amplicons.
- target amplicon specifically amplified target PCR products
- non-specific amplicons which include single primer products, primer-dimers, and other aberrant amplicons.
- a method for detecting in real time the amount of a target nucleic acid molecule in a sample, wherein the melting of the target nucleic acid molecule starts at a temperature T ⁇ g and completes at a temperature TME > me method comprising: A.
- the inventive method described above may also be used to detect multiple targets in a sample. Specifically, when the sample contains n target nucleic acid molecules, wherein n is an integer greater than one, wherein the melting of the first target nucleic acid molecules starts at a temperature T MS1 and completes at a temperature T ME ⁇ .
- the method for multiplex detection comprises:
- the first and the second target nucleic acid molecules reside on the same genome of an organisms, and the copy number per genome for the first target nucleic acid molecule is Icnown, whereby the copy number per genome for the second target nucleic acid molecule is determined.
- the first target nucleic acid is an invertase gene or a lectin gene
- the second target nucleic acid is selected from the group consisting of the 35 S promoter of CaMV, a Cry9C gene, and an GA21 gene.
- the target nucleic acid molecule is selected from the group consisting of SEQ ID NO:l 1, SEQ ID NO: 12, SEQ ID NO:9, and SEQ ID NO: 10.
- the target nucleic acid molecule is a nucleic acid fragment is part of a transgene contained in a genetically modified organism.
- Figure 2 Real-time quantitative PCR amplification of the amplicon in Figure 1, showing the relationship between template starting concentration and the delta slope difference (S M -S B ).
- Figure 3 Standard Curve for the amplicon of Figure 1, showing the relationship between the log of initial target concentrations and threshold cycle number in PCR.
- Figure 4 Melting profile of the lectin Amplicon, showing the relationship between temperature and relative fluorescence intensity, and the baseline slopes (S B ) and the amplicon slope (S M ).
- Figure 5 Standard Curve for the amplicon of Figure 3, showing the relationship between the log of initial target concentrations and threshold cycle number in PCR.
- Figure 6 Melting profile of E. coli O17:H7 Amplicon, showing the relationship between temperature and relative fluorescence intensity, and the baseline slopes (S B ) and the amplicon slope (S M ).
- Figure 7 Real-time quantitative PCR amplification of the amplicon in Figure 6, showing the relationship between template starting concentration and the delta slope difference (S M -S B ).
- Figure 8 Standard Curve for the amplicon of Figure 6, showing the relationship between the log of initial target concentrations and threshold cycle number in PCR.
- Figure 9 Melting profile of the SV40 amplicon, showing the relationship between the temperature and relative fluorescence intensity, and the baseline slopes (S B ) and the amplicon slope (S M ).
- Figure 10 Standard Curve for the amplicon of Figure 9, showing the relationship between the log of initial target concentrations and threshold cycle number in PCR.
- Figure 11 Melting profile of a mixture of both the E. coli O157:H7 and the SV40 amplicons. Mixture and slope determination.
- Figure 12 Standard Curve for the SV40 amplicon from a multiplex Q-PCR assay.
- Figure 13 Standard Curve for the E. coli O157:H7 amplicon from a multiplex Q-PCR Assay.
- Figure 14 35S CaMV melting profile and signal determination. This Figure shows the relationship between temperature and relative fluorescence intensity of a specific CaMV amplified product, and also demonstrates the difference between amplicon signal and background fluorescence.
- Figure 15 Real-time Quantitative PCR reaction of CaMV. This figure demonstrates the use of an average base line to set tlireshold, and shows the relationship between the number of thermal cycle of each concentration of CaMV samples and the fluorescence signal difference (specific - background).
- Figure 16 Determination of threshold cycle for an amplicon.
- the cycles used to define the initial fluorescence (F ) and the background threshold fluorescence (F B ) are shown.
- the threshold cycle (C ⁇ ) can be determined as a function of the amplicon production and F B .
- SEQ ID NO:l is the sequence of a synthetic oligonucleotide encoding a portion of the promoter region for the cauliflower mosaic virus 35S promoter (35S CaMV). When used in a PCR reaction with the oligonucleotide represented in SEQ ID NO:2 the fragment represented in SEQ ID NO: 9 is produced.
- SEQ ID NO:2 is the sequence of a synthetic oligonucleotide encoding a portion the promoter region for the cauliflower mosaic virus 35S promoter (35S CaMV). When used in a PCR reaction with the oligonucleotide represented in SEQ ID NO: 1 the fragment represented in SEQ ID NO: 9 is produced.
- SEQ ID NO: 3 is the sequence of a synthetic oligonucleotide encoding a portion of the soybean lectin gene Le-1.
- SEQ ID NO:4 is the sequence of a synthetic oligonucleotide encoding a portion of the soybean lectin gene Le-1.
- SEQ ID NO: 10 is produced.
- SEQ ID NO: 5 is the sequence of a synthetic oligonucleotide encoding a portion of the genome unique to Escherichia coli 0157:H7.
- SEQ ID NO: 6 is the sequence of a synthetic oligonucleotide encoding a portion of the genome unique to Escherichia coli 0157:H7.
- SEQ ID NO: 11 is produced.
- SEQ ID NO: 7 is the sequence of a synthetic oligonucleotide encoding a portion of the Large-T antigen from SV-40. When used in a PCR reaction with the oligonucleotide represented in SEQ ID NO:8 the fragment represented in SEQ ID NO: 12 is produced.
- SEQ ID NO: 8 is the sequence of a synthetic oligonucleotide encoding a portion of Large-T antigen from SV-40.
- SEQ ID NO: 7 When used in a PCR reaction with the oligonucleotide represented in SEQ ID NO: 7 the fragment represented in SEQ ID NO: 12 is produced.
- SEQ ID NO:9 is the nucleotide sequence portion of the 35-S CaMV promoter region amplified by primers represented in SEQ ID NOs:l and 2. This fragment has a melting temperature of 83-87.5°C.
- SEQ ID NO: 10 is the nucleotide sequence portion of the soybean lectin gene LE-1 that is amplified by primers represented in SEQ ID NOs:3 and 4. This fragment has a melting temperature of 81.5-83.5°C.
- SEQ ID NO:l 1 is the nucleotide sequence portion of a unique genome locus from Escherichia coli 0157:H7 that is amplified by primers represented in SEQ ID NOs:5 and 6. This fragment has a melting temperature of 82.6-89°C.
- SEQ ID NO: 12 is the nucleotide sequence portion of the SV-40 Large-T antigen amplified by primers represented in SEQ ID NOs:7 and 8. This fragment has a melting temperature of 77-79°C.
- the inventive, PCR-based method detects and quantifies double stranded nucleic acid molecule ("dsDNA” or “target”) by monitoring the fluorescence of the amplified target (“target amplicon”) during each amplification cycle at selected time points.
- dsDNA double stranded nucleic acid molecule
- target amplicon amplified target
- the two strands of a dsDNA separate or melt, when the temperature is higher than its melting temperature.
- Melting of a dsDNA molecule is a process, and under a given solution condition, melting starts at a temperature (designated T MS hereinafter), and completes at another temperature (designated T ME hereinafter).
- T MS temperature
- T ME temperature
- Tm designates the temperature at which melting is 50% complete.
- the melting curve characteristics of a target amplicon is predetermined.
- a typical PCR cycle involves a denaturing phase where the target dsDNA is melted, a primer annealing phase where the temperature optimal for the primers to bind to the now- single-stranded target, and a chain elongation phase (T E ) where the temperature is optimal for DNA polymerase to function.
- T ⁇ g should be higher than T E
- TMB should be lower (often substantially lower) than the temperature at which the DNA polymerase is heat-inactivated.
- Melting curve characteristics are, of course, the intrinsic properties of a given dsDNA molecule.
- a desirable melting curve is usually achieved by selecting the length and/or GC content of the target amplicon.
- Melting curve characteristics may also be altered by changing the PCR primers that are used to amply them. For example, adding GC-rich overhangs to the 5' end of the primers will increase the Tm for the amplified target.
- Double stranded nucleic acid molecules exhibits fluorescence under ultraviolet light when they are associated with certain dyes, and the intensity of the fluorescence may be proportionate to concentration of the dsDNA.
- Methods talcing advantage of such relationship to detect and quantify dsDNA are known in the art. Many dyes are known and used in the art for these purposes. The instant methods also takes advantage of such relationship. An example of such dyes includes intercalating dyes.
- dyes include, but are not limited to, S YBR Green-I®, ethidium bromide, propidium iodide, TOTO®-l ⁇ Quinolinium, l-r-[l,3-propanediylbis [(dimethyliminio) -3,1- propanediyl]]bis[4-[(3-methyl-2(3H)-benzothiazolylidene) methyl]]-, tetraiodide ⁇ , and YoPro® ⁇ Quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-l-[3- (trimethylammonio)propyl]-,diiodide ⁇ .
- Most preferred dye for the instant invention is a non- asymmetrical cyanide dye such as S YBR Green-I®, manufactured by Molecular Probes, Inc. (Eugene, OR.).
- SYBR Green®/DNA complex and SYBR Green® alone, has an inherent temperature dependent fluorescence. As the temperature increases, the fluorescence of the SYBR Green®/DNA complex will naturally decrease, even though the dsDNA strands are not separated. The rate of change increases proportionally to the concentration of DNA. In order to distinguish this temperature-dependent change from the change due to dsDNA strand separation, it is necessary that a threshold to be established before, and during, the measurements surrounding each amplicon in a reaction.
- the fluorescence (F) of the PCR reaction mixture is measured immediately before the temperature starts to increase from T E (F E at T E ), at any temperature point (T B ) in between T E and T MS (F B at T B ), immediately below the starting temperature of melting (F MS at and immediately above the completion of melting (F ⁇ at T ⁇ ).
- the temperature may need to be held steady for a period of time when making the various fluorescence measurements.
- any time periods between 0.5 to 60 seconds are suitable.
- a period of between 1-45 seconds, more preferably between 1-30 seconds, and still more preferably between 1-15 seconds are suitable.
- the most preferred time period for make a fluorescence measurement is 7 seconds.
- a baseline slope (S B ) is calculated.
- S B j s defined by negative (F B minus F E ), divided by (T B minus T E ), and a melting phase slope (Sj j), defined by negative (FME minus F MS ) divided by ME minus T M g), is also calculated.
- the concentration of the target amplicon increases and so does the value of F ⁇ g.
- the number of amplification cycles (the "threshold cycle number, C ") it takes for the first appearance of a positive change in the slope, where the quantity (S ⁇ - S B ) is greater than zero, is correlated with the starting concentration of the target amplicon (C).
- a standard curve for the target amplicon is established by starting with a series of dilutions of a solution of the target amplicon whose concentration is l ⁇ iown. By repeating the method above for each concentration in the dilution series, under identical PCR conditions, C- is determined for each of the Icnown concentration.
- the standard curve for the target amplicon under a given PCR condition is thus established by plotting C against C.
- the standard curve plots a suitable range of concentrations between 1-10 9 copies of the target amplicon, preferably a range of 10-10 8 , more preferably 10-10 7 , particularly preferably 10-10 6 copies.
- the sample is prepared in a suitable way such that it is suitable for amplification by PCR.
- the sample is then subject to a PCR amplification under the identical conditions under which its corresponding standard curve is established.
- the value of Cy for the sample is determined as discussed above and is compared with the standard curve to establish the corresponding concentration.
- the method according to the instant invention can also be used to detect simultaneously multiple target amplicons ("multiplex detection"). Referring to Figure 11, it is apparent that, when the sample contains more than one target amplicon, the respective S B and Sj i for each amplicon, F E at T E and F B at T B must be determined. Accordingly, for multiplex detection, the target amplicons should have distinguishable melting curve characteristics, such that F E at T E and F B at T B for each amplicon is determinable.
- the melting of the first target nucleic acid molecule starts at a temperature T j ⁇ g j and completes at a temperature TM E i
- the melting of the second target nucleic acid molecule starts at a temperature and completes at a temperature TM E 2.
- TMEI- I is recognized that under usual conditions, is not lower than 55°C, while T MS2 is not higher 95°C, and a 3-5°C difference is usually sufficient for multiplex amplification and quantification.
- amplicons including internal standard controls, other target amplicons, and non-specific products, e.g. primer dimers
- amplicons must be designed or selected to have non- overlapping melting temperatures with other possible products in the reaction. This is to ensure that the products melt at different temperatures, and the various fluorescence values can be differentiated and analyzed independently.
- the total fluorescence is additive and remain correlated to the concentrations of dsDNA products. This is to say that after a product dissociates or melts, it no longer contributes to the total florescence.
- a standard curve for each of the target nucleic acid molecule is established by: simultaneously PCR-amplifying, in the presence of a suitable fluorescent dye, the target nucleic acid molecules with a known starting concentration (Cj and C 2 ) as provided above.
- the fluorescence (F) is measured during each amplification cycle at the temperature immediately before the temperature starts to increase from T E (F E at T E ), at any temperature point (T B1 ) in between T E and T MS1 (F B1 at T B1 ), at T MS1 (F MS1 at T MS1 ), at T ME1 (F ⁇ at T ME1 ), at any time point (T B2 ) in between T ⁇ and T MS2 (F B2 at T B2 ), at T MS2 (F MS2 at T] jg 2 ), at TM E2 (FjviE 2 at TME2)»
- a baseline slope is calculated for the first target molecule (S B1 ), defined by negative (F B1 minus F E ), divided by (T B1 minus T E ), and a first amplicon melting phase slope is calculated for the first molecule (S j ⁇ j ), defined by negative (F MEj minus F S1 ) divided by (T ME1 minus T ⁇ gi); and a
- the above steps are then repeated for a sample suspected of containing an unknown concentration of the first and second target nucleic acid molecules, to obtain an Nj value and an N value for the sample, and to determine the target nucleic acid molecule concentrations via the standard curves.
- a threshold value is set arbitrarily. Any fluorescence level below such a threshold level is ignored and a determination is made that no meaningful or specific amplification is considered to have occurred. Only fluorescence level above the threshold level is measured and used for detection and quantification purposes. There is typically no detectable product during early cycles of PCR; therefore the first few cycles can be used to determine the threshold value.
- an Initial Fluorescence, Fj is defined as the average fluorescence for the first few PCR cycles ( Figure 16). According to the most preferred embodiment, initial cycles # 4 to 12 are used.
- Threshold Fluorescence, F B is the average fluorescence signal from cycle N-10 to cycle N-5 prior to exponential phase of PCR as threshold background signal.
- the detectable level of amplicon is pre-determined to be at cycle N when the delta slope (S M -S B ) fluorescence was 67% above baseline.
- the value of Ln[(F n - F B )/ F B ] is a linear function of the cycle number.
- the slope and R 2 for a linear regression is determined for F values in the range of 1.06 F B to 1.67 F B .
- a threshold may also be the average change in fluorescence (delta slope of S M -S B ) for the initial 10 cycles (#4 to 13) plus ten times the standard deviation of these values.
- Initial Fluorescence, F was defined as the average fluorescence for initial cycles # 4 to 12.
- the multiplex detection method of the instant invention is used to determine the copy number per genome of a target nucleic acid (Target A), using another target nucleic acid (Target B) as a reference point, wherein the copy number/genome for Target B is Icnown.
- Target A and Target B reside in the same genome, and the copy number/genome for Target B is known, when Target A and Target B are co-amplified and quantified using the multiplex detection method of the instant invention, the Target A copy number/genome can be readily calculated from the ratio between the amount of Target A and the amount of Target B, without the need of knowing or determining the genome size or the need to quantify the amount of genomic DNA used in the starting sample.
- An example of this embodiment is provided in Example 2.
- the instant detection method can be used to detect and quantify any target dsDNAs, from which the presence and level of target organisms can be determined. Examples of target organisms include pathogenic organisms including fungi, bacteria, infectious animals, viruses etc.
- the instant methods have been applied in the detection of Salmonella typhimurium, Salmonella enteriditis, Escherichia coli 0157 :W1 , Listeria spp., Listeria monocytogenes, Cryptosporidium parvum, Campylobacter jejuni, Campylobacter coli, Staphylococcus aureas, Pseudomonas aeruginosa, and SV-40 viral DNA.
- the instant methods can also be used for other clinical or non-clinical uses. For example, the methods can be used to determine the presence of genetically modified organisms in foods or feeds.
- the phosphoenolpyruvate carboxylase (PEPC) promoter in BT176 Corn
- the hsp70 promoter of CrylA(b) gene found in Mon 80100 corn
- CrylA(b) gene Mon 809 corn
- NOS gene Mon810 Corn
- the actin promoter gene G21 Corn
- the lectin gene, the invertase gene, and the aldolase gene may all be used, where appropriate.
- the instant method is very specific and sensitive. As few as 10 copies of the target dsDNA are detected.
- the PCR tablet for pathogenic organisms contains an internal positive control.
- the advantages of an internal positive control contained within the PCR reaction have been previously described (PCT Application
- control DNA will be of appropriate size and base composition to permit amplification in a primer directed amplification reaction.
- the control DNA sequence may be obtained from the target bacteria, or from another source, but must be reproducibly amplified under the same conditions that permit the amplification of the target amplicon DNA.
- the control DNA is similar in size and base composition to the target DNA to be detected.
- a control nucleic acid fragment was isolated from the genus Salmonella and was identical to the target to be detected, except that it was engineered to allow for amplification with a single primer (WO 97/11197).
- the control DNA is useful to validate the amplification reaction. Amplification of the control DNA is accomplished concurrently with the test sample containing the target DNA.
- a sample is subjected to the test PCR procedure in parallel with a control containing the control DNA as well as the sample.
- control shows amplification
- a suitable number of copies of the control DNA must be included in each amplification reaction. It is well known that sample matrix components, including food, can cause inhibition of PCR and therefore a resulting decrease in product formation and signal. Alternatively, the presence of certain food components in the PCR reaction has also been found to result in the opposite result, i.e. enhancement of the signal when fluorescent dye detection is employed. Use of the control as described herein eliminates such false positive results. Moreover, by calibrating the level of response in the control, it is possible to evaluate and compensate for any suppression or enhancement of the reaction in the test caused by extraneous material such as is found in many food-derived matrices.
- the instant method may also be used with other nucleic acid amplification methods such as strand displacement, ligase chain reaction(LCR) and nucleic acid sequence based amplification (NASBA) (See e.g. Food Microbiology Fundamentals and Frontiers, 1997, M.E. Doyle, L.R. Beucha, and TJ. Mondville, ASM Publication, pp. 723-724).
- LCR ligase chain reaction
- NASBA nucleic acid sequence based amplification
- an automated thermal cycler with fluorescence detection capabilities such as the Perkin-Elmer 7700 Sequence Detection System available from the Perkin-Elmer Corporation is used. Fluorescence data are exported and processed with the help of a data processing device such as a personal computer, with various transformations when necessary. Methods and instruments for such automated operation are apparent to a skilled person and are exemplified in the examples that follow.
- GM genetically modified
- These primers amplify a 206 bp fragment of the CaMV 35S promoter sequence, which is present in nearly all genetically modified organisms and thus used to screen samples for the GM product.
- the closed-tube homogeneous PCR process described below uses a commercial detection system and DNA intercalating dye, SYBR Green-I. During each thermal cycle, fluorescence data is collected at an intermediate temperature between the extension and denaturation steps. As the specific PCR product is generated, the dye intercalates into the product and the total fluorescence signal increases.
- the fluorescence value of the intercalating dye is inversely proportional to the temperature.
- DNA elution buffer 30 mM Tris/ 0. ImM EDTA, pH 8.35 • GM- Roundup ReadyTM Certified Reference Material IRMM410 (dried soy bean powder) (GM-RRTM) (Flulca, Retieseweg, Belgium)
- Gel filtration HPLC was performed with an aqueous buffer (0.1 M phosphate/0.3 M NaCl, Ph 7.0) as mobile phase at flow rate of 1 ml per minute. Quantifying DNA fragment concentration, by injecting known amount of 1000 base pair pure DNA (from 5 to 500 ng) per assay, then, plot the amount (ng) of DNA Vs HPLC peak area (mAU) to create a calibration curve. Use this calibration curve to determine the unknown sample DNA concentration.
- Bovine Serum Albumin (Roche Molecular Biochemicals, Indianapolis, IN)
- Reagent tablet Qualicon, Inc., Wilmington, DE 1.2 ⁇ M SYBR green 1, 4 mg/tablet BSA, all four d-NTP, 1.5 units TaqTM polymerase
- Sample #COC-BXJ539 (FUJI protein 545) first time appear at cycle 33.63, its CaMV amplicon melting slope is greater than baseline slope. Based on the linear regression from calibration curve of CaMV, the sample contents 18 coy of 35S CaMV promoter DNA in it. #M35-490 (isolated soy protein,) first time appear at cycle 30.72, its CaMV amplicon melting slope is greater than baseline slope. Based on the linear regression from calibration curve of CaMV, the sample contents 125 copy of 35S CaMV promoter DNA in it. #NAHX-61509 (soy flake) first time appear at cycle 33.35, its CaMV amplicon melting slope is greater than baseline slope.
- Lectin Primer rcl555 5'(TGG TGG AGG CAT CAT AGG TAA TGA GAA) 3'.
- Reagent tablet (Qualicon, Inc., Wilmington, DE):
- Sample #COC-BXJ539 (FUJI protein 545) first time appear at cycle 22.55, its Lectin amplicon melting slope is greater than baseline slope. Based on the linear regression from calibration curve of Lectin, the sample contents 29680 copy of Lectin DNA in it.
- the 35S CaMV content in the same sample was 21 copy.
- the GMO % Content is the ratio of CaMV level to Lectin level and it equals to 0.072%.
- #M35-490 isolated soy protein, lecithin first time appear at cycle 22.09, its Lectin amplicon melting slope is greater than baseline slope. Based on the linear regression from calibration curve of Lectin, the sample contents 40519 of Lectin DNA in it. Based on Example I, the 35S CaMV content in the same sample was 125 copy.
- the GMO % Content is the ratio of CaMV level to Lectin level and it equals to 0.31%
- Example II Based on Example I, the 35S CaMV content in the same sample was 18 copy.
- the GMO % Content is the ratio of CaMV level to Lectin level and it equals to 0.117%.
- Reference Vodkin, L. O., Rhodes, P. R., and Goldberg, R. B. Ca lectin gene insertion has the structural features of a transposable element. Cell 34: 1023-1031 (1983).
- EXAMPLE 3 SINGLE TARGET O-PCR ASSAY - DETECTION OF BACTERIAL DNA
- the assay uses PCR in the presence of SYBR Green I, a DNA intercalating dye.
- This method can quantify the initial copy number of pathogenic bacteria, e.g., E. coli 0157: H7, in the reaction.
- the method involves collecting data during each thermal cycle of PCR. Fluorescence data is collected at intermediate temperatures between the extension and denaturation steps. As the specific PCR product is generated, the dye intercalates into the product and the total fluorescence signal increases. The fluorescence value of the intercalating dye is inversely proportional to the temperature. We compare the change in slope of the fluorescence value of a specific amplicon with the baseline slope of intercalating dye. Then we record the thermal cycle at which the first appearance of a positive change in slope occurs, where the amplicon slope is greater than the baseline slope.
- the cycle at which the fluorescence rises above this value is the threshold cycle (C ).
- This value is inversely related to the starting target copy number.
- Standards of known concentration (1.25E+5 to 1.25E+1 E. coli genome/PCR) are run and a standard curve created by plotting the log concentration against C for the standard samples.
- the starting copy number of unlcnown samples is then determined from this standard curve.
- the method provides a specific and sensitive assay for quantifying starting copy number of a test sample.
- the cell counts of target E. coli O157:H7 in the final were approximately 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , and 0 CFU/ml respectively.
- One 5 ⁇ l sample of each diluted culture was removed and transferred to a lysis tube containing 195 ⁇ l of PCR buffer [3 ⁇ M SYBR Green I® (Molecular Probes, Inc.)., 200 ng/ ⁇ l Pronase-E (Sigma Chemical Co., St. Louis, MO), 50 mM Tris HCL, 3 mM MgCl2, 28 mM KCL, 0.1% Triton X 100, pH 8.3].
- lysis tubes were placed in a heating rack set at 37°C for 20 minutes.
- the lysed sample tubes were then placed in a 95°C heating block for 10 minutes to inactivate the Pronase-E.
- all samples were subject to PCR amplification followed by fluorescence detection in PE/ABI PRISM 7700 Sequence Detection System to determine the quantity of PCR product from different levels of E. coli 0157 :H7 for each PCR cycle.
- the PCR reagents consisted of BAX® for Screening IE.
- Duplicate aliquots of 50 ⁇ L of each lyzed sample were removed and transferred into a PCR tube containing one BAX/E. coli 0157:H7 tablet, then amplified in a P ⁇ /ABI PRISM 7700 Sequence Detection System.
- the selected primer pair P-538 and rc-1012 amplified a 475-base pair DNA fragment used to identify E. coli 0157:H7.
- Establishment of the threshold cycle value (C ⁇ ) for distinguishing samples containing various levels of E. coli 0157:H7 from those not containing target cells was based on the first appearance of a positive change of the target PCR product melting phase slope (S M j) from the baseline slope (Sgi).
- SV40 viral DNA was purchased from CIBCO BRL® Life Technology (Rockville, MD). It is purified from CsCl-banded SV40 vims (Strain 776) Propagated in BSC-1 cells. The molecular weight is about 3.5E6 daltons (5243 base pair dsDNA) supercoiled circular DNA.
- the SV40 viral DNA was used as a template for PCR amplification in the study. The stock SV40 DNA concentration was 500 ng/ul, then diluted one to 10,000-fold with distilled, de-ionized water to be 50 pg/ul working stock solution.
- coli 0157:H7 tablets that contained proven effective concentrations of 160mM of dATP, dCTP, dGTP, dTTP, 72nM of primer 5'(TAC CTG AGG CAG TAG CGA TAA TGA GC) 3'. 33-26-rcl012; 72nM of primer 5'(ATG CAG ACC CGC TGG AGT TTG AGA AA) 3'. 33-26-538 and 1.5 units of TaqTM polymerase.
- Duplicate aliquots of 50 ⁇ L of each SV40 DNA sample were removed and transferred into a PCR tube containing one BAX® for Screening/E. coli 0157:H7 tablet, then amplified in a P ⁇ /ABI PRISM 7700 Sequence Detection System.
- the reaction proceeded via an initial holding period of 2 min at 94°C, followed by 38 cycles of 94°C/15 seconds and 70°C/2.53 minutes, then, (T ⁇ ) 70°C/7 seconds, (T B1 ) 76.5°C/7 seconds, (T MS1 ) 77.5°C/7 sec, and TMEI 79.5°C/7 seconds.
- the selected primer pair P-4158 and rc-4289 was used to amplify a 132-bp of DNA fragment, which can identify the SV40 target DNA.
- Establishment of the threshold cycle value (CT) for distinguishing samples containing various levels of SV40 from those not containing target cells was based on the first appearance of a positive change of the melting phase slope S i ⁇ fr° m me baseline slope (S B j).
- EXAMPLE 5 MULTIPLEX O-PCR ASSAY This Example demonstrates how the two previously described assays may be combined in a single reaction to test for both targets. The procedure is the same as for Example IV but in this study the samples include template DNA for E. coli 0157:H7 and SV40 thus amplicons for both targets will be produced and measured. Material And Methods Cell Culture E coli 0157:H7
- Example III Each process was the same as in Example III to prepare fresh culture and dilution.
- the cell counts of target E. coli 0157:H7 in the final were approximately 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , and 0 CFU/ml respectively.
- the cell lysis process was the same as in Example III.
- SV40 viral DNA spiked into E. coli 0157:H7 cell lysate The same SV40 DNA as in Example IV was used in this study.
- E. coli 0157:H7 SV40 from 50 pg/ul into 20, 10, 4, 0.8, 0.16 and 0 pg/50 ul were spiked into E. coli 0157:H7 at level of 0, 10 4 , 10 5 , 10 6 , 10 7 , and 10 s CFU/ml respectively. Finally, all samples were subject to PCR amplification followed by fluorescence detection in PE/ABI PRISM 7700 Sequence Detection System to determine the quantity of PCR product from different levels of E. coli 0157:H7 for each PCR cycle. The final E. coli 0157:H7 and SV40 DNA concentration per PCR reaction were set at 1.25 ⁇ +5 E. coli genome / 0 SV40, 1.25 E+4 E.
- the PCR reagents consisted of BAX® for Screening/E. coli 0157:H7 tablets (Qualicon, Inc.) that contained proven effective concentrations of 160mM of dATP, dCTP, dGTP, dTTP, 72nM of primer 5'(TAC CTG AGG CAG TAG CGA TAA TGA GC) 3'. 33-26-rcl012 ; 72nM of primer 5'(ATG CAG ACC CGC TGG AGT TTG AGA AA) 3'. 33-26-538 and 1.5units of TaqTM polymerase.
- Duplicate aliquots of 50 ⁇ L of each lysed sample spiked with SV40 DNA were removed and transferred into a PCR tube containing one BAX® for Screening/E. coli 0157:H7 tablet, then amplified in a P ⁇ /ABI PRISM7700 Sequence Detection System.
- reaction proceeded via an initial holding period of 2 min at 94°C, followed by 38 cycles of 94°C/8 seconds and 70°C/ 2.53 minutes, then, (T ⁇ ) 70°C/7 seconds, (T B1 ) 73.5°C/7 seconds, and (T MS1 ) 77.5°C/7 seconds, (T ⁇ ) 78.9°C/7, (T B2 ) 83°C/7 seconds, and (T ME2 ) 89.4°C/7 seconds.
- C ⁇ tlireshold cycle
- EXAMPLE 6 SINGLE TARGET QUANTITATIVE PCR ASSAY: ADDITIONAL THRESHOLD DETERMINATIONS
- the previous Examples I to IV compare the change in slope of the fluorescence value of a specific amplicon to that of the baseline of the intercalating dye.
- the threshold cycle is defined as the cycle in which the first positive change in the amplicon slope, with respect to the baseline slope, is detected.
- There are other approaches for determining the threshold for the first appearance of a specific amplicon in a PCR reaction One can assume there is no detectable production of target amplicon in the first ten PCR cycles.
- a threshold fluorescence value can be calculated by averaging the fluorescence values of the first ten cycles and adding to that fifteen times the standard deviation of this fluorescence value.
- This method is less specific for a target DNA since many amplicons have similar starting melting temperature. It can only apply to single target PCR quantification. It is less reproducible than the method employed in
- Examples 1-4 since it is affected by instrument to instrument variability, and well-to-well variability in PCR performance. It is also depends on fluorescence dye concentration in the assay which will shift the baseline signal.
- the threshold is determined as the average change in fluorescence value of the first ten cycles plus fifteen times the standard deviation of the ten fluorescence values (FE_ ⁇ 0 ave + 15XSD).
- the threshold cycle is defined as the cycle wherein the fluorescence of the sample exceeds FE_ ⁇ 0 ave + 15XSD. Quantification of unlcnown concentrations of target nucleic acid in the starting material can be extrapolated by comparing their threshold cycle (Ct) to a standard curve of Ct's generated from controls of known concentration.
- Stage II Run 40 cycles with: 94°C for 20 seconds
- Stage III 72°C for 3 minutes Collect the fluorescence signal from each cycle of stage II at (T ⁇ ) 82°C, (T MT )
- T ⁇ Temperature in a PCR reaction of total melting temperature for All ds-DNA.
- TMS Temperature in a PCR reaction of the beginning of the melting temperature for amplicon.
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AU2001250803A AU2001250803A1 (en) | 2000-03-07 | 2001-03-07 | Real time quantitative pcr with intercalating dye for single and multiplex target dna |
US10/204,889 US20040053230A1 (en) | 2001-03-07 | 2001-03-07 | Real time quantitative pcr with intercalating dye for single and multiplex target dna |
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EP1335027A1 (en) * | 2000-10-26 | 2003-08-13 | National Food Research Institute | Method of quantifying genetic modification and standard molecule to be used therein |
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DE60140804D1 (en) * | 2000-06-27 | 2010-01-28 | Nat Inst Of Advanced Ind Scien | NEW NUCLEIC ACID EASTS AND METHOD FOR TESTING NUCLEIC ACIDS UNDER USE |
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- 2001-03-07 JP JP2001565402A patent/JP2004533801A/en active Pending
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AU2001250803A1 (en) | 2001-09-17 |
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