U.S. patent application number 13/655698 was filed with the patent office on 2013-02-28 for quantification of nucleic acid molecules using multiplex pcr.
This patent application is currently assigned to PRIMERADX, INC.. The applicant listed for this patent is Michael Bristol, Vladimir Dancik, Elizabeth Garcia, Kyle Hart, Vladimir I. Slepnev, John Unger, Patrick Wells. Invention is credited to Michael Bristol, Vladimir Dancik, Elizabeth Garcia, Kyle Hart, Vladimir I. Slepnev, John Unger, Patrick Wells.
Application Number | 20130053274 13/655698 |
Document ID | / |
Family ID | 40261968 |
Filed Date | 2013-02-28 |
United States Patent
Application |
20130053274 |
Kind Code |
A1 |
Hart; Kyle ; et al. |
February 28, 2013 |
QUANTIFICATION OF NUCLEIC ACID MOLECULES USING MULTIPLEX PCR
Abstract
Described are novel quantification methods and systems that
permit, within the context of multiplex PCR, the quantification of
all targets within a single reaction tube. The methods employ
quantitation algorithms applied to the amplification profiles of
internal calibration controls or standards utilizing a plurality of
nucleic acid templates that are amplified within the same reaction
tube as the nucleic acid target(s) interrogated.
Inventors: |
Hart; Kyle; (Belmont,
MA) ; Dancik; Vladimir; (North Andover, MA) ;
Bristol; Michael; (Foxboro, MA) ; Wells; Patrick;
(North Attleboro, MA) ; Garcia; Elizabeth;
(Barrington, RI) ; Slepnev; Vladimir I.;
(Cincinnati, OH) ; Unger; John; (Norwood,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hart; Kyle
Dancik; Vladimir
Bristol; Michael
Wells; Patrick
Garcia; Elizabeth
Slepnev; Vladimir I.
Unger; John |
Belmont
North Andover
Foxboro
North Attleboro
Barrington
Cincinnati
Norwood |
MA
MA
MA
MA
RI
OH
MA |
US
US
US
US
US
US
US |
|
|
Assignee: |
PRIMERADX, INC.
Mansfield
MA
|
Family ID: |
40261968 |
Appl. No.: |
13/655698 |
Filed: |
October 19, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12266133 |
Nov 6, 2008 |
8321140 |
|
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13655698 |
|
|
|
|
61002149 |
Nov 7, 2007 |
|
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Current U.S.
Class: |
506/9 ;
702/19 |
Current CPC
Class: |
C12Q 1/6851 20130101;
C12Q 1/6851 20130101; C12Q 2537/165 20130101 |
Class at
Publication: |
506/9 ;
702/19 |
International
Class: |
G06F 19/00 20110101
G06F019/00; C40B 30/04 20060101 C40B030/04 |
Claims
1. A method of determining an initial concentration (Q.sub.0) of a
target nucleic acid template in a sample, the method comprising the
steps of: a) amplifying said target nucleic acid template and a
plurality of standard templates, each of said standard templates
present at a different, known copy number, in a nucleic acid
amplification reaction regimen comprising a plurality of cycles of
primer annealing, primer elongation and strand dissociation,
wherein said reaction is performed using a reaction mixture
comprising said target nucleic acid template and said plurality of
standard templates; b) measuring, at plural cycles of said
amplifying regimen, signals from said target template and each of
said plurality of standard templates after nucleic acid species in
an aliquot taken from the reaction mixture are separated, wherein
said measuring generates a set of measurements for each said
template; c) estimating a cycle threshold (C.sub.t) value for each
of said plurality of standard templates and said target template,
wherein the C.sub.t value is estimated by a method comprising the
steps, for each set of measurements for each said template, of: i)
compiling a candidate list of all sets of consecutive signal
measurements with cardinality 3 or greater; ii) removing outliers
based on those signal measurements from said candidate list; iii)
computing a best-fitting line for an amplification curve for each
set of measurements, wherein the best-fitting line correlates a
signal measurement to a cycle number; iv) computing a fitness score
for each of said target template and said plurality of standard
templates, wherein the fitness score is computed as a weighted sum
of quality attributes comprising a correlation coefficient
determined from the best-fitting line, the cardinality of the
candidate data set, and proximity to the signal threshold; v)
selecting the set with the highest fitness score for each of said
target template and said plurality of standard templates; and vi)
computing C.sub.t from the selected set of each of said target
template and said plurality of standard templates, whereby C.sub.t
is computed by: A) choosing a threshold value for a signal
measurement; and B) determining the intercept of the threshold
value on the best-fitting line, wherein the C.sub.t is equal to the
corresponding cycle number at the intercept. d) generating a
standard curve, by plotting C.sub.t values estimated in step (c) on
the y axis for each of said plurality of said standard templates
versus the log of said known copy number on the x axis for each
said standard template; and e) calculating an initial concentration
(Q.sub.0) from the standard curve generated in step (d) for said
target nucleic acid template.
2. The method of claim 1, wherein the efficiency of amplification
is similar for said plurality of standards and said target
template.
3. The method of claim 1, wherein the difference in the efficiency
of amplification between said plurality of standards and said
target template is greater than or equal to 0.1.
4. The method of claim 3, wherein a normalized estimate of the
initial concentration (Q.sub.0') for said target nucleic acid
template is computed using:
log(Q.sub.0')=log(Q.sub.0)*(E.sub.R/E.sub.T).sup.Ct, where E.sub.T
is the efficiency of said target nucleic acid template and E.sub.R
is the efficiency of said plurality of standards.
5. The method of claim 1, wherein the outliers comprise the signal
measurements from said candidate list for which peak area
measurement is less than peak area measurement at the previous
cycle.
6. The method of claim 1, wherein said measuring comprises
measurement of a fluorescent signal.
7. The method of claim 1, wherein said nucleic acid species are
separated by capillary electrophoresis.
8. The method of claim 1, wherein the initial concentration
(Q.sub.0) for said target template is calculated by solving the
equation C t ( target ) = [ I - log ( Q 0 ) ] log ( E R )
##EQU00003## for Q.sub.0, wherein I is the X intercept of said
standard curve and E.sub.R is the efficiency obtained from said
standard curve.
9. The method of claim 1, wherein said measurements of step (b) are
entered into a computer-readable physical memory, and wherein steps
(c)-(e) are performed by a computer processor executing
instructions, encoded on a computer-readable physical memory, for
performing such steps.
10. The method of claim 1, wherein each of said standard templates
and said target nucleic acid are amplified by the same pair of
oligonucleotide primers.
11. The method of claim 1, wherein said measuring step (b) is
performed on a plurality of aliquots taken from said reaction
mixture at respective plural cycles during said amplifying
regimen.
12. A method of determining initial concentrations (Q.sub.0) of a
plurality of different target nucleic acid templates in a sample,
the method comprising the steps of: a) amplifying the plurality of
different target nucleic acid templates and a plurality of sets of
standard templates, each of said standard templates in a set
present at a different, known copy number, in a nucleic acid
amplification reaction regimen comprising a plurality of cycles of
primer annealing, primer elongation and strand dissociation,
wherein said reaction is performed using a reaction mixture
comprising said target nucleic acid templates and said plurality of
standard templates, wherein the efficiency of amplification of at
least one of the target nucleic acid templates is different from
the efficiency of amplification of the other different target
nucleic acid templates, and wherein the efficiency of amplification
of each of the different target nucleic acid templates is similar
to the efficiency of amplification of at least one set of the
standard templates; b) measuring, at plural cycles of said
amplifying regimen, signals from said target templates and each of
said standard templates after nucleic acid species in an aliquot
taken from the reaction mixture are separated, wherein said
measuring generates a set of measurements for each said template;
c) estimating a cycle threshold (C.sub.e) value for each of said
standard templates in each set and said target templates; d) for
each set of said standard templates, generating a standard curve,
by plotting C.sub.t values estimated in step (c) on the y axis for
each of said standard templates in the set versus the log of said
known copy number on the x axis for each said standard template in
the set; and e) for each of said target templates, calculating an
initial concentration (Q.sub.0) from the standard curve generated
in step (d) based on a set of said standard templates having the
efficiency of amplification similar to that of the target
template.
13. The method of claim 12, wherein the C.sub.t value is estimated
by a method comprising the steps, for each set of measurements for
each said template, of: i) compiling a candidate list of all sets
of consecutive signal measurements with cardinality 3 or greater;
ii) removing outliers based on those signal measurements from said
candidate list; iii) computing a best-fitting line for an
amplification curve for each set of measurements, wherein the
best-fitting line correlates a signal measurement to a cycle
number; iv) computing a fitness score for each of said target
templates and said standard templates, wherein the fitness score is
computed as a weighted sum of quality attributes comprising a
correlation coefficient determined from the best-fitting line, the
cardinality of the candidate data set, and proximity to the signal
threshold; v) selecting the set with the highest fitness score for
each of said target templates and said standard templates; and vi)
computing C.sub.t from the selected set for each of said target
templates and said standard templates, wherein C.sub.t is computed
by: A) choosing a threshold value for a signal measurement; and B)
determining the intercept of the threshold value on the
best-fitting line, whereby the C.sub.t is equal to the
corresponding cycle number at the intercept.
14. The method of claim 13, wherein the outliers comprise the
signal measurements from said candidate list for which peak area
measurement is less than peak area measurement at the previous
cycle.
15. The method of claim 13, wherein the best-fitting line is
computed by linear regression, said line representing a log-linear
amplification curve described by the equation (1): log(measured
value)=C.sub.0+EC wherein C is the cycle number, C.sub.0 is the X
intercept and E is the slope of the line; and wherein C.sub.t is
computed by: A) choosing a threshold value for log(measured value)
of equation (1), B) solving equation (1) for C when log(measured
value) equals the chosen threshold value, and C) setting C.sub.t
equal to the solved value of C.
16. The method of claim 12, wherein said measuring comprises
measurement of a fluorescent signal.
17. The method of claim 12, wherein said nucleic acid species are
separated by capillary electrophoresis.
18. The method of claim 12, wherein the initial concentration
(Q.sub.0) for each of said target templates is calculated by
solving the equation C t ( target ) = [ I - log ( Q 0 ) ] log ( E R
) ##EQU00004## for Q.sub.0, wherein I is the X intercept of said
standard curve generated in step (d) based on a set of said
standard templates having the efficiency of amplification similar
to that of the target template and E.sub.R is the efficiency
obtained from said standard curve.
19. The method of claim 12 wherein said measurements of step (b)
are entered into a computer-readable physical memory, and wherein
steps (c)-(e) are performed by a computer processor executing
instructions, encoded on a computer-readable physical memory, for
performing such steps.
20. A non-transitory computer-readable physical storage medium
comprising instructions, that when executed by a processor, cause
the processor to perform a procedure comprising the steps of: a)
receiving a plurality of measurements obtained from a plurality of
standard templates; b) receiving a plurality of measurements
obtained from a target template; c) estimating a cycle threshold
(C.sub.t) value for each of said plurality of standard templates
and said target template, wherein the C.sub.t value is estimated by
a method comprising the steps, for each set of measurements for
each said template, of: i) compiling a candidate list of all sets
of consecutive signal measurements with cardinality 3 or greater;
ii) removing outliers based on those signal measurements from said
candidate list; iii) computing a best-fitting line for an
amplification curve for each set of measurements, wherein the
best-fitting line correlates a signal measurement to a cycle
number; iv) computing a fitness score for each of said target
template and said plurality of standard templates, wherein the
fitness score is computed as a weighted sum of quality attributes
comprising a correlation coefficient determined from the
best-fitting line, the cardinality of the candidate data set, and
proximity to the signal threshold; v) selecting the set with the
highest fitness score for each of said target template and said
plurality of standard templates; and vi) computing C.sub.t from the
selected set of each of said target template and said plurality of
standard templates, whereby C.sub.t is computed by: A) choosing a
threshold value for a signal measurement; and B) determining the
intercept of the threshold value on the best-fitting line, wherein
the C.sub.t is equal to the corresponding cycle number at the
intercept. d) generating a standard curve by plotting C.sub.t
values estimated in step (c) on the y axis for each of said
plurality of said standard templates versus the log of said known
copy number on the x axis for each said standard template; and e)
calculating an initial concentration (Q.sub.0) from the standard
curve generated from step (d) for said target nucleic acid
template.
21. A system comprising a computer processor, and instructions that
cause the processor to perform a procedure comprising the steps of:
a) receiving a plurality of measurements obtained from a plurality
of standard templates; b) receiving a plurality of measurements
obtained from a target template; c) estimating a cycle threshold
(C.sub.t) value for each of said plurality of standard templates
and said target template, wherein the C.sub.t value is estimated by
a method comprising the steps, for each set of measurements for
each said template, of: i) compiling a candidate list of all sets
of consecutive signal measurements with cardinality 3 or greater;
ii) removing outliers based on those signal measurements from said
candidate list; iii) computing a best-fitting line for an
amplification curve for each set of measurements, wherein the
best-fitting line correlates a signal measurement to a cycle
number; iv) computing a fitness score for each of said target
template and said plurality of standard templates, wherein the
fitness score is computed as a weighted sum of quality attributes
comprising a correlation coefficient determined from the
best-fitting line, the cardinality of the candidate data set, and
proximity to the signal threshold; v) selecting the set with the
highest fitness score for each of said target template and said
plurality of standard templates; and vi) computing C.sub.t from the
selected set of each of said target template and said plurality of
standard templates, whereby C.sub.t is computed by: A) choosing a
threshold value for a signal measurement; and B) determining the
intercept of the threshold value on the best-fitting line, wherein
the C.sub.t is equal to the corresponding cycle number at the
intercept. d) generating a standard curve by plotting C.sub.t
values estimated in step (c) on the y axis for each of said
plurality of said standard templates versus the log of said known
copy number on the x axis for each said standard template; and e)
calculating an initial concentration (Q.sub.0) from the standard
curve generated in step (d) for said target nucleic acid template.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 12/266,133 filed on Nov. 6, 2008, which
claims benefit under 35 U. S. C. .sctn.119(e) of U.S. Provisional
Application No. 61/002,149 filed Nov. 7, 2007, the contents of
which are incorporated herein by reference in their entireties.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Oct. 16, 2012, is named
20121019_SequenceListing-TextFile.sub.--046264.sub.--060507_C and
is 4,111 bytes in size.
BACKGROUND OF THE INVENTION
[0003] Since the advent of the polymerase chain reaction (PCR) it
was recognized that amplification can be used to estimate the
initial concentration of a template nucleic acid. However,
variations in the efficiency or other aspects of an amplification
can and do result from a number of different influences on the
reactions. Only if such variations are accounted for or neutralized
can one hope to obtain quantitative results. A number of approaches
have been used or proposed to address this fundamental issue for
quantitative PCR. Multiplex quantitative PCR, or quantitative PCR
in which a plurality of target nucleic acids is amplified and
quantified in one reaction mixture presents additional
challenges.
SUMMARY OF THE INVENTION
[0004] Disclosed herein are methods for the determination or
estimation of an initial amount of a target nucleic acid, or,
preferably, a plurality of different target nucleic acids in a
sample. The methods described use a plurality of internal
calibration controls, or standards, co-amplified with target
nucleic acids. Measurement of signal corresponding to each of the
calibration controls at different cycles of a cyclic amplification
regimen permits the generation of a standard curve, which is then
applied to estimate the initial amount of a target of unknown
quantity.
[0005] Accordingly, in one aspect, disclosed herein is a method of
determining an initial concentration (Q.sub.0) of a target nucleic
acid template in a sample, the method comprising the steps of: (a)
amplifying the target nucleic acid template and a plurality of
standard templates, each of the standard templates present at a
different, known copy number, in a nucleic acid amplification
reaction regimen comprising a plurality of cycles of primer
annealing, primer elongation and strand dissociation, wherein the
reaction is performed using a reaction mixture comprising the
target nucleic acid template and the plurality of standard
templates, and wherein the efficiency of amplification, E, is
similar for the plurality of standards and the target template; (b)
measuring, at plural cycles of the amplifying regimen, signals from
the target and each of the plurality of standard templates, wherein
the measuring generates a set of measurements for each the
template; (c) estimating a cycle threshold (C.sub.t) value for each
of the plurality of standard templates and the target template; (d)
generating a standard curve, by plotting C.sub.t values estimated
in step (c) on the y axis for each of the plurality of the standard
templates versus the log of the known copy number on the x axis for
each the standard template; and (e) calculating an initial
concentration (Q.sub.0) for the target nucleic acid template by
solving the equation C.sub.t(target)=[I-log(Q.sub.0)]/log(E) for
Q.sub.0, wherein I is the X intercept of the standard curve and E
is the slope of the standard curve.
[0006] In one embodiment of this and other aspects described
herein, the measurements of step (b) are entered into a
computer-readable physical storage medium or memory, and steps
(c)-(e) are performed by a computer processor executing
instructions, encoded on a computer-readable physical storage
medium or memory, for performing such steps. In particular
embodiments, the computer-readable memory comprising the
instructions can be the same as or different from the memory on
which the measurements of step (b) are stored.
[0007] In another embodiment of this and other aspects described
herein, C.sub.t is estimated by a method comprising the steps, for
each set of measurements for each template: a) compiling a
candidate list of all sets of consecutive signal measurements with
cardinality 3 or greater; b) removing those points from the
candidate list for which the area measurement is less than the area
measurement at the previous cycle; c) computing a best-fitting line
for each set of measurements, the line representing a log-linear
amplification curve described by equation (1):
log(measured value)=C.sub.0+E*C, [equation (1)]
wherein C is the cycle number, C.sub.0 is the X intercept and E is
the slope of the line; d) removing candidate data sets having an
R-squared correlation coefficient less than 0.8, a slope less than
1.45 or a slope greater than 2.1; e) computing a fitness score for
each of the target nucleic acid and the plurality of standards; f)
selecting the set with the highest fitness score as the best set
for each of the target nucleic acid and the plurality of standards;
and g) computing C.sub.t from the best set from each of the target
nucleic acid and the plurality of standards, wherein C.sub.t is the
fractional cycle number at the intercept of T(measured value) on
the line described by equation (1).
[0008] In another embodiment of this and other aspects described
herein, each of the standard templates and the target nucleic acid
are amplified by the same pair of oligonucleotide primers.
[0009] In another embodiment of this and other aspects described
herein, the measuring comprises measurement of a fluorescent
signal.
[0010] In another embodiment of this and other aspects described
herein, the measuring step (b) is performed on a plurality of
aliquots taken from the reaction mixture at respective plural
cycles during the amplifying regimen.
[0011] In another embodiment of this and other aspects described
herein, prior to the measuring step, nucleic acid species in the
plurality of aliquots are separated. The nucleic acid species can
be separated, e.g., by capillary electrophoresis.
[0012] In another aspect, a computer-readable physical storage
medium or memory is provided, the memory storing a program or set
of instructions sufficient to implement a process, employing a
computer, for determining an initial concentration (Q.sub.0) of a
target nucleic acid template. The template is present in a sample
amplified in a nucleic acid amplification reaction regimen
comprising a plurality of cycles of primer annealing, primer
elongation and strand dissociation, and the reaction is performed
using a reaction mixture comprising the target nucleic acid
template and a plurality of standard templates, wherein each of the
plurality of standard templates is present at a different, known
copy number, and wherein the efficiency of amplification, E, is
similar for the plurality of standards and the target template. The
program stored in the memory comprises: a) instructions for
inputting values for signals obtained from the target and each of
the plurality of standard templates, at each of a plurality of
cycles of the regimen, into the program; (b) instructions for
estimating a cycle threshold (C.sub.t) value for each of the
plurality of standard templates and the target template using the
values entered according to the instructions of (a); (c)
instructions for generating a standard curve, by plotting C.sub.t
values estimated in step (b) on the y axis for each of the
plurality of the standard templates versus the log of the known
copy number on the x axis for each the standard template; (d)
instructions for calculating an initial concentration (Q.sub.0) for
the target nucleic acid template by solving the equation
C.sub.t(target)=[I-log(Q.sub.0)]/log(E) for Q.sub.0, wherein I is
the X intercept of the standard curve and E is the slope of the
standard curve; and (e) instructions for outputting a calculated Qo
for the target nucleic acid template to a memory, display or other
user interface.
[0013] In another aspect, a computer system is provided for
determining an initial concentration (Q.sub.0) of a target nucleic
acid template in a sample, the computer system comprising a
computer processor, a physical memory or storage medium, a user
interface and a display. The memory comprises a program or set of
instructions sufficient to implement a process, employing the
computer processor, for determining an initial concentration
(Q.sub.0) of a target nucleic acid template in a sample amplified
in a nucleic acid amplification reaction regimen comprising a
plurality of cycles of primer annealing, primer elongation and
strand dissociation, wherein the reaction is performed using a
reaction mixture comprising the target nucleic acid template and a
plurality of standard templates, wherein each of the plurality of
standard templates is present at a different, known copy number,
and wherein the efficiency of amplification, E, is similar for the
plurality of standards and the target template. The program on the
memory comprises: a) instructions for inputting values for signals
obtained from the target and each of the plurality of standard
templates, at each of a plurality of cycles of the regimen, into
the program; (b) instructions for estimating a cycle threshold
(C.sub.t) value for each of the plurality of standard templates and
the target template using the values entered according to the
instructions of (a); (c) instructions for generating a standard
curve, by plotting C.sub.t values estimated in step (b) on the y
axis for each of the plurality of the standard templates versus the
log of the known copy number on the x axis for each standard
template; (d) instructions for calculating an initial concentration
(Q.sub.0) for the target nucleic acid template by solving the
equation C.sub.t(target)=[I-log(Q.sub.0)]/log(E) for Q.sub.0,
wherein I is the X intercept of the standard curve and E is the
slope of the standard curve; and (e) instructions for outputting a
calculated Q.sub.0 for the target nucleic acid template to a
memory, display or other user interface.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 shows a standard curve plot of cycle threshold (Ct)
versus log of the concentration of reference target or calibrator
target (Log10 Concentration).
[0015] FIG. 2 shows the extension of the standard curve for low
concentration templates by pairwise linear fit with low
concentration reference calibrators.
[0016] FIG. 3 shows standard curves used to evaluate the efficiency
of quantitative standards. Three separate reactions were examined
with respect to the efficiency of the quantitative standard curve.
These standard curves were used to quantify the unknown virus in
the same reaction. In this example the delta efficiency can be
shown by the equations: Eff sampleA-Eff sampleB, Eff sampleA-Eff
sampleC, and EffsampleB-EffsampleC. The delta Eff is 0.05, 0.04,
and 0.01 respectively. The slope, R2, and amplification efficiency
are listed in the table.
[0017] FIGS. 4A-4C show the Log Log plots of copy number input and
copy number derived over a linear range of target input. The copy
number was estimated using the quantitative standards that are in
each reaction. FIGS. 4A-4C show that the use of the quantitative
standards is linear over a large dynamic range. The thick line in
each figure is the fitted regression shown for comparison against
the observations to judge goodness of fit. The two thin lines in
each figure indicate the range around the fitted regression line
likely to contain the population regression line.
[0018] The table and graph of FIG. 5 represent the Efficiency of
each target in a reaction tube. The table and graph shows that in
the same reaction tube the quantitative standards have the same
efficiency as the target virus.
DETAILED DESCRIPTION
[0019] Multiplex PCR can permit detection and monitoring of the
amplification of dozens of nucleic acid targets within the same
reaction tube. Described herein are novel quantification methods
that permit, within the context of increased multiplex capacity,
the quantification of all targets within a single reaction tube.
The methods are based on the amplification profile of internal
calibration controls or standards utilizing at least two, but
preferably three to five, nucleic acid templates that are amplified
within the same reaction tube as the nucleic acid target(s)
interrogated.
[0020] In one aspect of the methods described herein, each
interrogated target and calibrator is labeled with a fluorophore,
preferably 5,6-FAM, and identity of each is based on unique size.
The concentration of the targets is preferably measured by
capillary electrophoresis; however, other methods that monitor DNA
concentration are also applicable. Quantification of the targets is
based on amplification of calibrators within the same reaction in
order to control for variability between reactions that may be due,
for example, to contaminants introduced during sample preparation,
environmental factors, and operator or instrument non-uniformity.
In principle, similar quantification algorithms are applicable to
real-time PCR methods given that a sufficient number of
fluorophores are available that can be accurately monitored and
quantified within the same reaction without significant overlap in
spectra. In such an embodiment the calibrators should have unique
(different from each other) sequences which would be targeted by
the color-encoded probes. Accordingly, in such an embodiment there
is no requirement for unique size for each calibrator.
[0021] In one embodiment, each calibration control is a DNA
molecule which contains an amplifiable unique nucleotide sequence
of varying length. The length must be unique for each calibrator
with respect to all potentially amplified targets within the
reaction. The amplified length of each calibrator is preferably
within 250 bases of each other, but may differ in size up to 1000
bases. The amplifiable sequences of each calibrator are flanked by
a set of sequences at their 5'- and 3'-ends that are used for
priming PCR amplification. These sequences can be the same in all
calibrators, or they can have different sequence, but common
composition (defined as % CG content varying by no more than 10%)
or similar melting temperatures (calculated using standard methods,
such as next neighbor calculation and being within 5.degree. C.).
Importantly, the efficiency of PCR amplification using primers
targeting selected sequences in calibrating molecules should be the
same or similar (as used in this context, the efficiencies are
"similar" if the difference in the amplification efficiency for all
calibrators is less than 0.1). Calibrators are seeded in the
reaction at minimally two, preferably three to five, concentrations
ranging from 10-100,000 fold excess over the concentration of the
least abundant calibrator. In the case when all calibrators are
amplified by a common set of primers, the concentration of all
calibrators is set such that the difference is no greater than
10,000-fold, preferably 100 to 1,000 fold to avoid competitive
inhibition during PCR amplification. The concentration of
calibrators is also dictated by variability of the assay such that
the calculated copy number demonstrates a coefficient of variation
between 0 and 50%, preferably <30%. It should be noted that
where ranges are described herein, the ranges include and describe
all integer values therebetween, as well as all sub-ranges, as if
they were specifically recited herein. Thus, for example, the range
0 to 50% includes, not only 1%, 2%, 3%, 4% . . . 50%, but also, as
non-limiting examples, 0-40%, 0-30%, 5-45%, 5-40%, 5-10%, 10-30%,
and 40-45%.
[0022] In another embodiment the calibrators are RNA molecules.
This is particularly useful if the target molecules being
quantified are also RNAs. In this embodiment targets and
calibrators are converted to DNA prior to PCR amplification using
standard methods of reverse transcription known in art (for example
RT transcription using random primers or target-specific
primers).
[0023] In another embodiment the calibrators and targets to be
quantified show amplification efficiencies within 0.1 of the
calibrators. Then the assumption that amplification efficiency is
the same for all calibrators and targets can be applied to simplify
quantification algorithms as described below. Alternatively, the
amplification efficiency of the targets can be different from
amplification efficiency of the calibrators. In this case, a
correction for calculation of targets' quantities can applied as
also described below.
[0024] It is also well known that PCR amplification of very low
level target results in poor amplification efficiencies. Additional
algorithms are described herein to address quantification of these
rare targets. Essentially, additional calibrators are seeded within
the reaction at low levels, 5 to 100 copies per reaction. Targets
amplifying with low efficiency, typically 1.4 to 1.6, are
quantified to calibrators exhibiting amplification efficiencies
within 0.1, preferably, 0.05 of the interrogated target. Low level
calibrators exhibit the same characteristics as described for
calibrators above
Data Analysis
[0025] Quantification of DNA fragment abundance is done through
analysis of the label signal (preferably fluorescent signal)
measurements modeled as amplification curves. Signal is measured
after a plurality of cycles of an amplification regimen. In
preferred embodiments the signal is measured in aliquots of
amplification reaction mixture taken at a plurality of cycles of
the amplification regimen. The signal for each different target and
calibrator is measured at each measurement point (e.g., in each
aliquot, the amount of signal corresponding to each target and each
calibrator is measured, to provide a data set corresponding to the
amplified amounts of each target and calibrator in the reaction
mixture). The data analysis steps are: [0026] 1. Exponential Phase
Identification; [0027] 2. Limit of Detection; [0028] 3. Standard
Curve Quantification; [0029] 4. Low Concentration Quantification
(alternative method); and [0030] 5. Efficiency Normalization.
Exponential Phase Identification
[0031] Exponential phase identification involves the following
steps:
Step 1. Enumerate Candidate Point Sets
[0032] From a series of fluorescent measurements (RFU units) of
increasing cycle number for each target, compile a candidate list
of all sets of consecutive measurement points with cardinality 3 or
greater.
Step 2. Remove Outliers
[0033] Remove internal points from each candidate set whenever the
area measurement of a point is less than the area measurement at
the previous cycle. Candidate sets of cardinality less than 3 are
dropped from further consideration.
Step 3. Compute Linear Regression
[0034] Compute the best-fitting line for each set using linear
regression. Use Log(RFU) and cycle number (C) for each point to
create a log-linear amplification curve having equation:
Log(RFU)=C.sub.0+E.sub.T*C where C.sub.0 is the intercept and
E.sub.T is the slope of the line. (Equation 1)
Step 4. Remove Low Quality Sets
[0035] Disqualify candidate sets in which the R-squared correlation
coefficient value is less than 0.80, the slope (E) is less than
1.45, or the slope is greater than 2.1. If no set passes this
quality threshold, then the target is considered not present and
reported as not amplified (not detected).
Step 5. Compute Fitness Score
[0036] The fitness of a candidate set is computed as a weighted sum
of quality attributes. These attributes are (1) linearity
(R-squared correlation coefficient), (2) number of data points used
(cardinality), and (3) proximity to the fluorescence threshold. A
set that is closer to the fluorescence threshold is considered
higher quality because errors in the linear regression line cause
an increase in C.sub.t estimation error that is proportional to
distance.
[0037] Given a Log(RFU) threshold T.sub.RFU, minimum log(RFU) value
in the set Min.sub.RFU, maximum Log(RFU) in the set Max.sub.RFU,
set cardinality Num, and correlation coefficient RSQ, compute the
fitness score using Equation 2:
Fitness Score = RSQ * 100 + max ( 5 , Num ) * 1.0 + if Min RFU
.ltoreq. T RFU .ltoreq. Max RFU then 1.0 else if Max RFU < T RFU
then ( T RFU - Max RFU ) * - 0.75 else if Min RFU > T RFU then (
Min RFU - T RFU ) * - 0.75 ( Equation 2 ) ##EQU00001##
Step 6. Select Best Set
[0038] Select the set with the highest fitness score as the best
set for the target.
Step 7. Compute Cycle Threshold
[0039] From the best set for each target, compute cycle threshold
(Ct)) as the fractional cycle number at the intercept of T.sub.RFU
on the regression line (defined by Equation 1).
Limit of Detection
[0040] Although poor quality amplification curves are identified in
Step 4 of the Exponential Phase Identification method, additional
acceptance criteria have been established through a comparison of
the low-concentration samples and no template control samples
(negatives). The supervised learning method of Decision Trees [1]
is used to discriminate between these samples to establish limit of
detection (LOD) criteria. The attributes used to assess quality are
described in Table 1 and the inferred rules for determining when
samples are not amplified are described in Table 2. If none of the
failure criteria described in Table 2 are met, then the sample is
considered to be detected (above LOD).
TABLE-US-00001 TABLE 1 Limit of Detection Attributes Attribute
Definition Purpose Maximum Peak The maximum height Assessment of
Height of the target peak maximum fragment from any measurement
abundance of the amplified target Maximum Peak The maximum area of
Assessment of Area the target peak from maximum fragment any
measurement abundance of the amplified target Peak Variance
Variance of the peak Assessment of the position in bases. quality
of target peak selection Fitness Score Amplification curve
Assessment of fitness score from amplification curve Equation 2
quality
TABLE-US-00002 TABLE 2 Limit of Detection Algorithm Criteria
Explanation Result Criteria 1 Maximum Peak Height <=3313
Failure: Low Peaks Criteria 2 Maximum Peak Height <=7828
Failure: High Variance and Maximum Peak Height >3313 and Peak
Variance <=0.538697 Criteria 3 Maximum Peak Height >=7828
Failure: Low Quality and Peak Variance >0.921343 and Fitness
Score <=20.649301 and Peak Area >257247
Standard Curve Quantification--Method of Determining Nucleic Acid
Template Quantities
[0041] Using amplified reference targets of known concentration, a
standard curve is created to estimate the initial concentration of
other amplified targets.
[0042] For each reference target, the cycle threshold (Ct) is
estimated using the Exponential Phase Identification method as
described above. Each standard data point is obtained by
associating Ct (plotted along the y-axis) with the initial template
concentration (expressed as the log of the template copy number and
plotted along the x-axis) (see FIGS. 1 and 3). Linear regression
using the standard data points yields an approximation line that
indicates a substantial degree of linearity in the standard curve.
The linear relationship between Ct and initial concentration
(Q.sub.0) is described using Equation 3:
Ct=[I-log(Q.sub.0)]/log(E.sub.R) where I is the x intercept and
E.sub.R is the slope (efficiency) (Equation 3)
[0043] Standard curve quality is assessed by evaluation of the
r-squared value and the slope. The acceptable minimum value for
r-squared is determined by modeling historical values using a
Chi-squared distribution of (1-R.sup.2). Acceptable minimum and
maximum values for the slope are modeled using a Gaussian
distribution. These threshold values are set based on a 99.9%
expected rate of acceptance in their respective distributions.
[0044] The cycle thresholds for measured targets are determined in
the same manner as the reference targets using the Exponential
Phase Identification method. The estimate of initial concentration
of measured targets is determined using the standard curve
described by Equation 3 which is rearranged in terms of log(Qo) in
Equation 4:
log(Q.sub.0)=I-(Ct*log(E.sub.R)) (Equation 4)
Low Concentration Quantification
[0045] Due to the gradual reduction in amplification efficiency at
the end of a PCR reaction, the assumption of constant efficiency
used by the standard curve model may no longer be valid. The use of
extrapolation from the standard curve can therefore introduce
measurement errors.
[0046] To correct for such changes in efficiency, the standard
curve is extended with piecewise-linear segments using additional
low concentration reference targets. These reference targets are
not used in the linear regression that determines the original
standard curve. The use of these targets for quantification is
described in Equation 5 (and results are depicted, for example, in
FIG. 2):
Let Ct be the cycle threshold of the measured target. (Equation 5)
[0047] Let Ct-S.sub.n be the cycle threshold of the smallest
concentration reference target used in the standard curve. [0048]
Let Ct-L.sub.l, . . . , Ct-L.sub.n be the cycle thresholds of
additional low concentration reference targets
[0049] Initial concentration is estimated using linear
interpolation or extrapolation (Equation 4) [0050] Case 1: If
Ct.ltoreq.Ct-S.sub.n, then use previously defined standard curve
method [0051] Case 2: If Ct-S.sub.n<Ct.ltoreq.Ct-L.sub.1, then
use linear interpolation using the line between calibrators S.sub.n
and L.sub.1 [0052] Case 3: If
Ct-L.sub.i<Ct.ltoreq.Ct-L-.sub.i+1, then use linear
interpolation using the line between calibrators L, and L.sub.i+1
[0053] Case 4: If Ct>Ct-L.sub.n, then use linear extrapolation
using the line between calibrators L.sub.n and L.sub.n-1
Efficiency Variation Normalization
[0054] In the simplest application of described quantification
method it is assumed that the efficiency of the reference targets
(calibrators) E.sub.R is identical to the efficiency of the
measured target E.sub.T for all cycles in the reaction. For
measurements in which the efficiency difference is greater than or
equal to 0.1, a normalized estimate of initial concentration is
computed using Equation 6:
log(Q.sub.0)'=log(Q.sub.0)*(E.sub.R/E.sub.T).sup.Cr where Ct is the
cycle threshold of the measured target (Equation 6)
[0055] whenever |E.sub.R-E.sub.T|.gtoreq.0.01
For example, if E.sub.R=1.81, E.sub.T=1.80 and Ct=32.0, the
normalized estimate would be
log ( Q 0 ) ' = log ( Q 0 ) * ( 1.81 / 1.80 ) 32.0 = log ( Q 0 ) *
1.194 ( Illustration 1 ) ##EQU00002##
[0056] A variety of methods can be used to estimate E.sub.R,
E.sub.T, and E.sub.R/E.sub.T. For individual efficiency estimates,
the slope of the linear region of the amplification curve can be
used (e.g. by Exponential Phase Identification, the E.sub.T in
Equation 1). The slope of a dilution (standard) curve from multiple
concentrations of a target can also be used (e.g. by Standard Curve
Quantification, the E.sub.R in Equation 3).
[0057] Efficiencies and efficiency ratios may be estimated using
(1) only the targets measured in a single PCR reaction, (2) using
historical measurements from many PCR reactions, or (3) using both
historical measurements and measurements from a single
reaction.
[0058] The methods described herein are illustrated by, but not
intended to be limited by the following Example.
EXAMPLES
Example 1
[0059] The following conditions are used to amplify viral targets
using quantitative standards in a multiplex PCR reaction. A PCR
reaction is set up in a single tube containing the analyte, common
PCR reagents (eg, DNA polymerase, dNTPs), and, for each target to
be amplified, viral-specific primers where at least one of each
pair is labeled with a fluorophore. The PCR reaction is assembled
with three quantitative standards that are amplified from a single
plant based gene using one primer pair for each of the quantitative
standards in a competitive nature used to quantify the viral
targets. The quantitative standards have similar amplification
efficiencies to ensure accurate estimation of viral load of the
unknown targets.
[0060] Template DNA was prepared by spiking plasma matrix with
known copies of Cytomegalovirus (CMV), BK virus (BK), and Human
Herpesvirus 7 (HHV7). The plasma was spiked with a dose of viral
particles from 3e6 copies/ml to 3e2 copies/ml from three virus';
Cytomegalovirus (CMV), BK virus (BK), and Human Herpesvirus 7
(HHV7) to demonstrate linearity of the system. A sample processing
control virus was added to the plasma to monitor that the
extraction procedure. The nucleic acid was extracted from 400 ul of
the spiked plasma samples with the use of the NucliSENS.RTM.
easyMAG sample processing instrument and eluted in 25 ul of buffer.
PCR reactions were set up to quantify the viral targets in the
system. The PCR reaction contained; 1.) 2.times. Qiagen Multiplex
PCR master mix, containing HotStarTaq DNA polymerase, Multiplex PCR
Buffer, dNTP Mix 2.) 25.times. Multiplex primer mix, containing a)
viral specific primers for each virus tested, primers for the
amplification control, sample processing control, and
quantification controls b) viral specific sensitivity controls and,
quantification control targets. 3.) 20 ul of extracted sample.
[0061] The PCR reaction is placed in a MJ research PTC-200
thermocycler that is integrated with a PerkinElmer Multiprobe II
Plus liquid handling system. The reaction is amplified under the
following conditions: 1) 95.degree.-15 minutes, 2) 95.degree.-30
seconds, 62.degree.-90 seconds, 72.degree.-60 seconds--for 3 cycles
3) 95.degree.-30 seconds, 60.degree.-90 seconds, 72.degree.-60
seconds--for 3 cycles 4) 95.degree.-30 seconds, 58.degree.-90
seconds, 72.degree.-60 seconds--for 3 cycles 5) 95.degree.-30
seconds, 57.degree.-90 seconds, 72.degree.-60 seconds--for 33
cycles. Starting at cycle 20, the thermocycler pauses and the lid
opens to take a 2 ul aliquot of the PCR reaction and dispenses it
into 13 ul formamide/rox standard mixture. The integrated
thermocycler and liquid handler takes an aiquot of the PCR reaction
from cycle 20 up to an including cycle 42. After all of the
aliquots are taken the formamide rox mixture that now contains the
PCR sample is placed at 95.degree. C. for 5 minutes to denature the
sample. The denatured samples are loaded into the Applied
BioSystems 3730x1 DNA Analyzer for analysis of each sample at each
cycle taken.
[0062] The samples are analyzed to determine the initial
concentration of a nucleic acid as outlined in "Method of
Determining Nucleic Acid Template Quantities" section of this
disclosure.
TABLE-US-00003 TABLE 3 Summary of STAR multiplex primer sequence
Primer Primer Sequence Rubisco Forward 5'-CTT TGT CGG GTT TTC TCC
GTA TCC-3' (SEQ ID NO: 1) Rubisco Reverse 5'-/56-FAM/TTC GCT CGT
AGT CGA ACG CC-3' (SEQ ID NO: 2) CMV Forward 5'-/56-FAM/TCC GGC GAT
GTT TAC TTT ATC AAC C-3' (SEQ ID NO: 3) CMV Reverse 5'-CCG TGA TAA
AAC ACA AAC TGG CAA A-3' (SEQ ID NO: 4) HHV7 Forward 5'-/56-FAM/ATA
TTG TGC CTT GCA GCT CTA TGT TTC TC-3' (SEQ ID NO: 5) HHV7 Reverse
5'-ACC GAG ATG CGG CTT TTA TAG TTG A-3' (SEQ ID NO: 6) BK Forward
5'-/56-FAM/GCT TGA TCC ATG TCC AGA GTC TTC A-3' (SEQ ID NO: 7) BK
Reverse 5'-GGA AGG AAA GGC TGG ATT CTG AGA T-3' (SEQ ID NO: 8)
Sample ctrl Forward 5'-/56-FAM/TGC TTT TGT AAT TGG CTT CTG ACC A-3'
(SEQ ID NO: 9) Sample ctrl Reverse 5'-CGC AAT CCA ATA ACT TGG AAC
GAA T-3' (SEQ ID NO: 10)
TABLE-US-00004 TABLE 4 Summary of Quantitative Standards Amplified
Region Standard Amplified Region DNA2
5'-CGTCGGGTTTTCTCCGTATCCCGTCAATTCAAAAGTCTCAAAACCCTTCCTTT
CTCTTTTCTCGACCCAAGAAATCTTTGGTGAGACCCATTTCAGCTTCAAGCTCAGAGT TGC-3'
(SEQ ID NO: 11) DNAB
5'-GTCGTATCCGGGAAGTCTGTGGCAGAACCTGCGGTGGTACCGGAAGGT
TTGGGACTTGTAGCCAGGCGTGATATTGGAAGAAACGAGGTCGTATTG
GAGATTCCCAAGCGATTGTGGATAAACCCAGAGACAGTGACTGCTTCC
AAGATTGGACCTTTATGCGGCGGATTAAAGCCGTGGGTTTCAGTAGCT
CTGTTTTTGATCAGAGAGAAGTATGAAGAAGAGTCTTCATGGAGAGTT
TATCTTGATATGCTTCCTCAATCTACTGATTCTA (SEQ ID NO: 12) DNAC
5'-TCTACTGATTCTACTGTCTTCTGGTCAGAAGAGGAGCTT
GCTGAGCTTAAAGGGACTCAACTGTTGAGCACCACATTGGGTGTGAAA
GAGTATGTGGAGAATGAATTCTTGAAACTGGA
ACAAGAGATATTACTGCCTAACAAAGATCTCTTCTCATCCCGCATAAC
ACTTGATGACTTCATATGGGCGTTTGGGATCC
TCAAGTCGAGGGCTTTTTCTCGTCTCCGTGGCCAAAACCTTGTCTTGAT
CCCTCTTGCAGACTTGATAAACCATAACCCC GCGATAAAGACAGAAGATTATGCATACGA-3'
(SEQ ID NO: 13)
[0063] As is apparent to one of skill in the art, various
modification and variations of the above embodiments can be made
without departing from the spirit and scope of this invention.
These modifications and variations are within the scope of this
invention.
Sequence CWU 1
1
13124DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1ctttgtcggg ttttctccgt atcc 24220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2ttcgctcgta gtcgaacgcc 20325DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 3tccggcgatg tttactttat caacc
25425DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 4ccgtgataaa acacaaactg gcaaa 25529DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5atattgtgcc ttgcagctct atgtttctc 29625DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
6accgagatgc ggcttttata gttga 25725DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 7gcttgatcca tgtccagagt
cttca 25825DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 8ggaaggaaag gctggattct gagat 25925DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
9tgcttttgta attggcttct gacca 251025DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
10cgcaatccaa taacttggaa cgaat 2511114DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
11cgtcgggttt tctccgtatc ccgtcaattc aaaagtctca aaacccttcc tttctctttt
60ctcgacccaa gaaatctttg gtgagaccca tttcagcttc aagctcagag ttgc
11412274DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 12gtcgtatccg ggaagtctgt ggcagaacct
gcggtggtac cggaaggttt gggacttgta 60gccaggcgtg atattggaag aaacgaggtc
gtattggaga ttcccaagcg attgtggata 120aacccagaga cagtgactgc
ttccaagatt ggacctttat gcggcggatt aaagccgtgg 180gtttcagtag
ctctgttttt gatcagagag aagtatgaag aagagtcttc atggagagtt
240tatcttgata tgcttcctca atctactgat tcta 27413308DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
13tctactgatt ctactgtctt ctggtcagaa gaggagcttg ctgagcttaa agggactcaa
60ctgttgagca ccacattggg tgtgaaagag tatgtggaga atgaattctt gaaactggaa
120caagagatat tactgcctaa caaagatctc ttctcatccc gcataacact
tgatgacttc 180atatgggcgt ttgggatcct caagtcgagg gctttttctc
gtctccgtgg ccaaaacctt 240gtcttgatcc ctcttgcaga cttgataaac
cataaccccg cgataaagac agaagattat 300gcatacga 308
* * * * *