Analytical method and instrument
Subjects of the invention are a method for determination of an analyte using a particular algorithm based on a mathematical model for transforming measurement data into a growth curve and an analytical instrument comprising a computing unit for more precise determination of an analyte.
1. A method for determining the presence of a nucleic acid in a sample, comprising:
providing a reaction mixture suspected of containing a nucleic acid, wherein said reaction mixture is in a form suitable for eliciting a detectable signal from said suspected nucleic acid,
detecting said signals from said reaction mixture in known intervals,
transforming said signals into measurement data, and
creating from said measurement data a growth curve through a mathematical algorithm,
wherein said mathematical algorithm comprises a mathematical growth curve model formula
f
(
x
)
=
p
1
·
(
1
+
p
2
·
x
)
+
p
3
[
1
+
exp
{
-
p
4
·
(
x
-
p
5
)
}
]
·
[
1
+
exp
{
-
p
6
·
(
x
-
p
7
)
}
]
or its mathematic equivalents, wherein
x
is a measure of the time or sequence of the measurements,
p 1
is the intercept of the growth curve for the start of the reaction,
p 2
is the relative drift of the base line,
p 3
is the distance between the base line and the saturation line,
p 4
is the slope at the inflection point of a first multiplicative
sigmoid function representing exponential growth,
p 5
is the inflection point of a first multiplicative sigmoid function,
p 6
is the slope at the inflection point of a second multiplicative
sigmoid function representing saturation growth, and
p 7
is the inflection point of a second multiplicative sigmoid function,
wherein optimal values of the parameters are determined by
a non-linear aggression fitting algorithm to said measurement data,
determining from said growth curve whether said nucleic acid is present or absent in said reaction mixture.
2. The method according to claim 1 , wherein said parameters of the said growth curve model formula are determined using a non-linear regression fitting algorithm, wherein
the starting values for said parameters are generated using said measurement data,
the range of said parameters are checked, and
automatic algorithm stopping criteria are employed.
3. The method according to claim 1 , wherein for further calculations said measurement data are partially or fully replaced by data generated using said growth curve model.
4. The method according to claim 1 , further comprising comparing one or more data characteristic for said growth curve to corresponding reference values.
5. The method according to claim 1 , further comprising determining a value from said growth curve indicative of the concentration of said nucleic acid present in said sample.
6. The method according to claim 1 , wherein a statistical test is used for comparison with said background curve estimation.
7. The method according to claim 1 , wherein the quality of components leading to the final result is determined.
8. A method for determining the presence of a nucleic acid in a sample, comprising:
providing a reaction mixture suspected of containing a nucleic acid, wherein said reaction mixture is in a form suitable for eliciting a detectable signal from said suspected nucleic acid,
detecting said signals from said reaction mixture in known intervals,
transforming said signals into measurement data, and
creating from said measurement data a growth curve through a mathematical algorithm,
wherein said mathematical algorithm comprises a mathematical growth curve model formula
f
(
x
)
=
p
1
·
(
1
+
p
2
·
x
)
+
p
3
[
1
+
exp
{
-
p
4
·
(
x
-
p
5
)
}
]
·
[
1
+
exp
{
-
p
6
·
(
x
-
p
7
)
}
]
-
p
8
2
[
1
+
sign
{
s
-
x
+
0.5
}
]
or its mathematic equivalents wherein
p 1
is the intercept of the growth curve for the start of the reaction,
p 2
is the relative drift of the base line,
p 3
is the distance between the base line and the saturation line,
p 4
is the slope at the inflection point of a first multiplicative
sigmoid function representing exponential growth,
p 5
is the inflection point of a first multiplicative sigmoid function,
p 6
is the slope at the inflection point of a second multiplicative
sigmoid function representing saturation growth,
p 7
is the inflection point of a second multiplicative sigmoid function,
wherein optimal values of the parameters are determined by a
non-linear aggression fitting algorithm to said measurement data,
p 8
is the signal step size at a point where measurement
conditions change, and sign means the function
sign (x) := x/{square root over ((x 2 ))}, x<>0; sign (0) := 0 and s a pre-set step
cycle number from which on a measurement condition has changed,
determining from said growth curve whether said nucleic acid is present or absent in said reaction mixture.