U.S. patent application number 12/281540 was filed with the patent office on 2009-06-18 for apparatus for detecting nucleic acid amplification product in real time.
Invention is credited to Akifumi Iwama, Yasuaki Sonoda, Yuichi Tamaoki.
Application Number | 20090155891 12/281540 |
Document ID | / |
Family ID | 38474831 |
Filed Date | 2009-06-18 |
United States Patent
Application |
20090155891 |
Kind Code |
A1 |
Tamaoki; Yuichi ; et
al. |
June 18, 2009 |
APPARATUS FOR DETECTING NUCLEIC ACID AMPLIFICATION PRODUCT IN REAL
TIME
Abstract
There is provided an apparatus for detecting a nucleic acid
amplification product in real time, which is capable of effectively
excluding or reducing apparatus error factors without using a
second fluorescence signal used for correction. A plurality of
wells 7A are given with temperature cycles and fluorescence
strength from a nucleic acid amplification product is detected in
real time in each well 7A. A fluorescence measurement value
[DNA]raw obtained from the well 7A and a fluorescence measurement
value [DNA]bg obtained from a-connection wall near the well 7A are
detected, and the fluorescence measurement value [DNA]bg is
subtracted from the fluorescence measurement value [DNA]raw to
determine fluorescence strength [DNA]real of the well 7A.
Inventors: |
Tamaoki; Yuichi; (Gunma,
JP) ; Iwama; Akifumi; (Ibaraki, JP) ; Sonoda;
Yasuaki; (Gunma, JP) |
Correspondence
Address: |
KRATZ, QUINTOS & HANSON, LLP
1420 K Street, N.W., Suite 400
WASHINGTON
DC
20005
US
|
Family ID: |
38474831 |
Appl. No.: |
12/281540 |
Filed: |
March 1, 2007 |
PCT Filed: |
March 1, 2007 |
PCT NO: |
PCT/JP2007/053949 |
371 Date: |
December 2, 2008 |
Current U.S.
Class: |
435/287.2 |
Current CPC
Class: |
C12Q 1/6851 20130101;
G01N 21/6452 20130101; C12Q 1/686 20130101 |
Class at
Publication: |
435/287.2 |
International
Class: |
C12M 1/34 20060101
C12M001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 6, 2006 |
JP |
2006-059381 |
Claims
1. An apparatus for detecting fluorescence strength from a nucleic
acid amplification product in each of a plurality of reaction
regions given with temperature cycles in real time, wherein a
fluorescence measurement value [DNA]raw obtained from the reaction
region and a fluorescence measurement value [DNA]bg obtained from
regions other than the reaction region adjacent to the reaction
region are detected, and the fluorescence measurement value [DNA]bg
is subtracted from the fluorescence measurement value [DNA]raw to
determine fluorescence strength [DNA]real of the reaction
region.
2. The apparatus according to claim 1, wherein the fluorescence
measurement value [DNA]bg is a simple average value of fluorescence
measurement values obtained from the regions other than the
plurality of reaction regions adjacent to the reaction region, or
an average value of fluorescence measurement values after the
fluorescence measurement values are weighted.
3. The apparatus according to claim 1 or 2, wherein the
fluorescence measurement value [DNA]bg is detected every detection
of the fluorescence measurement value [DNA]raw, and the
fluorescence measurement value [DNA]bg is subtracted from the
fluorescence measurement value [DNA]raw to determine the
fluorescence strength [DNA]real.
4. An apparatus for detecting fluorescence strength from a nucleic
acid amplification product in each of a plurality of reaction
regions given with temperature cycles in real time, wherein
fluorescence strengths [DNA]n obtained from the reaction regions
every temperature cycles n are normalized using the maximum value
of the fluorescence strengths [DNA]n or related value [DNA]max, and
a threshold cycle number Ct is calculated by setting a threshold Th
in an exponential amplification region of an amplification curve
drawn using a normalized fluorescence strength [DNA]nN.
5. The apparatus according to claim 4, wherein the fluorescence
strength [DNA]nN is calculated by dividing the fluorescence
strength [DNA]n for each reaction region by a value obtained by
adding a value common to the reaction regions to be compared to the
maximum value or related value [DNA]max.
6. The apparatus according to claim 4 or 5, wherein the
amplification curve is drawn after fluorescence strength [DNA]base
before the exponential amplification region is subtracted from the
fluorescence strength [DNA]n for each reaction region and the
maximum value or related value [DNA]max.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to an apparatus for real-time
detecting a polynucleotide product obtained from a polymerase chain
reaction (PCR).
[0003] 2. Description of the Related Art
[0004] A PCR is a cyclic enzyme reaction to replicate a DNA chain.
As the PCR is used as a template of a cycle in which PCR products
(nucleic acid amplification products) replicated in previous cycles
are consecutive, arrayed target molecules can be exponentially
amplified. A real time PCR is to excite fluorescent material by
irradiating a PCR product with excitation light using, for example,
array-specific probes (TaqMan probes) marked with two different
kinds of fluorescent pigments interfering with each other, measure
the strength of the fluorescence and monitor amplification of the
PCR products in real time.
[0005] In quantitative use, a threshold ((6) in FIG. 7) is set in
an exponential amplification region of an amplification curve for
existing samples, and a point (threshold cycle number (Ct). (8) in
FIG. 8) at which the threshold intersects the amplification curve
is calculated. There is a linear relation between the threshold
cycle number (Ct) and the initial amount of DNA of a test sample
measured in terms of log value, and a calibration curve
representing this linear relation can be prepared. The initial
amount of DNA of the test sample is estimated based on the
calibration curve. This enables correct quantitativeness based on a
PCR amplification speed theory.
[0006] Here, since an actual PCR efficiency is not 100%, the
concentration of an amplified PCR product is expressed by the
following Equation 1.
[DNA]=[DNA].sub.0(1+e).sup.c . . . (1)
[0007] Where, [DNA]: Concentration of PCR product
[0008] [DNA].sub.0: Initial concentration of target Template
[0009] e: Average PCR efficiency
[0010] c: Cycle number
[0011] That is, if the average PCR efficiency (e) is 100% (i.e.,
e=1 in the above Equation (1)), although the concentration [DNA] of
the PCR product is exponentially amplified with 2.sup.c, since the
efficiency (e) is slowly lowered from the initial stage, through
the middle stage, to the late state of the cycle, an amplification
curve is as shown in FIG. 7. In FIG. 7, a horizontal axis
represents the cycle number and a vertical axis represents the
fluorescence strength. As shown in the figure, the fluorescence
strength is exponentially amplified ((5) in FIG. 7) at the cycle
initial stage, linearly amplified ((6) in FIG. 7) at the cycle
middle stage, and not amplified ((7) in FIG. 7) by a plateau effect
at the cycle late stage.
[0012] Chemical-reactive factors for this plateau effect are as
follows.
[0013] Hydrolysis of DNTP and primer
[0014] Deactivation of DNA polymerase (DNA synthase to make a copy
of a template (cast)) by heat.
[0015] Lowering of primer annealing efficiency by reassociation of
one chain PCR fragment
[0016] Competitive material by non-specific PCR product
[0017] Accumulation of PCR inhabitation material such as
pyrophosphate
[0018] Hydrolysis of PCR product by exonuclease activation of DNA
polymerase
[0019] Accordingly, the measurement in the exponential
amplification region satisfying the relation of the Equation (1) is
a precondition for the real time PCR (see Patent Document 1)
[0020] [Patent Document 1] Japanese Patent Application Publication
No. 2005-516630
[0021] [Patent Document 2] Japanese Patent No. 2909216
[0022] As a reactive vessel used for the real time PCR, a vessel
called a micro plate having a plurality (for example, 96) of wells
(reactive regions constituted by concave portions) is being used in
common and reactive solution having a predetermined initial DNA
concentration is divisionally poured in the wells. However, an
amplification curve for each of the wells of the reactive vessel
becomes unbalanced due to the following apparatus error factors
[0023] Error of optical system
[0024] Concentration error of correction solution
[0025] Divisional pour error of correction solution
[0026] Light transmission error of cap of reactive vessel or seal
film
[0027] Contamination error of reactive vessel
[0028] Divisional pour error of reactive vessel
[0029] Here, the reactive vessel mainly uses a cheap method in
which the above-mentioned seal film with an adhesive is attached to
the entire region of a single side and the wells are cover by a
cap. In addition, the wells are irradiated with excitation light
through the seal film and fluorescence generated from the PCR
product (reaction product) is detected by a light detecting part
such as a CCD camera through the seal film (these components
constitute an optical system). In this manner, although the seal
film and the body of the reactive vessel constitute important
factors of the optical system in measurement of the fluorescence
strength, these components are consumable parts, it is difficult to
expect optical performance with high uniformity and precision.
[0030] FIG. 4 shows an actual image of a reactive vessel before
PCR, which is detected by an optical detecting part. While the
circumference of wells of the reactive vessel appears to be black
as a whole, the brightness of pixels of the image as a background
is not necessarily constant and there occurs a spot due to
contamination of the optical system or way-out light as indicated
by (1) in the figure. When a PCR reaction is initiated, this spot
overlaps with images (96 images appearing to be round in FIG. 5) of
the wells as indicated by (2) in FIG. 5, wastefully adding to the
fluorescence strength of the wells.
[0031] So, in the prior art, empty reactive vessels containing no
DNA are initially prepared, and fluorescence strengths for wells
are measured in such an empty state and are stored as standard
correction values. Then, by performing a correcting process in
which the stored correction values are subtracted from measurement
values of actual fluorescence strengths, such correction of the
optical system is performed. However, since errors due to
contamination of the empty reactive vessels are inherent to the
respective reactive vessels, if correction values by other standard
empty reactive vessels are used, there occurs a problem of errors
in measurement values.
[0032] In addition, in Patent Document 2, although a first
fluorescence signal is corrected with a second fluorescence signal,
since a solution that generates second fluorescence for reference
must be added to a solution that generates first fluorescence to be
originally measured, work becomes complicated and costs are raised.
In addition, the solution that generates the second fluorescence
can not give any effect if it can not be divisionally poured and
measured with very high precision. In addition, since an especial
optical filter has to be used to measure the second fluorescence
and has to be exchanged for the solution that generates the first
fluorescence and the solution that generates the second
fluorescence every measurement, there is a problem that it takes
extra time to acquire and process data.
[0033] The present invention has made to overcome the above
technical problems and it is an object of the invention to provide
an apparatus for detecting a nucleic acid amplification product in
real time, which is capable of effectively excluding or reducing
apparatus error factors without using a second fluorescence signal
used for correction.
SUMMARY OF THE INVENTION
[0034] According to a first aspect of the invention, there is
provided an apparatus for detecting fluorescence strength from a
nucleic acid amplification product in each of a plurality of
reaction regions given with temperature cycles in real time,
wherein a fluorescence measurement value [DNA]raw obtained from the
reaction region and a fluorescence measurement value [DNA]bg
obtained from regions other than the reaction region adjacent to
the reaction region are detected, and the fluorescence measurement
value [DNA]bg is subtracted from the fluorescence measurement value
[DNA]raw to determine fluorescence strength [DNA]real of the
reaction region.
[0035] According to a second aspect of the invention, the
fluorescence measurement value [DNA]bg is a simple average value of
fluorescence measurement values obtained from the regions other
than the plurality of reaction regions adjacent to the reaction
region, or an average value of fluorescence measurement values
after the fluorescence measurement values are weighted.
[0036] According to a third aspect of the invention, the
fluorescence measurement value [DNA]bg is detected every detection
of the fluorescence measurement value [DNA]raw, and the
fluorescence measurement value [DNA]bg is subtracted from the
fluorescence measurement value [DNA]raw to determine the
fluorescence strength [DNA]real.
[0037] According to a fourth aspect of the invention, there is
provided an apparatus for detecting fluorescence strength from a
nucleic acid amplification product in each of a plurality of
reaction regions given with temperature cycles in real time,
wherein fluorescence strengths [DNA]n obtained from the reaction
regions every temperature cycles n are normalized using the maximum
value of the fluorescence strengths [DNA]n or related value
[DNA]max, and a threshold cycle number Ct is calculated by setting
a threshold Th in an exponential amplification region of an
amplification curve drawn using a normalized fluorescence strength
[DNA]nN.
[0038] According to a fifth aspect of the invention, the
fluorescence strength [DNA]nN is calculated by dividing the
fluorescence strength [DNA]n for each reaction region by a value
obtained by adding a value common to the reaction regions to be
compared to the maximum value or related value [DNA]max.
[0039] According to a sixth aspect of the invention, the
amplification curve is drawn after fluorescence strength [DNA]base
before the exponential amplification region is subtracted from the
fluorescence strength [DNA]n for each reaction region and the
maximum value or related value [DNA]max.
[0040] According to the first aspect of the invention, in the
apparatus for detecting fluorescence strength from a nucleic acid
amplification product in each of a plurality of reaction regions
given with temperature cycles in real time, since a fluorescence
measurement value [DNA]raw obtained from the reaction region and a
fluorescence measurement value [DNA]bg obtained from regions other
than the reaction region adjacent to the reaction region are
detected, and the fluorescence measurement value [DNA]bg is
subtracted from the fluorescence measurement value [DNA]raw to
determine fluorescence strength [DNA]real of the reaction region,
it is possible to obtain the original fluorescence strength
[DNA]real of the DNA product of the reaction region except for the
fluorescence measurement value of a background by errors or
contamination of the reaction region and its circumferences and
way-out light for each reaction region. Accordingly, it is possible
to realize preparation and quantitativeness of a correct
amplification curve. In this case, with no need to use the second
fluorescence signal in the prior art, it is possible to reduce time
taken to acquire and process data without increase of costs and
deterioration of workability.
[0041] According to the second aspect of the invention, in addition
to the first aspect, since the fluorescence measurement value
[DNA]bg is a simple average value of fluorescence measurement
values obtained from the regions other than the plurality of
reaction regions adjacent to the reaction region, or an average
value of fluorescence measurement values after the fluorescence
measurement values are weighted, it is possible to calculate more
correct fluorescence strength of the background to determine the
fluorescence strength [DNA]real with higher precision.
[0042] According to the third aspect of the invention, in addition
to the first aspect or the second aspect, since the fluorescence
measurement value [DNA]bg is detected every detection of the
fluorescence measurement value [DNA]raw, and the fluorescence
measurement value [DNA]bg is subtracted from the fluorescence
measurement value [DNA]raw to determine the fluorescence strength
[DNA]real, although the fluorescence strength of the background is
varied during reaction, it is possible to always obtain the
original fluorescence strength [DNA]real of the DNA product in real
time with high precision.
[0043] According to the fourth aspect of the invention, in the
apparatus for detecting fluorescence strength from a nucleic acid
amplification product in each of a plurality of reaction regions
given with temperature cycles in real time, since fluorescence
strengths [DNA]n obtained from the reaction regions every
temperature cycles n are normalized using the maximum value of the
fluorescence strengths [DNA]n or related value [DNA]max, and a
threshold cycle number Ct is calculated by setting a threshold Th
in an exponential amplification region of an amplification curve
drawn using a normalized fluorescence strength [DNA]nN, it is
possible to correct and reduce unbalance of the fluorescence
strength of each reaction region due to the apparatus error factors
such as optical system errors, correction solution concentration
errors, correction solution divisional pour errors, reaction
solution divisional pour errors, etc., thereby enabling calculation
of threshold cycle numbers Ct with high reliability.
[0044] According to the fifth aspect of the invention, in addition
to the fourth aspect, since the fluorescence strength [DNA]nN is
calculated by dividing the fluorescence strength [DNA]n for each
reaction region by a value obtained by adding a value common to the
reaction regions to be compared to the maximum value or related
value [DNA]max, behavior of data occurring due to factors other
than the apparatus error factors can be easily grasped by
approximating amplification curves from the middle stage and the
late stage of the cycle to actual data while suppressing the
normalization effect and sufficiently securing the correction
effect at the threshold.
[0045] According to the sixth aspect of the invention, in addition
to the fourth aspect or the fifth aspect, since the amplification
curve is drawn after fluorescence strength [DNA]base before the
exponential amplification region is subtracted from the
fluorescence strength [DNA]n for each reaction region and the
maximum value or related value [DNA]max, it is possible to grasp
the situation of the fluorescence strength from the PCR product
itself, excluding the fluorescence strength generated from the
reaction solution itself in the reaction region.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1 is a configurational view of a real time detecting
apparatus according to an embodiment of the invention.
[0047] FIG. 2 is an end side view of a reaction detecting apparatus
constituting the real time detecting apparatus shown in FIG. 1.
[0048] FIG. 3 is a plan sectional view of the reaction detecting
apparatus shown in FIG. 2.
[0049] FIG. 4 is a view showing a fluorescence strength state of a
background of a reaction vessel.
[0050] FIG. 5 is a view showing a fluorescence strength state after
reaction.
[0051] FIG. 6 is a view showing another fluorescence strength state
after reaction.
[0052] FIG. 7 shows a DNA amplification curve of a well.
[0053] FIG. 8 shows DNA amplification curves of all wells.
[0054] FIG. 9 shows a DNA amplification curve when the data of FIG.
8 are normalized.
[0055] FIG. 10 shows a DNA amplification curve when the data of
FIG. 8 are incompletely normalized.
[0056] FIG. 11 is a functional block diagram of a processing
apparatus constituting the real time detecting apparatus shown in
FIG. 1.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0057] Hereinafter embodiments of the present invention will be
described in detail with reference to the drawings.
Embodiment 1
[0058] FIG. 1 is a configurational view of a real time detecting
apparatus R according to an embodiment of the invention, FIG. 2 is
an end side view of a reaction detecting apparatus 1 constituting
the real time detecting apparatus R shown in FIG. 1, and FIG. 3 is
a plan sectional view of the reaction detecting apparatus 1 shown
in FIG. 2. The real time detecting apparatus R of the invention
includes the reaction detecting apparatus 1 and a processing
apparatus C such as a computer for processing detected data from
the reaction detecting apparatus 1 in real time.
[0059] In this embodiment, the reaction detecting apparatus 1 is an
apparatus for proliferating chromosome DNA as reaction samples and
detecting a reaction state related to the proliferation by means of
an optical measurement method. The reaction detecting apparatus 1
includes a body 3 having a reaction chamber 4 formed thereon, and a
reaction detecting part 5 that is disposed on the body 3 in the
rear of the reaction chamber 4. Within the reaction chamber 4 is
provided a reaction block 6 made of thermal conductive material
such as aluminum. The reaction block 6 is provided with a plurality
of support holes 8 for supporting a reaction vessel 7 having a
plurality of wells 7A . . . receiving a reactive solution including
DNA (target template: .lamda.DNA, etc), reagent, a medium solution,
etc.
[0060] The reaction vessel 7 used in this embodiment is a micro
plate In which the wells 7A . . . as 96 (12.times.8) reaction
regions are integrally formed and are connected to respective
connection wells (regions other than the wells 7A (reaction
regions)). The reaction vessel is not limited to a vessel having
the integrally formed wells but may be a vessel having a plurality
line of tubes. The number of wells 7A is not limited to this but
may be, for example, 384 for ease handling. The wells 7A of the
reaction vessel 7 have its opened top side attached with a seal cap
9 for preventing a reaction solution from being evaporated due to
temperature treatment of the reaction solution. In this embodiment,
light transmissive synthetic resin material is used for detection
of fluorescence strength since the detection can be achieved when
light passes through the cap 9.
[0061] Within the body 2 is provided a peltier device 10 for
heating and cooling the reaction block 6. The peltier device 10 is
temperature-controlled by a controller (not shown) and heats and
cools the reaction block 6 cyclically, thereby cultivating
(amplifying) DNA (reaction samples) within the wells 7A of the
reaction vessel 7.
[0062] In this embodiment, a dark chamber component part 12 is
provided in the other end from the reaction detecting part 5
provided on the rear top side of the body 2, that is, over the
front top side of the body 2 in which the reaction chamber 4 is
formed. The front side of the dark chamber component part 12 is
forward opened, and the cover 2 inclined low toward the front is
provided in this forward opening in a feely opened/closed manner.
The cover 2 is moveable backward and forward by a rail member 13
formed from the front side of the dark chamber component part 12,
that is, the top front side of the body 3 to an inner rear side of
the dark chamber component part 12. In a state where the cover 2 is
moved backward, the cover 2 is received in the dark chamber
component part 12.
[0063] Within the cover 2, a pressing member 15 for pressing the
reaction vessel 7 against the reaction block 6 of the reaction
chamber 4 is movably provided to face the reaction block 6, with
the cover 2 blocked. The pressing member 15 Is a plate made of
aluminum having good thermal conductivity and is provided with a
plurality of transparent holes in correspondence to the top of the
wells 7A.
[0064] On the top side of the pressing member 15 is disposed a
Fresnel screen 21 as an optical lens. The Fresnel screen 21
generally has a plurality of grooves which are formed on its plane
and reflect and extend incident light. At this time, the Fresnel
screen 21 has an optical property to collimate the incident light
to be completely or nearly parallelized when the incident light is
reflected and extended, thereby allowing the incident light to be
transmitted along its optical path corrected for distortion.
[0065] In the meantime, a reflecting plate 22 is disposed on a
surface constituting the reaction chamber 4 side of the cover 2
blocking the front top side of the reaction chamber 4 in the top of
the reaction block 6. In this embodiment, the reflecting plate 22
is formed of a flat mirror or the like and serves to reflect light
from a light source lamp 23, which will be described in detail
later, toward the Fresnel screen 21.
[0066] In the meantime, the reaction detecting part 5 contains the
light source lamp 23, a filter unit 35 having a plurality of band
pass filters, a reflecting plate 26, a CCD camera 27 and a filter
driver 28 for rotating the filter unit 35.
[0067] The light source lamp 23 is a lamp for emitting light
including excitation light for exciting fluorescence from a
reaction solution depending on the amount of a DNA product to be
detected in the reaction solution. A halogen lamp is typically used
as the light source lamp 23. The reflecting plate 26 reflects light
having a predetermined wavelength, which is emitted from the light
source lamp 23, at a predetermined angle, and polarizes the
reflected light t the reflecting plate 22. The reflecting plate 26
has a property to transmit predetermined fluorescence. In this
embodiment, when the light from the light source lamp 23 disposed
in the side of the reaction detecting part 5 is forward reflected
by the reflecting plate 26, the reflecting plate 22 is irradiated
with the light from the light source lamp 23.
[0068] The filter unit 35 is a unit configured by arranging various
kinds of band pass filters in the form of a wheel. This filter unit
35 is rotated by the filter driver 28. The band pass filters are
selected and positioned between the light source lamp 23 and the
reflecting plate 26 or between the reflecting plate 22 and the
camera 27. In the figure, a band pass filter 24 is positioned
between the light source lamp 23 and the reflecting plate 26 and a
band pass filter 25 is positioned between the reflecting plate 22
and the camera 27.
[0069] The band pass filter 24 is an optical filer having a
property to pass only light having a wavelength, which is required
to excite fluorescence from the reaction solution, of components of
the light from the light source lamp 23. The light passed the
filter 24 becomes excitation light for exciting the fluorescence
from a specified component of the reaction solution.
[0070] The band pass filter 25 is an optical filter having a
property to pass fluorescence generated from the reaction solution
in the wells 7A of the reaction vessel 7 and a predetermined
fluorescence component from reflected light through the reflecting
plate 22. Here, reflected light components other than the
predetermined fluorescence component are intercepted.
[0071] The camera 27 is a device for detecting the fluorescence
passed the band pass filter 25. A fluorescence image detected by
the camera 27 is inputted to the controller and is sent to the
processing apparatus C for analysis of concentration, i.e., amount
of amplification, of the reaction solution. In addition, these band
pass filers 24 and 25 may be selectively used in any combination
thereof based on the reaction solution to be detected and the kind
of fluorescence pigment used corresponding to the reaction
solution.
[0072] With the above configuration, the controller controls the
peltier device 10 to set the reaction solution in the reaction
vessel 7 supported by the support holes 8 of the reaction block 6
to be, for example, a thermal deformation temperature of
+95.degree. C., and then performs a thermal deformation process to
thermally deform the reaction solution. Subsequently, the
controller controls the peltier device 10 to cool the reaction
block 6 to, for example, +60.degree. C., and then performs an
annealing process and an expansion process for DNA in the
thermally-deformed reaction solution that is received in the
reaction vessel 7. The controller performs cultivation
(amplification) of DNA and the like according a PCR method by
repeating one cycle including the thermal deformation process, the
annealing process and the expansion process several times, for
example, 40 times.
[0073] During or after this cultivation, the reaction detecting
part 5 performs a detection operation regularly, 5 such as after
one cycle, in order to detect an amplification state of DNA of the
reaction solution in the reaction vessel 7. In the detection
operation, first, light emitted from the light source lamp 23
reaches the reflecting plate 26 through the band pass filter 24.
The band pass filter 24 passes only light having a wavelength
required to excite fluorescence, that is, excitation light, of the
light from the light source lamp 23. The reflecting plate 26
reflects the excitation light toward the reflecting plate 22
through the dark chamber component part 12. The reflecting plate 22
again reflects the excitation light toward the Fresnel screen 21
provided in the reaction block 6, that is, from the top to the
bottom.
[0074] The excitation light Impacting on the Fresnel screen 21 is
condensed by the lens 21 and is changed In its incident angle to an
angle which is parallel or nearly parallel to the wells 7A of the
reaction vessel 7 received in the reaction block 6. Accordingly,
the excitation light passed the lens 21 is incident into the wells
7A with an incident angle which is parallel or nearly parallel
through the transparent holes formed in the pressing member 15.
[0075] When the DNA in the well 7A beforehand added with
predetermined fluorescence pigment is irradiated with the
excitation light incident at the parallel or nearly parallel angle
in the wells 7A, fluorescence is generated depending on the amount
of PCR product. The generated fluorescence and other reflected
light from the PCR product reach the reflecting plate 22 through
the transparent holes formed in the pressing member 15 formed in
the pressing member 15 and the Fresnel screen 21 as well.
[0076] Thereafter, the fluorescence and the other reflected light
that reached the reflecting plate 22 form an optical path in a
substantial horizontal direction within the dark chamber component
part 12 by the action of the reflecting plate 22 and reaches the
camera 27 through the band pass filter 25 facing the reflecting
plate 22. In this case, since the cover 2 is blocked within the
dark chamber component part 12, a dark chamber Is formed, thereby
avoiding attenuation of fluorescence.
[0077] At this time, since the reflecting plate 26 is made of
fluorescence transmissive material, the reflected light and the
fluorescence that passes the reflecting plate 26 reaches the band
pass filter 25. Only specified fluorescence can be passed through
the band pass filter 25 depending on the kind of the filter 25, as
described above, only the specified fluorescence can reach the
camera 27 disposed in the rear of the filter 25.
[0078] When the camera 27 take an image of the received
fluorescence, a fluorescence state of the PCR products In the wells
7A of the reaction vessel 7 is detected. Detection data (image data
shown in FIGS. 4 to 6) on the detected fluorescence state of the
PCR products are sent from the controller to the processing
apparatus C. The processing apparatus C analyzes the detection data
to detect the concentration of the samples, that is, the amount of
amplification of DNA and the like. Since the positional relation
between the positions of the wells 7A and the image is known in
advance, by obtaining the brightness of pixels of the image of the
wells 7A, fluorescence strength of the wells 7A can be measured and
the amount of PCR products can be detected from the measured
fluorescence strength.
[0079] The processing apparatus C includes an arithmetic processing
part (CPU) 31 for performing the detection data sent from the
reaction detecting apparatus 1, a storage device (storing means) 32
connected to the arithmetic processing part 31, a keyboard (or
input means such as a mouse) 33, a display (output means) 34, a
printer (output means) 36, an external storing device 37 such as
FD, CD, DVD, memory or the like, etc., as shown in FIG. 11. The
detection data sent from the reaction detection apparatus 1 is
stored in the storing device 32 and is displayed on the display 34
and so on after being subjected to the following process by the
arithmetic processing part 31.
[0080] Next, a process sequence of the detection data in the
processing apparatus C will be described. FIGS. 5 and 6 show images
of the detection data sent from the reaction detecting apparatus 1
to the processing apparatus C. 96 annular images appearing to be
white are fluorescence generated from the PCR products in the wells
7A. In this embodiment, with SYBR Premix Ex Taq (registered
trademark) as a base, a reaction solution is adjusted from PCR
Forward Primer, PCR reverse Primer, Template (KDNA given as an
initial value) and dH.sub.2O. In FIGS. 5 and 6, a reaction solution
having the concentration of 0.2 pg/.mu.L is divisionally poured in
48 upper half wells 7A (X group) and a reaction solution having the
concentration of 0.4 pg/.mu.L is divisionally poured in 48 lower
half wells 7A (Y group). The temperature cycle number is 40.
[0081] When the temperature cycle number progresses from reaction
initiation, the fluorescence strength increases according to the
amount of amplification of DNA. Plotting this procedure is the
amplification curve of FIG. 7 as described above. This
amplification curve is obtained for each well 7A and is displayed
on the display 34 (FIG. 8). When the keyboard (or mouse) 33 is used
to set a threshold Th ((11) in FIG. 7) in an exponential
amplification region ((5) in FIG. 7), cycle number Ct at that point
(threshold cycle number Ct) is read to 24.5 in this figure. Since
there is a correlation between the threshold cycle number Ct and
the initial amount of DNA of a test sample, a calibration curve
representing this linear relation can be prepared. The arithmetic
processing part 31 estimates the initial amount of DNA of the test
sample based on this calibration. This enables correct
quantitativeness based on a PCR amplification speed theory.
[0082] (A) Removal of Apparatus Error Factors
[0083] As described above, before the reaction initiation, the
brightness of pixels in the image data obtained by the camera 27 of
the reaction detecting apparatus 1 is not necessarily constant and
there occurs a spot due to contamination of the optical system or
way-out light as indicated by (1) in FIG. 4. When the PCR reaction
is progressed, this spot overlaps with the images of the wells 7A
as shown in FIG. 5, wastefully adding to the fluorescence strength
of the wells 7A.
[0084] So, the arithmetic processing part 31 of the processing
apparatus C performs the following process in order to obtain the
original fluorescence strength of the wells. That is, for example,
in case of a well 7A lying in a stage (B11) second from the right
side and second from the top side in FIG. 6, a fluorescence
measurement value [DNA]rawB11 of the well 7A ((4) in FIG. 6) of the
B11 stage is measured and stored in the storing device 32. Next, 4
adjacent fluorescence measurement values [DNA]bg1, [DNA]bg2,
[DNA]bg3 and [DNA]bg4 in a connection wall portion near the well 7A
of the B11 stage are measured and an average value of the 4
fluorescence measurement values (a fluorescence measurement value
of a background) is obtained and stored in the storing device 32.
Then, according to the following Equation (2), by subtracting the
average value from the fluorescence measurement value [DNA]rawB11,
the original fluorescence strength [DNA]realB1 of the well 7A of
the B11 stage is calculated.
[DNA]realB11=[DNA]rawB11-(([DNA]bg1+[DNA]bg2+[DNA]bg3+[DNA]bg4)/4)
. . . (2)
[0085] This process is performed for all 96 wells every detection
of the fluorescence measurement value [DNA]raw to determine the
original fluorescence strength [DNA]real of all wells 7A. Thus, it
is possible to obtain the original fluorescence strength [DNA]real
of the DNA product of the well 7A except for the fluorescence
measurement value [DNA]bg of the background by errors or
contamination of the well 7A and its circumferences and way-out
light for each well 7A (reaction region).
[0086] Accordingly, it is possible to realize preparation and
quantitativeness of a correct amplification curve. In this case,
with no need to use the second fluorescence signal in the prior
art, since an arithmetic process may have only to be performed in
the processing apparatus C, it is possible to reduce time taken to
acquire and process data without increase of costs and
deterioration of workability.
[0087] In this embodiment, although the 4 adjacent fluorescence
measurement values [DNA]bg1, [DNA]bg2, [DNA]bg3 and [DNA]bg4 near
the well 7A are measured and their average value is subtracted from
the fluorescence measurement value [DNA]raw, the present invention
is not limited to this but the average value may be one point value
or an average value of two or three points. However, when the 4
point average value is used as in this embodiment, it is possible
to calculate more correct fluorescence strength of the background
to determine the fluorescence strength [DNA]real with higher
precision. In addition, although a simple average of the 4 point
fluorescence measurement values near the well 7A is used in this
embodiment, the present invention Is not limited to this, but
points may be weighted and averaged in consideration of spots of
contamination conditions.
[0088] Variation (drift) of the fluorescence strength of the
background before and after reaction is not clear. However, in this
embodiment, since the fluorescence measurement values [DNA]bg1 to
[DNA]bg4 are detected every detection of the fluorescence
measurement value [DNA]raw and the average value of the
fluorescence measurement values [DNA]bg1 to [DNA]bg4 is subtracted
from the fluorescence measurement value [DNA]raw to determine the
fluorescence strength [DNA]real, although the fluorescence strength
of the background is varied during reaction, it is possible to
always obtain the original fluorescence strength [DNA]real of the
DNA product in real time with high precision.
[0089] (B) Normalization 1
[0090] Next, FIG. 8 shows DNA amplification curves of all 96 wells
7A. In the figure, a horizontal axis represents temperature cycle
number and a vertical axis represents fluorescence strength. As
described above, since the reaction solutions having different
concentrations divided into the upper half X group and the lower
half Y group in FIGS. 5 and 6 are divisionally poured, a reaction
curve have to appear as two lines originally. In addition, the
threshold cycle number Ct will also be two. However, due to
apparatus error factors such as the above-mentioned optical system
errors, correction solution concentration errors, correction
solution divisional pour errors, reaction solution divisional pour
errors, etc., the amplification curve of each well 7A may be
unbalanced and a plurality of threshold cycle numbers Ct may occurs
as indicated by (8) and (9) in FIG. 8 (unbalance).
[0091] So, the arithmetic processing part 31 normalizes data based
on a predetermined selection command from the keyboard (or mouse)
33. That is, the arithmetic processing part 31 obtains fluorescence
strength [DNA]nN normalized by the following Equation (3) using the
fluorescence strength [DNA]n (the original fluorescence strength
[DNA]real at an n-th temperature cycle number) determined every
temperature cycle numbers n and stored in the storing device 32,
the fluorescence strength maximum value [DNA]max (the maximum of
fluorescence strengths [DNA]real up to the temperature cycle
numbers n, this is stored in the storing device 32) for each well
7A, and a value Z common to wells 7A (all wells 7A in this
embodiment) to be compared.
[DNA]nN=[DNA]n/([DNA]max+Z) . . . (3)
[0092] By this process, the fluorescence strength of each well 7A
is normalized. FIG. 9 shows a case of Z=0. Since unbalance of the
fluorescence strength due to the apparatus error factors of each
well 7A is corrected and reduced by this normalization, unbalance
of the threshold cycle numbers Ct in the X group and the Y group
can be further reduced as compared to FIG. 8. This enables
calculation of threshold cycle numbers with high reliability. In
addition, FIG. 9 shows multiplication of [DNA]nN by a value common
to all wells in order to set the whole scale to be a proper
value.
[0093] (C) Normalization 2
[0094] Here, the fluorescence strength in a plateau region in the
late of cycle may be unbalanced due to unbalance of chemical
reaction in addition to the above-mentioned apparatus error
factors. It is believed that such a chemical reaction factor has no
proportional effect on the fluorescence strength in an exponential
amplification region. That is, In some cases, fluorescence
strengths in regions having the plateau effect had better not to be
completely matched each other.
[0095] In this case, the keyboard (or mouse) 33 is used to increase
the Z value of the above Equation (3). FIG. 10 shows amplification
curves for wells 7A in case of Z=20000. Increase of the Z value
means weakening of the normalization effect. However, it can be
seen from FIG. 10 that behavior of the amplification curves from
the middle stage to the late state of the cycle approximates to
behavior of actual fluorescence strength (see FIG. 8). In the
meantime, it can be seen from this figure that the correction
effect at the threshold is sufficiently secured. Accordingly, by
approximating the amplification curves from the middle stage and
the late stage of the cycle to actual data while suppressing the
normalization effect and sufficiently securing the correction
effect at the threshold, behavior of data occurring due to factors
other than the apparatus error factors can be easily grasped.
[0096] Here, when such incomplete normalization is made, the Z
value may be determined according to the following Equation
(4).
Z=.alpha.[DNA]max+.beta. . . . (4)
[0097] Where, .alpha. and .beta. are coefficients common to the
wells 7A.
[0098] In addition, the arithmetic processing part 31 calculates
the above-described maximum value [DNA]max as a moving average of
fluorescence strengths. This is because the fluorescence strength
[DNA]real shows a saw shape actually. However, the maximum value
used for the normalization may be a peak value of the saw shape or,
for example, 90% of the peak value (either being related to the
maximum value).
[0099] (D) Correction of Baseline
[0100] Here, although (10) in FIG. 8 is a region of a level at
which a reaction result can not be detected at the initial stage of
cycle, since the reaction solution itself in the wells 7A initially
generates predetermined fluorescence, the fluorescence strength is
not generally zero in this region. So, the arithmetic processing
part 31 corrects a baseline based on an instruction from the
keyboard (or mouse) 33. That is, the arithmetic processing part 31
sets an average value [DNA]base of the fluorescence strengths of
the well 7A in the region 10 at the initial stage of cycle to be a
baseline and performs the above-described normalization process
after subtracting the average value from the above-mentioned [DNA]n
and [DNA]max. The reason for this process is that the initial
fluorescence strength is set to be zero in FIGS. 9 and 10
([DNA]base may use the minimum value of [DNA]n).
[0101] Accordingly, it is possible to grasp the situation of the
fluorescence strength from the PCR product itself, excluding the
fluorescence strength generated from the reaction solution itself
in the wells 7A.
[0102] It should be understood that material, amount and number
shown in this embodiment are not particularly limited.
* * * * *