JPH0474950A - Emission detecting type detector of genetic material - Google Patents

Abstract

PURPOSE:To obtain a means of measuring the quantity and the position of genetic material such as DNA fixed on a membrane, quantitatively and automatically with chemical emission used, by providing a planar holding means for holding the membrane and a detecting means of converting the quantity of emission from the membrane into the quantity of electricity and detecting it. CONSTITUTION:A recessed place 3 formed substantially in the same shape with that of a membrane 2 is provided in the upper surface of a membrane holding table 1, and the depth of the recessed place 2 is so set that the upper surface of the holding table 1 can be positioned above the upper surface of the membrane 2 when the membrane 2 is fitted in the recessed place. On the other hand, a device converting the quantity of light into the quantity of electricity is positioned above the holding table 1. This device comprises a condensing optical system, a photoelectric conversion system and a moving device thereof, when sorted roughly, and a moving device thereof, when sorted roughly, and the condensing optical system is an ordinary condensing system and has a plurality of sets of lenses and reflectors. A condensed light is transferred to the photoelectric conversion system having a photomultiplier tube and is converted into the quantity of electricity. Both of this light detecting element and the holding table 1 are moved in the direction perpendicular to the surface of this paper by a driving device.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to a device for detecting genetic material such as DNA or RNA by chemiluminescence or bioluminescence, and is applicable in the field of molecular biology or genetic diagnosis. It is something that will be done.

[Conventional technology]

A conventional method using chemiluminescence as a means of detecting genetic material is described in Clinical Chemistry Vol. 35, No. 9 (1989), pp. 1856-1857 (CIin, Chem,
35 (9°pp, 1856-1857 (1989
))It is described in. In this report, DNA derived from herpes simplex virus was immobilized in spots on a membrane, and an oligonucleotide probe for the virus labeled with alkaline phosphatase was hybridized to this.
In addition, 3-(2'-spiroadamantane), which is a chemiluminescent substrate for alkaline phosphatase,
-4-methroxy -4-(3”-phosp
horyloxy) phenyl-1,2-dioxe
React the tane. At this time, chemiluminescence is generated from the dissociation product resulting from dephosphorylation of the substrate. This luminescence intensity is originally proportional to the amount of DNA immobilized on the membrane. In the above report, the herpes simplex virus with up to 1.4 XIO' copies was detected by transferring this luminescence to a photosensitive film such as an instant photosensitive film.

In addition, for DNA detection, DNA digested with restriction enzymes is separated by molecular weight by agarose gel electrophoresis, then immobilized on a membrane such as nitrocellulose, and hybridized with a labeled probe. There is a Southern method for detecting DNA fragments (Lab Manual Genetic Engineering (Maruzen, 1988), pp. 67-69).

The conventional technique of transferring onto a photosensitive film described above can also be applied to this detection method.

[Problem to be solved by the invention]

In the above-mentioned conventional technology, in order to measure the amount of target DNA in a sample, the brightness of a spot on a photosensitive film must be determined and quantified by a person, which has a problem with the accuracy.

The dynamic range of measurements was also limited. Furthermore, no consideration was given to the automation of measurement.

Furthermore, when the above-mentioned conventional technology is applied to the Southern method, it is necessary to manually measure the migration distance of each DNA fragment arranged in a ladder shape on a photosensitive film, which is time-consuming and also poses problems in measurement accuracy. Ta.

In addition, in the above conventional technology, the luminescent reagent tends to diffuse to the surroundings during measurement, and the luminescent reagent adheres to the film, which prevents unnecessary signals from appearing in the luminescent pattern transferred to the film. Therefore, it was necessary to wrap the membrane tightly in something like a transparent vinyl chloride film before taking measurements. Furthermore, this method has problems in that it is not suitable for automation, such as the inability to support the membrane in a flat plane and the inability to set the membrane in the correct position with good reproducibility.

Furthermore, the prior art does not consider adding a luminescent reagent after setting the membrane in the device.

The present invention aims to solve the above-mentioned problems and provide a means for quantitatively and automatically measuring the amount or position of genetic material such as DNA or RNA fixed on a membrane using chemiluminescence. There is.

[Means to solve the problem]

In order to achieve the above object, in the present invention, DNA
Alternatively, in a genetic material detection device that detects RNA by chemical or bioluminescence, there is a planar holding means for holding a membrane on which DNA or RNA is immobilized in a spot or ladder shape, and the amount of light emitted from the membrane is converted into an amount of electricity. and a detection means for detecting the detection result.

As a means for converting the amount of luminescence into an amount of electricity and detecting it, it is possible to use a means consisting of a condensing optical system and a photomultiplier tube.
This is a preferable form from the viewpoint of measurement accuracy.

Furthermore, by further providing a mechanism for moving each of the holding means and the detecting means two-dimensionally, or by further providing a mechanism for moving both of them in a right angle direction, various It becomes easy to measure the fixed image of the pattern.

Furthermore, so that only the amount of light of the desired optical image can be detected,
In the optical system until the light emitted from the membrane is converted into an electric quantity, there is a means to prevent light outside the target plate from passing through, such as a shape corresponding to the shape fixed on the membrane of DNA or RNA. A shaped slit can also be inserted.

In the present invention, the same function can also be achieved by making the detection means itself capable of two-dimensional image measurement instead of making the membrane support stand and/or the detection means movable. .

Furthermore, by using a holding means that has a recess that has almost the same shape as the membrane to be measured and whose recess depth is greater than the thickness of the membrane, it is possible to hold the membrane to which the luminescent reagent has been added. This allows for setting in the correct position and addition of luminescent reagent after setting.

[For production]

The membrane holding means can support the membrane on which DNA or RNA is immobilized in a flat shape, so it is possible to support the membrane on which DNA or RNA is immobilized.
Alternatively, the RNA pattern will not be distorted, and the positional relationship of the optical system up to the detection means that converts the amount of light emitted from the membrane into an amount of electricity and detects it will not shift. Therefore, it is possible to realize quantitative and highly accurate measurement of luminescence intensity and luminescence pattern.

By using a detector such as a photomultiplier tube that measures luminescence photoelectrically as a detection means that converts the amount of light emitted from the membrane into an amount of electricity, it is possible to obtain a photoelectric pulse proportional to the amount of light emitted. By measuring it using direct current or pulse counting, quantitative luminescence measurement with a wide dynamic range can be realized.

DNA or RNA is two-dimensionally spread out on the membrane as a number of spots or a ladder-like pattern. Therefore, to measure these patterns, it is necessary to scan the membrane two-dimensionally. This can be realized by providing a mechanism that two-dimensionally moves either the membrane holding stand side or the detection means side that converts the amount of light emission into an amount of electricity and detects it. Further, this can also be realized by providing a mechanism for moving the membrane holding stand and the detection means for converting and detecting the amount of light emission into an amount of electricity in the directions of axes that intersect perpendicularly to each other.

From a membrane to which a luminescent reagent has been added, chemiluminescence is generated to a greater or lesser extent from locations other than those where DNA or RNA to which a label that catalyzes chemiluminescence is attached is present.

Therefore, in order to detect the light emitted from the sample portion with high sensitivity and accuracy, a means for transmitting only the light emitted from the sample portion is required. This involves forming an image of the light emission pattern in a condensing optical system that guides the light to a detection means that converts the light emission into an amount of electricity and detecting it, and then inserting a slit that matches the shape of the light emission on the imaging surface. This can be achieved by Alternatively, this can be realized by providing a slit having the same shape as the light emitting pattern as close as possible to the membrane.

On the other hand, the light emission pattern is spot-like DNA or R
When the NA is fixed, it is circular, and when it is fixed in a ladder shape using the Southern method, each one is a long and narrow rectangle. Therefore, by preparing circular and rectangular slits corresponding to each of the above cases and providing a mechanism that allows them to be freely exchanged, it becomes possible to measure according to each pattern.

A two-dimensional pattern of DNA or RNA developed on a membrane can also be realized by using a detection means that converts the amount of light emission into an amount of electricity and is capable of measuring two-dimensional images.

By making the membrane holding means have a recess having almost the same shape as the membrane to be measured, and the depth of the recess being greater than the thickness of the membrane, it is possible to hold the membrane on which DNA or RNA is immobilized. It is possible to set the membrane in the correct position with good reproducibility, and the luminescent reagent on the membrane does not diffuse to the surrounding area, and it is also possible to add the luminescent reagent so as to cover the membrane surface after setting the membrane in the holding means. .

〔Example〕

An embodiment of the present invention will be described below with reference to FIG. As sample DNA, λ phage was digested with the restriction enzyme Hind■. Furthermore, a nitrocellulose membrane was used as the membrane.

First, a description will be given of the dot plot method, that is, the procedure for fixing a DNA sample on a membrane in the form of a spot. 5
A sample solution of approximately 20 μm containing sample DNA from 00 pg to Hlg was placed in a microtiter-like 8 rows x 12 rows of 9
Inject into a dot plot device with 6 sample injection slots. By suctioning through the nitrocellulose membrane with an aspirator over a period of about 10 minutes, the DNA in the sample solution adheres to the nitrocellulose membrane.

The nitrocellulose membrane was previously immersed in water and then immersed in toxssc buffer (sodium citrate 0.15M, NaC 11.5M) (IXssc: sodium citrate 0.015M).
, NaC10,15M, the same hereinafter).

The nitrocellulose membrane removed from the dot plot device is subjected to the following immobilization treatment. First, 0.5M N
aOH, 1.5M NaCl aqueous solution soaked paper, then 0.5M Tris-HCI (pH 7
, 5), Neutralize on a paper soaked with 1.5M NaCl aqueous solution.

Further wash with 2XSSC buffer. Finally, 80
Dry heat treatment is performed at ℃ for about 2 hours.

Next, a description will be given of the Southern blot method, that is, the procedure for fixing a DNA sample onto a membrane in a ladder shape. First, DNA samples from 5001)g to 1100p are electrophoresed in a 1% agarose gel to create a ladder pattern in which DNA fragments are arranged in order of molecular weight in the gel. Electrophoresis was carried out on a 20 cm gel at a voltage of 60 V at both ends for about 4 hours. After electrophoresis, the gel was shaken in a 250mM HCI aqueous solution, and
Denature in 1,0,5M NaOH solution. Wash with distilled water followed by 0.5 M Tris-HCI.

Neutralize with 1.5M HCl solution (pH 7.5).

Transfer of DNA from the gel to the nitrocellulose membrane is performed as follows. Place the gel on a triple layer of paper with both ends soaked in 20XSSC buffer. Place a nitrocellulose membrane of the same size as the gel on top of the gel. In this case as well, the nitrocellulose membrane was previously immersed in water and then immersed in 10XSSC buffer.

Furthermore, three pieces of slip paper of the same size as the gel are soaked in 20×SSC buffer and placed on top of the nitrocellulose membrane. Paper towels approximately 5 cm thick were stacked on top of this, and a glass plate and weight were placed on top of this, and the mixture was left overnight. Thereafter, the membrane with the gel attached is placed on the oral paper, and the gel is peeled off. After shaking in 6xSSC buffer,
Air-dry on paper and heat dry at 80°C for 2 hours.

Hereinafter, the procedure leading to the generation of chemiluminescence is the same for the above two methods. Amersh as a chemiluminescence reagent kit
The ECL gene detection system manufactured by am was used.

First, HRP (Horseradish peroxy)
dase complex) is prepared. DNA solution as a probe (concentration 0.2μ
720 μI) in boiling water for 5 minutes. Next, this is rapidly cooled in an ice water bath and briefly centrifuged. This is followed by an equal amount of D
Add NA labeling reagent and glutaraldehyde solution and mix.

After centrifugation, a final incubation is performed at 37°C for 10 minutes.

Each DNA-immobilized nitrocellulose membrane was incubated at 910°C at 42°C in hybridization buffer y-(0.25ml/car) supplemented with NaCl.
After shaking for a minute, the labeled probe prepared in the above procedure (final concentration 20 ng/ml) is added, mixed, and a hybridization reaction is carried out overnight while shaking. Then, the primary wash buffer (6M urea, 0.4%
Two 20 min washes in SDS, 20X SSC) and a second wash buffer (20X 5SC) at room temperature.
Perform two 5 minute washes.

The nitrocellulose membrane prepared by the above procedure was sprinkled with an appropriate amount (0,125 ml/car) of a mixture of detection reagents 1 and 2 in the Amersham ECL gene detection system, and after incubation for 1 minute, the present invention is set on the membrane holder of the detection device.

FIG. 1 shows, in partial cross-section, the main parts of the luminescent detection type genetic material detection device according to the present invention. In the figure, the membrane holder 1 has a substantially rectangular shape as a whole, and its upper surface is a horizontal surface.

A recess 3 having almost the same shape as the membrane 2 is provided on its upper surface. The depth is such that it can be located above the surface.

The holding stand 1 has a bearing 5 attached to its back surface, and is movably attached via the bearing 5 to a horizontal first support rod 4 fixed to a machine frame (not shown). Further, a screw shaft 6 is provided parallel to the first support rod 4, and the screw shaft 6 is connected to a motor 8 fixed to the machine frame via an appropriate joint 7, and is driven by the motor 8. Ru.

The holding table 1 and the screw shaft 6 are linked by a ball screw bearing 9 fixed to the back surface of the holding table 1, and the screw shaft 6
The support stand l moves in the left-right direction in the figure depending on the direction of rotation.

A device (photodetector) 20 for converting the amount of light into an amount of electricity is located above the holding table 1. This device can be broadly divided into
It consists of a condensing optical system, a photoelectric conversion system, and a moving device for the condensing optical system. The collected light is transferred to a photoelectric conversion system having a photomultiplier tube 23 and converted into an amount of electricity.

This photodetector 20 is also configured to move in a direction perpendicular to the plane of the paper in the drawing by a drive device similar to that of the holding table 1.

That is, the photodetector 20 is connected to a second support rod 25 fixed to the machine frame in a state extending perpendicularly to the first support rod 4 via a bearing 24 installed therein. It is movably engaged with.

Furthermore, a screw shaft 27 connected to a motor 26 fixed to the machine frame is provided parallel to the second support rod 25, and the screw shaft 27 engages with a ball screw 28 on the photodetector 20. There is. Therefore, similarly to the support base 1, the photodetector 20 also moves in the direction perpendicular to the plane of the paper in the figure according to the rotating direction of the screw shaft 27 due to the rotation of the motor 26. The directions of movement are perpendicular to each other.

In this example, the condensing optical system is configured so that there is a surface on which the emission image on the membrane is imaged at an image magnification xl, and the fixed DNA pattern is located at this imaging position. A slit shaped according to the shape, that is, a circular slit 31
and a disc-shaped shutter plate 30 with a rectangular slit 32
is provided.

The shutter plate 30 is connected to a motor 34 via a gear box 33, and as the motor 34 rotates, one of the shutter plates 31 and 32 is positioned at the image forming position.

Note that the holding table l and the photodetector 20 can be moved continuously along their respective axes, or can be moved sequentially to preset points. These movements are controlled by a controller 43.

Chemiluminescence emitted from the portion of the membrane where the DNA is immobilized is focused onto the photomultiplier tube 23 by a focusing optical system consisting of a lens 219, a reflecting mirror 22, and a lens 21. The current output of the photomultiplier tube is subjected to current-to-voltage conversion by an amplifier 40, A-D conversion by an A/D converter 41, and then integrated processing by a data processing unit 42, and the peak intensity is calculated by subtraction with background light, etc. Required data processing such as quantification, smoothing processing, calculation of peak position, etc.

Note that the condensing optical system, photomultiplier tube, amplifier, data processing section, and controller used here are those that have been used individually in the past. Since the method used is a known data processing method, no particular explanation will be given here.

In this embodiment, the shape of the slit in the shutter plate 30 is a circular slit 31 in the case of the dot plot method, and a rectangular slit 32 in the case of the Southern plot method. The circular or rectangular dimensions are the size of the DNA on the membrane.
Alternatively, it is determined as appropriate depending on the size of the fixed shape of RNA. Furthermore, the shape of the slit itself can be freely selected in consideration of experimental conditions, sensitivity, electrophoretic separation ability, etc. In this case, it is also possible to prepare a plate having slits of more shapes and/or sizes at the same time and use them selectively.

Since the injection slot diameter of the dot plot device used in this example was 3 mm, a circle 31 with a diameter of 3 mm was used as the slit shape. In addition, the slot for injecting DNA samples for agarose gel electrophoresis used in Southern blotting is 5 m wide.
m, a rectangle 32 of 5 mm x 1 mm was used as the slit shape.

As mentioned above, in this embodiment, the holding table l can be moved in the left-right direction in FIG. Similarly, the photodetector 20 also
It is configured to be able to move in a direction perpendicular to the plane of the paper in FIG. 1 by a similar l-axis moving mechanism. Therefore, the pattern of the DNA sample developed on the membrane can be detected two-dimensionally by moving the membrane holder l and the photodetector 20 in directions perpendicular to each other.

Furthermore, it is preferable to install the membrane holding stand and the photodetector in this example in a dark box (not shown) in order to improve measurement precision.

FIG. 2 shows the results measured using the apparatus of the present invention using the dot plot method and the Southern plot method to measure the luminescence pattern from DNA derived from λ phage fixed.

Figure 2 (a) shows the results of the dot plot method, in which 500 pg was placed in the second slot in the 12th column direction of a microplate-like 8 rows x 12 rows of sample slots, and too much was added to the fourth slot.
pg, 50.10 each from 6th to 12th.
5゜4, 3. 2. 1 pg of DNA was injected, fixed, and measured by moving the detector coaxially. The whole was divided into 210 points, and the output of the photodetector was integrated for 3.5 seconds at each point.

Down to 1 pg of DNA could be clearly detected. Results were obtained in which the intensity of each peak was approximately proportional to the injected DNA sample.

In this case, it is not necessarily necessary to sequentially scan each column corresponding to the sample slot as shown in Figure 2 (a), but the detector can be sequentially moved to each fixed point on the DNA sample to measure the luminescence intensity. If measured, the value will be proportional to the amount of DNA in the sample and can be quantified. At that time, we measured the intensity of background light around each point,
If compared with that value, the accuracy of quantification will be even higher.

FIG. 2(b) shows the results of measuring the molecular weight separation pattern by agarose gel electrophoresis of a DNA fragment obtained by cleaving the loopg λ phage DNA with the restriction enzyme HindIII using the Southern blot method. The measurement was performed by moving the detection means that converts the amount of luminescence into the amount of electricity and detects it in the electrophoresis direction, and six types of disconnections (fragment length 23.1 kb, 9.4
kb, 6.5kb, 4.4kb, 2.3kb,
2.1 kb) could be clearly detected. Each peak position can be determined by data processing such as smoothing and differential processing. This makes it possible to determine the molecular weight of unknown DNA or to identify it with a reference sample based on the above-mentioned peak positions of λDNA cut with HindI [having a known molecular weight.

In this example, the noise caused by thermionic electrons from the photomultiplier tube was about IO times the background light emission from the membrane surface, that is, the light emission from the part where the DNA sample was not present.

This noise can be reduced by about one order of magnitude by cooling the photomultiplier tube from 0°C to about -20°C or by removing low-current noise photocurrent pulses by employing a photon counting method.
It is possible to make the signal intensity comparable to the background light emission from the membrane. This makes it possible to further improve detection sensitivity.

Another embodiment of the present invention will be described with reference to FIG.

In this embodiment, the two-dimensional luminescence pattern of DNA fixed on the membrane 2 set on the membrane holding table l is measured by a detection means that converts the two-dimensional luminescence amount into an electrical quantity and detects it. In this detection means, light on the membrane is transmitted through an imaging lens 50 and an image intensifier 5.
1. CCD camera 5 via optical fiber coupling element 52
3 as a two-dimensional light emission pattern. The detected two-dimensional light emission pattern is transferred to the data processing unit 55 via the frame memory 54, where the light emission spot position is determined, the light emission intensity is quantified, and the like.

According to this embodiment, there is an advantage that a membrane holding stand or a moving mechanism for a detection means that converts the amount of light emission into an amount of electricity and detects it is not necessary. Furthermore, since it can be measured as a two-dimensional image, it also has the effect of being able to deal with irregularly shaped light emission patterns.

In the present invention, the means for moving the holding table or the photodetector may be any means as long as the entire surface of the membrane on the holding table can be scanned by the photodetector by movement of both, It is not limited to what is shown as an example. Furthermore, it is clear that any means can be used as the driving source.

Furthermore, the means provided in the optical system until the light emitted from the membrane is converted into an electric quantity to prevent light from passing through the front plate to be measured is not limited to a rotating shutter plate type. It is also easy to understand that the mounting position may be any position from the light emitting position to the photomultiplier tube as long as it can block light other than the light emitted image on the membrane.

〔Effect of the invention〕

According to the present invention, a membrane on which genetic material such as DNA or RNA is immobilized is held in a flat shape, and the luminescence emitted from the immobilized site is detected photoelectrically, so that the substance can be quantified with high precision and in a wide dynamic range. This can be done in the microwave.

Furthermore, since two-dimensional measurement of the light emission pattern on the membrane becomes possible, automation of measurement and data analysis according to the dot plot method or the Southern plot method can be realized.

Furthermore, the membrane can be set in the correct position,
Alternatively, since a luminescence reagent can be added after setting, highly accurate luminescence measurement can be achieved.

[Brief explanation of the drawing]

FIG. 1 is a block diagram of one embodiment of the present invention, FIG. 2 is an example of measurement data obtained by the apparatus shown in FIG. 1, and FIG. 3 is a block diagram of another embodiment. l... Membrane holding stand, 2... Membrane, 3.
... recess, 4... first support rod, 6.27... screw shaft, 8.26.34... motor, 20... light detection unit, 21... lens, 22... ...Reflector, 23...Photomultiplier tube, 25...Second support rod, 30...Shutter plate, 31.32...Slit, 40...Amplifier, 41...A /D exchanger, 42... data processing section, 43... controller, 50... imaging lens, 5
1... Image intensifier, 52... Optical fiber coupling element, 53... CCD camera, 54...
Claim memory, 55...Data processing section Fig. 1

Claims (1)

[Claims] 1. In an apparatus for detecting DNA or RNA by chemical or bioluminescence, a planar holding means for holding a membrane on which DNA or RNA is immobilized in a spot shape or a ladder shape, and luminescence from the membrane. 1. A genetic material detection device comprising a detection means for converting a quantity into an electrical quantity and detecting the same. 2. The detection device according to claim 1, wherein the detection means includes a condensing optical system and a photomultiplier tube. 3. The detection device according to claim 1 or 2, further comprising a mechanism for two-dimensionally moving the holding means. 4. The detection device according to claim 1 or 2, further comprising a mechanism for two-dimensionally moving the detection means. 5. The detection device according to claim 1 or 2, further comprising a mechanism for moving both the holding means and the detection device in a perpendicular direction, respectively. 6. Claim 1 or 2, characterized in that the optical system until the light emitted from the membrane is converted into an electric quantity is provided with means for blocking light other than the light to be measured. 5. The detection device according to 5. 7. The means for blocking light other than the light to be measured is a slit having a shape corresponding to the shape fixed on the DNA or RNA membrane.
The detection device according to claim 6. 8. The detection device according to claim 1, wherein the detection means is capable of measuring two-dimensional images. 9. Claim 9, wherein the holding means has a recess having substantially the same shape as the membrane to be measured, and the recess has a depth greater than the thickness of the membrane. 9. The detection device according to items 1 to 8.