JP4017604B2 - Ultrasonic automatic reaction equipment - Google Patents

Description

  The present invention relates to a reaction apparatus that amplifies and detects abnormal proteins and the like by ultrasonic waves, and particularly relates to a reaction apparatus that automatically performs a reaction detection operation.

The PMCA method has been devised as a technique for increasing detection sensitivity by amplifying a protein when detecting an abnormal prion protein or the like in a test tube. In this PMCA method, the detection sensitivity is increased by repeating the process of disrupting the aggregates of abnormal proteins by ultrasonic treatment and the conversion between them by culture.
As a conventional ultrasonic reaction apparatus used for this, there was one described in Patent Document 1. This is a crossed ultrasonic crusher, which crushes a sample in a sealed tube arranged in a processing tank with ultrasonic waves oscillated from an ultrasonic vibrator provided outside the processing tank.
Utility model registration No. 3092396

However, in these operations, if the sample is irradiated with ultrasonic waves continuously for a long time, the sample is destroyed. Therefore, after irradiation for a certain period of time, the irradiation must be stopped for a certain period of time. Therefore, in the conventional apparatus as described above, a researcher has to manually turn the switch on and off by operating the ultrasonic generator while measuring time.
Usually, a reaction work requires 24 hours or more, so two or three researchers were working under unfavorable conditions of working overnight. For this reason, not only is the researcher's mental and physical burdens high, but there are also problems that data may be inaccurate due to simple mistakes during work.

  The present invention has been made in view of the above points, and is a simple and efficient ultrasonic reaction apparatus that automates the reaction process by a reaction apparatus, can stably amplify and detect abnormal proteins, etc., and obtain accurate data. Is to provide.

The gist of the present invention is that
A processing tank having a sealed tube containing a sample disposed therein, an ultrasonic oscillation device, and a reaction unit equipped with a motor for vibrating the sealed tube are formed in the lower part, and the upper part oscillates ultrasonic waves by the ultrasonic oscillation device. It is an ultrasonic automatic reaction device that constitutes an operation unit equipped with a timer and a counter for controlling time,
The operation of oscillating the ultrasonic wave oscillated from the ultrasonic oscillating device for 0.1 second to 999 seconds and stopping for 0.1 second to 999 seconds by the timer and counter of the operation unit is repeated 1 to 999 times continuously. After that, while the oscillation of the ultrasonic wave is stopped for 1 minute to 99 hours, the sealed tube is vibrated by the rotation of the motor to prevent the precipitation of the sample, and this series of repeated operations is performed for 24 hours or more. It is a continuous ultrasonic automatic reaction device.

  Since the present invention is configured as described above, the configuration of the apparatus is simple and easy to operate, and since the reaction process of the reaction apparatus is automated during operation, the burden on the researcher is reduced, and abnormal proteins, etc. There are effects such as stable amplification and detection of the sample, and accurate detection data can be obtained.

In the best mode for carrying out the present invention, a reaction tank provided with a processing tank having a sealed tube containing a sample disposed therein and an ultrasonic oscillator is formed in the lower part, and the upper part is formed by the ultrasonic oscillator. It is an ultrasonic automatic reaction device that comprises an operation unit with a timer and counter that controls the oscillation time of the ultrasonic wave,
The operation of oscillating the ultrasonic wave oscillated from the ultrasonic oscillating device for 10 seconds to 60 seconds and stopping for 1 second to 10 seconds continuously by the timer and counter of the operation unit is repeated 5 to 10 times continuously, and then for 1 hour to This is an automatic ultrasonic oscillating apparatus that stops for 2 hours and continuously performs this series of repetitive operations for 24 to 48 hours.

  FIG. 1 is a schematic front view showing an embodiment of the automatic ultrasonic reaction apparatus of the present invention, FIG. 2 is a plan view showing a form of a reaction portion of the automatic ultrasonic reaction apparatus of the present invention, and FIG. 4 is a schematic side view showing an embodiment of the reaction part of the ultrasonic automatic reaction apparatus of the present invention, FIG. 4 is a flowchart of an embodiment of the ultrasonic automatic reaction apparatus of the present invention, and FIG. 5 is data amplified by detecting abnormal proteins. It is.

A configuration of the ultrasonic automatic reaction device 1 of the present embodiment will be described with reference to the drawings.
1 to 3, reference numeral 1 denotes an ultrasonic automatic reaction device. Reference numeral 10 denotes an operation unit of the apparatus, and reference numeral 20 denotes a reaction unit provided at a lower part of the operation unit 10. The reaction tank 20 contains a processing tank 21, an ultrasonic transducer 26, and the like.

The operation unit 10 incorporates an ultrasonic oscillation control circuit therein, and the oscillation time of the ultrasonic transmitter 26 can be sequence-controlled by the timers 11, 12, 13 and the counter 14 formed on the surface. ing.
Among these, the timer 11 is a timer for controlling the oscillation time of the ultrasonic wave, and an arbitrary time between 0.1 and 999 seconds can be set, but in this embodiment, it is set to 10 seconds.
The timer 12 is a timer for controlling the stop time of the ultrasonic oscillation, and an arbitrary time can be set, but in this embodiment, it is set to 1 second.
The timer 13 is also a timer for controlling the ultrasonic oscillation stop time, and an arbitrary time can be set, but in this embodiment, it is set to 1 hour.
The counter 14 counts the number of times the timers 11, 12, and 13 are activated.
Reference numeral 18 denotes a main changeover switch, which is an intermittent / pulse / continuous transmission changeover switch, and reference numeral 19 denotes an ultrasonic strength changeover switch.

Next, the structure of the reaction unit 20 will be described.
The processing tank 21 is a substantially rectangular parallelepiped water tank, and has a structure in which a container 30 of a sealed tube 31 described later can be placed on the upper part. The substantially central portions of the vertical side plates 22 and 22 on both sides are bent 45 degrees inward to form inclined plates 23 and 23, and the lower ends of the inclined plates 23 and 23 are connected to the horizontal bottom plate 24. .

On the outer surfaces of the two inclined plates 23, ultrasonic transducers 26 are attached at right angles to the inclined plate 23, respectively, and a so-called crossing type is provided on the inner side of the inclined plate 23. ing. Therefore, the ultrasonic waves intersect each other at right angles to make the energy stronger, and an effective crushing operation can be performed even using the sealed tube 31 made of a plastic container made of a flexible material.
In this embodiment, the inclined plates 23 are formed at two locations on the left and right side plates, but can also be attached to the front and rear side plates. In this case, there are four inclined plates 23, and therefore there are four ultrasonic transducers 26. It becomes. Further, when more powerful crushing is required, the number of ultrasonic vibrators 26 is increased by adding the ultrasonic vibrators 26 to the bottom plate 24.

  The container 30 is a rack for fixing the sealed tube 31 to the processing tank 21, and a holder 32 is attached inside. The holder 32 fixes the sealed tube 31 with its tip immersed in the water 29. The holder 32 is disposed at a position where the ultrasonic wave is most easily received in the central portion of the processing tank 21.

The operation of the present embodiment configured as described above will be described with reference to the flowchart of FIG.
First, the sealed tubes 31, 31... Are fixed to the holder 32 of the container 30, and then the container 30 is mounted in the processing tank 21. The sealed tube 31 is filled with a sample 40 containing an aqueous solution and an abnormal protein that is an object to be crushed, and its tip is immersed in water 29. This is because the ultrasonic waves are propagated through the water 29. The water 29 is always kept at 37 ° C. by a circulation device (not shown).

Thereafter, when the main switch 18 of the operation unit 10 is turned on and the switch 41 is turned on, the ultrasonic transducers 26 and 26 start operating and oscillate ultrasonic waves for 10 seconds (S10). The ultrasonic wave passes through the diaphragms 25 and 25 and becomes strong, and advances toward the sealed tubes 31 and 31.
At this time, since the ultrasonic waves travel in a direction perpendicular to the respective inclined plates 23, the ultrasonic waves cross each other at a right angle. And this crossing point is set beforehand in the vicinity of the water surface at the center of the processing tank 21, and the sealed tubes 31, 31,. The ultrasonic wave passes through the water 29 in the processing tank 21 and reaches the sealed tube 31, further passes through the aqueous solution in the sealed tube 31, reaches the sample 40, and is pulverized.

The process proceeds to S12 10 seconds after the start of the ultrasonic oscillation, the timer 11 is turned off and the operation of the ultrasonic transducer 26 is stopped. At the same time, the timer 12 is turned on, and the operation of the ultrasonic transducer 26 is activated. Hold stopped for 1 second.
The process proceeds to S14, where the value C of the counter 14 (initial value is zero) is incremented by 1, and the process proceeds to S16 to determine whether the value C of the counter 14 is 5. If not, the process proceeds to the first S10, and the above process is repeated.
As described above, the ultrasonic wave is oscillated for 10 seconds and stopped for 1 second, and the counter value C becomes 5 by repeating the operation five times in succession. Stop oscillation for 1 hour. Thereafter, the process proceeds to S20, the counter value C is reset to zero, the process returns to S10, and the above processing is repeated.
In this way, the detection sensitivity is increased by repeatedly disrupting the abnormal protein in the sample and changing it between the cultures. The reaction operation is completed by performing this operation continuously for 48 hours.

In the above reaction operation, the oscillation time of the ultrasonic wave by the timer 11 can be arbitrarily determined. However, since the protein is destroyed at 30 seconds or more, the time is suitably 1 to 30 seconds, and 10 seconds is most desirable.
The number of operations of each timer is counted by the counter 14, and after counting a predetermined number of times, the counter 14 gives control to each timer. Although the number of times is 5 in this embodiment, it can be arbitrarily determined and can be changed depending on the type of sample.
Moreover, while the oscillation of the ultrasonic wave is stopped for 1 hour, the sample 40 inside the sealed tube 31 is prevented from being precipitated by vibration due to the rotation of the motor 39. That is, the rotation of the motor 39 causes the container 30 and the holder 32 to vibrate slightly, thereby imparting vibration to the sealed tube 31 to prevent the sample from precipitating.

  As described above, according to the present embodiment, the reaction work of automatically amplifying and detecting a sample can be efficiently performed with a simple configuration and an easy operation, which only reduces the burden on researchers. In addition, operational errors are avoided and accurate data can be obtained quickly.

An experimental example of this example is shown below.
Using the ultrasonic automatic reaction apparatus 1 of this example, the reaction was performed using a sample obtained by diluting a hamster prion strain Sc237-infected brain emulsion to 1/500 with a normal hamster brain emulsion. After Proteinase K (PK) treatment, abnormal prion protein was detected by Western blotting (see FIG. 5).
When the ultrasonic output of the present reaction apparatus 1 was set to be strong, 10 seconds of oscillation every hour and 1 second pause pulse oscillation was repeated 5 times and incubated at 37 ° C. for 48 hours, a PK resistance band was clearly observed. Signal enhancement was observed and the amplification factor was 30 times or more.
As a result, the automatic ultrasonic reaction apparatus 1 of the present invention is useful for labor saving and efficiency improvement such as high-sensitivity detection of abnormal prion protein, elucidation of the conversion mechanism of abnormal protein, and screening of infection preventive drugs.

Front schematic diagram showing one embodiment of the automatic ultrasonic reaction apparatus of the present invention. The top view which abbreviate | omitted one part which shows one form of the reaction part of the ultrasonic automatic reaction apparatus of this invention Side surface schematic diagram which shows one form of the reaction part of the ultrasonic automatic reaction apparatus of this invention The flowchart of the Example of the ultrasonic automatic reaction apparatus of this invention Data amplified by detecting abnormal proteins

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Ultrasonic automatic reaction apparatus 10 Operation part 11 Timer 12 Timer 13 Timer 14 Counter 20 Reaction part 21 Processing tank 25 Diaphragm 26 Ultrasonic vibrator 30 Container 31 Sealed tube 32 Holder

Claims (1)

A processing tank having a sealed tube containing a sample disposed therein, an ultrasonic oscillation device, and a reaction unit equipped with a motor for vibrating the sealed tube are formed in the lower part, and the upper part oscillates ultrasonic waves by the ultrasonic oscillation device. It is an ultrasonic automatic reaction device that constitutes an operation unit equipped with a timer and a counter for controlling time,
The operation of oscillating the ultrasonic wave oscillated from the ultrasonic oscillating device for 0.1 second to 999 seconds and stopping for 0.1 second to 999 seconds by the timer and counter of the operation unit is repeated 1 to 999 times continuously. After that, while the oscillation of the ultrasonic wave is stopped for 1 minute to 99 hours, the sealed tube is vibrated by the rotation of the motor to prevent the precipitation of the sample, and this series of repeated operations is performed for 24 hours or more. Continuous automatic ultrasonic reaction equipment.