KR101268411B1 - Non destructive method and apparatus for detection of gourd seeds contaminated with virus using optical scattering amplitude per depth - Google Patents

Abstract

The present invention relates to a method and apparatus for nondestructive screening of virally infected sperm seeds using light scattering size according to depth, and more specifically, to obtain light scattering profiles for depths of the sperm seed to be selected; Obtaining a light scattering value of the seed from the obtained profile; And determining the healthy seed when the average light scattering value of the seed is lower than the average light scattering value of the virally infected seed, and determining the viral infected seed when the average light scattering value is higher than the average light scattering value of the healthy seed. The present invention relates to a method for screening virally infected sesame seeds.
According to the present invention, the average optical tomographic scattering size can be measured by acquiring an optical tomographic scattering profile for each scanning point in the optical tomographic image or the obtained optical tomographic image of the scabbard seed to be diagnosed through a signal processing process using an optical interferometer. The average value of the measured optical tomographic scattering size can be used to easily distinguish between healthy and seed infected virus.

Description

Non destructive method and apparatus for detection of gourd seeds contaminated with virus using optical scattering amplitude per depth}

The present invention relates to a method and apparatus for nondestructive screening of virally infected sperm seeds using light scattering size according to depth, and more specifically, to obtain light scattering profiles for depths of the sperm seed to be selected; Obtaining a light scattering value of the seed from the obtained profile; And determining the healthy seed when the average light scattering value of the seed is lower than the average light scattering value of the virally infected seed, and determining the viral infected seed when the average light scattering value is higher than the average light scattering value of the healthy seed. The present invention relates to a method for screening virally infected sesame seeds.

Diseased Strained Seeds infected with Cucumber mosaic virus (CMV) and Cucumber green mottle mosaic virus (CGMMV) are most likely not to germinate. It causes a lot of loss of productivity. Fast and accurate screening of healthy seeds and early diagnosis of diseased seeds are an essential skill in the field of agricultural production, and are also important for the plant quarantine of seeds imported and exported both at home and abroad.

To date, the CMV and CGMMV test methods in most seedling companies and plant quarantine agencies around the world mainly use the biological diagnosis by sowing and the serological diagnosis using antiserum. However, since these methods have a detection limit and are not easy to diagnose a virus, molecular biological diagnosis by RT-PCR is widely used. However, these diagnostic methods not only require long test periods, use expensive reagents and expensive equipment, but are also unable to recycle seeds after testing. In addition, the detection sensitivity is not constant, so the reliability is considerably lowered. Moreover, due to the time limit, it is impossible to perform a full inspection on a large amount of seeds.

1) Biological diagnostic method: It is possible to determine whether or not good seed should be more than a certain time after growing seed by diagnosis by sowing. It takes too much time and is not applicable to large quantities of seeds.

2) Serological diagnostic method: Diagnosis using antiserum takes a long time, and the seeds are destroyed and diluted with the diagnostic antiserum to determine whether the good seed. It is expensive and not applicable to many seeds. Therefore, they are randomly selected and classified in a specific group.

3) RT-PCT Molecular Biology Diagnosis: As the most widely used method, it is easily used as a normal seed detection method, but it takes about a day to produce results, and it is necessary to destroy seeds to extract RNA information, especially cucumber seeds. In the case of the same test should be performed twice, but it can be determined whether the good seed.

Accordingly, the present inventors have made intensive studies to overcome the problems of the prior art, and when comparing the optical monolayer scattering size of the healthy snail seed and the non-healthy stalk seed with the virus latent infection, the virus-infected scab seed is not destroyed. As a result, the present invention was confirmed to be easily distinguishable.

Therefore, the main object of the present invention is to non-destructively select virus-infected scabbard seeds, which can be easily distinguished from diseased scabbard seeds infected with viruses by investigating the light scattering size of the seed from the light-scattering profile of the scabbard seeds. The present invention provides a method for screening healthy seeds or virally infected seeds.

It is another object of the present invention to provide a sorting device for healthy seed or virus infected seed using the above method.

According to one aspect of the present invention, the present invention provides a method for screening whole seed or virally infected seed, comprising the following steps:

a) obtaining a light scattering profile for each depth of the seed to be screened;

b) obtaining a light scattering value of the seed from the obtained profile; And

c) determining the seed as a healthy seed if the average light scattering value of the seed is 20% or more lower than the average light scattering value of the virally infected seed. .

The present invention is a technology that can easily distinguish between diseased scabbard seeds and healthy scabbard seeds infected with a virus, and for the first time it can be easily distinguished between diseased scabbard seeds infected with a virus and healthy scam seeds by only one depth light scattering profile. It was. In the present invention, the light scattering includes an optical acoustic signal due to scattering, diffusion, and absorption generated by light energy incident on a sample.

In the present invention, the "depth light scattering profile for each depth" is to measure the light monolayer scattering size by injecting light energy non-invasively without physical cutting to the seed to be screened, Optical Coherence Domain Reflectometry (OCDR) The light scattering size for each depth inside the seed can be measured. Preferably, an optical tomography apparatus using an optical interferometer, such as an optical coherence tomography (OCT) such as a time-domain OCT, a spectral-domain OCT, a swept source OCT, or an optical diffuse tomography apparatus using multiple light scattering characteristics ( Diffuse Optical Tomography (DOT), Photoacoustic Tomography, and the like can be used to obtain the light scattering profile of the present invention.

Optical tomography or optical coherence tomography (hereinafter, referred to as "OCT") is a new technique for obtaining high resolution cross-sectional images of non-invasive biological tissues using low-interference light. The OCT system is an extension of Optical Coherence Domain Reflectometry (OCDR), developed in 1987, and was first developed by David Huang of M.I.T in 1991. OCDR is a system that obtains the intensity of backscattering light in one dimension by using the Michelson low interferometer. OCT is a method of acquiring two-dimensional images by adding transverse scanning in such an OCDR system and corresponds to a B-mode scanning method of ultrasound images.

이라 할 때 간섭신호는

상에서 주기가

인 정현파가 된다. 여기서

는 optical wavenumber이다. 통신에서

와

가 Fourier Transform pair 관계에 있는 것처럼

와

도 서로 Fourier Transform pair 관계에 있다. 따라서 FD-OCT 시스템에서는 간섭신호를

상에서 바로 얻기가 어렵다. 일반적으로 FD-OCT에서는 Spectrometer 구조를 사용하므로 파장 도메인(

)상에서 간섭신호가 얻어진다. 따라서 rescaling 계산과정을 통하여 이를

상에서의 간섭신호로 바꾸어야 한다. 만일 샘플단상에 여러개의 반사체가 있다면 각 반사체에 대하여 각각 다른 형태의 간섭신호가 서로 중첩된 형태로 파장 도메인상에서 존재한다. 이를 위하여 설명한 것처럼

상으로 다시 rescaling 하면 각 반사체에 대하여 각각 다른 주기의 정현파 간섭신호가 중첩된 형태로 존재한다. 이를 inverse Fourier Transform(IFFT)하게 되면 각 반사체에 대한 거리 정보를 얻을 수 있다.Optical coherence tomography (TD-OCT) and frequency domain optical tomography (FD-OCT) technology for the time domain are largely dependent on the method of obtaining the interference signal. Divided. The TD-OCT technology scans the reference distance of the Miegelson interferometer to obtain the distance information by envelope detection of the interference signal obtained when the distance between the reflector existing in the sample stage coincides with each other and extracts the elapsed time from its peak. On the other hand, FD-OCT, with a fixed base, was first introduced in the mid-1990s. In this method, the distance difference between the reflector existing in the reference stage and the sample stage

Interference signal is

Cycle on

To be a sinusoid. here

Is the optical wavenumber. In communication

Wow

Is in a Fourier Transform pair relationship

Wow

Are also in a Fourier Transform pair relationship with each other. Therefore, in FD-OCT system,

Difficult to get right on the bed In general, FD-OCT uses a spectrometer structure, so the wavelength domain (

), An interference signal is obtained. Therefore, through the rescaling process

It should be replaced with the interference signal on the top. If there are multiple reflectors on the sample stage, different types of interference signals for each reflector exist in the wavelength domain in an overlapping manner. As explained for this

When rescaling onto the phase, sinusoidal interference signals of different periods exist for each reflector. Inverse Fourier Transform (IFFT) can get distance information about each reflector.

The optical tomography (OCT) has a structure of TD-OCT as shown in FIG. 3, Swept-Source OCT (hereinafter referred to as SS-OCT) as a type of FD-OCT as shown in FIG. 4, and Specral domain OCT as shown in FIG. SD-OCT).

By default, OCT has a broadband light source. The wider the full width at half maximum (FWHM) of the broadband light source, the higher the axial resolution of the image. Light emitted from the light source is divided into a reference stage and a sample stage through an optical splitter, and when the optical paths of each reference stage and the sample stage are the same, a single-layer interference signal of the sample stage is generated. The generated interference signal goes back to the optical splitter and enters the photodetector as the receiving end to acquire the signal.

The TD-OCT of FIG. 3 and the SS-OCT of FIG. 4 use a photodetector at a receiving end. However, in the case of the SD-OCT of FIG. 5, a spectrometer is installed at a receiver to acquire interference information of a signal for each wavelength. In the case of the TD-OCT of FIG. 3 and the SS-OCT of FIG. 4, the structure looks the same, but in the case of the TD-OCT, an interference signal is generated by quickly moving the optical path at the reference stage physically, and in the case of the SS-OCT Without motion, the Swept Source quickly scans a wide band of narrow band optical signals, producing the same effect as a TD-OCT moving the reference stage quickly to produce an interference signal. In general, in case of TD-OCT, the scanning speed in the depth direction depends on the change of the physical optical path of the reference stage, but the SS-OCT has a maximum speed of 400 because the speed depends on how fast the Swept-Source moves. Speeds above kHz have been reported to the Society. In the case of SD-OCT, the speed is determined by the line rate of the scan camera used in the spectrometer. 140 kHz cameras are in use today and are limited by computer performance.

In a preferred embodiment of the invention, the light scattering profile is the light scattering profile of the scabbard seeds obtained using Time-domain OCT.

A high-resolution two-dimensional tomographic image can be obtained using the optical tomography camera described above. The obtained 2D image may acquire a 1D profile in a depth direction as shown in FIG. 6. The obtained one-dimensional profile has light scattering information for each depth of the tomographic image. The x-axis of the profile according to the invention represents the light scattering magnitude for each depth measured by the optical tomography, and the y-axis represents the depth of the sample. Therefore, when the sum of the scattering values for each depth is obtained, the optical tomographic scattering values of the entire sample are obtained. The total optical tomographic scattering values can be obtained simply by integration by depth.

In the present invention, the light scattering value of the healthy seed, or the light scattering value of the virus-infected seed, the average value is calculated from the scattering size by depth of the seed measured for a number of known or non-healthy seed, or probability The distribution can be obtained by calculating interval values with 90% or 95% confidence.

In the present invention, the “healthy seed” refers to a normal seed that is not infected with any plant virus that infects the plant seed and affects the germination of the seed. In addition, as a contrasting concept, “non-whole seed” or “disease seed” refers to a seed that is infected with the plant virus and does not germinate.

In the method of the present invention, the virus is Cucumber mosaic virus (CMV) or Cucumber green mottle mosaic virus (CGMMV) is characterized in that. In the embodiment of the present invention, the light scattering profiles of healthy and non-healthy seeds were obtained using scabbard seeds, and the light scattering average value of the seeds was confirmed in the light scattering profile, so that the light scattering average value of the virus-infected seeds was larger. It confirmed that it appeared (refer FIG. 1 and Table 1). RT-PCR was performed on the non-healthy seeds to confirm that the infected virus was a cucumber nocturnal mosaic virus (see FIG. 10).

In a preferred embodiment of the present invention, microscopic examination of the cross-sections of healthy and non-healthy seed is found that a lot of holes in the seed of the healthy seed and fine holes in the seed of the non-healthy seed. (See FIG. 2). Compared with the results of light scattering fault size experiments, healthy seeds have wider pores than diseased seeds, so that light energy is less scattered when entering the seed, and conversely, non-healthy seeds do not have larger pores than healthy seeds. This results in a lot of light energy scattered on the seed particles. This difference in light scattering size makes it possible to distinguish between healthy seeds and non-healthy seeds.

In the method of the present invention, the light scattering profile in step a) can be obtained by analyzing the light scattering size for each depth by using optical coherence tomography (OCT). Preferably, the light scattering profile can be obtained by using an optical tomography apparatus of all regions including time-domain OCT, Spectral-domain OCT, Swep source OCT, in the embodiment of the present invention using Time-domain OCT Light scattering profiles were obtained.

The light scattering size for each depth represents interference information for each depth of the crunch seed, and a tomographic image of the seed may be made using the light scattering size information for each depth. The OCT acquires interference information for each depth of the area scanned by the beam through B-mode scanning (beam moves in one axis) at the sample stage. In the embodiment of the present invention, the average depth of interference information of the sperm seeds through the method of analyzing the light scattering size by depth, the average value of the interference signals at the same position by the depth of the center of the highest floating signal of the seedlings It was. Therefore, the light scattering magnitude value for each depth identified in the embodiment of the present invention may be referred to as the unique depth-specific interference signal information of the sample moth seed.

In the present invention, the x-axis of the light scattering profile represents the light scattering size for each depth measured by the optical tomography, and the y-axis represents the depth of the sample. Therefore, when the sum of the scattering values for each depth is obtained, the total optical tomographic scattering value of the sample can be obtained (see FIG. 1).

In the method of the present invention, in step c), the mean light scattering value of the healthy seed or the viral light scattering mean value of the seed seeding is the DB (database) or known virus infection of the light scattering value of the known healthy seed It is characterized in that it is calculated statistically from the DB of the light scattering values of the seeds.

The DB refers to a DB storing light scattering sizes of depths of seeds measured by analyzing light scattering profiles of depths for a plurality of known or non-healthy seeds. Calculate the average value from the light scattering size contained in each DB, or calculate the interval value with 90% or 95% reliability through probability distribution, and calculate the mean value of the healthy light seed or the average light intensity of viral infected starch seeds. Use as scattering value.

In a preferred embodiment of the present invention, the average light scattering value of the healthy seed is 90% interval (light scattering size range is 29-36; see FIG. 9A) in the normal probability distribution graph of known healthy seeds, The average light scattering value of may be in a 90% interval (38-45 light scattering size range; see FIG. 9B) in a normal probability distribution graph of known virus infected seeds.

According to another aspect of the present invention, the present invention provides an optical tomography unit for acquiring the light scattering size for each depth of the seed to be selected; A signal processing unit processing a signal output from the optical tomography unit to obtain a light scattering profile of the scabbard seed; And a data analysis unit for determining whether or not a viral infection of the selected target scab seed is obtained by obtaining an average value of scattering size for each depth of the profile obtained by the signal processing unit, wherein the data analysis unit includes the depth of the profile obtained by the signal processing unit. Calculate the mean value of the scattering size to determine if the average light scattering value of the seeds is 20% or more lower than the average light scattering value of the virally impaired seed, and if it is 20% or more higher than the mean light scattering value of the healthy seed. Provided is a sorting device for healthy stalk seed or virally infected stalk seed, characterized in that it is determined as a seed.

In the sorting device of the healthy rapeseed seed or virus-infected rapeseed seed of the present invention, the optical tomography unit includes: a broadband light source for generating a broadband light source for injecting light energy to obtain light scattering energy; It includes a first to fourth terminal, receives the light generated from the light source through the first terminal, is divided into two light and output to the second and third terminals, and is input from the second and third terminals An optical splitter for coupling the light to the fourth terminal; A reference terminal connected to the second terminal of the optical splitter and reflecting the light source transmitted through the optical splitter as it is; A sample stage connected to the third terminal of the optical splitter and radiating the light source transmitted through the optical splitter to the target seed scab seed, and outputting the reflected light reflected from the seed target chop seed; A signal coupled to a fourth terminal of the optical splitter and receiving a combined signal of the reflected light of the reference stage and the sampled stage coupled by the optical splitter, detecting the interference signal of the two reflected light, and converting the signal into an electrical signal for outputting; A photodetector implemented with a balanced detector having an input and an input; And an optical circulator for transmitting the light returned from the optical splitter to the broadband light source to the input terminal of the photodetector.

The optical tomography apparatus described above is a description of the time domain optical tomography apparatus and at the same time a description of the optical tomography apparatus used in the embodiment of the present invention. In addition to the time domain optical tomography apparatus, the frequency domain optical tomography apparatus also exhibits the same performance.

Hereinafter, with reference to the accompanying drawings, the screening device of the healthy rapeseed seed or virus-infected sperm seed of the present invention will be described in more detail.

Figure 7 is a block diagram showing the configuration of the sorting device of healthy seed or virus infected seed according to the present invention.

Referring to FIG. 7, the apparatus for sorting healthy seed or virus-infected seed of the present invention includes an optical tomography unit, a signal processor, and a data analyzer.

The optical tomography unit measures light monolayer scattering size by injecting light energy non-invasively without physical cutting to the seed of the seed to be screened, and irradiates a broadband light source with the seed of seed to be screened. Outputs an interference signal by a Michelson interferometer capable of obtaining the light scattering profile of the sack seed. More specifically, the optical tomography unit is implemented by optical coherence tomography (OCT), and may use any kind of OCT, such as a time-domain OCT, a spectral-doamin OCT, a frequency-domain OCT, or a Swept source OCT.

In one embodiment of the present invention, the optical tomography unit is implemented with Time-domain OCT.

The signal processor may process the interference signal output from the optical tomography unit to obtain a light scattering profile of the scabbard seeds to be screened. More specifically, the signal processor outputs tomographic image data or a light scattering profile by performing filtering of DC components, envelope detection, and digital conversion on the interference signal.

The data analyzer analyzes the light scattering size of the sesame seed to be selected by the light scattering profile provided from the signal processor to determine whether the plant virus is infected. More specifically, in the present invention, the data analyzer calculates the light scattering value of the sperm seed by analyzing the light scattering profile obtained by the signal processing unit, so that the light scattering value of the seed is 20 If it is lower than%, it can be judged as a healthy seed, and if it is 20% or more higher than the average light scattering value of a healthy snail seed, it can be judged as a virus infection seed. The mean light scattering value of the virally infected sperm seeds or the mean light scattering value of the healthy scaber seeds can be calculated statistically from a database of light scattering size of known healthy seeds, or from a DB of light scattering size of known virally infected seeds. have.

The data analyzer may compare a program for calculating the average value of the light scattering size of the sperm seed from the light scattering profile obtained by the signal processor, and compare the calculated light scattering average value with a healthy seed or an unhealthy seed determination average light scattering value. It includes a program for determining whether or not the virus of the sperm seed virus, these programs can be installed and implemented in a personal computer (PC).

Next, with reference to FIG. 8, the system block diagram of the sorting apparatus of healthy seed | seed seed or virus-infected seed | strain seed | species which concerns on this invention is demonstrated. The optical tomography apparatus basically includes a broadband light source, an optical splitter, a reference stage, a sample stage, a photodetector, and an optical circulator.

First, the wide band light source used in the configuration of the sorting apparatus of the present invention uses a SLED (Super Luminescent Emitted Diode) having a full width at half maximum (FWHM) of 150 nm at 1310 nm. In general, the optical tomography apparatus improves the axial resolution by using a broadband source. (The time domain optical tomography apparatus and the spectrum-domain OCT in the frequency domain use a broadband light source, and the Swept-Source OCT In the case of a narrow band of wavelengths, the same effect is achieved by using a light source capable of irradiating broadband.

The light energy from the light source is divided into a sample stage and a reference stage through a 50:50 fiber coupler through a circulator. The shuffler serves to send the light energy returned to the light source in the other direction among the light energy reflected from the sample stage and the reference stage through the light splitter where the light energy is incident and returned through the light splitter. The optical splitter is used to create the optical interference phenomenon, and the light energy from the light source is distributed equally to the sample stage and the reference stage. Since the system according to the invention was fabricated using optical fibers, the optical splitter used an optical coupler.

The reference stage used Rapidly Scanning Optical Delayline (RSOD). The configuration was made by driving a commercially available diffraction grating and a galvo scanner moving at a speed of 300 Hz. In RSOD, the length of the reference stage is changed slightly to change the optical path. (The reference stage is fixed because the optical tomography in the frequency domain does not require the movement of the optical path.)

The sample stage was manufactured to enable B-mode scanning using a galvo scanner, and an objective lens having a focal length of 30 mm was used to beam-focus the collimated incident beam. When the optical path of the sample stage is at the interference distance by the galvo scanner of the reference stage moving finely, an interference signal is generated. The generated interference signal passes through the optical splitter to the optical receiver (but in the case of the frequency domain optical tomography camera, only the position within the interference distance between the sample stage and the reference stage is fixed).

In the case of the time-domain optical tomography camera, the light receiver uses a photodetector. In the present invention, a balanced photo detector is used to increase the SNR of the signal. Normally, the combined signal of the reference stage and the sample stage goes to the + terminal of the photodetector, and the signal returning to the light source unit goes back to the-terminal through the shuffler (in the case of Spectral-domain OCT The optical receiver enters the optical signal by a spectrometer manufactured by CCD and CMOS line scan cameras (a photodetector is used for the Swept-source OCT).

The interference signal introduced through the photodetector is converted into a voltage to enter the DAQ board. The DC voltage component is then filtered and envolpe-detected before being converted into a digital signal and then imaged (in the case of an optical tomography in the frequency domain, the signal from the spectrophotometer and photodetector is Fourier). After conversion, the signal is converted into a signal representing distance information, and the combination of these signals forms an image.

In the embodiment of the present invention, the sampling rate is 10,000,000 Hz, the number of A-scans is set to 200, and the number of pixels per A-scan is set to 15,000. In preparation for the experiment, the axial resolution of the OCT was 4.5 μm and 6 μm when measured in air.

When the image is implemented through the signal processing process by the optical tomography camera described above, the tomographic image of the scabbard seed to be diagnosed is acquired, and the image implementation can be omitted, and each scanning point in the obtained optical tomography image is obtained. Profiles of optical tomographic scattering sizes can be obtained.

As described above, according to the present invention, an average optical tomography is obtained by acquiring an optical tomographic scattering profile for each scanning point in an optical tomography image or an acquired optical tomography image of a scabbard seed to be diagnosed through a signal processing process using an optical tomography camera. Scattering size can be measured. The average value of the measured optical tomographic scattering size can be used to easily distinguish between healthy and seed infected virus.

1 is a diagram showing the light scattering profiles of healthy seeds and diseased seeds according to depth in the same graph. The red (Negative seeds) profile is healthy seeds and the blue (Positive seeds) profile shows the optical monolayer scattering size by depth of the diseased seeds.
FIG. 2 is a micrograph comparing the cross sections of healthy and seed disease Abnormal used in the experiment.
3 is a configuration diagram of a time-domain wideband tomography apparatus (TD-OCT).
4 is a configuration diagram of a Swept-Source tomography apparatus (SS-OCT).
5 is a schematic diagram of a Spectral-domain tomography apparatus (SD-OCT).
FIG. 6 illustrates the principle of acquiring a one-dimensional depth-specific optical tomographic scattering profile from a two-dimensional tomographic image using an optical tomography camera.
Figure 7 is a block diagram showing the configuration of the sorting device of healthy seed or virus infected seed according to the present invention.
8 is a system configuration diagram of a sorting device of healthy seed or virus infected seed in accordance with the present invention.
9A shows a 90% interval of the normal probability distribution graph of healthy crunch seeds.
9B shows a 90% interval of a normal probability distribution graph of non-healthy scabbard seeds.
FIG. 10 shows RT-PCR results of healthy and non-healthy sesame seeds.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example  1. Using optical interferometer Depth stars  Light scattering size measurement

In order to distinguish between virus-infected diseased seeds and healthy seeds, optical scattering size by depth was measured using optical coherence domain reflectometry (OCDR).

Specifically, Time Domain optical coherence tomography applied with an optical interferometer; self-made, axial resolution: 5.4 m, lateral resolution: 14 m, sample arm power: 5 mW) technology was used to measure the light scattering size by depth for 10 healthy bran seeds and 10 non-nutrition chard seeds supported by the seed company (Nongwoo Bio). When the optical tomography apparatus is implemented as an image through a signal processing process, a tomographic image of a scabbard seed to be diagnosed is obtained. Image implementation can be omitted. An optical tomographic scattering size profile was acquired for each scanning point in the obtained optical tomographic image. After the obtained optical tomographic scattering profile was obtained for each scanning point to obtain an average value, the average optical tomographic scattering size was measured by adding the optical tomographic scattering size by depth. The average value of the measured optical tomographic scattering size was used to distinguish between healthy and seed infected virus.

1 is a diagram showing the light scattering profiles of healthy seeds and diseased seeds according to depth in the same graph. The red (Negative Seeds) profile is the healthy seed and the blue (Positive Seeds) profile represents the optical monolayer scattering size by depth of the diseased seeds. As shown in Figure 1, healthy seeds and diseased seeds can be easily distinguished, the right picture is shown enlarged. The results of FIG. 1 are summarized in Table 1 below.

Table 1 shows numerical values and average values obtained by simply adding the measured optical tomographic scattering size for each depth. On the average, the depth of optical tomographic scattering by the depth of healthy seeds is about 31.7, and the size of the optical monolayer scattering by the depth of diseased seeds is about 41.04. That is, it was confirmed that the optical tomographic scattering size of diseased seeds was about 23% higher than that of healthy seeds.

Next, real microscopic images of healthy and diseased seeds were compared.

FIG. 2 is a micrograph comparing the cross sections of the healthy stalk seed and the diseased stalk seed used in the experiment with a sharp knife. In Figure 2, it was confirmed that a lot of holes in the seed for healthy seeds. In the case of diseased seeds, it was confirmed that there were minute pores in the epidermis. Compared with the results of light scattering fault size experiments, it can be predicted that the healthy seeds have a wider hole than the diseased seeds, so that light energy is less scattered upon incidence. On the other hand, it can be predicted that the light seed is scattered much in the seed particles because the diseased seeds do not have large pores compared to the healthy seeds.

Example 2 RT-PCR Verification of Healthy Seeds and Non-Healthy Seeds

After measuring the light scattering size of the sperm seed in Example 1, the integrity and non-healthiness of the seeds was verified by RNA detection using RT-PCT experiment, and each of the 10 grains was confirmed as healthy and non-healthy.

In order to confirm whether the seeds were infected with the virus, the primer set CGMM-C60 / CGMM-N30, which amplifies part of the CGMMV coat protein region and part of the movement protein region, was used for RT-PCR, respectively. After completion of the interference imaging experiment, total RNA was extracted from the shoot seeds and proceeded with RT-PCR. One mortar seed and 1,000 μl of Trizol (Invitrogen, USA) used in the experiment were completely ground in the mortar. The grinding solution was placed in a 1.5 ml tube and centrifuged at 15,000 rpm for 10 minutes. The supernatant was transferred to a new tube, stirred with the same amount of chloroform, and centrifuged at 15,000 rpm for 10 minutes. The supernatant was transferred back to a new tube, the same amount of 2-propanol was added and allowed to stand on ice for 10 minutes, followed by centrifugation at 15,000 rpm. The supernatant was removed and the precipitate was rinsed with 700 μl of 70% ethanol. The precipitate was completely dried and then dissolved in 100 μl RNase free water and used as RT-PCR template. RT-PCR was performed using RT / PCR premix (Bioneer, Korea). 0.5 μl of total RNA of seeds and CGMM-C60 (10 pM / μl) (5'- AAT TAA GTA AAG TCC TGA CG-3 ') and CGMM-N30 (10 pM / μl) (5'- ATG GAA CGT ACC GGA 0.5 μl of each ATC-3 ′) was added and RT-PCR was performed by adjusting the total volume to 20 μl with RNase free water. RT-PCR conditions were reverse-transcribed at 42 ° C. for 60 minutes to synthesize cDNA, and then amplified 35 times at 95 ° C. 45 seconds, 55 ° C. 1 minute, 72 ° C. 1 minute 30 seconds after pre-heating at 95 ° C. for 5 minutes. The reaction was carried out at 5 ° C for 5 minutes. 2 μl of the RT-PCR product was electrophoresed in 0.7% agarose gel and stained with ethidium bromide, and then detected by UV transilluminator.

10 shows the results of electrophoresis of the RT-PCR product. As shown in FIG. 10, it was confirmed that the target DNA fragment was not amplified in healthy scavenging seeds, and about 600 bp of the desired DNA fragment was amplified and infected by CGMMV in diseased (non-healthy) stalk seeds. Lane 1 is the result of RT-PCR of healthy sorghum seeds. In Lane 1, the DNA fragment of about 600 bp was not amplified, indicating that it was not infected with CGMMV. Lane 2 is the result of RT-PCR of crab seeds infected with CGMMV. It can be seen that about 600 bp of the desired DNA fragment was amplified and infected with CGMMV. Lane 3 confirmed the amplification of the desired DNA fragment of about 600 bp, which is the same as Lane 2, as a result of RT-PCR of diseased melon seeds expected to be infected with CGMMV.

Therefore, the seed was extracted from total RNA immediately after the image was taken using optical coherence tomography, and the presence of viral infection was confirmed by PCR products obtained after RT-PCR.

Claims (8)

A method of screening healthy seed or virally infected seed containing the following steps:
a) obtaining a light scattering profile for each depth of the seed to be screened;
b) obtaining an average light scattering value of the seed from the obtained profile; And
c) determining the seed as a healthy seed if the average light scattering value of the seed is 20% or more lower than the average light scattering value of the virally infected seed. .
The method of claim 1, wherein the virus is a cucumber nocturnal mosaic virus.
The method of claim 1, wherein the light scattering profile is obtained by performing intensity analysis of A-Scan using optical coherence tomography (OCT).
The method according to claim 1, wherein in step c), the average light scattering value of the healthy seed or the virally infected seed is the light scattering value of the known healthy seed or the DB of the known viral seed. And statistically calculated from the DB of scattering values.
5. The method of claim 4, wherein the average light scattering value of the healthy seed is in the range of 29-36 and the average light scattering value of the virally infected seed is in the range of 38-45.
An optical tomography unit for acquiring the light scattering size according to the depth of the seed to be selected;
A signal processing unit processing a signal output from the optical tomography unit to obtain a light scattering profile of the scabbard seed; And
And a data analyzer for determining whether or not a virus is infected by the selectable seed seeds by obtaining an average value of scattering size for each depth of the profile obtained by the signal processor.
The data analyzer calculates an average value of the scattering size for each depth of the profile obtained by the signal processor, and if the average light scattering value of the seed is less than 20% lower than the average light scattering value of the virus-infested seed, it is determined as a healthy seed. A screening device for healthy stalk seeds or virally infected stalk seeds, characterized in that it is determined to be a virus infected seed if it is 20% or more higher than the average light scattering value of the seed.
The method of claim 6, wherein the optical tomography unit
A broadband light source for generating a broadband light source for injecting light energy to obtain light scattering energy;
It includes a first to fourth terminal, receives the light generated from the light source through the first terminal, is divided into two light and output to the second and third terminals, and is input from the second and third terminals An optical splitter for coupling the light to the fourth terminal;
A reference terminal connected to the second terminal of the optical splitter and reflecting the light source transmitted through the optical splitter as it is;
A sample stage connected to the third terminal of the optical splitter and radiating the light source transmitted through the optical splitter to the target seed scab seed, and outputting the reflected light reflected from the seed target chop seed;
A signal coupled to a fourth terminal of the optical splitter and receiving a combined signal of the reflected light of the reference stage and the sampled stage coupled by the optical splitter, detecting the interference signal of the two reflected light, and converting the signal into an electrical signal for outputting; A photodetector implemented with a balanced detector having an input and an input; And
And an optical circulator for transmitting the light returned from the optical splitter to the broadband light source to the input terminal of the photodetector.
7. The healthy sham seed or virus according to claim 6, wherein the optical tomography unit is a time-domain optical coherence tomography device or a frequency-domain optical coherence tomography device. Screening device for infected seed.

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