KR20140126496A - Composition for diagnosing avellino corneal dystrophy and diagnostic method thereof - Google Patents

Composition for diagnosing avellino corneal dystrophy and diagnostic method thereof Download PDF

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KR20140126496A
KR20140126496A KR20130044730A KR20130044730A KR20140126496A KR 20140126496 A KR20140126496 A KR 20140126496A KR 20130044730 A KR20130044730 A KR 20130044730A KR 20130044730 A KR20130044730 A KR 20130044730A KR 20140126496 A KR20140126496 A KR 20140126496A
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corneal dystrophy
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이희정
이소영
박병준
김종훈
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주식회사 녹십자엠에스
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Abstract

The present invention relates to a composition for diagnosing Abelino corneal dystrophy and a method for diagnosing Abelino corneal dystrophy and a method for diagnosing Abelino corneal dystrophy using the primer set and the probe according to the present invention. The corneal dystrophy can be diagnosed and at the same time, the signal of the positive control group in the human can be used to discriminate the suitability of the nucleic acid used for the accurate determination and inspection of the Abelino corneal dystrophy.

Description

Technical Field [0001] The present invention relates to a composition for diagnosing Abelino corneal dystrophy and a method for diagnosing Abelino corneal dystrophy,

The present invention relates to a composition for diagnosing Abelino corneal dystrophy and a diagnostic method thereof, and more particularly, to a composition containing a real-time PCR primer set and a probe for the diagnosis of Abelino corneal dystrophy, comprising a human TGFB1 (371 G > A) mutation in the gene of the present invention.

Corneal dystrophy is one of the autosomal dominant inherited diseases that the opacity of the corneal center is aggravated by aging and the visual acuity decreases. The TGFBI gene (human transforming growth factor b-induced gene), which is most popular in corneal dystrophy, has been identified by Munier et al. In 1997 as being located in human bands 31 and 32 of the chromosome 5 of the human chromosome, and abnormal keratoepithelin Lt; RTI ID = 0.0 > exon. ≪ / RTI > Although these TGFBI genes have recently been reported to have various alterations, they are generally known to be associated with five corneal dystrophies of Abelino corneal dystrophy, Rice-Vikler's corneal dystrophy, lattice keratopathy, granular keratopathy and Thiel-Behnke corneal dystrophy . After genetic testing, it has become possible to accurately diagnose corneal dystrophy, which has been blurred according to the morphology so far. Most cases reported in Korea or Japan, which were diagnosed as corneal stromal dysplasia, granulosa corneal dystrophy, Diagnosis was changed to granular dystrophy type II.

Avellino corneal dystrophy belongs to type 2 granular keratopathy, which is a mutation of the TGFBI gene that causes an opaque layer on the cornea that damages the cornea, which causes vision loss or blindness. It is not known that the precise cause of the development or the cause is unknown, and it is known that many wounds occur in the cornea and UVB, which is the living ultraviolet ray. In patients with Abelino corneal dystrophy, heterozygotes show a significant loss of visual acuity as age increases, and homozygotes blind from age 6. As a result of genetic testing, it is estimated that there are about 40,000 patients in Korea, showing a prevalence rate of 1/340 ~ 1 / 1,000 in Korea (in case of heterozygote).

In particular, it has been reported that the corneal dystrophy is exacerbated after LASIK procedure. Therefore, mutation test of TGFBI gene is essential to accurately diagnose corneal dystrophy and to prevent deterioration of corneal dystrophy after LASIK procedure. Therefore, it is necessary to perform accurate diagnosis of patients with Abelino corneal dystrophy before LASIK surgery, and to prevent symptom progression by LASIK surgery. However, in conventional ophthalmology, an ophthalmologist diagnoses Abelino corneal dystrophy only by examining the ocular opacity with a microscope. The patient who does not progress to the symptom is not found, .

Sequncing, conventional PCR, DNA microarray, and so on are currently used as SNP test methods for the codon 124 of TGFBI gene, which is the gene responsible for Avelino corneal dystrophy. (SNaPShot) method in which a primer is attached to a PCR product of TGFBi gene followed by elongation analysis of ddNTP corresponding to each mutation, a primer is attached to a PCR product of TGFBI gene, and then various enzymes (DNA polymerase, A pyrosequencing method for analyzing mutations by detecting oxyluciferin released by using a fluorophore, sulfurylase, luciferase, and apyrase by a CCD camera. However, these methods are not only time-consuming and expensive, but also require a different procedure after purification of the PCR product.

Korean Patent Laid-Open Publication No. 10-2007-0076532 discloses oligonucleotides for the diagnosis of corneal dystrophy, more specifically, oligonucleotides for detecting TGFBI gene mutation for diagnosing corneal dystrophy including abelinosis which should be accurately diagnosed before the ophthalmic correction surgery A DNA chip for diagnosing corneal dystrophy is disclosed, wherein the oligonucleotide is immobilized. However, a diagnostic method using a DNA chip requires several steps such as amplifying DNA in a sample, hybridizing the amplified DNA with a DNA chip, washing the hybridized DNA chip, and detecting a positive reaction And there is a problem that the false positive result may appear depending on the probe used in the DNA chip.

Therefore, in the field of diagnosis of Abelino corneal dystrophy, it is necessary to provide a method of rapidly and easily diagnosing Abelino corneal dystrophy with high sensitivity and specificity.

It is an object of the present invention to provide a primer set and a probe for diagnosing Abelino corneal dystrophy using real-time PCR method more efficiently and accurately for diagnosing Abelino corneal dystrophy.

It is still another object of the present invention to provide a convenient and accurate determination method by providing a formula and a determination table useful for diagnosing Abelino corneal dystrophy.

In order to accomplish the above object, the present invention provides a real-time PCR primer set for the diagnosis of Abellino-corneal dystrophy, which is represented by the nucleotide sequences of SEQ ID NOs: 1 and 11, and a real-time PCR primer for human internal positive control diagnosis represented by the nucleotide sequences of SEQ ID NOs: A real-time PCR probe for the diagnosis of Abelino corneal dystrophy, which is represented by SEQ ID NO: 27, and a real-time PCR probe for human internal positive control diagnosis, which is shown by SEQ ID NO: 32.

The present invention relates to a primer set comprising a mutation sequence of TGFBI gene involved in Abelino corneal dystrophy and a primer set comprising a DNA polymerase, a reaction buffer solution, MgCl 2 , dATP, dTTP, dGTP, dCTP, A reaction composition for real-time PCR containing labeled probes is provided.

The present invention also provides a method of using formulas and judgment tables provided to conveniently and accurately diagnose Abelino corneal dystrophy.

In accordance with the diagnostic method of the present invention, the allele specific polymerase chain reaction (AS-PCR) and the hydrolysis probe method are used to determine the cause of Abelino corneal dystrophy The mutation status of the TGFBI gene can be confirmed in real time in a single tube and the mutation in the human positive control group can be amplified together with the advantage of being able to easily judge the mutation or not and to judge the suitability of the nucleic acid used for the diagnostic test .

Using the primer set and the probe according to the present invention, it is possible to diagnose Abelino corneal dystrophy more rapidly and more accurately than a conventional method of diagnosing Abelino corneal dystrophy using a DNA microarray or PCR which is an end-point detection method, Positive control signals can be used to determine the effectiveness of the PCR and the quality of the extracted DNA, so that the results can be more accurately determined.

According to Example 3 and FIG. 5 (A), the concentration and purity of the nucleic acid extracted using a UV-Visible spectrophotometer were 100 ng / ㎕ or more and A260 / 280> 1.8, There seemed to be no major problems in using it. However, when real-time PCR was performed using these DNAs as template DNAs, it can be seen that it is very difficult to read the results when a small amount of ethyl alcohol and chaotropic salts exist in the template DNA as shown in FIG. 5 (B). As a result, if the quality of the nucleic acid used as the template DNA can not be precisely known, the result of false negativity may appear, which is very dangerous. In the case of the present invention, when the human internal positive control is not amplified, it can be determined that there is a serious problem with the nucleic acid used as the template DNA. In this case, false nucleic acid generation is performed again and real- It is possible to block the possibility of the occurrence of the problem. In addition, the diagnosis of Abellino corneal dystrophy is to discriminate the presence or absence of heterozygotes of TGFBI gene. Using the allele specific primer and Taqman probe, the difference of expression between the heterozygote and normal human is clear So that it can be easily read.

1 shows the nucleotide sequence of TGFBI gene related to Abelino corneal dystrophy of the present invention.
2 is a real-time PCR result (sensitivity) using the primers set forth in SEQ ID NOS: 1, 11, 21 and 24 of the present invention and the probes described in SEQ ID NOS: 27 and 32, respectively.
Fig. 3 is a formula and judgment table for determining Abelino corneal dystrophy of the present invention.
FIG. 4 shows the results of the diagnosis of Abelino corneal dystrophy according to Example 2. FIG.
Fig. 5 shows the effect of the quality of the extracted nucleic acid on the test result on the Example 3. Fig.

The present invention relates to a primer set for the diagnosis of Abellino keratopathies, which is represented by the nucleotide sequences of SEQ ID NOS: 1 to 20, a primer set for positive control, represented by the nucleotide sequences of SEQ ID Nos: 21 to 26, A probe for the diagnosis of Abelino corneal dystrophy, a positive control diagnostic probe represented by the nucleotide sequences of SEQ ID NOS: 32 and 33, and a method for diagnosing Abelino corneal dystrophy using the same.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a primer set for the diagnosis of Abellino corneal dystrophy, which is represented by a base sequence selected from the group consisting of SEQ ID NOS: 1 to 20 below;

Ave_F 5'-CATGCCTCCTCGTCCTCTCCA-3 '(SEQ ID NO: 1)

Ave_F 5'-CATCCCTCCTTCTGTCTTCTGC-3 '(SEQ ID NO: 2)

Ave_F 5'-TGCAGCCCTACCACTCTCAAAC-3 '(SEQ ID NO: 3)

Ave_F 5'-TCTCTGTCAGAGAAGGGAGGGT-3 '(SEQ ID NO: 4)

Ave_F 5'-TGCTCTCTGTCAGAGAAGGGAG-3 '(SEQ ID NO: 5)

Ave_R 5'-AGGCCTCAGCTTCTCCGTCT-3 '(SEQ ID NO: 6)

Ave_R 5'-AGGCCTCAGCTTCTCCGTAT-3 '(SEQ ID NO: 7)

Ave_R 5'-AGGCCTCAGCTTCTCCGTTT-3 '(SEQ ID NO: 8)

Ave_R 5'-AGGCCTCAGCTTCTCCGAGT-3 '(SEQ ID NO: 9)

Ave_R 5'-GGCCTCAGCTTCTCCGGGT-3 '(SEQ ID NO: 10)

Ave_R 5'-GGCCTCAGCTTCTCCGCGT-3 '(SEQ ID NO: 11)

Ave_R 5'-AGGCCTCAGCTTCTCCCTGT-3 '(SEQ ID NO: 12)

Ave_R 5'-AGGCCTCAGCTTCTCCATGT-3 '(SEQ ID NO: 13)

Ave_R 5'-AGGCCTCAGCTTCTCCTTGT-3 '(SEQ ID NO: 14)

Ave_R 5'-GGCCTCAGCTTCTCCCCGT-3 '(SEQ ID NO: 15)

Ave_R 5'-AGGCCTCAGCTTCTCCTCGT-3 '(SEQ ID NO: 16)

Ave_R 5'-AGGCCTCAGCTTCTCCACGT-3 '(SEQ ID NO: 17)

Ave_R 5'-GGCCTCAGCTTCTCCGCCT-3 '(SEQ ID NO: 18)

Ave_R 5'-AGGCCTCAGCTTCTCCGCAT-3 '(SEQ ID NO: 19)

Ave_R 5'-AGGCCTCAGCTTCTCCGCTT-3 '(SEQ ID NO: 20)

A real-time PCR primer set for a positive control test for Abelino corneal dystrophy, which is selected from the group consisting of the following SEQ ID NOS: 21 to 26;

IPC_F 5'-TCCAAATGCTGAGCGCAGATCC-3 '(SEQ ID NO: 21)

IPC_F 5'-GTCAGGCCTTGATGGGATCTTC-3 '(SEQ ID NO: 22)

IPC_F 5'-TTGTCTTCTGTCCACCAGCACC-3 '(SEQ ID NO: 23)

IPC_R 5'-TCGTGGTGTGAGGAAGAGCGATG-3 '(SEQ ID NO: 24)

IPC_R 5'-CCTTCAGATTCCAGCTGCTGGT-3 '(SEQ ID NO: 25)

IPC_R 5'-CCCTAAGATGCAGACTCACGC-3 '(SEQ ID NO: 26)

A real-time PCR probe for the diagnosis of Abelino corneal dystrophy, which is represented by a base sequence selected from the group consisting of SEQ ID NOS: 27 to 31 below; And

Ave_Probe 5 'FAM-TGGTTGGGCTGGACCCCCAG-BHQ1 3' (SEQ ID NO: 27)

Ave_Probe 5 'FAM-TGTAGATGTACCGTGCTCTCTGT-BHQ1 3' (SEQ ID NO: 28)

Ave_Probe 5 'FAM-TCCGTGTACAGCTGAGTGGTGG-BHQ1 3' (SEQ ID NO: 29)

Ave_Probe 5 'FAM-CTGCAGCCCTACCACTCTCAAA-BHQ1 3' (SEQ ID NO: 30)

Ave_Probe 5 'FAM-CTCCTCGTCCTCTCCACCTGTAG-BHQ1 3' (SEQ ID NO: 31)

A real-time PCR probe for positive control of Abelino corneal dystrophy which is represented by the nucleotide sequence of SEQ ID NO: 32 or 33 below

IPC_Probe 5 'HEX-CCGGGTTGCCAGCTCCCATG-BHQ1 3' (SEQ ID NO: 32)

IPC_Probe 5 'HEX-AGGGAGACAATAGCCCTGTCTC-BHQ1 3' (SEQ ID NO: 33),

To a composition for the diagnosis of Abelino corneal dystrophy.

In one aspect of the present invention, the present invention provides a reverse primer comprising a forward primer represented by a base sequence selected from the group consisting of the following SEQ ID NOS: 1 to 5 and a reverse primer represented by a base sequence selected from the group consisting of the following SEQ ID NOS: 6 to 20 Set of real-time PCR primers for the diagnosis of Abellino-corneal dystrophy:

Ave_F 5'-CATGCCTCCTCGTCCTCTCCA-3 '(SEQ ID NO: 1)

Ave_F 5'-CATCCCTCCTTCTGTCTTCTGC-3 '(SEQ ID NO: 2)

Ave_F 5'-TGCAGCCCTACCACTCTCAAAC-3 '(SEQ ID NO: 3)

Ave_F 5'-TCTCTGTCAGAGAAGGGAGGGT-3 '(SEQ ID NO: 4)

Ave_F 5'-TGCTCTCTGTCAGAGAAGGGAG-3 '(SEQ ID NO: 5)

Ave_R 5'-AGGCCTCAGCTTCTCCGTCT-3 '(SEQ ID NO: 6)

Ave_R 5'-AGGCCTCAGCTTCTCCGTAT-3 '(SEQ ID NO: 7)

Ave_R 5'-AGGCCTCAGCTTCTCCGTTT-3 '(SEQ ID NO: 8)

Ave_R 5'-AGGCCTCAGCTTCTCCGAGT-3 '(SEQ ID NO: 9)

Ave_R 5'-GGCCTCAGCTTCTCCGGGT-3 '(SEQ ID NO: 10)

Ave_R 5'-GGCCTCAGCTTCTCCGCGT-3 '(SEQ ID NO: 11)

Ave_R 5'-AGGCCTCAGCTTCTCCCTGT-3 '(SEQ ID NO: 12)

Ave_R 5'-AGGCCTCAGCTTCTCCATGT-3 '(SEQ ID NO: 13)

Ave_R 5'-AGGCCTCAGCTTCTCCTTGT-3 '(SEQ ID NO: 14)

Ave_R 5'-GGCCTCAGCTTCTCCCCGT-3 '(SEQ ID NO: 15)

Ave_R 5'-AGGCCTCAGCTTCTCCTCGT-3 '(SEQ ID NO: 16)

Ave_R 5'-AGGCCTCAGCTTCTCCACGT-3 '(SEQ ID NO: 17)

Ave_R 5'-GGCCTCAGCTTCTCCGCCT-3 '(SEQ ID NO: 18)

Ave_R 5'-AGGCCTCAGCTTCTCCGCAT-3 '(SEQ ID NO: 19)

Ave_R 5'-AGGCCTCAGCTTCTCCGCTT-3 '(SEQ ID NO: 20).

In one aspect of the present invention, the present invention provides a reverse primer comprising a forward primer represented by a base sequence selected from the group consisting of the following SEQ ID NOS: 21 to 23, and a reverse primer represented by a base sequence selected from the group consisting of SEQ ID NOS: Lt; RTI ID = 0.0 > of Abelino < / RTI > corneal aberration positive control:

IPC_F 5'-TCCAAATGCTGAGCGCAGATCC-3 '(SEQ ID NO: 21)

IPC_F 5'-GTCAGGCCTTGATGGGATCTTC-3 '(SEQ ID NO: 22)

IPC_F 5'-TTGTCTTCTGTCCACCAGCACC-3 '(SEQ ID NO: 23)

IPC_R 5'-TCGTGGTGTGAGGAAGAGCGATG-3 '(SEQ ID NO: 24)

IPC_R 5'-CCTTCAGATTCCAGCTGCTGGT-3 '(SEQ ID NO: 25)

IPC_R 5'-CCCTAAGATGCAGACTCACGC-3 '(SEQ ID NO: 26).

In one embodiment of the present invention, the present invention provides a real-time PCR probe for the diagnosis of Abelino corneal dystrophy, the base sequence being selected from the group consisting of the following SEQ ID NOS: 27 to 31:

Ave_Probe 5 'FAM-TGGTTGGGCTGGACCCCCAG-BHQ1 3' (SEQ ID NO: 27)

Ave_Probe 5 'FAM-TGTAGATGTACCGTGCTCTCTGT-BHQ1 3' (SEQ ID NO: 28)

Ave_Probe 5 'FAM-TCCGTGTACAGCTGAGTGGTGG-BHQ1 3' (SEQ ID NO: 29)

Ave_Probe 5 'FAM-CTGCAGCCCTACCACTCTCAAA-BHQ1 3' (SEQ ID NO: 30)

Ave_Probe 5 'FAM-CTCCTCGTCCTCTCCACCTGTAG-BHQ1 3' (SEQ ID NO: 31).

In one aspect according to the present invention, the present invention provides a real-time PCR probe for the positive control of Abelino corneal dystrophy, which is represented by the nucleotide sequence of SEQ ID NO: 32 or 33:

IPC_Probe 5 'HEX-CCGGGTTGCCAGCTCCCATG-BHQ1 3' (SEQ ID NO: 32)

IPC_Probe 5 'HEX-AGGGAGACAATAGCCCTGTCTC-BHQ1 3' (SEQ ID NO: 33).

In one aspect of the present invention, there is provided a primer set for the diagnosis of Abelino corneal dystrophy, characterized in that it is a primer set represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 11.

In one embodiment of the present invention, the present invention provides a primer set for the positive control of Abelino corneal dystrophy, characterized in that it is a primer set represented by the nucleotide sequence of SEQ ID NO: 21 and SEQ ID NO: 24.

In one embodiment of the present invention, the present invention provides a real-time PCR probe for the diagnosis of Abelino corneal dystrophy, which is a probe represented by the nucleotide sequence of SEQ ID NO:

In one aspect of the present invention, there is provided a real-time PCR probe for the diagnosis of positive control of Abelino corneal dystrophy, which is a probe represented by the nucleotide sequence of SEQ ID NO: 32.

In one embodiment of the present invention, the present invention provides a reverse primer set comprising a forward primer represented by the nucleotide sequence of SEQ ID NO: 1, a reverse primer set represented by the nucleotide sequence of SEQ ID NO: 11, a forward primer represented by SEQ ID NO: 21, A reverse primer set represented by a sequence; And a probe represented by the nucleotide sequence of SEQ ID NO: 27 and SEQ ID NO: 32.

In one embodiment of the present invention, the 5'end of the probe is a fluorescent label selected from the group consisting of FAM, HEX, VIC, TET, JOE, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED And the 3 'end of the probe may be labeled with one type of fluorescent suppressor selected from the group consisting of BHQ-1, 2,3, 6-TAMRA and MGBNFQ. More specifically, the probe for the diagnosis of Abelino corneal dystrophy may be labeled with FAM at the 5 'terminus, with HEX at the human-positive internal control probe, and with BHQ-1 at the 3' terminus.

In another aspect according to the present invention, there is provided a method for detecting a protein comprising: (a) collecting a sample and separating DNA therefrom; (b) performing PCR using the composition of claim 1; And (c) measuring the fluorescence intensity to derive a Cp value.

In the present invention, the term "diagnosis" means confirming a pathological condition. For the purpose of the present invention, the diagnosis means confirming whether or not the Abellino keratopathy develops, Including whether or not the disease has occurred, development and alleviation.

The term "sample" as used herein refers to a biological sample, which is obtained from a patient who has developed or progressed to Abelino corneal dystrophy or suspected of being Abelino corneal dystrophy. The sample may include, for example, but not limited to, tissues, cells, blood, serum, plasma, saliva and urine. Also, the nucleic acid obtained from the sample may be DNA or RNA

At this time, the Cp (Crossing point) value is a cycle number at a point at which the amplification curve rises most rapidly, that is, the rate of change of the amplification speed is greatest. The Cp value is calculated automatically using the 2nd Derivative Maximum Analysis method (Abs Quant / 2nd Derivative Max), which is a second derivative program embedded in the real-time PCR instrument. Since the time point at which the rate of change of the amplification rate is the largest is set as the Cp value, the Cp value is not changed by the minimum activity that causes the reaction, and the influence of the detection error of the apparatus can be excluded.

In one aspect of the present invention, a method for diagnosing Abelino corneal dystrophy is as follows:

When the Cp value of the positive control group derived in the step (c) is 33 or less, the positive determination is made when the Cp value of the avelino allyl is less than 40 and the Cp value is less than 10, If Cp is over 10, it is judged as negative.

If the Cp value of the positive control group derived in step (c) is more than 33, it is determined that the reaction is not valid.

Using the primer set and probe according to the present invention, it is possible to diagnose Abelino corneal dystrophy more rapidly and more accurately than the method of diagnosing Abelino corneal dystrophy using DNA microarray or PCR, and at the same time, The validity and the quality of the extracted DNA can be known, and the result can be more accurately judged. According to Example 3 and FIG. 5 (A), the concentration and purity of the nucleic acid extracted using a UV-Visible spectrophotometer were 100 ng / ㎕ or more and A260 / 280> 1.8, There seemed to be no major problems in using it. However, real-time PCR was performed using these DNAs as template DNAs. As a result, it was found that it was very difficult to read out the results when trace amounts of ethyl alcohol and chaotropic salts existed in the template DNA as shown in FIG. 5 (B). As a result, if the quality of the nucleic acid used as the template DNA can not be precisely understood, the false negative result can be obtained. According to the present invention, when the human internal positive control is not amplified, In this case, it is possible to block the possibility of generating false negatives by performing a real-time PCR after a new nucleic acid extraction is performed. In addition, the diagnosis of Abellino corneal dystrophy is to discriminate the presence or absence of heterozygotes of TGFBI gene. Using the allele specific primer and Taqman probe, the difference of expression between the heterozygote and normal human is clear So that it can be easily read.

Hereinafter, the present invention will be described in more detail based on embodiments of the present invention. It should be understood, however, that the following examples of the present invention are provided only for the purpose of understanding the present invention and do not limit the scope of the present invention.

Example 1: Preparation of primers and probes for real-time PCR

Nucleotides substituted with adenine at the 3 'end of the TGFBI gene so that only the mutation R124H (371 G > A) of the TGFBI gene can be detected specifically, and a reverse mutation primer located 207 bp Directional primer was designed and the Tm was set to 63 ° C respectively.

The probe designed to obtain the fluorescence signal from the PCR amplification product was placed between the forward primer and the reverse primer, FAM dye at the 5 'end, BHQ1 at the 3' end, and designed to have a Tm of 68 ° C.

In addition, a forward and reverse primer set was designed to amplify the human Killer cell Ig-like receptor CD158z (KIR3DL7) gene for the detection of human positive internal control, and Tm was set to 65 ° C respectively.

The probe designed to acquire the fluorescence signal of the human positive internal control from the PCR amplification product was placed between the forward and reverse primers, and the HEX dye was attached to the 5 'end and BHQ1 was attached to the 3' end. Respectively.

Ave_F 5'-CATGCCTCCTCGTCCTCTCCA-3 '(SEQ ID NO: 1)

Ave_R 5'-GGCCTCAGCTTCTCCGCGT-3 '(SEQ ID NO: 11)

IPC_F 5'-TCCAAATGCTGAGCGCAGATCC-3 '(SEQ ID NO: 21)

IPC_R 5'-TCGTGGTGTGAGGAAGAGCGATG-3 '(SEQ ID NO: 24)

Ave_Probe 5 'FAM-TGGTTGGGCTGGACCCCCAG-BHQ1 3' (SEQ ID NO: 27)

IPC_Probe 5 'HEX-CCGGGTTGCCAGCTCCCATG-BHQ1 3' (SEQ ID NO: 32)

Example 2: Diagnosis of Abelino corneal dystrophy using real-time PCR

Samples were taken from the blood and oral epithelial cells of the subject for DNA isolation. For DNA extraction, QIAGEN DNA Blood Mini Kit was used. The separated DNA was dissolved in 200 μl of elution buffer, and 5 μl of template DNA was used. The primers (SEQ ID NOS: 1, 11, 21 and 24) and probes (SEQ ID NOS: 27 and 32) prepared in Example 1 were used for the real time PCR reaction.

The primers added to the reaction solution for real-time PCR were 500 nM (SEQ ID NOS: 1 and 11) and 125 nM (SEQ ID NOS: 21 and 24), respectively. The probes were 250 nM No. 32), 20 μl of a master mix was prepared and used.

The real-time PCR reaction was performed using LC480II (Roche, USA) and 45 cycles of 95 ° C for 15 minutes, 95 ° C for 20 seconds, and 68 ° C for 40 seconds as one cycle. Fluorescence of FAM and HEX was measured for each cycle.

The determination of the result is made by determining the value of DELTA Cp when the Cp value of the human positive internal control is in the effective range.

1) Valid results are obtained when the Cp value of the human-positive internal control is less than 33. If the Cp value is more than 33, it is judged to be invalid.

2) When the Cp value of the human positive internal control is 33 or less, the avellino allele Cp value of the specimen is less than 40, and the Cp value difference (ΔCp) is 10 or less.

3) When the Cp value of the human positive internal control group is 33 or less, it is judged as negative if the difference (Cp) of the Cp value (ΔCp) is over 10 regardless of the avellino allele Cp value of the specimen.

4) If the Cp value of the human-positive internal control exceeds 33, it is judged to be an invalid reaction and retest.

Example 3: Prediction of inaccuracy of result determination depending on the state of extracted nucleic acid

(0.125 M, 0.25 M, and 0.5 M), respectively, were added to the clone DNA in the presence of 1.25%, 2.5%, and 5.0% ethyl alcohol and a chaotropic salt (Gu-HCl) The absorbance was measured using a UV-Visible spectrophotometer and the concentration and purity were calculated (Fig. 5 (A)). Each of the experimental sets exhibited a concentration of 100 ng / μl or more and a purity (A260 / 280) of 1.8 or more. A high-concentration clone DNA was used for quantification, and a 1/1000 serial dilution of the clone DNA was used as a template to perform real-time PCR according to the experimental method of Example 2, and the results were summarized (Fig. 5 (B)).

<110> GreenCrossMS <120> Composition for diagnosing avellino corneal dystrophy and          diagnostic method thereof <130> P2013-0147 <160> 34 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-1 <400> 1 catgcctcct cgtcctctcc a 21 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-2 <400> 2 catccctcct tctgtcttct gc 22 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-3 <400> 3 tgcagcccta ccactctcaa ac 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-4 <400> 4 tctctgtcag agaagggagg gt 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-5 <400> 5 tgctctctgt cagagaaggg ag 22 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-6 <400> 6 aggcctcagc ttctccgtct 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-7 <400> 7 aggcctcagc ttctccgtat 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-8 <400> 8 aggcctcagc ttctccgttt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-9 <400> 9 aggcctcagc ttctccgagt 20 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-10 <400> 10 ggcctcagct tctccgggt 19 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-11 <400> 11 ggcctcagct tctccgcgt 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-12 <400> 12 aggcctcagc ttctccctgt 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-13 <400> 13 aggcctcagc ttctccatgt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-14 <400> 14 aggcctcagc ttctccttgt 20 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-15 <400> 15 ggcctcagct tctccccgt 19 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-16 <400> 16 aggcctcagc ttctcctcgt 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-17 <400> 17 aggcctcagc ttctccacgt 20 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-18 <400> 18 ggcctcagct tctccgcct 19 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-19 <400> 19 aggcctcagc ttctccgcat 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-20 <400> 20 aggcctcagc ttctccgctt 20 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-21 <400> 21 tccaaatgct gagcgcagat cc 22 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-22 <400> 22 gtcaggcctt gatgggatct tc 22 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-23 <400> 23 ttgtcttctg tccaccagca cc 22 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-24 <400> 24 tcgtggtgtg aggaagagcg atg 23 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-25 <400> 25 ccttcagatt ccagctgctg gt 22 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Ave_F-26 <400> 26 ccctaagatg cagactcacg c 21 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ave_Probe-27 <400> 27 tggttgggct ggacccccag 20 <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Ave_Probe-28 <400> 28 tgtagatgta ccgtgctctc tgt 23 <210> 29 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Ave_Probe-29 <400> 29 tccgtgtaca gctgagtggt gg 22 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Ave_Probe-30 <400> 30 ctgcagccct accactctca aa 22 <210> 31 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Ave_Probe-31 <400> 31 ctcctcgtcc tctccacctg tag 23 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IPC_Probe -32 <400> 32 ccgggttgcc agctcccatg 20 <210> 33 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IPC_Probe -33 <400> 33 agggagacaa tagccctgtc tc 22 <210> 34 <211> 800 <212> DNA <213> Artificial Sequence <220> <223> avellino <400> 34 ctgtaagact agtttgtaat taggatatct gtttccagtg tccattcctg cctctgttat 60 ctaaatgtct gggaacaaga gctgtgctct gctgtgttta aaatgattaa aaatcaccaa 120 ttagttgagt tcacgtagac aggcatttga cttattgagt tgttttaaga agactataac 180 aagccttaag ccccccagaa acagcctgtc tttgggcttt cccacatgcc tcctcgtcct 240 ctccacctgt agatgtaccg tgctctctgt cagagaaggg agggtgtggt tgggctggac 300 ccccagaggc catccctcct tctgtcttct gctcctgcag ccctaccact ctcaaacctt 360 tacgagaccc tgggagtcgt tggatccacc accactcagc tgtacacgga ccgcacggag 420 aagctgaggc ctgagatgga ggggcccggc agcttcacca tcttcgcccc tagcaacgag 480 gcctgggcct ccttgccagc tgtgagatga cctccgtctg cccgggggac tcttatgggg 540 aactgcctta cttccccgag gggtgggcat gatgaatggg agtctgcagt catttcctac 600 tgtttcagga agctttctcc ttaacccctt agaaaaggct gtggaacttg agctaaaata 660 tgtcttacca ggttgcgtct aatgcccccc gttccctact gggcagaaag acttgggtgc 720 ttcctgagga gggatccttg gcagaagaga ggcctgggct cacgagggct gagaacatgt 780 ttcccagagt tgcaaggacc 800

Claims (14)

A primer set for diagnosing Abelino corneal dystrophy which is represented by a base sequence selected from the group consisting of the following SEQ ID NOS: 1 to 20;
Ave_F 5'-CATGCCTCCTCGTCCTCTCCA-3 '(SEQ ID NO: 1)
Ave_F 5'-CATCCCTCCTTCTGTCTTCTGC-3 '(SEQ ID NO: 2)
Ave_F 5'-TGCAGCCCTACCACTCTCAAAC-3 '(SEQ ID NO: 3)
Ave_F 5'-TCTCTGTCAGAGAAGGGAGGGT-3 '(SEQ ID NO: 4)
Ave_F 5'-TGCTCTCTGTCAGAGAAGGGAG-3 '(SEQ ID NO: 5)
Ave_R 5'-AGGCCTCAGCTTCTCCGTCT-3 '(SEQ ID NO: 6)
Ave_R 5'-AGGCCTCAGCTTCTCCGTAT-3 '(SEQ ID NO: 7)
Ave_R 5'-AGGCCTCAGCTTCTCCGTTT-3 '(SEQ ID NO: 8)
Ave_R 5'-AGGCCTCAGCTTCTCCGAGT-3 '(SEQ ID NO: 9)
Ave_R 5'-GGCCTCAGCTTCTCCGGGT-3 '(SEQ ID NO: 10)
Ave_R 5'-GGCCTCAGCTTCTCCGCGT-3 '(SEQ ID NO: 11)
Ave_R 5'-AGGCCTCAGCTTCTCCCTGT-3 '(SEQ ID NO: 12)
Ave_R 5'-AGGCCTCAGCTTCTCCATGT-3 '(SEQ ID NO: 13)
Ave_R 5'-AGGCCTCAGCTTCTCCTTGT-3 '(SEQ ID NO: 14)
Ave_R 5'-GGCCTCAGCTTCTCCCCGT-3 '(SEQ ID NO: 15)
Ave_R 5'-AGGCCTCAGCTTCTCCTCGT-3 '(SEQ ID NO: 16)
Ave_R 5'-AGGCCTCAGCTTCTCCACGT-3 '(SEQ ID NO: 17)
Ave_R 5'-GGCCTCAGCTTCTCCGCCT-3 '(SEQ ID NO: 18)
Ave_R 5'-AGGCCTCAGCTTCTCCGCAT-3 '(SEQ ID NO: 19)
Ave_R 5'-AGGCCTCAGCTTCTCCGCTT-3 '(SEQ ID NO: 20)
A real-time PCR primer set for a positive control test for Abelino corneal dystrophy, which is selected from the group consisting of the following SEQ ID NOS: 21 to 26;
IPC_F 5'-TCCAAATGCTGAGCGCAGATCC-3 '(SEQ ID NO: 21)
IPC_F 5'-GTCAGGCCTTGATGGGATCTTC-3 '(SEQ ID NO: 22)
IPC_F 5'-TTGTCTTCTGTCCACCAGCACC-3 '(SEQ ID NO: 23)
IPC_R 5'-TCGTGGTGTGAGGAAGAGCGATG-3 '(SEQ ID NO: 24)
IPC_R 5'-CCTTCAGATTCCAGCTGCTGGT-3 '(SEQ ID NO: 25)
IPC_R 5'-CCCTAAGATGCAGACTCACGC-3 '(SEQ ID NO: 26)
A real-time PCR probe for the diagnosis of Abelino corneal dystrophy, which is represented by a base sequence selected from the group consisting of SEQ ID NOS: 27 to 31 below; And
Ave_Probe 5 'FAM-TGGTTGGGCTGGACCCCCAG-BHQ1 3' (SEQ ID NO: 27)
Ave_Probe 5 'FAM-TGTAGATGTACCGTGCTCTCTGT-BHQ1 3' (SEQ ID NO: 28)
Ave_Probe 5 'FAM-TCCGTGTACAGCTGAGTGGTGG-BHQ1 3' (SEQ ID NO: 29)
Ave_Probe 5 'FAM-CTGCAGCCCTACCACTCTCAAA-BHQ1 3' (SEQ ID NO: 30)
Ave_Probe 5 'FAM-CTCCTCGTCCTCTCCACCTGTAG-BHQ1 3' (SEQ ID NO: 31)
A real-time PCR probe for positive control of Abelino corneal dystrophy which is represented by the nucleotide sequence of SEQ ID NO: 32 or 33 below
IPC_Probe 5 'HEX-CCGGGTTGCCAGCTCCCATG-BHQ1 3' (SEQ ID NO: 32)
IPC_Probe 5 'HEX-AGGGAGACAATAGCCCTGTCTC-BHQ1 3' (SEQ ID NO: 33)
Or a pharmaceutically acceptable salt thereof, for the diagnosis of Abelino corneal dystrophy.
Comprising a forward primer represented by the nucleotide sequence selected from the group consisting of SEQ ID NOS: 1 to 5 below and a reverse primer set represented by the nucleotide sequence selected from the group consisting of the following SEQ ID NOS: 6 to 20, Primer set:
Ave_F 5'-CATGCCTCCTCGTCCTCTCCA-3 '(SEQ ID NO: 1)
Ave_F 5'-CATCCCTCCTTCTGTCTTCTGC-3 '(SEQ ID NO: 2)
Ave_F 5'-TGCAGCCCTACCACTCTCAAAC-3 '(SEQ ID NO: 3)
Ave_F 5'-TCTCTGTCAGAGAAGGGAGGGT-3 '(SEQ ID NO: 4)
Ave_F 5'-TGCTCTCTGTCAGAGAAGGGAG-3 '(SEQ ID NO: 5)
Ave_R 5'-AGGCCTCAGCTTCTCCGTCT-3 '(SEQ ID NO: 6)
Ave_R 5'-AGGCCTCAGCTTCTCCGTAT-3 '(SEQ ID NO: 7)
Ave_R 5'-AGGCCTCAGCTTCTCCGTTT-3 '(SEQ ID NO: 8)
Ave_R 5'-AGGCCTCAGCTTCTCCGAGT-3 '(SEQ ID NO: 9)
Ave_R 5'-GGCCTCAGCTTCTCCGGGT-3 '(SEQ ID NO: 10)
Ave_R 5'-GGCCTCAGCTTCTCCGCGT-3 '(SEQ ID NO: 11)
Ave_R 5'-AGGCCTCAGCTTCTCCCTGT-3 '(SEQ ID NO: 12)
Ave_R 5'-AGGCCTCAGCTTCTCCATGT-3 '(SEQ ID NO: 13)
Ave_R 5'-AGGCCTCAGCTTCTCCTTGT-3 '(SEQ ID NO: 14)
Ave_R 5'-GGCCTCAGCTTCTCCCCGT-3 '(SEQ ID NO: 15)
Ave_R 5'-AGGCCTCAGCTTCTCCTCGT-3 '(SEQ ID NO: 16)
Ave_R 5'-AGGCCTCAGCTTCTCCACGT-3 '(SEQ ID NO: 17)
Ave_R 5'-GGCCTCAGCTTCTCCGCCT-3 '(SEQ ID NO: 18)
Ave_R 5'-AGGCCTCAGCTTCTCCGCAT-3 '(SEQ ID NO: 19)
Ave_R 5'-AGGCCTCAGCTTCTCCGCTT-3 '(SEQ ID NO: 20).
Comprising a forward primer represented by the nucleotide sequence selected from the group consisting of the following SEQ ID NOS: 21 to 23 and a reverse primer represented by the nucleotide sequence selected from the group consisting of the following SEQ ID NOS: 24 to 26: aberrant corneal dystrophy: PCR primer set:
IPC_F 5'-TCCAAATGCTGAGCGCAGATCC-3 '(SEQ ID NO: 21)
IPC_F 5'-GTCAGGCCTTGATGGGATCTTC-3 '(SEQ ID NO: 22)
IPC_F 5'-TTGTCTTCTGTCCACCAGCACC-3 '(SEQ ID NO: 23)
IPC_R 5'-TCGTGGTGTGAGGAAGAGCGATG-3 '(SEQ ID NO: 24)
IPC_R 5'-CCTTCAGATTCCAGCTGCTGGT-3 '(SEQ ID NO: 25)
IPC_R 5'-CCCTAAGATGCAGACTCACGC-3 '(SEQ ID NO: 26).
A real-time PCR probe for the diagnosis of Abelino corneal dystrophy represented by a base sequence selected from the group consisting of the following SEQ ID NOS: 27 to 31:
Ave_Probe 5 'FAM-TGGTTGGGCTGGACCCCCAG-BHQ1 3' (SEQ ID NO: 27)
Ave_Probe 5 'FAM-TGTAGATGTACCGTGCTCTCTGT-BHQ1 3' (SEQ ID NO: 28)
Ave_Probe 5 'FAM-TCCGTGTACAGCTGAGTGGTGG-BHQ1 3' (SEQ ID NO: 29)
Ave_Probe 5 'FAM-CTGCAGCCCTACCACTCTCAAA-BHQ1 3' (SEQ ID NO: 30)
Ave_Probe 5 'FAM-CTCCTCGTCCTCTCCACCTGTAG-BHQ1 3' (SEQ ID NO: 31).
Real-time PCR probe for positive control Abelino corneal dystrophy represented by the nucleotide sequence of SEQ ID NO: 32 or 33:
IPC_Probe 5 'HEX-CCGGGTTGCCAGCTCCCATG-BHQ1 3' (SEQ ID NO: 32)
IPC_Probe 5 'HEX-AGGGAGACAATAGCCCTGTCTC-BHQ1 3' (SEQ ID NO: 33).
3. The method according to claim 1 or 2,
Wherein the primer set for diagnosing Abelino corneal dystrophy is a primer set represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 11.
The method according to claim 1 or 3,
Wherein the Abelino corneal dystrophy positive control diagnostic primer set is a primer set represented by the nucleotide sequences of SEQ ID NO: 21 and SEQ ID NO: 24.
The method according to claim 1 or 4,
Wherein the real-time PCR probe is a probe represented by the nucleotide sequence of SEQ ID NO: 27.
6. The method according to claim 1 or 5,
Wherein the real-time PCR probe for the positive control of Abelino corneal dystrophy is a probe represented by the nucleotide sequence of SEQ ID NO: 32.
A reverse primer set represented by the nucleotide sequence of SEQ ID NO: 11, a forward primer represented by SEQ ID NO: 21 and a reverse primer set represented by the nucleotide sequence of SEQ ID NO: 24; And
A kit for diagnosing Abelino corneal dystrophy comprising a probe represented by the nucleotide sequence of SEQ ID NO: 27 and SEQ ID NO: 32,
Ave_F 5'-CATGCCTCCTCGTCCTCTCCA-3 '(SEQ ID NO: 1)
Ave_R 5'-GGCCTCAGCTTCTCCGCGT-3 '(SEQ ID NO: 11)
IPC_F 5'-TCCAAATGCTGAGCGCAGATCC-3 '(SEQ ID NO: 21)
IPC_R 5'-TCGTGGTGTGAGGAAGAGCGATG-3 '(SEQ ID NO: 24)
Ave_Probe 5 'FAM-TGGTTGGGCTGGACCCCCAG-BHQ1 3' (SEQ ID NO: 27)
IPC_Probe 5 'HEX-CCGGGTTGCCAGCTCCCATG-BHQ1 3' (SEQ ID NO: 32).
The method according to claim 1,
Wherein the 5'end of the probe is labeled with one fluorescent marker selected from the group consisting of FAM, HEX, VIC, TET, JOE, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED,
Wherein the 3 'end of the probe is labeled with one kind of a fluorescent repressor selected from the group consisting of BHQ-1, 2,3, 6-TAMRA and MGBNFQ.
(a) collecting a sample and separating DNA therefrom;
(b) performing PCR using the composition of claim 1; And
(c) measuring fluorescence and deriving a Cp value.
Diagnosis of Abelino corneal dystrophy.
13. The method of claim 12,
When the Cp value of the positive control group derived in the step (c) is 33 or less,
When the Cp value of the abelino allyl is less than 40 and the value of DELTA Cp is 10 or less, it is judged to be positive,
A method for diagnosing Abelino corneal dystrophy characterized in that, when? Cp is more than 10 regardless of the Cp value of the avelino allyl, it is judged as negative.
13. The method of claim 12,
Wherein the Cp value of the positive control group derived in the step (c) is more than 33, and it is determined that the reaction is not effective.
KR1020130044730A 2013-04-23 2013-04-23 Composition for diagnosing avellino corneal dystrophy and diagnostic method thereof KR101577109B1 (en)

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