CN111041084A - Detection kit for small fat Willi syndrome and use method thereof - Google Patents

Detection kit for small fat Willi syndrome and use method thereof Download PDF

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CN111041084A
CN111041084A CN201811189458.0A CN201811189458A CN111041084A CN 111041084 A CN111041084 A CN 111041084A CN 201811189458 A CN201811189458 A CN 201811189458A CN 111041084 A CN111041084 A CN 111041084A
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sno
seq
artificial sequence
sequence shown
lncrna
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陈玲玲
吴煌
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Center for Excellence in Molecular Cell Science of CAS
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Shanghai Institutes for Biological Sciences SIBS of CAS
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Abstract

The invention belongs to the field of biotechnology detection, and particularly provides a detection kit for a small fat Willi syndrome and a use method thereof.sno‑lncRNA1、sno‑lncRNA2、sno‑lncRNA3、sno‑ lncRNA4 andsno‑lncRNA5, they are highly expressed in human embryonic stem cells and are abnormally stable. The detection kit prepared by using the gene for detection can detect the samples to be detected, such as saliva, urine and the like besides the commonly used blood, and provides guarantee for realizing the non-invasive detection of the small fat Willi syndrome. The using method is simple to operate and has low requirements on instruments and equipment; the required sample input amount is small, and the method has strong inclusiveness to the sample source; has the characteristics of high sensitivity, specificity and the like; the detection result is visual, concise and understandable; the detection method can complete the small fat Willi syndrome check in a short time.

Description

Detection kit for small fat Willi syndrome and use method thereof
Technical Field
The invention belongs to the field of small fat Willi syndrome diagnosis research, and particularly relates to a small fat Willi syndrome detection kit and a use method thereof.
Background
The small fat Willi syndrome (PWS) is a complex clinical syndrome of multisystem disorder, and the neonatal morbidity is 1/12000-1/15000. 11 th 5 th 2018, the contents of the first group of rare diseases, including the 121 diseases such as the small fat Willi syndrome, are formulated and released by five committees such as the national health and health Committee, the department of scientific and technology, the department of industry and informatization, the national drug administration, the national traditional Chinese medicine administration and the like. The main clinical features of the small fat willi syndrome include: the new-born or infant has low muscular tension, slow development, obvious facial features (narrow face, narrow forehead, long head and almond eyes), rapid weight increase at the beginning of childhood, delayed puberty, dysplasia of gonad and slow intelligence development. Clinically, the small fat Willi syndrome is easy to misdiagnose into cerebral palsy, myasthenia, simple obesity and the like or missed diagnosis. The main genetic types of PWS patients are: the method comprises the following steps of (1) deleting 15q11-13 regions of a father chromosome (65-75%), obtaining a maternal monad diploid (20-30%), carrying out micro deletion or mutation at the imprinting center (1-3%) and carrying out balanced translocation of chromosomes (< 1%).
The level of relevant medical research for rare diseases is significantly delayed compared to common diseases. The medical community has limited understanding on the pathogenesis of a plurality of rare diseases (such as the small fat Willi syndrome), insufficient detection means and lacks an effective treatment method, so that a plurality of patients and families thereof face the dilemma that the patients are subjected to long-term medical consultation and can not be clearly detected; meanwhile, due to the reasons of unbalanced medical resource distribution and the like, a plurality of patients with rare diseases (such as small fat Willi syndrome) are not diagnosed in time, and the treatment intervention time is delayed. Therefore, it is very important to establish a detection method which is simple, efficient, easy to operate and wide in coverage. Currently, methods for detecting the small-fat Willi syndrome include Methylation-specific amplification (MS-PCR) and Methylation-specific multiplex amplification (MS-MLPA). The MS-PCR and MS-MLPA technologies are used for detecting the obesity-Willi syndrome by utilizing the molecular characteristic that the methylation condition of the genetic Imprinting Center (IC) of the region q11-q13 of the 15 th chromosome is different on a father-origin chromosome and a mother-origin chromosome. The two methods have the defects of large sample input amount, complex operation, difficult detection of microdeletion and the like. The development of a new type of detection marker of the small boat-fruited Willi syndrome and a related kit is necessary, and the simplification of the using method of the kit has important significance for early detection and early intervention treatment of the small boat-fruited Willi syndrome.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention aims to provide a method for using a detection kit for the small fat Willi syndrome.
In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:
in a first aspect of the invention, there is provided a test kit for the detection of puffer syndrome, the kit comprising any one or more of the following features:
(1) sno-lncRNA1 recombinase polymerase amplification specific primer, gRNA specifically targeting sno-lncRNA1, and Cas13
Protein, biotin-fluorescent reporting reagent and colloidal gold immunochromatographic test paper; (2) sno-lncRNA2 recombinase polymerase amplification
Specificity-increasing primer, gRNA specifically targeting sno-lncRNA2, Cas13 protein, biotin-fluorescent reporter reagent and kit
Colloidal gold immunochromatographic test paper; (3) sno-lncRNA3 recombinase polymerase amplification specific primer and specific targeting
sno-lncRNA3 gRNA, (4) sno-lncRNA4 recombinase polymerase amplification specific primer, target sno-lncRNA4
The gRNA, the Cas13 protein, the biotin-fluorescent reporter reagent and the colloidal gold immunochromatographic test paper; (5) sno-lncRNA5
Recombinase polymerase amplification specific primer, gRNA specifically targeting sno-lncRNA5, Cas13 protein, biotin-
A fluorescent reporting reagent and colloidal gold immunochromatographic test paper.
A plurality of items refers to two, three, four or five items.
Further, the nucleotide sequence of sno-lncRNA1 is shown in SEQ ID NO. 1.
Further, the nucleotide sequence of sno-lncRNA2 is shown in SEQ ID NO. 2.
Further, the nucleotide sequence of sno-lncRNA3 is shown in SEQ ID NO. 3.
Further, the nucleotide sequence of sno-lncRNA4 is shown in SEQ ID NO. 4.
Further, the nucleotide sequence of sno-lncRNA5 is shown in SEQ ID NO. 5.
In one embodiment, the colloidal gold immunochromatographic test strip comprises a base plate, and a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water absorption pad which are arranged on the surface of the base plate in sequence from a sample addition end, wherein the gold-labeled pad contains a rabbit anti-FITC antibody labeled with colloidal gold, and the nitrocellulose membrane is coated with a quality control line C and a detection line T.
Preferably, the colloidal gold-labeled anti-fluorophore antibody is a colloidal gold-labeled rabbit-derived anti-FITC antibody.
In one embodiment, the nitrocellulose membrane has a control line C located closer to the sample addition end and a detection line T located farther from the sample addition end.
In one embodiment, the control line C is coated with a biotin ligand.
In one embodiment, the detection line T is coated with an antibody capable of binding to a colloidal gold-labeled anti-fluorophore antibody.
Preferably, the antibody capable of binding to the colloidal gold-labeled anti-fluorophore antibody is an anti-rabbit antibody.
In one embodiment, the kit further comprises any one or more of the following features:
(1) sno-lncRNA1 recombinase polymerase amplification specific primers comprise an upstream primer and a downstream primer; (2) sno-lncRNA2 recombinase polymerase amplification specific primers comprise an upstream primer and a downstream primer; (3) sno-lncRNA3 recombinase polymerase amplification specific primers comprise an upstream primer and a downstream primer; (4) sno-lncRNA4 recombinase polymerase amplification specific primers comprise an upstream primer and a downstream primer; (5) sno-lncRNA5 recombinase polymerase amplification specific primers include an upstream primer and a downstream primer.
In one embodiment, the kit further comprises any one or more of the following features:
(1) the recombinase polymerase amplification specific primer sequence of the sno-lncRNA1 is selected from primers of the following sequences: an upstream primer sno-lncRNA1-RP1F shown as a sequence SEQ ID NO.6, a downstream primer sno-lncRNA1-RP1R shown as a sequence SEQ ID NO.7, an upstream primer sno-lncRNA1-RP2F shown as a sequence SEQ ID NO.8, a downstream primer sno-lncRNA1-RP2R shown as a sequence SEQ ID NO.9, an upstream primer sno-lncRNA1-RP3F shown as a sequence SEQ ID NO.10, a downstream primer sno-lncRNA1-RP3R shown as a sequence SEQ ID NO.11, an upstream primer sno-lncRNA1-RP4F shown as a sequence SEQ ID NO.12, and a downstream primer sno-lncRNA1-RP4R shown as a sequence SEQ ID NO. 13;
(2) the recombinase polymerase amplification specific primer sequence of sno-lncRNA2 is selected from an upstream primer sno-lncRNA2-RP1F with the sequence shown in SEQ ID NO.14, a downstream primer sno-lncRNA2-RP1R with the sequence shown in SEQ ID NO.15, an upstream primer sno-lncRNA2-RP2F with the sequence shown in SEQ ID NO.16, a downstream primer sno-lncRNA2-RP 22 with the sequence shown in SEQ ID NO.17, an upstream primer 2-lncRNA 2-RP 32 with the sequence shown in SEQ ID NO.18, a downstream primer 2-lncRNA 2-RP 32 with the sequence shown in SEQ ID NO.19, an upstream primer 2-lncRNA 2-2 with the sequence shown in SEQ ID NO.20, a downstream primer 2-lncRNA 2-2 with the sequence shown in SEQ ID NO.21, and an upstream primer 2-lncRNA 2-2 with the sequence shown in SEQ ID NO.2, A downstream primer sno-lncRNA2-RP5R with the sequence shown in SEQ ID NO. 23;
(3) the recombinase polymerase amplification specific primer sequence of the sno-lncRNA3 is selected from primers of the following sequences: an upstream primer sno-lncRNA3-RP1F with a sequence shown in SEQ ID NO.24, a downstream primer sno-lncRNA3-RP1R with a sequence shown in SEQ ID NO.25, an upstream primer sno-lncRNA3-RP2F with a sequence shown in SEQ ID NO.26, a downstream primer sno-lncRNA sno-RP 2 sno with a sequence shown in SEQ ID NO.27, an upstream primer sno-lncRNA sno-RP 3 sno with a sequence shown in SEQ ID NO.28, a downstream primer sno-lncRNA sno-RP 3 sno with a sequence shown in SEQ ID NO.29, an upstream primer sno-lncRNA sno-RP 4 with a sequence shown in SEQ ID NO.30, a downstream primer sno-lncRNA sno-RP 4 sno with a sequence shown in SEQ ID NO.31, an upstream primer sno-lncRNA sno-RP 72 with a sequence shown in SEQ ID NO.32 and a downstream primer sno-lncRNA sno-sno with a sequence shown in SEQ ID NO. 72 are shown in SEQ ID NO. sno;
(4) the recombinase polymerase amplification specific primer sequence of the sno-lncRNA4 is selected from primers of the following sequences: an upstream primer sno-lncRNA4-RP1F with a sequence shown in SEQ ID NO.34, a downstream primer sno-lncRNA4-RP1R with a sequence shown in SEQ ID NO.35, an upstream primer sno-lncRNA4-RP2F with a sequence shown in SEQ ID NO.36, a downstream primer sno-lncRNA sno-RP 2 sno with a sequence shown in SEQ ID NO.37, an upstream primer sno-lncRNA sno-RP 3 sno with a sequence shown in SEQ ID NO.38, a downstream primer sno-lncRNA sno-RP 3 sno with a sequence shown in SEQ ID NO.39, an upstream primer sno-lncRNA sno-RP 4 with a sequence shown in SEQ ID NO.40, a downstream primer sno-lncRNA sno-RP 72 with a sequence shown in SEQ ID NO.41, an upstream primer sno-lncRNA sno-RP 72 with a sequence shown in SEQ ID NO.42, and a downstream primer sno-lncRNA sno-sno with a sequence shown in SEQ ID NO. 72, wherein a sequence shown in SEQ ID NO. sno are shown in SEQ ID NO. sno;
(5) the recombinase polymerase amplification specific primer sequence of the sno-lncRNA5 is selected from primers of the following sequences: an upstream primer sno-lncRNA5-RP1F with the sequence shown in SEQ ID NO.44, a downstream primer sno-lncRNA5-RP1R with the sequence shown in SEQ ID NO.45, an upstream primer sno-lncRNA5-RP2F with the sequence shown in SEQ ID NO.46, a downstream primer sno-lncRNA sno-RP 2 sno with the sequence shown in SEQ ID NO.47, an upstream primer sno-lncRNA sno-RP 3 sno with the sequence shown in SEQ ID NO.48, a downstream primer sno-lncRNA sno-RP 3 sno with the sequence shown in SEQ ID NO.49, an upstream primer sno-lncRNA sno-RP 4 with the sequence shown in SEQ ID NO.50, a downstream primer sno-lncRNA sno-RP 72 with the sequence shown in SEQ ID NO.51, an upstream primer sno-lncRNA sno-RP 72 with the sequence shown in SEQ ID NO.52, and a downstream primer sno-lncRNA sno with the sequence shown in SEQ ID NO. 72-sno, and a downstream primer sno-lncRNA sno with the sequence shown in SEQ ID NO. 72;
preferably, the recombinase polymerase amplification specific primers of sno-lncRNA1 are sno-lncRNA1-RP1F, sno-lncRNA1-RP 3R.
Preferably, the recombinase polymerase amplification specific primers of sno-lncRNA2 are sno-lncRNA2-RP1F, sno-lncRNA2-RP 5R.
Preferably, the recombinase polymerase amplification specific primers of sno-lncRNA3 are sno-lncRNA3-RP3F, sno-lncRNA3-RP 3R.
Preferably, the recombinase polymerase amplification specific primers of sno-lncRNA4 are sno-lncRNA4-RP4F, sno-lncRNA4-RP 5R.
Preferably, the recombinase polymerase amplification specific primers of sno-lncRNA5 are sno-lncRNA5-RP4F, sno-lncRNA5-RP 1R.
In one embodiment, the kit further comprises one or more of the following features:
(1) the gRNA sequence of specific target sno-lncRNA1 is selected from SEQ ID NO.54-SEQ ID NO. 62;
(2) the gRNA sequence of the specific target sno-lncRNA2 is selected from SEQ ID NO.63-SEQ ID NO. 94;
(3) the gRNA sequence of specific target sno-lncRNA3 is selected from SEQ ID NO.95-SEQ ID NO. 122;
(4) the gRNA sequence of specific target sno-lncRNA4 is selected from SEQ ID NO.123-SEQ ID NO. 141;
(5) the gRNA sequence specifically targeting sno-lncRNA5 is selected from SEQ ID NO.142-SEQ ID NO. 168.
Preferably, the biotin fluorescent reporter reagent comprises a fluorescent marker-RNA-biotin marker complex, wherein in the fluorescent marker-RNA-biotin marker complex, an RNA sequence is selected from SEQ ID NO.169-SEQ ID NO. 172.
Preferably, the amino acid sequence of the Cas13 protein is shown as SEQ ID No. 173.
The Cas13 protein can be combined with a gRNA specifically targeting sno-lncRNA1, a gRNA specifically targeting sno-lncRNA2, a gRNA specifically targeting sno-lncRNA3, a gRNA specifically targeting sno-lncRNA4 or a gRNA specifically targeting sno-lncRNA5, and is combined with a target gene sno-lncRNA1, sno-lncRNA2, sno-lncRNA3, sno-lncRNA4 and sno-lncRNA5 under the mediation of the combination, so that the Cas13 protein is activated to form a sno-lncRNA-gRNA complex which reacts with a biotin-fluorescence reporter reagent.
In one embodiment, the kit comprises one or more of the following features: an RNase inactivating agent, a DNA removing agent, a recombinase polymerase amplification reaction reagent, T7RNA polymerase, rNTP, Cas13 protein reaction buffer and yeastRNA. The RNase inactivator is used for inhibiting the activity of RNase in a sample, so that the RNase inactivator can be used for inhibiting the degradation of the RNase in the sample on RNA and effectively protecting the RNA to be detected. The DNA remover is used for removing the interfering genes in the RNA of the sample. The rNTP is ribonucleoside triphosphate, and plays a role of raw materials in RNA synthesis; the yeast tRNA is a yeast tRNA and is used for improving specificity.
Preferably, the RNase inactivation solution is TCEP/EDTA solution; the DNA remover is TURBO DNA-free Kit.
Preferably, the recombinase polymerase amplification reaction reagents comprise: amplification system dry powder and an amplification reaction buffer solution, wherein the amplification reaction buffer solution is PEG3000 aqueous solution.
Preferably, the immunochromatographic test strip is a HybriDetect test strip.
In a second aspect of the present invention, there is provided a method for using the detection kit for the small boat-fruited Willi syndrome as described above, comprising at least the following steps:
1) extracting RNA of a sample to be detected;
2) amplifying the sample RNA in step 1) with a recombinase polymerase of sno-lncRNA1, sno-lncRNA2, sno-lncRNA3, sno-lncRNA4, or sno-lncRNA5 to amplify the specific primer-mediated recombinase polymerase;
3) combining the amplified sample RNA obtained in the step 2) with one or more of gRNA specifically targeting sno-lncRNA1, gRNA specifically targeting sno-lncRNA2, gRNA specifically targeting sno-lncRNA3, gRNA specifically targeting sno-lncRNA4 and gRNA specifically targeting sno-lncRNA5 and Cas13 protein to activate the Cas13 protein, reacting with a biotin-fluorescence reporter reagent, dripping the reacted mixture onto an immunochromatographic test strip, and reflecting the expression condition of molecular markers sno-lncRNA1, sno-lncRNA2, sno-lncRNA3, sno-lncRNA4 or sno-lncRNA5 in the sample RNA according to the change condition of a detection band of the immunochromatographic test strip.
Specifically, the activated Cas13 protein reacts with the biotin-fluorescent reporter to cleave the biotin-fluorescent reporter, freeing the biotin group and the fluorophore. When the reacted mixture is dripped on the immunochromatographic test paper, the mixture moves forwards through capillary action to dissolve the colloidal gold labeled rabbit-derived anti-FITC antibody on the gold label pad, the colloidal gold labeled rabbit-derived anti-FITC antibody can be combined with FITC fluorescent group and then moves to the nitrocellulose membrane, the biotin group is intercepted on the quality control line C containing biotin ligand, the combined substance of the reacted mixture and the gold labeled reagent is specifically combined with the anti-rabbit antibody on the detection line T to be intercepted and gathered on the detection line T, and the chromogenic result can be observed through naked eyes.
Further, any one or more of the following features are included in the method:
(1) sno-lncRNA1 recombinase polymerase amplification specific primers comprise an upstream primer and a downstream primer; (2) sno-lncRNA2 recombinase polymerase amplification specific primers comprise an upstream primer and a downstream primer; (3) sno-lncRNA3 recombinase polymerase amplification specific primers comprise an upstream primer and a downstream primer; (4) sno-lncRNA4 recombinase polymerase amplification specific primers comprise an upstream primer and a downstream primer; (5) sno-lncRNA5 recombinase polymerase amplification specific primers include an upstream primer and a downstream primer.
Further, any one or more of the following features are included in the method:
(1) the recombinase polymerase amplification specific primer sequence of the sno-lncRNA1 is selected from primers of the following sequences: an upstream primer sno-lncRNA1-RP1F shown as a sequence SEQ ID NO.6, a downstream primer sno-lncRNA1-RP1R shown as a sequence SEQ ID NO.7, an upstream primer sno-lncRNA1-RP2F shown as a sequence SEQ ID NO.8, a downstream primer sno-lncRNA1-RP2R shown as a sequence SEQ ID NO.9, an upstream primer sno-lncRNA1-RP3F shown as a sequence SEQ ID NO.10, a downstream primer sno-lncRNA1-RP3R shown as a sequence SEQ ID NO.11, an upstream primer sno-lncRNA1-RP4F shown as a sequence SEQ ID NO.12, and a downstream primer sno-lncRNA1-RP4R shown as a sequence SEQ ID NO. 13;
(2) the recombinase polymerase amplification specific primer sequence of sno-lncRNA2 is selected from an upstream primer sno-lncRNA2-RP1F with the sequence shown in SEQ ID NO.14, a downstream primer sno-lncRNA2-RP1R with the sequence shown in SEQ ID NO.15, an upstream primer sno-lncRNA2-RP2F with the sequence shown in SEQ ID NO.16, a downstream primer sno-lncRNA2-RP 22 with the sequence shown in SEQ ID NO.17, an upstream primer 2-lncRNA 2-RP 32 with the sequence shown in SEQ ID NO.18, a downstream primer 2-lncRNA 2-RP 32 with the sequence shown in SEQ ID NO.19, an upstream primer 2-lncRNA 2-2 with the sequence shown in SEQ ID NO.20, a downstream primer 2-lncRNA 2-2 with the sequence shown in SEQ ID NO.21, and an upstream primer 2-lncRNA 2-2 with the sequence shown in SEQ ID NO.2, A downstream primer sno-lncRNA2-RP5R with the sequence shown in SEQ ID NO. 23;
(3) the recombinase polymerase amplification specific primer sequence of the sno-lncRNA3 is selected from primers of the following sequences: an upstream primer sno-lncRNA3-RP1F with a sequence shown in SEQ ID NO.24, a downstream primer sno-lncRNA3-RP1R with a sequence shown in SEQ ID NO.25, an upstream primer sno-lncRNA3-RP2F with a sequence shown in SEQ ID NO.26, a downstream primer sno-lncRNA sno-RP 2 sno with a sequence shown in SEQ ID NO.27, an upstream primer sno-lncRNA sno-RP 3 sno with a sequence shown in SEQ ID NO.28, a downstream primer sno-lncRNA sno-RP 3 sno with a sequence shown in SEQ ID NO.29, an upstream primer sno-lncRNA sno-RP 4 with a sequence shown in SEQ ID NO.30, a downstream primer sno-lncRNA sno-RP 4 sno with a sequence shown in SEQ ID NO.31, an upstream primer sno-lncRNA sno-RP 72 with a sequence shown in SEQ ID NO.32 and a downstream primer sno-lncRNA sno-sno with a sequence shown in SEQ ID NO. 72 are shown in SEQ ID NO. sno;
(4) the recombinase polymerase amplification specific primer sequence of the sno-lncRNA4 is selected from primers of the following sequences: an upstream primer sno-lncRNA4-RP1F with a sequence shown in SEQ ID NO.34, a downstream primer sno-lncRNA4-RP1R with a sequence shown in SEQ ID NO.35, an upstream primer sno-lncRNA4-RP2F with a sequence shown in SEQ ID NO.36, a downstream primer sno-lncRNA sno-RP 2 sno with a sequence shown in SEQ ID NO.37, an upstream primer sno-lncRNA sno-RP 3 sno with a sequence shown in SEQ ID NO.38, a downstream primer sno-lncRNA sno-RP 3 sno with a sequence shown in SEQ ID NO.39, an upstream primer sno-lncRNA sno-RP 4 with a sequence shown in SEQ ID NO.40, a downstream primer sno-lncRNA sno-RP 72 with a sequence shown in SEQ ID NO.41, an upstream primer sno-lncRNA sno-RP 72 with a sequence shown in SEQ ID NO.42, and a downstream primer sno-lncRNA sno-sno with a sequence shown in SEQ ID NO. 72, wherein a sequence shown in SEQ ID NO. sno are shown in SEQ ID NO. sno;
(5) the recombinase polymerase amplification specific primer sequence of the sno-lncRNA5 is selected from primers of the following sequences: an upstream primer sno-lncRNA5-RP1F with the sequence shown in SEQ ID NO.44, a downstream primer sno-lncRNA5-RP1R with the sequence shown in SEQ ID NO.45, an upstream primer sno-lncRNA5-RP2F with the sequence shown in SEQ ID NO.46, a downstream primer sno-lncRNA sno-RP 2 sno with the sequence shown in SEQ ID NO.47, an upstream primer sno-lncRNA sno-RP 3 sno with the sequence shown in SEQ ID NO.48, a downstream primer sno-lncRNA sno-RP 3 sno with the sequence shown in SEQ ID NO.49, an upstream primer sno-lncRNA sno-RP 4 with the sequence shown in SEQ ID NO.50, a downstream primer sno-lncRNA sno-RP 72 with the sequence shown in SEQ ID NO.51, an upstream primer sno-lncRNA sno-RP 72 with the sequence shown in SEQ ID NO.52, and a downstream primer sno-lncRNA sno with the sequence shown in SEQ ID NO. 72-sno, and a downstream primer sno-lncRNA sno with the sequence shown in SEQ ID NO. 72;
further, any one or more of the following features are included in the method:
(1) the gRNA sequence of specific target sno-lncRNA1 is selected from SEQ ID NO.54-SEQ ID NO. 62;
(2) the gRNA sequence of the specific target sno-lncRNA2 is selected from SEQ ID NO.63-SEQ ID NO. 94;
(3) the gRNA sequence of specific target sno-lncRNA3 is selected from SEQ ID NO.95-SEQ ID NO. 122;
(4) the gRNA sequence of specific target sno-lncRNA4 is selected from SEQ ID NO.123-SEQ ID NO. 141;
(5) the gRNA sequence specifically targeting sno-lncRNA5 is selected from SEQ ID NO.142-SEQ ID NO. 168.
Further, the biotin-fluorescent reporter reagent comprises a fluorescent marker-RNA-biotin marker complex, wherein in the fluorescent marker-RNA-biotin marker complex, an RNA sequence is selected from SEQ ID NO.169 to SEQ ID NO. 172.
The invention has the following advantages and beneficial effects:
(1) sno-lncRNAs protected by a protein complex snoRNP at two ends are a novel biomarker, and are different from a traditional detection marker for the small fat Willi syndrome, namely DNA methylation. The novel RNA marker has the characteristics of stability, easiness in detection and the like. These features will improve the sensitivity and specificity of detection of the obesity willi syndrome. The successful development of the RNA markers is beneficial to the detection of the small fat Willi syndrome, and meanwhile, reference significance is provided for the research and development of biomarkers of other diseases.
(2) The invention realizes the amplification of the substrate RNA to be detected by utilizing recombinase polymerase isothermal amplification nucleic acid segments and combining an in vitro transcription technology, so that the detection of trace samples becomes possible. In addition, the method greatly enriches the sources of the samples to be detected, and the samples to be detected can detect saliva, urine and other samples besides the commonly used blood, thereby providing guarantee for realizing the non-invasive examination of the small fat Willi syndrome.
(3) The invention utilizes the auxiliary cleavage characteristic of Cas13 protein and combines with lateral chromatography test paper, so that the result of detecting substrate RNA is visualized, and an interpretation instruction of the detection result is provided. The presentation of the detection result of the small fat Willi syndrome becomes very simple, clear, popular and easy to understand.
(4) The invention provides a method for detecting a small fat Willi syndrome. The detection method is simple to operate and has low requirements on instruments and equipment; the required sample input amount is small, and the method has strong inclusiveness to the sample source; has the characteristics of high sensitivity, specificity and the like; the detection result is visual, concise and understandable; the detection of the small fat Willi syndrome can be completed in a short time by using the kit. The above innovations are beneficial to the common use of the detection kit for the small fat Willi syndrome in the primary medical unit, and the rapid detection of the small fat Willi syndrome of the newborn is greatly promoted, so that the doctor can take intervention measures in time.
Drawings
FIG. 1: and (3) detecting the inhibition of the RNase activity in the blood sample by the RNase inactivating agent.
FIG. 2: sno-lncRNA1, sno-lncRNA2, sno-lncRNA3, sno-lncRNA4 and sno-lncRNA5 recombinase polymerase amplification primer screening results.
FIG. 3: results of the screening for the auxiliary efficiency of the gRNA mediated Cas13 specifically targeting sno-lncRNA1, sno-lncRNA2, sno-lncRNA3, sno-lncRNA4 and sno-lncRNA 5.
FIG. 4: sno-lncRNA1, sno-lncRNA2, sno-lncRNA3, sno-lncRNA4 and sno-lncRNA5 molecular markers are combined to be used as biomarkers and PCR product detection results.
FIG. 5: and detecting the detection result of the expression quantity of the corresponding molecular marker in the positive control substance and the negative control substance of the Pickuri syndrome by using the specific PCR primer.
FIG. 6: and detecting the detection result of the expression quantity of the corresponding molecular marker in the blood sample of the patient with the small fat Willi syndrome by using a specific PCR primer.
FIG. 7: the sensitivity and specificity of sno-lncRNA1, sno-lncRNA2, sno-lncRNA3, sno-lncRNA4 and sno-lncRNA5 in the normal group and the small fat Willi syndrome patient group are analyzed by using a ROC curve.
FIG. 8: the expression condition of the molecular marker in the samples (blood) of normal people and patients with the small fat Willi syndrome and the description schematic diagram of the detection result are detected by using the small fat Willi syndrome detection kit.
FIG. 9: the use flow chart of the kit.
Detailed Description
Long noncoding RNAs (lncRNAs) are a class of RNAs that are greater than 200 nucleotides in length and lack the ability to encode proteins. LncRNA can participate in a series of gene expression regulation, has complex biological functions, and plays an important role in biological processes, such as chromatin modification, X chromosome inactivation, regulation of gene transcription, protein translation, and the like. Abnormal expression regulation of LncRNA can lead to the development of diseases such as tumor, amyotrophic lateral sclerosis, alzheimer's disease. The long noncoding RNA sno-lncRNAs are transcribed from a PWS core region, small nucleolar RNA (snorRNA) cap structures are arranged at two ends, and snoRNP and snoRNA are combined to form a stable complex, so that sno-lncRNAs have extremely high stability. sno-lncRNAs are widely present in various tissues and cells of human body, and the expression deletion of sno-lncRNAs can cause PWS diseases. These characteristics make sno-lncRNAs have wide prospects in the development and application of methods for detecting and treating the small fat Willi syndrome.
The inventors have found, through long-term studies, lncRNA (sno-lncRNAs) from introns ending at both ends with small nucleolar RNA (snorRNA). Five sno-lncRNAs exist in the human disease small fat Willi syndrome close association region, are respectively named as sno-lncRNA1, sno-lncRNA2, sno-lncRNA3, sno-lncRNA4 and sno-lncRNA5, and have extremely high expression amount and abnormal stability in human embryonic stem cells. The gene expression of the region q11-q13 of chromosome 15 in a small fat Willi syndrome patient is deleted, and the minimum core deletion region is 108 kb. Normally, long noncoding RNAs such as sno-lncRNAs are produced from this region.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
EXAMPLE 1 Rapid extraction of nucleic acids from clinical samples (e.g., blood, saliva, urine)
To achieve rapid extraction of nucleic acids, the inventors added rnase inactivator to the sample, followed by 95 ℃ lysis of the cells, releasing the nucleic acids. The method comprises adding RNase inactivator (TCEP/EDTA with final concentration of 100mM/1 mM) into sample (such as blood, saliva, urine), blowing and mixing with sterile gun head (Thermo QSP), heating at 95 deg.C for 10min, centrifuging at 14000rpm for 10min, and collecting supernatant. RNase alert Lab Test Kit v2(Thermo) is used for detecting the activity of RNA enzyme in the supernatant, real-time fluorescence change is monitored by a multifunctional microplate detector (BioTek synergy NEO) (shown in figure 1), the RNA enzyme inactivator can effectively inhibit the activity of RNA enzyme in a sample (blood), so that the RNA degradation is inhibited, the RNA to be detected is effectively protected, and further, the DNA in the supernatant is effectively removed by a TURBO DNA-free Kit (Thermo). Clinical samples were collected from the subsidiary children hospital of Zhejiang university medical college.
Example 2 screening of efficient and specific primers for recombinase polymerase amplification against molecular markers of sno-lncRNAs
2.1 design and Synthesis of specific recombinase polymerase amplification primers
The sno-lncRNAs molecular marker combination of small fat Willi syndrome comprises 5 long noncoding RNAs, namely sno-lncRNA1, sno-lncRNA2, sno-lncRNA3, sno-lncRNA4 and sno-lncRNA 5. The sequence is in NCBI GeneExpression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/(ii) a access number gse 38541). In order to realize micro-detection, the invention utilizes recombinase polymerase to amplify sno-lncRNAs molecular markers. The recombinase polymerase amplification primers of the molecular marker combination of the Pictures wirley syndrome sno-lncRNAs are multi-pair specific primers, and the primers are designed by an NICB Primer-BLAST online Primer design tool (https:// www.ncbi.nlm.nih.gov/tools/primer-blast/) Designed, the primer is synthesized by Beijing Optimalaceae New Biotechnology Co. The nucleotide sequence of the specific recombinase polymerase amplification primer is as follows:
TABLE 1
Figure BDA0001827100700000111
Figure BDA0001827100700000121
Figure BDA0001827100700000131
Figure BDA0001827100700000141
2.2 recombinase polymerase amplification primers with high semi-orthogonal screening efficiency and strong specificity
By using
Figure BDA0001827100700000142
The Basic RT kit (twist Dx) is used for screening recombinase polymerase amplification primers with high efficiency and strong specificity aiming at the molecular marker. The recombinase polymerase amplification reaction system and the process are as follows:
Figure BDA0001827100700000143
the amplification products were separated by electrophoresis on a 2.5% TAE agarose gel (containing ethidium bromide) and the results are shown in FIG. 2.
As can be seen from the results, the efficiency of amplification using the following primers was high:
sno-lncRNA1, sno-lncRNA1-RP1F, sno-lncRNA1-RP 3R.
sno-lncRNA2, sno-lncRNA2-RP1F, sno-lncRNA2-RP 5R.
sno-lncRNA3, sno-lncRNA3-RP3F, sno-lncRNA3-RP 3R.
sno-lncRNA4, sno-lncRNA4-RP4F, sno-lncRNA4-RP 5R.
sno-lncRNA4, sno-lncRNA5-RP4F, sno-lncRNA5-RP 1R.
Example 3 screening of gRNA (CRISPR guide RNA) for molecular markers of sno-lncRNAs with high efficiency and specificity
The nucleotide sequence of gRNA is as follows:
TABLE 2
Figure BDA0001827100700000151
Figure BDA0001827100700000161
Figure BDA0001827100700000171
Figure BDA0001827100700000181
Figure BDA0001827100700000191
Figure BDA0001827100700000201
Figure BDA0001827100700000211
Figure BDA0001827100700000221
Figure BDA0001827100700000231
Figure BDA0001827100700000241
Figure BDA0001827100700000251
Screening the gRNA of the molecular marker of high-efficiency and specific target sno-lncRNAs:
under the "guide" of the gRNA, Cas13a binds to the targeting RNA to exert its cleavage activity, and the "activated" Cas13a, in addition to cleaving the targeting RNA, also nonspecifically cleaves other RNAs. Therefore, a gRNA with high efficiency was selected by Cas 13's ability to attach cleavage of fluorescent RNA substrates. The fluorescent RNA substrate RNAse Alert v2 was purchased from Inc. (Integrated DNAtechnologies). Cas13 cleavage RNA reaction system is as follows:
Figure BDA0001827100700000252
Figure BDA0001827100700000261
the reaction was carried out at 37 ℃ for 3h, and the change in fluorescence of the substrate RNA was monitored in real time using a multifunctional microplate detector (BioTek SynergyNEO) (as shown in FIG. 3).
Example 4 sno-IncRNA 1, sno-IncRNA 2, sno-IncRNA 3, sno-IncRNA 4 and sno-IncRNA 5 molecular markers in combination as biomarker PCR product specific assays
4.1 culture of Stem cells H9 and extraction of RNA
Stem cells H9 were cultured on Martigel coated dishes using CM (fibroblast-conditioner) medium, which was the culture supernatant of MEF cells supplemented with 4ng/mL fibroblast growth factor (bFGF, Life technologies).
After washing the adherently growing H9 cells in a 3.5cm dish twice with PBS, 1mL of TRIzol reagent (Invitrogen) was added, and the pipetting was repeated until all the cells were lysed. Then 0.2mL of chloroform was added, the RNA was in the upper aqueous phase after centrifugation and transferred to a 1.5mL EP tube with RNase-free, an equal volume of isopropanol was added and mixed well and centrifuged, and the RNA was pelleted at the bottom of the tube. Rinsing twice with 75% ethanol, air drying at room temperature, and dissolving with RNase-free water.
4.2 reverse transcription of RNA, RT-PCR and gel electrophoresis
During reverse transcription, DNaseI (Ambion) is used to remove genome pollution, then PrimeScript reverse transcription kit (Takara) is used to carry out reverse transcription to obtain cDNA, the cDNA is diluted by 3-4 times by ultrapure water and stored at-20 ℃. The specific DNaseI treatment and reverse transcription process is as follows:
Figure BDA0001827100700000262
homogenizing for 2min, centrifuging at highest rotation speed for 5min, collecting supernatant, and storing at-80 deg.C.
10μl
5×PrimeScript Buffer 2μl
PrimeScript RT Enzyme 0.5μl
Oligo dT Primer 0.5μl
Random 6mers 2μl
DNaseI treated RNA 500ng
DEPC-H20 up to 10μl
The reaction is carried out for 15min at 37 ℃ and for 10min at 85 ℃.
Using cDNA as template, utilizing primer pair for detecting sno-lncRNA1 characteristic
sno-lncrRNA 1-qF 1: 5'-aaaattcgcatcctactgattg-3' (SEQ ID NO.174) and
sno-lncRNA1-qR1：5’gagtgagtgtgtctatgtgtat-3’(SEQ ID NO.175)、
primer pair for detecting biomarker sno-lncRNA2 characteristic
sno-lncrRNA 2-qF 4: 5'-tatggcaggaacacacaggt-3' (SEQ ID NO.176) and
sno-lncRNA2-qR4：5’-ctcacaaaagctacagggcc-3’(SEQ ID NO.177)、
primer pair for detecting biomarker sno-lncRNA3 characteristic
sno-lncrRNA 3-qF 4: 5'-gttgtggtcagcaggcatag-3' (SEQ ID NO.178) and
sno-lncRNA3-qR4：5’-acaccgggatcaggataagc-3’(SEQ ID NO.179)、
primer pair for detecting biomarker sno-lncRNA4 characteristic
sno-lncrRNA 4-qF 4: 5'-tttgtgcccgtgttgtatcc-3' (SEQ ID NO.180) and
sno-lncrRNA 4-qR 4: 5'-gcaatgccactcacaaaagc-3' (SEQ ID NO.181) and
primer pair for detecting biomarker sno-lncRNA5 characteristic
sno-lncrRNA 5-qF 1: 5'-attggaaaactaacctacccat-3' (SEQ ID NO.182) and
sno-lncRNA5-qR1：5’-gaaataacgtcccatgaatctc-3’(SEQ ID NO.183)
and amplifying the DNA segment to be detected. The PCR reaction system is as follows:
10μl
2×Taq Mater Mix(Vazyme) 5μl
cDNA Template 0.5μl
Forward Primer(10μM) 0.4μl
Reverse Primer(10μM) 0.4μl
ddH2O up to 10μl
the reaction conditions were as follows:
Figure BDA0001827100700000271
the PCR products were separated by electrophoresis on a 1.5% TAE agarose gel (containing ethidium bromide) and the results are shown in FIG. 4.
Example 5 determination of differential expression of molecular markers in a Positive control and a negative control for Willi's syndrome
WT H9 cells were used as positive control, and H9P-KO cells were used as negative control, both of which were derived from previous results of the inventors (see below) (1)http://dx.doi.org/10.1016/j.molcel.2016.10.007). The inventor uses the two cells for the related experiments of the detection of the small fat Willi syndrome for the first time.
The culture conditions and methods, RNA extraction method, and RNA reverse transcription reaction system and conditions of the above two cells were the same as those in example 4. Using the cDNAs as templates, respectively adopting a sno-lncRNA1 characteristic primer pair sno-lncRNA1-qF1(SEQ ID NO.174) and sno-lncRNA1-qR1(SEQ ID NO.175), a sno-lncRNA2 characteristic primer pair sno-lncRNA2-qF4(SEQ ID NO.176) and sno-lncRNA2-qR4(SEQ ID NO.177), a probe biomarker sno-lncRNA3 characteristic primer pair sno-lncRNA 3-3 (SEQ ID NO.178) and 3-lncRNA 3-qR 3 (SEQ ID NO.179), a probe biomarker 3-lncRNA 3 characteristic primer pair 3-lncRNA 3 (SEQ ID NO.180) and a 3-lncRNA 3 (SEQ ID NO.181) and a probe biomarker 3-lncRNA 3 (SEQ ID NO. 72) as templates, and carrying out real-time fluorescent quantitative PCR analysis. The real-time fluorescent quantitative PCR reaction system and conditions were the same as in example 4. As can be seen from FIG. 5, the expression levels of sno-IncRNA 1, sno-IncRNA 2, sno-IncRNA 3, sno-IncRNA 4 and sno-IncRNA 5 in the negative control were significantly lower than those in the positive control.
Example 6 analysis of sno-lncRNAs clinical examination of Pangolin syndrome
6.1 Whole blood sample Collection and RNA extraction
The whole blood sample is collected from a child hospital affiliated to Zhejiang university medical college, and the collection time is 7 months in 2018. 7 samples of whole blood of patients with the small fat Willi syndrome and 4 samples of whole blood of normal people are used as a small fat Willi syndrome group and a normal control group. All of the above specimens were obtained with subject consent and with tissue ethics committee approval.
Adding blood RNA stabilizer (TIANGEN) into fresh anticoagulated blood at a ratio of 1:3 (such as adding 900 μ l blood RNA stabilizer into 300 μ l fresh whole blood of healthy people), covering tube cover immediately, mixing by turning upside down for 8-10 times, and standing at room temperature for 2 hr. Whole blood RNA was extracted using RNAprep Pure blood total RNA extraction kit (TIANGEN) (see RNAprep Pure blood total RNA extraction kit for detailed procedures and precautions).
6.2 reverse transcription of RNA
The reaction system and conditions for reverse transcription of RNA were the same as in example 4.
6.3 real-time fluorescent quantitative PCR
The real-time fluorescent quantitative PCR reaction was carried out using the above cDNA as a template, and the other reaction system and conditions were the same as those in example 4.
As can be seen from FIG. 6, the expression levels of sno-lncRNA2, sno-lncRNA3 and sno-lncRNA4 in the small fat Willi syndrome group are obviously lower than those of the normal control group, and the P values of sno-lncRNA1, sno-lncRNA2, sno-lncRNA3, sno-lncRNA4 and sno-lncRNA5 are less than 0.05 by using T test (T-test), which proves that the differences are significant. As shown in FIG. 7, we further evaluated sno-lncRNA1, sno-lncRNA2, sno-lncRNA3 and sno-lncRNA4 for the sensitivity and specificity in differentiating the small fat Willi syndrome group from the normal group by the Receiver operating characteristic Curve (ROC), and the results showed that their Area under the Curve (Area under Curve Roc, AUC) was 1, and sno-lncRNA5 had an Area under the Curve of 0.96, which was very high in sensitivity and specificity, and was similar to the "gold standard". The experiment shows that the markers sno-lncRNA1, sno-lncRNA2, sno-lncRNA3, sno-lncRNA4 and sno-lncRNA5 provided by the invention have excellent detection performance, high accuracy and sensitivity and strong specificity when used for detecting the small fat Willi syndrome, and can be used for developing a detection kit for the small fat Willi syndrome.
Example 7 detection of Normal and Pangolin patients Using the novel Pangolin syndrome detection method
Adding the sample supernatant after RNase inactivation into a tubular recombinase polymerase amplification reaction system, wherein the reaction system comprises sno-lncRNA1 recombinase polymerase amplification specific primers sno-lncRNA1-RP1F and sno-lncRNA1-RP 3R;
sno-lncRNA2, sno-lncRNA2-RP1F, sno-lncRNA2-RP 5R;
sno-lncRNA3, sno-lncRNA3-RP3F, sno-lncRNA3-RP 3R;
sno-lncRNA4, sno-lncRNA4-RP4F, sno-lncRNA4-RP 5R;
sno-lncRNA5, sno-lncRNA5-RP4F, sno-lncRNA5-RP 1R.
Reacting at 37 ℃ for 30min, wherein the reaction system and conditions are as follows:
Figure BDA0001827100700000291
then adding the recombinase polymerase amplification reaction product into a one-tube type small fat Willi syndrome detection reaction system, and reacting for 30min at 37 ℃, wherein the reaction system is as follows:
Figure BDA0001827100700000292
Figure BDA0001827100700000301
wherein the fluorescent marker-RNA-biotin marker complex is synthesized by Shanghai biological engineering (Shanghai) GmbH, wherein the RNA sequence information is shown as follows:
sequence name Sequence (5 'to 3') Numbering
polyAU AUAUAUAUAUAUAU SEQ ID NO.169
polyGU GUGUGUGUGUGUGU SEQ ID NO.170
polyCU CUCUCUCUCUCUCU SEQ ID NO.171
polymixU AUGUCUUAUGUCU SEQ ID NO.172
The structure of the fluorescent marker-RNA-biotin marker complex is shown as follows:
sequence name Sequence (5 'to 3')
LF-polyAU 6-FAM-AUAUAUAUAUAUAU-Bio
LF-polyGU 6-FAM-GUGUGUGUGUGUGU-Bio
LF-polyCU 6-FAM-CUCUCUCUCUCUCU-Bio
LF-polymixU 6-FAM-AUGUCUUAUGUCU-Bio
Mu.l of HybriDetect Assay Buffer was added to the reaction tube, mixed well, and HybriDetectDipsiticks was added to insert the reaction tube for 5min, the results and instructions are shown in FIG. 8. The experiment shows that the detection kit for the small fat Willi syndrome provided by the invention has the following characteristics when used for detecting the small fat Willi syndrome: excellent detection and operation performance, high accuracy and sensitivity and strong specificity.
The advantages are favorable for the general popularization and use of the detection method for the small fat Willi syndrome in the basic medical unit, which provides strong and powerful support for the detection of the small fat Willi syndrome newborn and provides possibility for timely intervention.
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.
Sequence listing
<110> Shanghai Life science research institute of Chinese academy of sciences
<120> detection kit for small fat Willi syndrome and use method thereof
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tggatcgatg atgagtcctc caaaaaaaac attccttgga aaagctgaac aaaatgagtg 60
aaaactcata ccgtcattct catcggaact gaggtccagc acattgcttc ctctggggcc 120
tttactagtg agggacaact tccactagtc cctataggtt ctaacaccca tggcgttcac 180
aggtgaaggt ctcctgtcat attacaaaca ttccccccac tcccctccca aaaaaaggaa 240
catttcctct gtgtcaggta cacaggtcag agagtcatag ggtgactggc tgctctgtga 300
tggcatagtg gccgtggaag tggatggaaa tgtcgcaggc ttaggggagt gtggggctgg 360
cctcggcttg gtgagctccc aggaaggggg acctggaaat cgctgcgggc atccttgcat 420
agaggaccag gtacgtgttc cagttctgct ttcaaggaga gcatgaggcc caggcctcag 480
acagtccttg gtcttgatct ttggttagct gaggacaggg gagtgccaag ggtgataggg 540
gagagcgcac agcaatggat gttgtggcag acagctaaag cctttctgtc cctcctttca 600
cggtcagtcc cagggggccc ccagcacaga cttttttcac ctctcgagct gtgagattcc 660
tgtggtgttg tggccaagcc tgggcaggaa ggtaccccag agaaaaggtt gtagagggct 720
cacgtggaca tgcatgccat cagtggtctt gggctgtcat ttggtggtga tgggctttcc 780
agggatttct ctggtggatg acaggttggc cattgggggt cttcaagggc aggtcctagg 840
gagcatgagg gtcgggctga tgcctgtggt cctgggtcac tccattcttc ctggcagagt 900
agtagaagtg tccatcttcc tggaaatggt ctcttggccc gccattgcca ggcggcccca 960
cctgctgcag ctcgtgtgag caaatactgc tctggatccc atttcccgct ttgaaagcag 1020
cttccatccc aaaggcgtcc agagggaatg tgccctggcc ctggccctgg cctgggactc 1080
tccgatgggg tccagtcaca cttggcgtat tcatggaggt ccttgtgcat tccaggcaac 1140
acatgtgcaa taccaggggc actgccgtgg agagtctctt gatttgggat gcctcctcgc 1200
ctttgtgtgt tggcgatgtt ccagtattgg attctgctac atcctcaggg caatgaagcg 1260
tttcagctca cttagccggt ttgacatcat agtgcagcca ggacagccgt gccacattgg 1320
catggcacat agttgactta ggacagtcgg aaagggttgg tggttctatg tctcgcagct 1380
cacttcatag ctgtttgcat gtaaacctgg tgatggtgtt gagatggtgt tgacttgctg 1440
acgttggggt ggttggtagt gcagttaagt tggctgcatg atgggatgtt gcctcagatg 1500
tcggtgcagc gaagccctgt aggtgtgtcc aatgcaagga aaatgaaagg tggtgtgtcc 1560
tgtccacctt gtctttaggg gttcaggcag gaatgtcctg ttggaaaact agccattccc 1620
tttgtgtgtc ctgaaggcat ggtgcgggca tgtgaactga ttggaggaag gaagtgtgtg 1680
tcttgaggtc ctttggctgg gttcagaaga ctgtctctgg ccttgatgcc gtgtgccgtg 1740
tttaagacca acgtggtggc cattgcaaag gtgccttcca gtgcaagttt tggttggttt 1800
ggtgtcaggt gtgccttacc tgggagagcg gcgagagaca gtgtctggtc acgacattat 1860
atgccccgta aggaggactg tgtgcttgca ccgccagggg tgctggctgt tagtcgaggg 1920
acatcaggcc aatgtctgtc tgacccctag gtcaaaattc gcatcctact gattgaaggc 1980
cttgggtctc gagccttttg gttgcaggtg tggggatggt ttccaggtct ggaggctgcc 2040
aactatgaca acccgttgtg ctgttgggaa acatctgtgt gggggagaca catacacata 2100
gacacactca ctctgacata cacacctaca catgcacagg cgtgcacagc cacaggcaga 2160
tagatgcagc aatgggtgtg tgctattgtt gtggtcacat gtgcttccta ggtgtccaca 2220
gagtgacatg ggtcaggtat attcatgaag atggaggtct gtcatggagc ccaaggagaa 2280
gccggtgtgc aagtttccct gtgggaaaga atgggattga agtggtgacc agtggacttg 2340
ggcccgtttc tcaagcatgc atttgatttc tttacacttc tcagcagcag ttgtcatgag 2400
aggtctgccg cttgctggca aggaagatgg tcaattcttc tgaccaacag aggctgcctt 2460
gagtcctgat ttggtgtggc ctggcatcta tggtgcaccc aatgctggtg agtggctcag 2520
ggtggtaagc attcctctgc cctgtgtgca gaggtgtctt ttgaattttg agacctgcaa 2580
agagctgttt ctcagcaggc cactaatggc atggcgagtt ccactcccag agctgattgt 2640
ctgtgttcat tgctcagtgg tgtggctctg ggttcccccc aggatgctga aggtgtttaa 2700
ggtcatcagc tctcccactg cccagcctgt ggtgtcaaat gttctgcctc ttcgaacgtg 2760
cttggatcga tgatgagtcc cccataaaaa cattccttgg aaaagctgaa caaaatgagt 2820
gagaactcat accgtcgttc tcatcagaac tgaggtccag cacgttgcct cctctgggga 2880
ctgtaggagc gagggacaac ttcca 2905
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tggaccaatg atgacttcca tacatgcatt ccttggaaag ctgaacaaaa tgagtgggaa 60
ctctgtacta tcatcttagt tgaactgagg tccaccgggg gctatagagg gacaactgtc 120
attttttttg atgcccgttg catgaagcat tcttccaggc ttaggaaggg attccgtttg 180
ggtgaaggag agtcacaggg gagttgaggc tatgtgcaca ggacttctgt ctacaaggct 240
ggggtagcac acatgcagtc cgtgggacta ggtgaatgtg caggttgctc agggagagtt 300
aacctgtagc ctgcctctgg gtggccaaga ttgtccttga ctcctggctc tgggttccca 360
caacccctgg aggaagacaa gtaattgtgc ttgggaagag agaacaaggc catacttttg 420
cctttctttt tttcattctt agtttgagaa cagtggagtt ccaatcacct ggcatagctt 480
tcatgttgtg ttccatgcat tgtgagtaca tgcatatggc aggaacacac aggttcccac 540
tgcaagctga gcctgatagg caaggagtct ctagcttaac cccagcccag tgtacgtcaa 600
tggcttcctc attaaaattg catggccctg tagcttttgt gagtgacatt ataggtatcg 660
atgtaagggt gttgagaggg atgctcaatg tcagtgctct ttagtgtgct gatgcactac 720
tacaagggca cctgagccct tgtccatgca tttctgtgtc caaaatcagg taaagccaga 780
gttgggtatc aaccagatac actgcagggg atcagaggac atggcctttg tgtactgcac 840
atctgtcaga ttttctcaag cacacccgtg tttcctcttc acctcccacg tggaagtgat 900
gaattggcca ggaccatgtt ggtgagggac aatcatcatc tcatgttaac ctcagagaag 960
ccaggaagct cctggacaca catggtgtag gcttccttgg aggctgttgg atctctcctg 1020
aatgtaagca attccttccc agtgcaccct gattttcctc atttgcagaa acaaagattg 1080
tgtgtggatc gatgatgact tccatatata cattccttgg aaagctgaac aaaatgagtg 1140
aaaactctat accgtcattc tcgtcgaact gaggtccagc acattactcc a 1191
<210>3
<211>1249
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
tggatcgatg atgacttcct tatatacatt ccttggaaag ctgaacaaaa tgagtgaaaa 60
ctctataccg tcatcctcgt cgaactgagg tccagcacat tgctcttaca ggggctagag 120
agagagggac aaatttcatt tgatgatgcc cattgcacca aggggttctg tccaggctta 180
ggatggggtc tcgtttgggc aaaggagaat ggcaggggag tggaggctat gtacacagga 240
gattccttgt ttgaaggact ctatttgtga ggccagggta ccacacatgc tgtccgcagg 300
agtaggtgaa tgtgcagttg cccaggaaga gttagcctgt agcctgcctc tgcatggcag 360
tttgtccttg ggtcctggct ctggatttcc atgttccttg gaggaggata ggtgattttg 420
cttgagaaga cagcacagta ccatactttt gttgtttttc tgctcatttc atcgtccatt 480
gaggacaatg aagttgtggt cagcaggcat agctttcagg ccagcgtgcc catgttgtgt 540
cccatgcatt gtgagcacat gcatgtggca tgaacacata ggctgccact ccaagctgag 600
tctgataggc aatgagactc tggcttatcc tgatcccggt gtagatcaaa gtcttcccag 660
taggattgca tggccccgag gctattgtga gctgcattgc aggtgtggaa gcaagggtgt 720
tgagagggat gctcaacatt agtgctcttt agcgagatga tgcactataa gggcaccctg 780
aacccagacg tgcatcccta tgtacgtgca tttctgtgtc cataaatagt tgaagccaga 840
cagccagatt ccagatgtat cgcagggggc tggatgacat ggcccttgtc acctgtgtac 900
ctgtctgcct ttctgaagca cgcttgtgtt tcctctacac ctcccaggta gcattggcat 960
ggaaggcagg cccatgttgg tgagggacaa ttgttatctt gtgtgagccg cagggatacc 1020
aggaaacccc tggacacaaa tggcaaaggc ttctttggaa gttgttggat cccttctgca 1080
tgtaagcagt tctttcccag agcgctctga ttttcctcat ttgcagggac aaacactgtg 1140
cgtggatcga tgatgacttc catatataca ttccttggaa agctgaacaa aatgagtgaa 1200
aactctatac cgtcatcctc gtcgaactga ggtccagcac attactcca 1249
<210>4
<211>1249
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
tggatcgatg atgacttcca tatatacatt ccttggaaag ctgaacaaaa tgagtgaaaa 60
ctctatactg tcatcctcgt cgaactgagg tccagcacat tactccaaca ggggctagac 120
agagaaggcc aacatccgtt tgttgacatg ggttatatca aggcgtctgt tcaggcttag 180
aatgtggtct cttatgggtg atgggggtca caggagagtg gtggctccca tgtataggaa 240
atttcttgtt tgaaggactg tcagtgaggg tgggtaacac atgcattgtc tgcaggacta 300
ggtgaatgtc catgtggcct agcaagagtt agctggtagc ccgcctctgg ttgccaattt 360
gttcttgagt ccttgttctg ggttctcagg tcccacggag gaaaacagat ctgtgtggtt 420
gagaggtggg tacaaggccg catctttgtc atttgttggc taactttgtc cttggttgag 480
gacattagag ttttggtcac caggcatagc ctatgtgcct ttgtgcccgt gttgtatccc 540
acgtgttttg aggacatgta ttttgcacgt aaaggtgagc tcctgctcca agctggttct 600
gataccaaag gagtccctgg cttatcctaa actcatggta ggttaaagcc ttcctcctta 660
ggggttcagg gccgcaaggc ttttgtgagt ggcattgcag gcgttgaagc agtgatgttg 720
agagggatgg tcaatgtcag tgctctttag caggatggtg tactgcaggg gcccccagcc 780
ccgagacgag catccctgca tccatgcatt tctgcctcca tgaacagggg aggccagaga 840
caggcagata gtagataaat tgcaggggac tggatgacat ggccctcgtg acctgtgcac 900
ctgtctgtct ttctgaagca cgcctgtgtt aactctgcac ctcccaggta gcactggcat 960
ggagggcagg cacatgttgg tgagggacaa ttgttacctt gtgtgagctg cggagatacc 1020
aggaagcccc tggacacaaa tggcaaaggc tccttcggaa gttgttggat cccttctgaa 1080
tgtaagcact tctttcccag agcactctga gtttcctcat ttgcagggac aaatactgtg 1140
cgtggatcga tgatgacttc cacatataca ttccttggaa agctgaacaa aatgagtgaa 1200
aactctatac cgtcatcctc gtcgaactga ggtccagcac attactcca 1249
<210>5
<211>2187
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
tggatcgatg atgactataa aaaaaatgga tctcatcgga atctgaacaa aatgagtgac 60
caaatcattt ctgtgccact tctgtgagct gaggtccagc actgggacca tgccaggcaa 120
acatgaggag ggagtttaca gggactaggg tattggaaaa ctaacctacc catatatatt 180
agagcaaatg aattgttctc agttctgtaa atatccagct gtggtttgac atagcaactc 240
tctttgtatt tgcttggcag catttataga aacagctggt agaccctggg gaaccagggc 300
atagtgagat tcatgggacg ttatttcacc tactcttgag ccattcacac atttgagctc 360
gcagaattat ctggtcaatg agcagtctct tgagggtaat gagatggtca acatccaaat 420
ccaaagatac taatgagatt ttactttttt ttttgagatg gagtctcatt ctgttgccca 480
ggctggagtg cagtggcata atctcggctc actgcaacct ccacctcctg ggttcaagag 540
attctcccgc cccagcatcc tgagtagctg ggattacagg cgcccgccac cacccctggc 600
taatttttgt gtttgtagta gagatggagt ttcaccatat tggccaagct ggtctcgaac 660
tcccaacctc aggtgatcca gccactgcag cctctcaaag tgttgggatt acaggcatga 720
gccgccgtgc ccggccttac tttttatttt caattaagtg aaacaattac atggaaattg 780
aaaaaaaaaa aaggtactct aagtagacta ctctagtgta ttttaatttt cttgccaatt 840
cagccactag gattgtgtca cttgtttttt tgattttagt ttttcgtatc aaaaagcaaa 900
ccactgttta tatttgagtg ctggtttcaa ctgtatgctg cctcaaataa acttagatta 960
tattacaaat tcttttttac atagatttca ttggctatat tcatatatcc tcaaaggttt 1020
ccattgtgtt tgaagaggta tcctaattgt aataaatgct tttgattggg gatgccaggg 1080
aagctttgag actaatgtct cagatttttt ttaaaaaagt ttatattcta tagtgatcaa 1140
agatgtaaga ataatttttc ttgtcattca ttggatagtc cagtgagtgt ctactgtgaa 1200
ttcttatatt cacttgtaac taaacattcc atttggcaaa tttcgtttca taacttccct 1260
aatttcctct acactttgta gattgggtga gatacaattc catggaattt taccactgaa 1320
ccagtggagg gctaattttg tcatttgtac cagttaacta acttgggagg aaacagaaca 1380
acacactcct gaggtagtat ccaattttta gtaaatttat ttttattatt tttcaaactg 1440
tcagtgattt ggtttggtga acaataggtt aatacagaca atgttaggaa ttattaatct 1500
gatgcgctat ctgaagcatc agtttccttt atttttaaga tttttttctt ctctgtagtt 1560
tggataatat ttaaaacatt tccaagagtt ataaattagt atattttcta tgctgattaa 1620
taatgaaacc ttttgtctta tacagatttt taaaaaatgg acgtgtgcgt gcatgtgtat 1680
atatataaat atatatatat ataaaatctc ttctcacaag atgatgcaga ctaaaactgc 1740
attttacatt ttatatggct aagtttattg tttccttgtg ttgtaatatg atggaaagta 1800
atgggaataa ttctgcttcc tgtccttagg gaccagtagg aattttccca tgataagcat 1860
tttgtcctta aaatactgac atcctgttaa gagatcatga cactccaatg accatgagac 1920
cctggctatg ctagtcagcc agggggctga ccattgacag tgatgctggt ctccatgatt 1980
aaatagtgtg ggtcagcatg tcccagggtg acagaaagct gtgtgatcag tgtctgtcac 2040
atatctcaga ctcagggttc ctttttaaaa tgtgcctgga tcgatgatga cttaaagatt 2100
tatctaattt aaatctgaac aaaatgagtg accaaaacac ttctgtacca cttctgtgag 2160
ctgaggtcca gcacagagac cactcca 2187
<210>6
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
gaaattaata cgactcacta taggggtgaa ggtctcctgt catattacaa acattcc 57
<210>7
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
cagtcaccct atgactctct gacctgtgta 30
<210>8
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
gaaattaata cgactcacta tagggggtga aggtctcctg tcatattaca aacattc 57
<210>9
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
cctatgactc tctgacctgt gtacctgaca 30
<210>10
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
gaaattaata cgactcacta tagggaggtg aaggtctcct gtcatattac aaacatt 57
<210>11
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
ccctatgact ctctgacctg tgtacctgac 30
<210>12
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
gaaattaata cgactcacta tagggggcct ttactagtga gggacaactt ccacta 56
<210>13
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
ggggaatgtt tgtaatatga caggagacct tc 32
<210>14
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
gaaattaata cgactcacta tagggcatta taggtatcga tgtaagggtg ttgagag 57
<210>15
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
tctggcttta cctgattttg gacacagaaa tg 32
<210>16
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
gaaattaata cgactcacta tagggttgtg agtgacatta taggtatcga tgtaagg 57
<210>17
<211>31
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
ctgattttgg acacagaaat gcatggacaa g 31
<210>18
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
gaaattaata cgactcacta taggggacat tataggtatc gatgtaaggg tgttgag 57
<210>19
<211>31
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>19
ctggctttac ctgattttgg acacagaaat g 31
<210>20
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
gaaattaata cgactcacta tagggacatt ataggtatcg atgtaagggt gttgaga 57
<210>21
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
ttacctgatt ttggacacag aaatgcatgg ac 32
<210>22
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>22
gaaattaata cgactcacta tagggattat aggtatcgat gtaagggtgt tgagag 56
<210>23
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>23
tggctttacc tgattttgga cacagaaatg 30
<210>24
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>24
gaaattaata cgactcacta tagggtagag agagagggac aaatttcatt tgatgat 57
<210>25
<211>31
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>25
caaacaagga atctcctgtg tacatagcct c 31
<210>26
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
gaaattaata cgactcacta tagggagaga gagagggaca aatttcattt gatgatg 57
<210>27
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>27
ttcaaacaag gaatctcctg tgtacatagc ct 32
<210>28
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>28
gaaattaata cgactcacta tagggctaga gagagaggga caaatttcat ttgatg 56
<210>29
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>29
aaacaaggaa tctcctgtgt acatagcctc 30
<210>30
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>30
gaaattaata cgactcacta tagggctaga gagagaggga caaatttcat ttgatgat 58
<210>31
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
tcaaacaagg aatctcctgt gtacatagcc tc 32
<210>32
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>32
gaaattaata cgactcacta taggggagag agagggacaa atttcatttg atgatg 56
<210>33
<211>31
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>33
tcaaacaagg aatctcctgt gtacatagcc t 31
<210>34
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>34
gaaattaata cgactcacta tagggaacat ccgtttgttg acatgggtta tatcaag 57
<210>35
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>35
acagtccttc aaacaagaaa tttcctatac at 32
<210>36
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>36
gaaattaata cgactcacta tagggcatcc gtttgttgac atgggttata tcaag 55
<210>37
<211>31
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>37
cagtccttca aacaagaaat ttcctataca t 31
<210>38
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>38
gaaattaata cgactcacta tagggttata tcaaggcgtc tgttcaggct tagaatg 57
<210>39
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>39
gacagtcctt caaacaagaa atttcctata ca 32
<210>40
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>40
gaaattaata cgactcacta tagggacatc cgtttgttga catgggttat atcaag 56
<210>41
<211>31
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>41
acagtccttc aaacaagaaa tttcctatac a 31
<210>42
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>42
gaaattaata cgactcacta taggggttat atcaaggcgt ctgttcaggc ttagaat 57
<210>43
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>43
cttcaaacaa gaaatttcct atacatggga gc 32
<210>44
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>44
gaaattaata cgactcacta tagggtgttg taatatgatg gaaagtaatg ggaataa 57
<210>45
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>45
tcatgatctc ttaacaggat gtcagtattt ta 32
<210>46
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>46
gaaattaata cgactcacta tagggagttt attgtttcct tgtgttgtaa tatgatg 57
<210>47
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>47
tattttaagg acaaaatgct tatcatggga aa 32
<210>48
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>48
gaaattaata cgactcacta tagggtttta tatggctaag tttattgttt ccttgtg 57
<210>49
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>49
tcagtatttt aaggacaaaa tgcttatcat gg 32
<210>50
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>50
gaaattaata cgactcacta taggggtgtt gtaatatgat ggaaagtaat gggaata 57
<210>51
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>51
cttaacagga tgtcagtatt ttaaggacaa aa 32
<210>52
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>52
gaaattaata cgactcacta tagggaagtt tattgtttcc ttgtgttgta atatgat 57
<210>53
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>53
taacaggatg tcagtatttt aaggacaaaa tg 32
<210>54
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>54
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc cccacucccc ucccaaaaaa 60
aggaacau 68
<210>55
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>55
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc ccacuccccu cccaaaaaaa 60
ggaacauu 68
<210>56
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>56
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc cacuccccuc ccaaaaaaag 60
gaacauuu 68
<210>57
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>57
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc acuccccucc caaaaaaagg 60
aacauuuc 68
<210>58
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>58
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc uccccuccca aaaaaaggaa 60
cauuuccu 68
<210>59
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>59
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacu ccccucccaa aaaaaggaac 60
auuuccuc 68
<210>60
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>60
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc cccucccaaa aaaaggaaca 60
uuuccucu 68
<210>61
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>61
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc cucccaaaaa aaggaacauu 60
uccucugu 68
<210>62
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>62
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc cccacucccc ucccaaaaaa 60
aggaacau 68
<210>63
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>63
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc acuaaagagc acugacauug 60
agcaucc 67
<210>64
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>64
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc acacuaaaga gcacugacau 60
ugagcau 67
<210>65
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>65
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg cacacuaaag agcacugaca 60
uugagca 67
<210>66
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>66
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc agcacacuaa agagcacuga 60
cauugag 67
<210>67
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>67
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacu cagcacacua aagagcacug 60
acauuga 67
<210>68
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>68
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc aucagcacac uaaagagcac 60
ugacauu 67
<210>69
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>69
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg caucagcaca cuaaagagca 60
cugacau 67
<210>70
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>70
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacu gcaucagcac acuaaagagc 60
acugaca 67
<210>71
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>71
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg ugcaucagca cacuaaagag 60
cacugac 67
<210>72
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>72
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca gugcaucagc acacuaaaga 60
gcacuga 67
<210>73
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>73
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacu agugcaucag cacacuaaag 60
agcacug 67
<210>74
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>74
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg uagugcauca gcacacuaaa 60
gagcacu 67
<210>75
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>75
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca guagugcauc agcacacuaa 60
agagcac 67
<210>76
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>76
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacu aguagugcau cagcacacua 60
aagagca 67
<210>77
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>77
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg uaguagugca ucagcacacu 60
aaagagc 67
<210>78
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>78
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacu guaguagugc aucagcacac 60
uaaagag 67
<210>79
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>79
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc ccuuguagua gugcaucagc 60
acacuaa 67
<210>80
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>80
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg cccuuguagu agugcaucag 60
cacacua 67
<210>81
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>81
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacu gcccuuguag uagugcauca 60
gcacacu 67
<210>82
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>82
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg ugcccuugua guagugcauc 60
agcacac 67
<210>83
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>83
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg gugcccuugu aguagugcau 60
cagcaca67
<210>84
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>84
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc aggugcccuu guaguagugc 60
aucagca 67
<210>85
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>85
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc ucaggugccc uuguaguagu 60
gcaucag 67
<210>86
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>86
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg cucaggugcc cuuguaguag 60
ugcauca 67
<210>87
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>87
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg gcucaggugc ccuuguagua 60
gugcauc 67
<210>88
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>88
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg ggcucaggug cccuuguagu 60
agugcau 67
<210>89
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>89
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca gggcucaggu gcccuuguag 60
uagugca 67
<210>90
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>90
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc aagggcucag gugcccuugu 60
aguagug 67
<210>91
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>91
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca caagggcuca ggugcccuug 60
uaguagu 67
<210>92
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>92
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg acaagggcuc aggugcccuu 60
guaguag 67
<210>93
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>93
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg gacaagggcu caggugcccu 60
uguagua 67
<210>94
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>94
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacu ggacaagggc ucaggugccc 60
uuguagu 67
<210>95
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>95
acuaccccaa aaacgaaggg gacuaaaaca cagaaccccu uggugcaaug ggcauca 57
<210>96
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>96
acuaccccaa aaacgaaggg gacuaaaacg acagaacccc uuggugcaau gggcauc 57
<210>97
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>97
acuaccccaa aaacgaaggg gacuaaaacg gacagaaccc cuuggugcaa ugggcau 57
<210>98
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>98
acuaccccaa aaacgaaggg gacuaaaacc cuggacagaa ccccuuggug caauggg 57
<210>99
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>99
acuaccccaa aaacgaaggg gacuaaaacg ccuggacaga accccuuggu gcaaugg 57
<210>100
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>100
acuaccccaa aaacgaaggg gacuaaaaca gccuggacag aaccccuugg ugcaaug 57
<210>101
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>101
acuaccccaa aaacgaaggg gacuaaaaca agccuggaca gaaccccuug gugcaau 57
<210>102
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>102
acuaccccaa aaacgaaggg gacuaaaacc cuaagccugg acagaacccc uuggugc 57
<210>103
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>103
acuaccccaa aaacgaaggg gacuaaaacu ccuaagccug gacagaaccc cuuggug 57
<210>104
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>104
acuaccccaa aaacgaaggg gacuaaaacc cccauccuaa gccuggacag aaccccu 57
<210>105
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>105
acuaccccaa aaacgaaggg gacuaaaaca ccccauccua agccuggaca gaacccc 57
<210>106
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>106
acuaccccaa aaacgaaggg gacuaaaacg accccauccu aagccuggac agaaccc 57
<210>107
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>107
acuaccccaa aaacgaaggg gacuaaaaca gaccccaucc uaagccugga cagaacc 57
<210>108
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>108
acuaccccaa aaacgaaggg gacuaaaacc gagaccccau ccuaagccug gacagaa 57
<210>109
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>109
acuaccccaa aaacgaaggg gacuaaaaca cgagacccca uccuaagccu ggacaga 57
<210>110
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>110
acuaccccaa aaacgaaggg gacuaaaaca acgagacccc auccuaagcc uggacag 57
<210>111
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>111
acuaccccaa aaacgaaggg gacuaaaacc ccaaacgaga ccccauccua agccugg 57
<210>112
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>112
acuaccccaa aaacgaaggg gacuaaaacg cccaaacgag accccauccu aagccug 57
<210>113
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>113
acuaccccaa aaacgaaggg gacuaaaacu gcccaaacga gaccccaucc uaagccu 57
<210>114
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>114
acuaccccaa aaacgaaggg gacuaaaacu ugcccaaacg agaccccauc cuaagcc 57
<210>115
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>115
acuaccccaa aaacgaaggg gacuaaaacc cuuugcccaa acgagacccc auccuaa 57
<210>116
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>116
acuaccccaa aaacgaaggg gacuaaaacc uccuuugccc aaacgagacc ccauccu 57
<210>117
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>117
acuaccccaa aaacgaaggg gacuaaaacu cuccuuugcc caaacgagac cccaucc 57
<210>118
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>118
acuaccccaa aaacgaaggg gacuaaaacu ucuccuuugc ccaaacgaga ccccauc 57
<210>119
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>119
acuaccccaa aaacgaaggg gacuaaaacc cauucuccuu ugcccaaacg agacccc 57
<210>120
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>120
acuaccccaa aaacgaaggg gacuaaaacg ccauucuccu uugcccaaac gagaccc 57
<210>121
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>121
acuaccccaa aaacgaaggg gacuaaaacc cccugccauu cuccuuugcc caaacga 57
<210>122
<211>57
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>122
acuaccccaa aaacgaaggg gacuaaaacc uccccugcca uucuccuuug cccaaac 57
<210>123
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>123
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg accacauucu aagccugaac 60
agacgcc 67
<210>124
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>124
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca gaccacauuc uaagccugaa 60
cagacgc 67
<210>125
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>125
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg agaccacauu cuaagccuga 60
acagacg 67
<210>126
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>126
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca gagaccacau ucuaagccug 60
aacagac 67
<210>127
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>127
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca agagaccaca uucuaagccu 60
gaacaga 67
<210>128
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>128
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacu aagagaccac auucuaagcc 60
ugaacag 67
<210>129
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>129
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc ccauaagaga ccacauucua 60
agccuga 67
<210>130
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>130
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc acccauaaga gaccacauuc 60
uaagccu 67
<210>131
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>131
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacu cacccauaag agaccacauu 60
cuaagcc 67
<210>132
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>132
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc ccccaucacc cauaagagac 60
cacauuc 67
<210>133
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>133
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca cccccaucac ccauaagaga 60
ccacauu 67
<210>134
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>134
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg acccccauca cccauaagag 60
accacau 67
<210>135
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>135
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacu gacccccauc acccauaaga 60
gaccaca 67
<210>136
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>136
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg ugacccccau cacccauaag 60
agaccac 67
<210>137
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>137
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc cugugacccc caucacccau 60
aagagac67
<210>138
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>138
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc uccugugacc cccaucaccc 60
auaagag 67
<210>139
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>139
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc ucuccuguga cccccaucac 60
ccauaag 67
<210>140
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>140
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc cacucuccug ugacccccau 60
cacccau 67
<210>141
<211>67
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>141
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacc caccacucuc cugugacccc 60
caucacc 67
<210>142
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>142
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca cuggucccua aggacaggaa 60
gcagaauu 68
<210>143
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>143
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacu acuggucccu aaggacagga 60
agcagaau 68
<210>144
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>144
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacu ccuacugguc ccuaaggaca 60
ggaagcag 68
<210>145
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>145
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacu uccuacuggu cccuaaggac 60
aggaagca 68
<210>146
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>146
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca uuccuacugg ucccuaagga 60
caggaagc 68
<210>147
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>147
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca auuccuacug gucccuaagg 60
acaggaag 68
<210>148
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>148
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca aauuccuacu ggucccuaag 60
gacaggaa 68
<210>149
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>149
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca aaauuccuac uggucccuaa 60
ggacagga 68
<210>150
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>150
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg aaaauuccua cuggucccua 60
aggacagg 68
<210>151
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>151
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg gaaaauuccu acuggucccu 60
aaggacag 68
<210>152
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>152
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg ggaaaauucc uacugguccc 60
uaaggaca 68
<210>153
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>153
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacu gggaaaauuc cuacuggucc 60
cuaaggac 68
<210>154
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>154
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca ugggaaaauu ccuacugguc 60
ccuaagga 68
<210>155
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>155
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacu caugggaaaa uuccuacugg 60
ucccuaag 68
<210>156
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>156
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca ucaugggaaa auuccuacug 60
gucccuaa 68
<210>157
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>157
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacu aucaugggaa aauuccuacu 60
ggucccua 68
<210>158
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>158
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacu uaucauggga aaauuccuac 60
uggucccu 68
<210>159
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>159
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg cuuaucaugg gaaaauuccu 60
acuggucc 68
<210>160
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>160
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacu gcuuaucaug ggaaaauucc 60
uacugguc 68
<210>161
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>161
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca ugcuuaucau gggaaaauuc 60
cuacuggu 68
<210>162
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>162
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca augcuuauca ugggaaaauu 60
ccuacugg 68
<210>163
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>163
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca aaugcuuauc augggaaaau 60
uccuacug68
<210>164
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>164
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca aaaugcuuau caugggaaaa 60
uuccuacu 68
<210>165
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>165
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca caaaaugcuu aucaugggaa 60
aauuccua 68
<210>166
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>166
ggggauuuag acuaccccaa aaacgaaggg gacuaaaacg acaaaaugcu uaucauggga 60
aaauuccu 68
<210>167
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>167
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca ggacaaaaug cuuaucaugg 60
gaaaauuc 68
<210>168
<211>68
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>168
ggggauuuag acuaccccaa aaacgaaggg gacuaaaaca aggacaaaau gcuuaucaug 60
ggaaaauu 68
<210>169
<211>14
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>169
auauauauau auau 14
<210>170
<211>14
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>170
gugugugugu gugu 14
<210>171
<211>14
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>171
cucucucucu cucu14
<210>172
<211>13
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>172
augucuuaug ucu 13
<210>173
<211>1152
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>173
Met Lys Val Thr Lys Val Asp Gly Ile Ser His Lys Lys Tyr Ile Glu
1 5 10 15
Glu Gly Lys Leu Val Lys Ser Thr Ser Glu Glu Asn Arg Thr Ser Glu
20 25 30
Arg Leu Ser Glu Leu Leu Ser Ile Arg Leu Asp Ile Tyr Ile Lys Asn
35 40 45
Pro Asp Asn Ala Ser Glu Glu Glu Asn Arg Ile Arg Arg Glu Asn Leu
50 55 60
Lys Lys Phe Phe Ser Asn Lys Val Leu His Leu Lys Asp Ser Val Leu
65 70 75 80
Tyr Leu Lys Asn Arg Lys Glu Lys Asn Ala Val Gln Asp Lys Asn Tyr
85 90 95
Ser Glu Glu AspIle Ser Glu Tyr Asp Leu Lys Asn Lys Asn Ser Phe
100 105 110
Ser Val Leu Lys Lys Ile Leu Leu Asn Glu Asp Val Asn Ser Glu Glu
115 120 125
Leu Glu Ile Phe Arg Lys Asp Val Glu Ala Lys Leu Asn Lys Ile Asn
130 135 140
Ser Leu Lys Tyr Ser Phe Glu Glu Asn Lys Ala Asn Tyr Gln Lys Ile
145 150 155 160
Asn Glu Asn Asn Val Glu Lys Val Gly Gly Lys Ser Lys Arg Asn Ile
165 170 175
Ile Tyr Asp Tyr Tyr Arg Glu Ser Ala Lys Arg Asn Asp Tyr Ile Asn
180 185 190
Asn Val Gln Glu Ala Phe Asp Lys Leu Tyr Lys Lys Glu Asp Ile Glu
195 200 205
Lys Leu Phe Phe Leu Ile Glu Asn Ser Lys Lys His Glu Lys Tyr Lys
210 215 220
Ile Arg Glu Tyr Tyr His Lys Ile Ile Gly Arg Lys Asn Asp Lys Glu
225 230 235 240
Asn Phe Ala Lys Ile Ile Tyr Glu Glu Ile Gln Asn Val Asn Asn Ile
245 250 255
Lys Glu Leu Ile Glu LysIle Pro Asp Met Ser Glu Leu Lys Lys Ser
260 265 270
Gln Val Phe Tyr Lys Tyr Tyr Leu Asp Lys Glu Glu Leu Asn Asp Lys
275 280 285
Asn Ile Lys Tyr Ala Phe Cys His Phe Val Glu Ile Glu Met Ser Gln
290 295 300
Leu Leu Lys Asn Tyr Val Tyr Lys Arg Leu Ser Asn Ile Ser Asn Asp
305 310 315 320
Lys Ile Lys Arg Ile Phe Glu Tyr Gln Asn Leu Lys Lys Leu Ile Glu
325 330 335
Asn Lys Leu Leu Asn Lys Leu Asp Thr Tyr Val Arg Asn Cys Gly Lys
340 345 350
Tyr Asn Tyr Tyr Leu Gln Val Gly Glu Ile Ala Thr Ser Asp Phe Ile
355 360 365
Ala Arg Asn Arg Gln Asn Glu Ala Phe Leu Arg Asn Ile Ile Gly Val
370 375 380
Ser Ser Val Ala Tyr Phe Ser Leu Arg Asn Ile Leu Glu Thr Glu Asn
385 390 395 400
Glu Asn Asp Ile Thr Gly Arg Met Arg Gly Lys Thr Val Lys Asn Asn
405 410 415
Lys Gly Glu Glu Lys Tyr Val SerGly Glu Val Asp Lys Ile Tyr Asn
420 425 430
Glu Asn Lys Gln Asn Glu Val Lys Glu Asn Leu Lys Met Phe Tyr Ser
435 440 445
Tyr Asp Phe Asn Met Asp Asn Lys Asn Glu Ile Glu Asp Phe Phe Ala
450 455 460
Asn Ile Asp Glu Ala Ile Ser Ser Ile Arg His Gly Ile Val His Phe
465 470 475 480
Asn Leu Glu Leu Glu Gly Lys Asp Ile Phe Ala Phe Lys Asn Ile Ala
485 490 495
Pro Ser Glu Ile Ser Lys Lys Met Phe Gln Asn Glu Ile Asn Glu Lys
500 505 510
Lys Leu Lys Leu Lys Ile Phe Lys Gln Leu Asn Ser Ala Asn Val Phe
515 520 525
Asn Tyr Tyr Glu Lys Asp Val Ile Ile Lys Tyr Leu Lys Asn Thr Lys
530 535 540
Phe Asn Phe Val Asn Lys Asn Ile Pro Phe Val Pro Ser Phe Thr Lys
545 550 555 560
Leu Tyr Asn Lys Ile Glu Asp Leu Arg Asn Thr Leu Lys Phe Phe Trp
565 570 575
Ser Val Pro Lys Asp Lys Glu Glu Lys AspAla Gln Ile Tyr Leu Leu
580 585 590
Lys Asn Ile Tyr Tyr Gly Glu Phe Leu Asn Lys Phe Val Lys Asn Ser
595 600 605
Lys Val Phe Phe Lys Ile Thr Asn Glu Val Ile Lys Ile Asn Lys Gln
610 615 620
Arg Asn Gln Lys Thr Gly His Tyr Lys Tyr Gln Lys Phe Glu Asn Ile
625 630 635 640
Glu Lys Thr Val Pro Val Glu Tyr Leu Ala Ile Ile Gln Ser Arg Glu
645 650 655
Met Ile Asn Asn Gln Asp Lys Glu Glu Lys Asn Thr Tyr Ile Asp Phe
660 665 670
Ile Gln Gln Ile Phe Leu Lys Gly Phe Ile Asp Tyr Leu Asn Lys Asn
675 680 685
Asn Leu Lys Tyr Ile Glu Ser Asn Asn Asn Asn Asp Asn Asn Asp Ile
690 695 700
Phe Ser Lys Ile Lys Ile Lys Lys Asp Asn Lys Glu Lys Tyr Asp Lys
705 710 715 720
Ile Leu Lys Asn Tyr Glu Lys His Asn Arg Asn Lys Glu Ile Pro His
725 730 735
Glu Ile Asn Glu Phe Val Arg Glu Ile Lys Leu GlyLys Ile Leu Lys
740 745 750
Tyr Thr Glu Asn Leu Asn Met Phe Tyr Leu Ile Leu Lys Leu Leu Asn
755 760 765
His Lys Glu Leu Thr Asn Leu Lys Gly Ser Leu Glu Lys Tyr Gln Ser
770 775 780
Ala Asn Lys Glu Glu Thr Phe Ser Asp Glu Leu Glu Leu Ile Asn Leu
785 790 795 800
Leu Asn Leu Asp Asn Asn Arg Val Thr Glu Asp Phe Glu Leu Glu Ala
805 810 815
Asn Glu Ile Gly Lys Phe Leu Asp Phe Asn Glu Asn Lys Ile Lys Asp
820 825 830
Arg Lys Glu Leu Lys Lys Phe Asp Thr Asn Lys Ile Tyr Phe Asp Gly
835 840 845
Glu Asn Ile Ile Lys His Arg Ala Phe Tyr Asn Ile Lys Lys Tyr Gly
850 855 860
Met Leu Asn Leu Leu Glu Lys Ile Ala Asp Lys Ala Lys Tyr Lys Ile
865 870 875 880
Ser Leu Lys Glu Leu Lys Glu Tyr Ser Asn Lys Lys Asn Glu Ile Glu
885 890 895
Lys Asn Tyr Thr Met Gln Gln Asn Leu His Arg Lys Tyr AlaArg Pro
900 905 910
Lys Lys Asp Glu Lys Phe Asn Asp Glu Asp Tyr Lys Glu Tyr Glu Lys
915 920 925
Ala Ile Gly Asn Ile Gln Lys Tyr Thr His Leu Lys Asn Lys Val Glu
930 935 940
Phe Asn Glu Leu Asn Leu Leu Gln Gly Leu Leu Leu Lys Ile Leu His
945 950 955 960
Arg Leu Val Gly Tyr Thr Ser Ile Trp Glu Arg Asp Leu Arg Phe Arg
965 970 975
Leu Lys Gly Glu Phe Pro Glu Asn His Tyr Ile Glu Glu Ile Phe Asn
980 985 990
Phe Asp Asn Ser Lys Asn Val Lys Tyr Lys Ser Gly Gln Ile Val Glu
995 1000 1005
Lys Tyr Ile Asn Phe Tyr Lys Glu Leu Tyr Lys Asp Asn Val Glu Lys
1010 1015 1020
Arg Ser Ile Tyr Ser Asp Lys Lys Val Lys Lys Leu Lys Gln Glu Lys
1025 1030 1035 1040
Lys Asp Leu Tyr Ile Arg Asn Tyr Ile Ala His Phe Asn Tyr Ile Pro
1045 1050 1055
His Ala Glu Ile Ser Leu Leu Glu Val Leu Glu Asn Leu Arg LysLeu
1060 1065 1070
Leu Ser Tyr Asp Arg Lys Leu Lys Asn Ala Ile Met Lys Ser Ile Val
1075 1080 1085
Asp Ile Leu Lys Glu Tyr Gly Phe Val Ala Thr Phe Lys Ile Gly Ala
1090 1095 1100
Asp Lys Lys Ile Glu Ile Gln Thr Leu Glu Ser Glu Lys Ile Val His
1105 1110 1115 1120
Leu Lys Asn Leu Lys Lys Lys Lys Leu Met Thr Asp Arg Asn Ser Glu
1125 1130 1135
Glu Leu Cys Glu Leu Val Lys Val Met Phe Glu Tyr Lys Ala Leu Glu
1140 1145 1150
<210>174
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>174
aaaattcgca tcctactgat tg 22
<210>175
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>175
gagtgagtgt gtctatgtgt at 22
<210>176
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>176
tatggcagga acacacaggt 20
<210>177
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>177
ctcacaaaag ctacagggcc 20
<210>178
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>178
gttgtggtca gcaggcatag 20
<210>179
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>179
acaccgggat caggataagc 20
<210>180
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>180
tttgtgcccg tgttgtatcc 20
<210>181
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>181
gcaatgccac tcacaaaagc 20
<210>182
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>182
attggaaaac taacctaccc at 22
<210>183
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>183
gaaataacgt cccatgaatc tc 22

Claims (11)

1. A detection kit for the syndrome of small inflated willi, comprising any one or more of the following features:
(1) sno-lncRNA1 recombinase polymerase amplification specific primers, a gRNA specifically targeting sno-lncRNA1, a Cas13 protein, a biotin-fluorescent reporting reagent and colloidal gold immunochromatographic test paper; (2) sno-lncRNA2 recombinase polymerase amplification specific primers, a gRNA specifically targeting sno-lncRNA2, a Cas13 protein, a biotin-fluorescent reporting reagent and colloidal gold immunochromatographic test paper; (3) sno-lncRNA3 recombinase polymerase amplification specific primers, gRNAs specifically targeting sno-lncRNA3, (4) sno-lncRNA4 recombinase polymerase amplification specific primers, gRNAs targeting sno-lncRNA4, Cas13 protein, biotin-fluorescent reporter reagent and colloidal gold immunochromatographic test paper; (5) sno-lncRNA5 recombinase polymerase amplification specific primers, a gRNA specifically targeting sno-lncRNA5, a Cas13 protein, a biotin-fluorescent reporter reagent and colloidal gold immunochromatographic test paper.
2. The detection kit for puffiness syndrome according to claim 1, wherein the colloidal gold immunochromatographic test strip comprises a base plate, and a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water absorbent pad which are arranged on the surface of the base plate in this order from the sample addition end, wherein the gold-labeled pad contains a colloidal gold-labeled anti-fluorophore antibody, and the nitrocellulose membrane is coated with a quality control line C and a detection line T.
3. The detection kit for puffiness syndrome according to claim 2, wherein the nitrocellulose membrane has a quality control line C located closer to the sample addition end and a detection line T located farther from the sample addition end.
4. The reagent kit for detecting the puffiness syndrome as set forth in claim 2, wherein the quality control line C is coated with a biotin ligand.
5. The detection kit for puffiness syndrome according to claim 2, wherein the detection line T is coated with an antibody capable of binding to a colloidal gold-labeled anti-fluorophore antibody.
6. The detection kit for the syndrome of small inflated Willi of claim 1, wherein the kit further comprises any one or more of the following features:
(1) sno-lncRNA1 recombinase polymerase amplification specific primers comprise an upstream primer and a downstream primer; (2) sno-lncRNA2 recombinase polymerase amplification specific primers comprise an upstream primer and a downstream primer; (3) sno-lncRNA3 recombinase polymerase amplification specific primers comprise an upstream primer and a downstream primer; (4) sno-lncRNA4 recombinase polymerase amplification specific primers comprise an upstream primer and a downstream primer; (5) sno-lncRNA5 recombinase polymerase amplification specific primers include an upstream primer and a downstream primer.
7. The detection kit for the syndrome of small inflated Willi of claim 1, wherein the kit further comprises any one or more of the following features:
(1) the recombinase polymerase amplification specific primer sequence of the sno-lncRNA1 is selected from primers of the following sequences: an upstream primer sno-lncRNA1-RP1F shown as a sequence SEQ ID NO.6, a downstream primer sno-lncRNA1-RP1R shown as a sequence SEQ ID NO.7, an upstream primer sno-lncRNA1-RP2F shown as a sequence SEQ ID NO.8, a downstream primer sno-lncRNA1-RP2R shown as a sequence SEQ ID NO.9 and an upstream primer shown as a sequence SEQ ID NO.10
sno-lncRNA1-RP3F, a downstream primer sno-lncRNA1-RP3R with the sequence shown in SEQ ID NO.11, an upstream primer sno-lncRNA1-RP4F with the sequence shown in SEQ ID NO.12 and a downstream primer sno-lncRNA1-RP4R with the sequence shown in SEQ ID NO. 13;
(2) the recombinase polymerase amplification specific primer sequence of sno-lncRNA2 is selected from an upstream primer sno-lncRNA2-RP1F with the sequence shown in SEQ ID NO.14, a downstream primer sno-lncRNA2-RP1R with the sequence shown in SEQ ID NO.15, an upstream primer sno-lncRNA2-RP2F with the sequence shown in SEQ ID NO.16, a downstream primer sno-lncRNA2-RP 22 with the sequence shown in SEQ ID NO.17, an upstream primer 2-lncRNA 2-RP 32 with the sequence shown in SEQ ID NO.18, a downstream primer 2-lncRNA 2-RP 32 with the sequence shown in SEQ ID NO.19, an upstream primer 2-lncRNA 2-2 with the sequence shown in SEQ ID NO.20, a downstream primer 2-lncRNA 2-2 with the sequence shown in SEQ ID NO.21, and an upstream primer 2-lncRNA 2-2 with the sequence shown in SEQ ID NO.2, A downstream primer sno-lncRNA2-RP5R with the sequence shown in SEQ ID NO. 23;
(3) the recombinase polymerase amplification specific primer sequence of the sno-lncRNA3 is selected from primers of the following sequences: an upstream primer sno-lncRNA3-RP1F with a sequence shown in SEQ ID NO.24, a downstream primer sno-lncRNA3-RP1R with a sequence shown in SEQ ID NO.25, an upstream primer sno-lncRNA3-RP2F with a sequence shown in SEQ ID NO.26, a downstream primer sno-lncRNA sno-RP 2 sno with a sequence shown in SEQ ID NO.27, an upstream primer sno-lncRNA sno-RP 3 sno with a sequence shown in SEQ ID NO.28, a downstream primer sno-lncRNA sno-RP 3 sno with a sequence shown in SEQ ID NO.29, an upstream primer sno-lncRNA sno-RP 4 with a sequence shown in SEQ ID NO.30, a downstream primer sno-lncRNA sno-RP 4 sno with a sequence shown in SEQ ID NO.31, and an upstream primer sno-lncRNA 68672-RP 72 with a sequence shown in SEQ ID NO.32, and a downstream primer sno-lncRNA sno-sno with a sequence shown in SEQ ID NO. 72;
(4) the recombinase polymerase amplification specific primer sequence of the sno-lncRNA4 is selected from primers of the following sequences: an upstream primer sno-lncRNA4-RP1F with a sequence shown in SEQ ID NO.34, a downstream primer sno-lncRNA4-RP1R with a sequence shown in SEQ ID NO.35, an upstream primer sno-lncRNA4-RP2F with a sequence shown in SEQ ID NO.36, a downstream primer sno-lncRNA sno-RP 2 sno with a sequence shown in SEQ ID NO.37, an upstream primer sno-lncRNA sno-RP 3 sno with a sequence shown in SEQ ID NO.38, a downstream primer sno-lncRNA sno-RP 3 sno with a sequence shown in SEQ ID NO.39, an upstream primer sno-lncRNA sno-RP 4 with a sequence shown in SEQ ID NO.40, a downstream primer sno-lncRNA sno-RP 72 with a sequence shown in SEQ ID NO.41, an upstream primer sno-lncRNA sno-RP 72 with a sequence shown in SEQ ID NO.42, and a downstream primer sno-lncRNA sno-sno with a sequence shown in SEQ ID NO. 72, wherein a sequence shown in SEQ ID NO. sno-sno are shown in SEQ ID NO. sno;
(5) the recombinase polymerase amplification specific primer sequence of the sno-lncRNA5 is selected from primers of the following sequences: an upstream primer sno-lncRNA5-RP1F with the sequence shown in SEQ ID NO.44, a downstream primer sno-lncRNA5-RP1R with the sequence shown in SEQ ID NO.45, an upstream primer sno-lncRNA5-RP2F with the sequence shown in SEQ ID NO.46, a downstream primer sno-lncRNA sno-RP 2 sno with the sequence shown in SEQ ID NO.47, an upstream primer sno-lncRNA sno-RP 3 sno with the sequence shown in SEQ ID NO.48, a downstream primer sno-lncRNA sno-RP 3 sno with the sequence shown in SEQ ID NO.49, an upstream primer sno-lncRNA sno-RP 4 sno with the sequence shown in SEQ ID NO.50, a downstream primer sno-lncRNA sno with the sequence shown in SEQ ID NO.51, an upstream primer sno-lncRNA 6865-RP 72 with the sequence shown in SEQ ID NO.52 and a downstream primer sno-lncRNA sno-sno with the sequence shown in SEQ ID NO. 72.
8. The detection kit for the syndrome of small inflated Willi of claim 1, wherein the kit further comprises one or more of the following features:
(1) the gRNA sequence of specific target sno-lncRNA1 is selected from SEQ ID NO.54-SEQ ID NO. 62;
(2) the gRNA sequence of the specific target sno-lncRNA2 is selected from SEQ ID NO.63-SEQ ID NO. 94;
(3) the gRNA sequence of specific target sno-lncRNA3 is selected from SEQ ID NO.95-SEQ ID NO. 122;
(4) the gRNA sequence of specific target sno-lncRNA4 is selected from SEQ ID NO.123-SEQ ID NO. 141;
(5) the gRNA sequence specifically targeting sno-lncRNA5 is selected from SEQ ID NO.142-SEQ ID NO. 168.
9. The detection kit for the picowilli syndrome according to claim 1, wherein the biotin-fluorescent reporter reagent comprises a fluorescent marker-RNA-biotin marker complex, wherein in the fluorescent marker-RNA-biotin marker complex, the RNA sequence is selected from the group consisting of SEQ ID No.169 to SEQ ID No. 172.
10. The detection kit for the syndrome of small inflated Willi of claim 1, wherein the kit comprises one or more of the following features: an RNase inactivating agent, a DNA removing agent, a recombinase polymerase amplification reaction reagent, T7RNA polymerase, rNTP, Cas13 protein reaction buffer and yeast tRNA.
11. The use of the detection kit for the picowilli syndrome according to any one of claims 1 to 10, at least comprising the following steps:
1) extracting RNA of a sample to be detected;
2) amplifying the sample RNA in step 1) with a recombinase polymerase of sno-lncRNA1, sno-lncRNA2, sno-lncRNA3, sno-lncRNA4, or sno-lncRNA5 to amplify the specific primer-mediated recombinase polymerase;
3) combining the amplified sample RNA obtained in the step 2) with one or more of gRNA specifically targeting sno-lncRNA1, gRNA specifically targeting sno-lncRNA2, gRNA specifically targeting sno-lncRNA3, gRNA specifically targeting sno-lncRNA4 and gRNA specifically targeting sno-lncRNA5 and Cas13 protein to activate the Cas13 protein, reacting with a biotin-fluorescence reporter reagent, dripping the reacted mixture onto a colloidal gold immunochromatographic strip, and reflecting the expression condition of molecular markers sno-lncRNA1, sno-lncRNA2, sno-lncRNA3, sno-lncRNA 39 4 or sno-lncRNA5 in the sample RNA according to the change condition of a colloidal gold immunochromatographic strip detection line.
CN201811189458.0A 2018-10-12 2018-10-12 Detection kit for small fat Willi syndrome and use method thereof Pending CN111041084A (en)

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