WO2020024222A1 - 包含通用荧光消减探针核酸分子的基因检测试剂盒及其应用 - Google Patents

包含通用荧光消减探针核酸分子的基因检测试剂盒及其应用 Download PDF

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WO2020024222A1
WO2020024222A1 PCT/CN2018/098338 CN2018098338W WO2020024222A1 WO 2020024222 A1 WO2020024222 A1 WO 2020024222A1 CN 2018098338 W CN2018098338 W CN 2018098338W WO 2020024222 A1 WO2020024222 A1 WO 2020024222A1
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sequence
molar ratio
complementary
hybridization
probe
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French (fr)
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李孝锦
夏庆杰
康焰
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四川大学华西医院
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    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation

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  • the invention relates to the field of gene detection, in particular to a gene detection kit containing a universal fluorescent subtraction probe nucleic acid molecule and application thereof.
  • Nucleic acid molecule hybridization means that under certain conditions (suitable temperature and humidity, ionic strength, etc.), two nucleic acid single strands with a certain homology can be combined into a double strand by complementary bases.
  • Nucleic acid molecular hybridization can be further divided into Southern hybridization, Northern hybridization, in situ hybridization (ISH), fluorescent in situ hybridization (FISH), chip hybridization (which belongs to solid-liquid phase hybridization) and the like. Each hybridization technology has its own characteristics. Because of different probe selections, many related technologies can be derived, which play an important role in different fields.
  • FISH is a non-radioactive in situ hybridization technique. Its basic principle is: if the target DNA or RNA on the chromosome or tissue section to be detected is homologous and complementary to the nucleic acid probe used, the two can form the target nucleic acid and nucleic acid probe through denaturation-annealing-renaturation. Needle hybrid. A reporter molecule such as biotin and digoxin is labeled with a certain nucleotide of a nucleic acid probe, and the immunochemical reaction between the reporter molecule and specific avidin labeled with fluorescein can be used. Perform qualitative, quantitative, or relative localization analysis of the test DNA.
  • a reporter molecule such as biotin and digoxin is labeled with a certain nucleotide of a nucleic acid probe, and the immunochemical reaction between the reporter molecule and specific avidin labeled with fluorescein can be used. Perform qualitative, quantitative, or relative localization analysis of the test DNA.
  • FISH technology is not only used in clinical and scientific research of cytogenetics, but also widely used in the study of tissue cell localization of gene expression.
  • the traditional FISH technology is complicated in operation, has many influencing factors, takes a long time, is difficult in technology, and is difficult to promote in a large area.
  • subtractive probe technologies because each gene must synthesize its specific fluorescently labeled probe and fluorescent quenching probe, the cost is high, and hybridization conditions are not easy to standardize.
  • the present invention provides a new kit and method.
  • the invention provides a special double-stranded fluorescence reduction probe for simple and fast technical methods for fluorescent in situ hybridization, thereby significantly reducing the technical difficulty and detection cost of in situ hybridization.
  • the gene detection kit of the present invention includes the following probes:
  • N 1 N 2 N 3 N 4 N 5 ... N 6 N 7 N 8 N 9 N 10 M 3 M 4 CTGAAGCGCTTCGCGAGCC-3'; where N 1 N 2 N 3 N 4 N 5 ... N 6 N 7 N 8 N 9 N 10 is the complementary sequence of the gene or fragment to be tested, and M 1 M 2 and M 3 M 4 are linker sequences;
  • the B sequence, C sequence and A sequence are combined, and the amount of B sequence and C sequence can be combined with all A sequences;
  • the D sequence is combined with A sequence, and the amount of D sequence can be combined with all A sequences, ie Yes;
  • the D sequence is combined with the E sequence, and the amount of the E sequence can be combined with all the D sequences.
  • the molar ratio of the A sequence to the B sequence is 1: (2-7); the molar ratio of the A sequence to the C sequence is 1: (2-7); the molar ratio of the A sequence to the D sequence is 1: (2-7); the molar ratio of the D sequence to the E sequence is 1: (2-5);
  • the molar ratio of A sequence, B sequence, C sequence, D sequence and E sequence is 1: 5: 5: 3: 6.
  • the invention also provides a kit, which includes the following probes:
  • N 1 N 2 N 3 N 4 N 5 ... N 6 N 7 N 8 N 9 N 10 M 3 M 4 CTGAAGCGCTTCGCGAGCC-3'; where N 1 N 2 N 3 N 4 N 5 ... N 6 N 7 N 8 N 9 N 10 is the complementary sequence of the gene or fragment to be tested, and M 1 M 2 and M 3 M 4 are linker sequences;
  • (2)B sequence 5'-n 5 n 4 n 3 n 2 n 1 m 2 m 1 TAGGCTCGCGAAGC-3 '; n 1 n 2 n 3 n 4 n 5 is complementary to the sequence of N 1 N 2 N 3 N 4 N 5 m 1 m 2 is complementary to the sequence of M 1 M 2 ;
  • (3)C sequence 5'-GCGAAGCGCTTCAGm 4 m 3 n 10 n 9 n 8 n 7 n 6 -3 '; n 6 n 7 n 8 n 9 n 10 is complementary to the sequence of N 6 N 7 N 8 N 9 N 10 , m
  • the sequences of 3 m 4 and M 3 M 4 are complementary;
  • the B sequence, C sequence is combined with the A sequence, and the amount of the B sequence and the C sequence can be combined with all the A sequences;
  • the F sequence is combined with the A sequence, and the amount of the F sequence can be combined with all the A sequences.
  • F-R1 sequence, F-R2 sequence is combined with F sequence, the amount of F-R1 sequence, F-R2 sequence can be combined with all F sequences;
  • G sequence is combined with F sequence, and the amount of G sequence can be combined with all F sequences.
  • the F-sequence is sufficient; the G-R1 sequence and the G-R2 sequence are combined with the G sequence, and the amount of the G-R1 sequence and the G-R2 sequence can be combined with all the G sequences; The amount can be combined with all the G sequences; the I sequence can be combined with the H sequence, and the amount of the I sequence can be combined with all the H sequences.
  • the molar ratio of the A sequence to the B sequence is 1: (2-7); the molar ratio of the A sequence to the C sequence is 1: (2-7); the molar ratio of the A sequence to the F sequence is 1: (2-7); the molar ratio of the F sequence to the F-R1 sequence is 1: (2-7); the molar ratio of the F sequence to the F-R2 sequence is 1: (2-7);
  • the molar ratio of the F sequence to the G sequence is 1: (2-7); the molar ratio of the G sequence to the G-R1 sequence is 1: (2-7); the molar ratio of the G sequence to the G-R2 sequence is The molar ratio is 1: (2-7); the molar ratio of the G sequence to the H sequence is 1: (2-7); the molar ratio of the H sequence to the I sequence is 1: (2-5);
  • the molar ratio of the A sequence, B sequence, C sequence, F sequence, F-R1 sequence, F-R2 sequence, G sequence, G-R1 sequence, G-R2 sequence, H sequence, I sequence is 1 : 2: 2: 2.4: 4: 4: 4: 8: 8: 16.
  • the M 1 M 2 is TA or AC
  • / or the M 3 M 4 is AT, CA, or TC.
  • the gene to be tested or a partial fragment sequence of the gene to be tested indicated by "N1N2N3N4N5 ... N6N7N8N9N10" may have any length, and preferably has a length of 10 bp or more.
  • the gene to be tested is any human, rat, or mouse gene.
  • the fluorescent molecules are FAM (6-carboxyfluorescein), TET (tetrachloro-6carboxyfluorescein), HEX (hexachloro-6methylfluorescein), JOE (2,7, -dimethyl-4 , 5, dichloro-6-6 carboxyfluorescein).
  • the fluorescence quenching molecule is TAMRA (6-carboxy-4 methylrhodamine) or BHQ (black hole quenching group).
  • the kit also includes a buffer.
  • the buffer solution includes TE buffer solution and pre-hybridization solution; TE buffer solution (including 10 mmol / L tritris (ammonium tromethamine), 1 mmol / L EDTA, pH value 8.0); pre-hybridization solution (compared with the hybridization solution, no Contains probes and other salt solutions of the same composition, including: 60mmol / L Tritris (ammonium tromethamine), 50mmol / L NaCl, 1mmol / L EDTA, 0.5% SDS (sodium dodecylsulfonate), 5% Formamide, pH 7.4.
  • TE buffer solution including 10 mmol / L tritris (ammonium tromethamine), 1 mmol / L EDTA, pH value 8.0
  • pre-hybridization solution compared with the hybridization solution, no Contains probes and other salt solutions of the same composition, including: 60mmol / L Tritris (ammonium tromethamine), 50
  • the invention also provides a method for gene detection, the steps are as follows (protection from light during the whole process):
  • the slide is heated to 95 ° C to 98 ° C for denaturation for 3 to 5 minutes, and then rapidly cooled to 45 ° C to 70 ° C for 20 to 60 minutes to allow the universal fluorescent probe to complete molecular hybridization with the target nucleic acid, and then cooled to 30 ° C -50 °C for 40 to 60min;
  • step (4) is: denaturing the glass slide at 95 ° C or 98 ° C for 3min, then quickly lowering the temperature to 68 ° C for 30min, naturally lowering the temperature to 40 ° C for 50min, and then naturally lowering to room temperature;
  • Step (5) is as follows: adding an anti-quenching sealer, placing a cover glass, observing under a fluorescence microscope at 488 nm, 405 nm, and 670 nm and taking photos.
  • the beneficial effect of the present invention is that the fluorescence reduction probe technology of the present invention enables the fluorescence of a fluorescently labeled probe that has not hybridized with a target nucleic acid to be quenched by the quenching group of a universal quenching probe, so that it is not complicated and difficult to control
  • the washing step can remove non-hybridized fluorescent signals only by controlling the hybridization temperature, which greatly improves the efficiency of nucleic acid hybridization, saves time and effort, can quickly complete fluorescence in situ hybridization, and detects the effect of specific genes or their expression distributions.
  • This method requires It is completed in 2 hours, which is more than 10 hours longer than the traditional FISH method, which is an improvement of the FISH method.
  • a set of probes for detection of one site were synthesized.
  • the total number of probe bases was 240 bases, and the two ODs were 1.5 yuan.
  • Two fluoresceins were labeled, each 600 yuan, and two quenched groups were labeled.
  • 500 yuan, a total of 2,560 yuan, 40 yuan for other conventional reagents, a total of 2,600 yuan, can complete the target gene detection 400 times, can complete the production of 4 kits (100 times / box).
  • the cost of each kit is 650 yuan, and the cost of each gene test is 6.5 yuan.
  • the in situ hybridization reagents currently on the market are 4,000 yuan / 25 times, and each gene test is 160 yuan. Our kit has obvious cost advantages. .
  • the traditional FISH method takes a long time and takes more than ten hours.
  • the invention only needs 2 hours to complete the experiment, and the time cost of the invention is lower.
  • the fixed sequence in the kit will not bind with any gene of human, rat, and mouse, so it can be used to detect any gene fragment of human, rat, and mouse, and overcome the traditional method Problems that require specific design when testing each gene.
  • the kit and method of the invention can be applied to the detection of any gene in the human body, and the fluorescent sequence that plays a key role in the kit is a universal sequence, and the cost is low.
  • FIG. 13 Results of the negative group of telomere gene detection in Experimental Example 6;
  • FIG. 14 shows the results of the positive group of telomere gene detection in Experimental Example 6.
  • N 1 N 2 N 3 N 4 N 5 ... N 6 N 7 N 8 N 9 N 10 M 3 M 4 CTGAAGCGCTTCGCGAGCC-3'; where N 1 N 2 N 3 N 4 N 5 ... N 6 N 7 N 8 N 9 N 10 is the complementary sequence of the gene or fragment to be tested, and M 1 M 2 and M 3 M 4 are linker sequences;
  • M 1 M 2 is TA or AC
  • M 3 M 4 is AT, CA, or TC.
  • Buffer TE buffer (contains 10mmol / L tritris (ammonium tromethamine), 1mmol / L EDTA, pH 8.0); pre-hybridization solution (compared to the hybridization solution, it does not contain probes, and other components are the same salt solution , which contains: 60mmol / L tritris (ammonium tromethamine), 50mmol / L NaCl, 1mmol / L EDTA, 0.5% SDS (sodium dodecylsulfonate), 5% formamide, pH 7.4.)
  • N 1 N 2 N 3 N 4 N 5 ... N 6 N 7 N 8 N 9 N 10 M 3 M 4 CTGAAGCGCTTCGCGAGCC-3'; where N 1 N 2 N 3 N 4 N 5 ... N 6 N 7 N 8 N 9 N 10 is the complementary sequence of the gene or fragment to be tested, and M 1 M 2 and M 3 M 4 are linker sequences;
  • M 1 M 2 is TA or AC
  • M 3 M 4 is AT, CA, or TC.
  • (2)B sequence 5'-n 5 n 4 n 3 n 2 n 1 m 2 m 1 TAGGCTCGCGAAGC-3 '; n 1 n 2 n 3 n 4 n 5 is complementary to the sequence of N 1 N 2 N 3 N 4 N 5 m 1 m 2 is complementary to the sequence of M 1 M 2 ;
  • (3)C sequence 5'-GCGAAGCGCTTCAGm 4 m 3 n 10 n 9 n 8 n 7 n 6 -3 '; n 6 n 7 n 8 n 9 n 10 is complementary to the sequence of N 6 N 7 N 8 N 9 N 10 , m
  • the sequences of 3 m 4 and M 3 M 4 are complementary;
  • Buffer TE buffer (contains 10mmol / L tritris (ammonium tromethamine), 1mmol / L EDTA, pH 8.0); pre-hybridization solution (compared to the hybridization solution, it does not contain probes, and other components are the same salt solution , which contains: 60mmol / L tritris (ammonium tromethamine), 50mmol / L NaCl, 1mmol / L EDTA, 0.5% SDS (sodium dodecylsulfonate), 5% formamide, pH 7.4.)
  • nucleic acid detection technology can detect the target gene at 10 copies / ⁇ L, which translates into an FM sensitivity of 0.01667fM / L.
  • the samples to be tested were nerve cells of brain tissue in which rat brain edema appeared.
  • the method of the present invention is detected according to the method of Embodiment 1:
  • Negative control method The same as the method of the present invention except that the A sequence is not used.
  • the negative control has no red hybridization signal
  • the positive control has a relatively strong fluorescent hybridization signal, which indicates that the method of the present invention can accurately detect, but the specific sequence cannot be accurately detected.
  • the sample to be tested is rat liver tissue.
  • Negative control method The same as the method of the present invention except that the A sequence is not used.
  • the method of the present invention is detected according to the method of Embodiment 1:
  • HCR-A 5’CTGAAGCGCTTCGCGAGCCTAGGGTTAGGGTTAGGGTTAGGGTTAGGGATCTGAAGCGCTTCGCGAGCC-3 ’
  • HCR-B CCTAACCCTAGGCTCGCGAAGC
  • HCR-C GCGAAGCGCTTCAGATCCCTAACC
  • HCR-D 5 '.- FAM-GGCTCGCGAAGCGCTTCAG--FAM-3'
  • HCR-E 5 '-. TAMRA-CTGAAGCGCTTCGCGAGC–TAMRA-3';
  • Negative control method The same as the method of the present invention except that the A sequence is not used.
  • the negative control has almost no signal, while the positive control has a relatively strong blue fluorescent hybridization signal, indicating that the method of the present invention can accurately detect, but it cannot be detected by removing specific sequences.
  • the positive sample was the spleen tissue of mice with model sepsis after 24 hours, and the sample of the negative control was the spleen tissue of normal mice.
  • Sepsis can increase the expression of IL-6 gene in the spleen of mice. Compared with normal mice, the IL-6 gene in the spleen tissue of mice with sepsis should be significantly increased after 24 hours.
  • IL-10-B probe CTCCCCTCTGTGGCTCGCGAAGC
  • IL-10-C probe CGAAGCGCTTCAGTGAGGCGCTG
  • HCR-D 5'-FAM-GGCTCGCGAAGCGCTTCAG-FAM-3 '
  • HCR-E 5'-TAMRA-CTGAAGCGCTTCGCGAGC-TAMRA-3 '
  • the negative control has a very weak fluorescent signal, while the positive control has a strong green fluorescent signal.
  • the test was performed according to the method of Example 1.
  • the positive sample was the liver tissue of mice with sepsis (protective group injected with 120 mg / kg safflower yellow pigment) 24 hours later.
  • the sample of the negative control was liver tissue of normal mice .
  • IL-10-B probe CTCCCCTCTGTGGCTCGCGAAGC
  • IL-10-C probe CGAAGCGCTTCAGTGAGGCGCTG
  • HCR-D 5 '.- FAM-GGCTCGCGAAGCGCTTCAG--FAM-3'
  • E probe sequence HCR-E: 5 '-. TAMRA-CTGAAGCGCTTCGCGAGC–TAMRA-3'
  • the fluorescent signal of the negative control is very weak, while the positive control has a strong blue fluorescent signal, and the detection result is consistent with the nature of the tissue itself, indicating that the method of the present invention can effectively and accurately detect.
  • Experimental example 5 The method of the present invention is used to detect the IL-1 ⁇ gene
  • the positive sample was the lung tissue of mice with model sepsis after 24 hours, and the sample of the negative control was the lung tissue of normal mice.
  • IL-1 ⁇ -B probe CAGCACCACTAGGCTCGCGAAG
  • IL-1 ⁇ -C probe GAAGCGCTTCAGGAGTTCCCCAAC
  • HCR-D 5 '.- FAM-GGCTCGCGAAGCGCTTCAG--FAM-3'
  • E probe sequence HCR-E: 5 '-. TAMRA-CTGAAGCGCTTCGCGAGC–TAMRA-3'
  • Figs. 11-12 The results are shown in Figs. 11-12.
  • the two-photon confocal fluorescence microscope and ImageJ software were used to compare and analyze the imaging signals of the green part of the fluorograph.
  • the fluorescence signal of the negative control is very weak, while the fluorescence signal of the positive control is strong, the fluorescence intensity is statistically significant, and the test results are consistent with the nature of the tissue itself, indicating that the method of the present invention can effectively and accurately detect .
  • the sample to be tested is rat liver tissue.
  • Negative control method The same as the method of the present invention except that the A sequence is not used.
  • CRHCR-A-R1 CCTAACCCTAGGCTCGCGAAGC
  • CRHCR-B-R2 GCTCGCTCTGGTTCTGAAGC
  • the negative control has almost no signal, while the positive control has a relatively strong fluorescence hybridization signal, which indicates that the method of the present invention can accurately detect, but the specific sequence cannot be accurately detected.
  • kit and method of the present invention can effectively detect genes and have a good application prospect.

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Abstract

一种基因检测试剂盒以及基因检测方法。采用荧光消减探针技术，使未与靶核酸杂交的荧光标记探针的荧光被通用淬灭探针的淬灭基团所淬灭，达到不需要繁琐而不易控制的洗涤步骤，只通过控制杂交温度就可以除去非杂交的荧光信号，大大提高核酸杂交的效率，省时省力，能够快速完成荧光原位杂交，检测特定基因或其表达分布的效果，是传统FISH的改良，应用前景优良。

Description

包含通用荧光消减探针核酸分子的基因检测试剂盒及其应用 技术领域
本发明涉及基因检测领域，具体涉及一种包含通用荧光消减探针核酸分子的基因检测试剂盒及其应用。
背景技术
核酸分子杂交是指具有一定同源性的两条核酸单链在一定的条件下(适宜的温湿度及离子强度等)可按碱基互补结合成双链。核酸分子杂交又可分为Southern杂交，Northern杂交，原位杂交(ISH)，荧光原位杂交(FISH)，芯片杂交(属于固一液相杂交)等。每一种杂交技术都有其特点，因探针的选择不同又可以衍生出许多相关的技术，在不同领域发挥着重要作用。
FISH是一种非放射性原位杂交技术。它的基本原理是：如果被检测的染色体或组织切片上的靶DNA或RNA与所用的核酸探针是同源互补的，二者经变性-退火-复性，即可形成靶核酸与核酸探针的杂交体。将核酸探针的某一种核苷酸标记上报告分子如生物素、地高辛，可利用该报告分子与荧光素标记的特异亲和素之间的免疫化学反应，经荧光检测体系在镜下对待测DNA进行定性、定量或相对定位分析。FISH技术不仅应用于细胞遗传学临床与科研，而且还广泛应用于基因表达的组织细胞定位研究。但传统的FISH技术操作复杂，影响因素多，耗费时间长，技术难度大，很难大面积推广。其它消减探针技术则由于每个基因都要合成其特异的荧光标记探针及荧光淬灭探针，成本高，杂交条件不易标准化。
发明内容
为了解决上述问题，本发明提供了一种新的试剂盒和方法。本发明提供了一种特殊的双链荧光消减探针进行荧光原位杂交的操作简单快速的技术方法，从而显著降低原位杂交的技术难度、检测成本。
本发明基因检测试剂盒，它包括如下探针：
A序列:
5'–CTGAAGCGCTTCGCGAGCCM 1M 2N 1N 2N 3N 4N 5…N 6N 7N 8N 9N 10M 3M 4CTGAAGCGCTTCGCGAGCC-3'；其中，N 1N 2N 3N 4N 5…N 6N 7N 8N 9N 10为待检基因或其片段的互补序列，M 1M 2和M 3M 4为接头序列；
B序列:5'-n 5n 4n 3n 2n 1m 2m 1TAGGCTCGCGAAGC-3'；n 1n 2n 3n 4n 5与N 1N 2N 3N 4N 5的序列互补，m 1m 2与M 1M 2的序列互补；
C序列:5'-GCGAAGCGCTTCAGm 4m 3n 10n 9n 8n 7n 6-3'；n 6n 7n 8n 9n 10与N 6N 7N 8N 9N 10的序列互补，m 3m 4与M 3M 4的序列互补；
D序列:5'.-荧光分子-GGCTCGCGAAGCGCTTCAG–荧光分子-3'
E序列:5'-荧光淬灭分子-CTGAAGCGCTTCGCGAGC-荧光淬灭分子-3'。
本发明试剂盒中，B序列、C序列与A序列结合，B序列、C序列的用量能够结合所有的A序列即可；D序列与A序列结合，D序列的用量能够结合所有的A序列即可；D序列与E序列结合，E序列的用量能够结合所有的D序列即可。
优选所述A序列与B序列的摩尔比为1：(2～7)；所述A序列与C序列的摩尔比为1：(2～7)；所述A序列与D序列的摩尔比为1：(2～7)；所述D序列与E序列的摩尔比为1：(2～5)；
进一步优选地，A序列、B序列、C序列、D序列与E序列的摩尔比为1:5:5:3:6。
本发明还提供了一种试剂盒,它包括如下探针：
⑴A序列:
5'–CTGAAGCGCTTCGCGAGCCM 1M 2N 1N 2N 3N 4N 5…N 6N 7N 8N 9N 10M 3M 4CTGAAGCGCTTCGCGAGCC-3'；其中，N 1N 2N 3N 4N 5…N 6N 7N 8N 9N 10为待检基因或其片段的互补序列，M 1M 2和M 3M 4为接头序列；
⑵B序列:5'-n 5n 4n 3n 2n 1m 2m 1TAGGCTCGCGAAGC-3'；n 1n 2n 3n 4n 5与N 1N 2N 3N 4N 5的序列互补，m 1m 2与M 1M 2的序列互补；
⑶C序列:5'-GCGAAGCGCTTCAGm 4m 3n 10n 9n 8n 7n 6-3'；n 6n 7n 8n 9n 10与N 6N 7N 8N 9N 10的序列互补，m 3m 4与M 3M 4的序列互补；
⑷F序列:
5'-CCAGAGCGAGCAGTGTCAAGGCTCGCGAAGCGCTTCAGAACCAGAGCGAGCAGTGTC-3'
⑸F-R1序列:5'-CGAGCCTTGACACTGCTCG-3'
⑹F-R2序列:5'-GCTCGCTCTGGTTCTGAAGC-3'
⑺G序列
5'-GAACAGTCGTGAACATCTGACACTGCTCGCTCTGGTGAACAGTCGTGAACATC-3'
⑻G-R1序列:
5'-GCAGTGTCAGATGTTCAC-3'
⑼G-R2序列:
5'-GACTGTTCACCAGAGCG-3'
⑽H序列:
5'-荧光分子-GATGTTCACGACTGTTC-荧光分子-3'
⑾I序列:
5'-淬灭分子-GAACAGTCGTGAACAT-淬灭分子-3'。
本发明试剂盒中，B序列、C序列与A序列结合，B序列、C序列的用量能够结合所有的A序列即可；F序列与A序列结合，F序列的用量能够结合所有的A序列即可；F-R1序列、F-R2序列与F序列结合，F-R1序列、F-R2 序列的用量能够结合所有的F序列即可；G序列与F序列结合，G序列的用量能够结合所有的F序列即可；G-R1序列、G-R2序列与G序列结合，G-R1序列、G-R2序列的用量能够结合所有的G序列即可；H序列与G序列结合，H序列的用量能够结合所有的G序列即可；I序列与H序列结合，I序列的用量能够结合所有的H序列即可。
优选所述A序列与B序列的摩尔比为1：(2～7)；所述A序列与C序列的摩尔比为1：(2～7)；所述A序列与F序列的摩尔比为1：(2～7)；所述F序列与F-R1序列的摩尔比为1：(2～7)；所述F序列与F-R2序列的摩尔比为1：(2～7)；所述F序列与G序列的摩尔比为1：(2～7)；所述G序列与G-R1序列的摩尔比为1：(2～7)；所述G序列与G-R2序列的摩尔比为1：(2～7)；所述G序列与H序列的摩尔比为1：(2～7)；所述H序列与I序列的摩尔比为1：(2～5)；
优选地，所述A序列、B序列、C序列、F序列、F-R1序列、F-R2序列、G序列、G-R1序列、G-R2序列、H序列、I序列的摩尔比为1:2:2:2.4:4:4::8:8:8:16。
前述试剂盒中，所述M 1M 2为TA或AC，和/或，所述M 3M 4为AT、CA或TC。
本发明中“N1N2N3N4N5…N6N7N8N9N10”所示待检基因或者待检基因的部分片段序列，可以为任意长度，优选长度为10个bp以上。
所述待检基因为任意人体和大鼠、小鼠基因。
所述荧光分子为FAM(6-羧基荧光素)、或者TET(四氯-6羧基荧光素)、HEX(六氯-6甲基荧光素)、JOE(2，7，-二甲基-4,5，二氯-6-6羧基荧光素)。
所述荧光淬灭分子为TAMRA(6-羧基-4甲基罗丹明)、或者BHQ(黑洞淬灭集团)。
所述试剂盒还包括缓冲液。
所述缓冲液包括TE缓冲液和预杂交液；TE缓冲液(包含10mmol/L tris(缓血酸氨),1mmol/L EDTA，PH值8.0)；预杂交液(与杂交液相比，没有包含探针，其他成分相同的盐溶液，其中包含：60mmol/L tris(缓血酸氨),50mmol/L NaCl,1mmol/L EDTA,0.5％SDS(十二烷基磺酸钠)，5％的甲酰胺，PH值7.4。
本发明还提供了一种基因检测方法，步骤如下(全程避光，)：
(1)取前述试剂盒中的探针，溶解于TE缓冲液中，得到包含所有探针序列的混合液PM1；
(2)取混合液PM1加入到预杂交液配制成杂交缓冲液PM2；
(3)全程避光，取杂交缓冲液PM2滴加到待测的染色体滴片或组织切片上，盖上盖玻片，用封胶封固；
(4)将玻片加热到95℃～98℃变性3到5min，再迅速降温至45℃-70℃保温20到60min，使通用荧光探针与靶核酸完成分子杂交，然后再降温至30℃-50℃保温40到60min；
(5)加入防淬灭封片剂，置荧光显微镜下观察杂交信号。
优选地，步骤(4)为：将玻片在95℃或者98℃环境下变性3min，再迅速降温至68℃保温30min，自然降温至40℃保温50min，再自然降至室温；
步骤(5)为：加入防淬灭封片剂，放上盖玻片，置荧光显微镜下488nm、405nm、670nm激发光下观察并照相。
本发明的有益效果是：本发明荧光消减探针技术，使未与靶核酸杂交的荧光标记探针的荧光被通用淬灭探针的淬灭基团所淬灭，达到不需要繁琐而不易控制的洗涤步骤，只通过控制杂交温度就可以除去非杂交的荧光信号，大大提高核酸杂交的效率，省时省力，能够快速完成荧光原位杂交，检测特定基因或其表达分布的效果，本方法需要2小时完成，较传统FISH法提高了10多小时，是FISH法的一种改进。
检测一个位点的探针，合成一套，探针碱基共有240个碱基，每两个OD为1.5元，标记两个荧光素，每个600元，标记两个淬灭集团，每个500元，共计2560元，其他常规试剂40元，合计2600元，能够完成400次检测目标基因，可以完成4个试剂盒(100次/盒)的生产。每个试剂盒的成本为650元，每次基因检测为6.5元，目前市面上出售的原位杂交试剂4000元/25次，每次基因检测为160元，我们的试剂盒具有明显的成本优势。
传统的FISH法，消耗时间长，需要十多小时，本发明只需要2小时完成实验，本发明时间成本更低。
本发明通过特定设计，试剂盒中的固定序列，不会与人体、大鼠、小鼠的任何基因发生结合，因此可以用于人体、大鼠、小鼠的任何基因片段的检测，克服传统方法在检测每个基因时需要进行特定设计的问题。
本发明试剂盒和方法可以适用于人体任何基因的检测，而且试剂盒中，起到关键作用的荧光序列为通用序列，成本低廉。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1本发明基因检测方法的原理
图2本发明基因检测方法的原理
图3实验例1中AQP4基因检测的阴性对照结果；
图4实验例1中AQP4基因检测的阳性组结果；
图5实验例2中端粒基因检测的阴性对照结果；
图6实验例2中端粒基因检测的阳性组结果；
图7实验例3中IL-6基因检测的阴性对照结果；
图8实验例3中IL-6基因检测的阳性组结果；
图9实验例4中IL-10基因检测的阴性对照结果；
图10实验例4中IL-10基因检测的阳性组结果；
图11实验例5中IL-1β基因检测的阴性对照结果；
图12实验例5中IL-1β基因检测的阳性组结果；
图13实验例6中端粒基因检测的阴性组结果；
图14实验例6中端粒基因检测的阳性组结果。
具体实施方式
实施例1 本发明基因检测方法
1、检测试剂盒的构成
原理图如图1所示：
A序列:
5'–CTGAAGCGCTTCGCGAGCCM 1M 2N 1N 2N 3N 4N 5…N 6N 7N 8N 9N 10M 3M 4CTGAAGCGCTTCGCGAGCC-3'；其中，N 1N 2N 3N 4N 5…N 6N 7N 8N 9N 10为待检基因或其片段的互补序列，M 1M 2和M 3M 4为接头序列；
B序列:5'-n 5n 4n 3n 2n 1m 2m 1TAGGCTCGCGAAGC-3'；n 1n 2n 3n 4n 5与N 1N 2N 3N 4N 5的序列互补，m 1m 2与M 1M 2的序列互补；
C序列:5'-GCGAAGCGCTTCAGm 4m 3n 10n 9n 8n 7n 6-3'；n 6n 7n 8n 9n 10与N 6N 7N 8N 9N 10的序列互补，m 3m 4与M 3M 4的序列互补；
D序列:5'.-FAM-GGCTCGCGAAGCGCTTCAG–FAM-3'
E序列:5'-.TAMRA-CTGAAGCGCTTCGCGAGC–TAMRA-3'；
其中，M 1M 2为TA或AC，和/或，M 3M 4为AT、CA或TC。
缓冲液：TE缓冲液(包含10mmol/L tris(缓血酸氨),1mmol/L EDTA，PH值8.0)；预杂交液(与杂交液相比，没有包含探针，其他成分相同的盐溶液，其中包含：60mmol/L tris(缓血酸氨),50mmol/L NaCl,1mmol/L EDTA,0.5％SDS(十二烷基磺酸钠)，5％的甲酰胺，PH值7.4。)
2、检测方法
(1)将上述探针分别溶解于TE缓冲液中，得A序列的浓度为1umol/L、B序列的浓度为5umol/L、C序列的浓度为5umol/L、D序列的浓度为3umol/L、E序列的浓度为6umol/L的混合液PM1；取1ul混合液PM1加入到49ul预杂交液配制成杂交缓冲液PM2；
(2)全程避光，取待检样品，滴加25ul杂交液PM2，加盖玻片，以及封胶封固；
(3)将玻片在95℃环境下变性3min，再迅速降温至68℃保温30min，自然缓慢降温至40℃保温50min，再自然降至室温；
(4)将玻片取出后，加入防淬灭封片剂，放上盖玻片，置荧光显微镜下488nm、405nm、670nm激发光下观察并照相。
实施例2 本发明基因检测方法
1、检测试剂盒的构成
原理图如图2所示：
⑴A序列:
5'–CTGAAGCGCTTCGCGAGCCM 1M 2N 1N 2N 3N 4N 5…N 6N 7N 8N 9N 10M 3M 4CTGAAGCGCTTCGCGAGCC-3'；其中，N 1N 2N 3N 4N 5…N 6N 7N 8N 9N 10为待检基因或其片段的互补序列，M 1M 2和M 3M 4为接头序列；
其中，M 1M 2为TA或AC，和/或，M 3M 4为AT、CA或TC。
⑵B序列:5'-n 5n 4n 3n 2n 1m 2m 1TAGGCTCGCGAAGC-3'；n 1n 2n 3n 4n 5与N 1N 2N 3N 4N 5的序列互补，m 1m 2与M 1M 2的序列互补；
⑶C序列:5'-GCGAAGCGCTTCAGm 4m 3n 10n 9n 8n 7n 6-3'；n 6n 7n 8n 9n 10与N 6N 7N 8N 9N 10的序列互补，m 3m 4与M 3M 4的序列互补；
⑷F序列:
5'-CCAGAGCGAGCAGTGTCAAGGCTCGCGAAGCGCTTCAGAACCAGAGCGAGCAGTGTC-3'
⑸F-R1序列:5'-CGAGCCTTGACACTGCTCG-3'
⑹F-R2序列:5'-GCTCGCTCTGGTTCTGAAGC-3'
⑺G序列
5'-GAACAGTCGTGAACATCTGACACTGCTCGCTCTGGTGAACAGTCGTGAACATC-3'
⑻G-R1序列:
5'-GCAGTGTCAGATGTTCAC-3'
⑼G-R2序列:
5'-GACTGTTCACCAGAGCG-3'
⑽H序列:
FAM-GATGTTCACGACTGTTC-FAM
⑾I序列:
TAMRA-GAACAGTCGTGAACAT-TAMRA。
缓冲液：TE缓冲液(包含10mmol/L tris(缓血酸氨),1mmol/L EDTA，PH值8.0)；预杂交液(与杂交液相比，没有包含探针，其他成分相同的盐溶液，其中包含：60mmol/L tris(缓血酸氨),50mmol/L NaCl,1mmol/L EDTA,0.5％SDS(十二烷基磺酸钠)，5％的甲酰胺，PH值7.4。)
2、检测方法
(1)将上述11中探针分别溶解于TE缓冲液中，得浓度均为10ummol/L的探针溶液，取A序列溶液1uL、B序列2uL、C序列2uL、F序列2uL、F-R1序列4uL、F-R2序列4uL、G序列4uL、G-R1序列8uL、G-R2序列8uL、H序列8uL、I序列16uL，得混合液PM1；取1ul混合液PM1加入到49ul预杂交液配制成杂交缓冲液PM2；
(2)全程避光，取待检样品，滴加25ul杂交液PM2，加盖玻片，以及封胶封固；
(3)将玻片在98℃环境下变性3min，再迅速降温至68℃保温30min，自然缓慢降温至40℃保温50min，再自然降至室温；
(4)将玻片取出后，置荧光显微镜下488nm、405nm、670nm激发光下观察并照相。
主要性能指标：
Figure PCTCN2018098338-appb-000001
1.核酸检测的灵敏度达到fM/L级别：
Figure PCTCN2018098338-appb-000002
我们的核酸检测技术能够检测到10拷贝/μL的目标基因，换算成FM灵敏度为0.01667fM/L。
Figure PCTCN2018098338-appb-000003
2.方法特异性方面，实现有效识别核酸序列中的单碱基差异:
Figure PCTCN2018098338-appb-000004
我们严格按照核酸碱基配对原则设计荧光分子探针，进行原位杂交，实现了单碱基识别，无目标碱基序列，则会出现结合探针封闭A探针，无荧光杂交信号的现象。
以下用实验例的方式来说明本发明的有益效果：
实验例1 采用本发明方法检测脑细胞水肿基因AQP4基因
1、检测方法
待检样品为出现了大鼠脑水肿的脑组织神经细胞。
本发明方法，按照实施例1的方法检测：
(1)首先根据AQP4基因的mRNA序列设计合成一套荧光消减探针，其序列及标记分别为：
A序列:
5'-.CTGAAGCGCTTCGCGAGCCTAGCTACATGGAGGTGGAGGACAACCGATCTGAAGCGCTTCGCGAGCC-3'
B序列:5'.-CCATGTAGCTAGGCTCGCGAAGC-3'
C序列:5'.-GCGAAGCGCTTCAGATCGGTTGTC-3'
D序列:5'.-FAM-GGCTCGCGAAGCGCTTCAG–FAM-3'
E序列:5'-.TAMRA-CTGAAGCGCTTCGCGAGC–TAMRA-3'；
(2)将上述探针分别溶解于TE缓冲液中，使A序列的浓度为1umol/L、B序列的浓度为5umol/L、C序列的浓度为5umol/L、D序列的浓度为3umol/L、E序列的浓度为6umol/L的混合液PM1；取1ul混合液PM1加入到49ul预杂交液配制成杂交缓冲液PM2；
(3)取大鼠脑组织切片，全程避光，滴加25ul含荧光消减探针的杂交液PM2，加盖玻片，以及封胶封固；
(4)将玻片在95℃环境下变性3min，再迅速降温至68℃保温30min，自然缓慢降温至40℃保温50min，再自然降至室温；
(5)将玻片取出后置荧光显微镜下670nm激发光下观察并照相。
阴性对照的方法：除了不使用A序列，其余与本发明方法相同。
2、结果：
结果如图3～4所示，使用双光子共聚焦荧光显微镜和Image J(Fiji is just)软件对荧光照相的红色部分显像信号进行了比较分析。
可以看出，阴性对照无红色杂交信号，而阳性对照则有比较强烈的荧光杂交信号，说明本发明方法可以准确检测，而去除特异性序列则无法准确检测。
实验例2 采用本发明方法检测端粒基因
1、检测方法
待检样品为大鼠肝组织。
阴性对照的方法：除了不使用A序列，其余与本发明方法相同。
本发明方法，按照实施例1的方法检测：
(1)序列及标记分别为：
A序列
HCR-A:5’CTGAAGCGCTTCGCGAGCCTAGGGTTAGGGTTAGGGTTAGGGTTAGGGATCTGAAGCGCTTCGCGAGCC-3’
B序列：HCR-B:CCTAACCCTAGGCTCGCGAAGC
C序列：HCR-C:GCGAAGCGCTTCAGATCCCTAACC
D序列:HCR-D:5'.-FAM-GGCTCGCGAAGCGCTTCAG–FAM-3'
E序列:HCR-E:5'-.TAMRA-CTGAAGCGCTTCGCGAGC–TAMRA-3'；
(2)将上述探针分别溶解于TE缓冲液中，使A序列的浓度为1umol/L、B序列的浓度为5umol/L、C序列的浓度为5umol/L、D序列的浓度为3umol/L、E序列的浓度为6umol/L的混合液PM1；取1ul混合液PM1加入到49ul预杂交液配制成杂交缓冲液PM2；
(3)取小鼠肝组织切片，滴加25ul含荧光消减探针的杂交液PM2，加盖玻片，以及封胶封固；
(4)避光，将玻片在95℃环境下变性3min，再迅速降温至68℃保温30min，自然缓慢降温至40℃保温50min，再自然降至室温；
(5)将玻片取出后置荧光显微镜下405nm激发光下观察并照相。
阴性对照的方法：除了不使用A序列，其余与本发明方法相同。
2、结果
结果如图5～6所示，使用双光子共聚焦荧光显微镜和Image J(Fiji is just)软件对荧光照相的蓝色荧光部分显像信号进行了比较分析，与阴性对照比较，
可以看出，阴性对照几乎无信号，而阳性对照则有比较强烈的蓝色荧光杂交信号，说明本发明方法可以准确检测，而去除特异性序列则无法检测。
实验例3 采用本发明方法检测IL-6基因
1、检测方法
按照实施例1的方法检测，阳性样品为造模脓毒症的小鼠24小时后的脾脏组织，阴性对照的样品为正常小鼠的脾脏组织。
脓毒症会引起小鼠脾脏中IL-6基因的表达升高，与正常小鼠相比，脓毒症的小鼠24小时后的脾脏组织的IL-6基因应该显著提高。
(1)序列及标记分别为：
IL-10-A探针:
CTGAAGCGCTTCGCGAGCCACAGAGGGGAGAAATCGATGACAGCGCCTCACTGAAGCGCTTCGCGAGCC
IL-10-B探针:CTCCCCTCTGTGGCTCGCGAAGC
IL-10-C探针:CGAAGCGCTTCAGTGAGGCGCTG
D探针序列:HCR-D:5'-FAM-GGCTCGCGAAGCGCTTCAG-FAM-3'
E探针序列:HCR-E:5'-TAMRA-CTGAAGCGCTTCGCGAGC-TAMRA-3'
(2)将上述探针分别溶解于TE缓冲液中，使A序列的浓度为1umol/L、B序列的浓度为5umol/L、C序列的浓度为5umol/L、D序列的浓度为3umol/L、E序列的浓度为6umol/L的混合液PM1；取1ul混合液PM1加入到49ul预杂交液配制成杂交缓冲液PM2；
(3)取小鼠肝组织切片，滴加25ul含荧光消减探针的杂交液PM2，加盖玻片，以及封胶封固；
(4)避光，将玻片在95℃环境下变性3min，再迅速降温至68℃保温30min，自然缓慢降温至40℃保温50min，再自然降至室温；
(5)将玻片取出后置荧光显微镜下488nm激发光下观察并照相。
2、结果
结果如图7～8所示，使用双光子共聚焦荧光显微镜和ImageJ软件对荧光照相的绿色部分显像信号进行了比较分析。
可以看出，阴性对照的荧光信号非常弱，而阳性对照则绿色荧光信号强，
说明本发明方法可以准确检测，而去除特异性序列则无法准确检测。
实验例4 采用本发明方法检测IL-10基因
1、检测方法
按照实施例1的方法检测，阳性样品为造模脓毒症的小鼠(保护组注射120mg/kg的红花黄色素)24小时后的肝脏组织，阴性对照的样品为正常小鼠的肝脏组织。
脓毒症后，注射120mg/kg的红花黄色素可以维持小鼠肝脏中IL-10的高表达，与正常小鼠相比，脓毒症的小鼠24小时后的脾脏组织的IL-10应该显 著提高。
(1)序列及标记分别为：
IL-10-A探针:
CTGAAGCGCTTCGCGAGCCACAGAGGGGAGAAATCGATGACAGCGCCTCACTGAAGCGCTTCGCGAGCC
IL-10-B探针:CTCCCCTCTGTGGCTCGCGAAGC
IL-10-C探针:CGAAGCGCTTCAGTGAGGCGCTG
D探针序列:HCR-D:5'.-FAM-GGCTCGCGAAGCGCTTCAG–FAM-3'
E探针序列:HCR-E:5'-.TAMRA-CTGAAGCGCTTCGCGAGC–TAMRA-3'
(2)将上述探针分别溶解于TE缓冲液中，使A序列的浓度为1umol/L、B序列的浓度为5umol/L、C序列的浓度为5umol/L、D序列的浓度为3umol/L、E序列的浓度为6umol/L的混合液PM1；取1ul混合液PM1加入到49ul预杂交液配制成杂交缓冲液PM2；
(3)取小鼠肝组织切片，滴加25ul含荧光消减探针的杂交液PM2，加盖玻片，以及封胶封固；
(4)避光，将玻片在95℃环境下变性3min，再迅速降温至68℃保温30min，自然缓慢降温至40℃保温50min，再自然降至室温；
(5)将玻片取出后置荧光显微镜下405nm激发光下观察并照相。
2、结果
结果如图9～10所示，使用双光子共聚焦荧光显微镜和ImageJ软件对荧光照相的蓝色部分显像信号进行了比较分析。
可以看出，阴性对照的荧光信号非常弱，而阳性对照则蓝色荧光信号强，检测结果与组织本身的性质一致，说明本发明方法可以有效、准确地检测。
实验例5 采用本发明方法检测IL-1β基因
1、检测方法
按照实施例1的方法检测，阳性样品为造模脓毒症的小鼠24小时后的肺组织，阴性对照的样品为正常小鼠的肺组织。
(1)序列及标记分别为：
IL-1β-A探针:
Figure PCTCN2018098338-appb-000005
IL-1β-B探针:CAGCACCACTAGGCTCGCGAAG
IL-1β-C探针:GAAGCGCTTCAGGAGTTCCCCAAC
D探针序列:HCR-D:5'.-FAM-GGCTCGCGAAGCGCTTCAG–FAM-3'
E探针序列:HCR-E:5'-.TAMRA-CTGAAGCGCTTCGCGAGC–TAMRA-3'
(2)将上述探针分别溶解于TE缓冲液中，使A序列的浓度为1umol/L、B序列的浓度为5umol/L、C序列的浓度为5umol/L、D序列的浓度为3umol/L、E序列的浓度为6umol/L的混合液PM1；取1ul混合液PM1加入到 49ul预杂交液配制成杂交缓冲液PM2；
(3)取小鼠肝组织切片，滴加25ul含荧光消减探针的杂交液PM2，加盖玻片，以及封胶封固；
(4)避光，将玻片在95℃环境下变性3min，再迅速降温至68℃保温30min，自然缓慢降温至40℃保温50min，再自然降至室温；
(5)将玻片取出后置荧光显微镜下488nm激发光下观察并照相。
2、结果
结果如图11～12所示，使用双光子共聚焦荧光显微镜和ImageJ软件对荧光照相的绿色部分显像信号进行了比较分析。
可以看出，阴性对照的荧光信号非常弱，而阳性对照则荧光信号强则有比较，荧光强度定量有统计学意义，检测结果与组织本身的性质一致，说明本发明方法可以有效、准确地检测。
实验例6 采用本发明方法检测端粒基因
1、检测方法
按照实施例2的方法检测，待检样品为大鼠肝组织。
阴性对照的方法：除了不使用A序列，其余与本发明方法相同。
a、序列及标记分别为：
⑴HCR-A:
5
Figure PCTCN2018098338-appb-000006
⑵HCR-A-R1:CCTAACCCTAGGCTCGCGAAGC
⑶HCR-A-R2:GCGAAGCGCTTCAGTTCCCTAACC
⑷HCR-B:
5’CCAGAGCGAGCAGTGTCAAGGCTCGCGAAGCGCTTCAGAACCAGAGCGAGCAGTGTC
⑸HCR-B-R1:CGAGCCTTGACACTGCTCG
⑹HCR-B-R2:GCTCGCTCTGGTTCTGAAGC
⑺HCR-C
GAACAGTCGTGAACATCTGACACTGCTCGCTCTGGTGAACAGTCGTGAACATC
⑻HCR-C-R1:
GCAGTGTCAGATGTTCA⑼HCR-C-R2:
GACTGTTCACCAGAGCG
⑽HCR-D:
FAM-GATGTTCACGACTGTTC-FAM
⑾HCR-E:
TAMRA-GAACAGTCGTGAACAT-TAMRA
b、检测方法
(1)将上述11中探针分别溶解于TE缓冲液中，得浓度均为10ummol/L的探针溶液，取A序列溶液1uL、B序列2uL、C序列2uL、F序列2uL、F-R1序列4uL、F-R2序列4uL、G序列4uL、G-R1序列8uL、G-R2序列8uL、H序列8uL、I序列16uL，得混合液PM1；取1ul混合液PM1加入到49ul预杂交液配制成杂交缓冲液PM2；
(2)全程避光，取待检样品，滴加25ul杂交液PM2，加盖玻片，以及封胶封固；
(3)将玻片在98℃环境下变性3min，再迅速降温至68℃保温30min，自然缓慢降温至40℃保温50min，再自然降至室温；
(4)将玻片取出后置荧光显微镜下488nm激发光下观察并照相。
2、结果
结果如图13～14所示，使用双光子共聚焦荧光显微镜和ImageJ软件对荧光照相的绿色部分显像信号进行了比较分析。
可以看出，阴性对照几乎无信号，而阳性对照则有比较强烈的荧光杂交信号，说明本发明方法可以准确检测，而去除特异性序列则无法准确检测。
综上，本发明试剂盒和方法可以有效检测基因，应用前景良好。

Claims (10)

  1. 一种基因检测试剂盒，其特征在于：它包括如下探针：
    A序列:
    5'–CTGAAGCGCTTCGCGAGCCM 1M 2N 1N 2N 3N 4N 5…N 6N 7N 8N 9N 10M 3M 4CTGAAGCGCTTCGCGAGCC-3'；其中，N 1N 2N 3N 4N 5…N 6N 7N 8N 9N 10为待检基因或其片段的互补序列，M 1M 2和M 3M 4为接头序列；
    B序列:5'-n 5n 4n 3n 2n 1m 2m 1TAGGCTCGCGAAGC-3'；n 1n 2n 3n 4n 5与N 1N 2N 3N 4N 5的序列互补，m 1m 2与M 1M 2的序列互补；
    C序列:5'-GCGAAGCGCTTCAGm 4m 3n 10n 9n 8n 7n 6-3'；n 6n 7n 8n 9n 10与N 6N 7N 8N 9N 10的序列互补，m 3m 4与M 3M 4的序列互补；
    D序列:5'-荧光分子-GGCTCGCGAAGCGCTTCAG–荧光分子-3'
    E序列:5'-荧光淬灭分子-CTGAAGCGCTTCGCGAGC-荧光淬灭分子-3'。
  2. 一种基因检测试剂盒，其特征在于：它包括如下探针：
    ⑴A序列:
    5'–CTGAAGCGCTTCGCGAGCCM 1M 2N 1N 2N 3N 4N 5…N 6N 7N 8N 9N 10M 3M 4CTGAAGCGCTTCGCGAGCC-3'；其中，N 1N 2N 3N 4N 5…N 6N 7N 8N 9N 10为待检基因或其片段的互补序列，M 1M 2和M 3M 4为接头序列；
    ⑵B序列:5'-n 5n 4n 3n 2n 1m 2m 1TAGGCTCGCGAAGC-3'；n 1n 2n 3n 4n 5与N 1N 2N 3N 4N 5的序列互补，m 1m 2与M 1M 2的序列互补；
    ⑶C序列:5'-GCGAAGCGCTTCAGm 4m 3n 10n 9n 8n 7n 6-3'；n 6n 7n 8n 9n 10与N 6N 7N 8N 9N 10的序列互补，m 3m 4与M 3M 4的序列互补；
    ⑷F序列:
    5'-CCAGAGCGAGCAGTGTCAAGGCTCGCGAAGCGCTTCAGAACCAGAGCGAGCAGTGTC-3'
    ⑸F-R1序列:5'-CGAGCCTTGACACTGCTCG-3'
    ⑹F-R2序列:5'-GCTCGCTCTGGTTCTGAAGC-3'
    ⑺G序列
    5'-GAACAGTCGTGAACATCTGACACTGCTCGCTCTGGTGAACAGTCGTGAACATC-3'
    ⑻G-R1序列:
    5'-GCAGTGTCAGATGTTCAC-3'
    ⑼G-R2序列:
    5'-GACTGTTCACCAGAGCG-3'
    ⑽H序列:
    5'-荧光分子-GATGTTCACGACTGTTC-荧光分子-3'
    ⑾I序列:
    5'-淬灭分子-GAACAGTCGTGAACAT-淬灭分子-3'。
  3. 根据权利要求1或3所述的检测试剂盒，其特征在于：所述M 1M 2为TA或AC，和/或，所述M 3M 4为AT、CA或TC。
  4. 根据权利要求1或3所述的检测试剂盒，其特征在于：所述A序列与B序列的摩尔比为1：(2～7)；所述A序列与C序列的摩尔比为1：(2～7)；所述A序列与D序列的摩尔比为1：(2～7)；所述D序列与E序列的摩尔比为1：(2～5)；
    优选地，A序列、B序列、C序列、D序列与E序列的摩尔比为1:5:5:3:6。
  5. 根据权利要求2或3所述的检测试剂盒，其特征在于：所述A序列与B序列的摩尔比为1：(2～7)；所述A序列与C序列的摩尔比为1：(2～7)；所述A序列与F序列的摩尔比为1：(2～7)；所述F序列与F-R1序列的摩尔比为1：(2～7)；所述F序列与F-R2序列的摩尔比为1：(2～7)；所述F序列与G序列的摩尔比为1：(2～7)；所述G序列与G-R1序列的摩尔比为1：(2～7)；所述G序列与G-R2序列的摩尔比为1：(2～7)；所述G序列与H序列的摩尔比为1：(2～7)；所述H序列与I序列的摩尔比为1：(2～5)；
    优选地，所述A序列、B序列、C序列、F序列、F-R1序列、F-R2序列、G序列、G-R1序列、G-R2序列、H序列、I序列的摩尔比为1:2:2:2.4:4:4::8:8:8:16。
  6. 根据权利要求1～5任意一项所述的试剂盒，其特征在于：所述待检基因为任意人体、大鼠和小鼠基因。
  7. 根据权利要求1～5任意一项所述的试剂盒，其特征在于：所述荧光分子为6-羧基荧光素、四氯-6羧基荧光素、六氯-6甲基荧光素或者2，7，-二甲基-4,5，二氯-6-6羧基荧光素；
    和/或，所述荧光淬灭分子为6-羧基-4甲基罗丹明或者黑洞淬灭集团。
  8. 根据权利要求1～4任意一项所述的试剂盒，其特征在于：所述试剂盒还包括TE缓冲液和预杂交液；
    所述TE缓冲液是含有10mmol/L tris和1mmol/L EDTA的溶液；
    所述预杂交液是含有60mmol/L tris、50mmol/L NaCl、1mmol/L EDTA、0.5％SDS(w/v)和5％(w/v)的甲酰胺的溶液。
  9. 一种基因检测方法，其特征在于：步骤如下：
    (1)取权利要求1～6任意一项所述试剂盒中的探针，溶解于TE缓冲液 中，得到包含所有探针序列的混合液PM1；
    (2)取混合液PM1加入到预杂交液配制成杂交缓冲液PM2；
    (3)全程避光，取杂交缓冲液PM2滴加到待测的染色体滴片或组织切片上，盖上盖玻片，用封胶封固；
    (4)将玻片加热到95℃～98℃变性3到5min，再迅速降温至45℃-70℃保温20到60min，使通用荧光探针与靶核酸完成分子杂交，然后再降温至30℃-50℃保温40到60min；
    (5)加入防淬灭封片剂，置荧光显微镜下观察杂交信号。
  10. 根据权利要求9所述的方法，其特征在于：步骤如下：步骤(4)为：将玻片在95℃或者98℃环境下变性3min，再迅速降温至68℃保温30min，自然降温至40℃保温50min，再自然降至室温；
    步骤(5)为：加入防淬灭封片剂，放上盖玻片，置荧光显微镜下488nm、405nm、670nm激发光下观察并照相。
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