CN102660634A - MRNA-level in-situ hybridization detection kit for KLOTHO gene in pancreas precancerous period of KLOTHO gene in prophase of pancreas cancer detecting method and application - Google Patents

Abstract

The invention discloses a in-situ hybridization detection kit, comprising a hybridization probe and a marker. The invention also discloses an mRNA-level method for in-situ hybridization detection of a Klotho gene which is closely related to early pathologic evolution of pancreatic cancers by using the kit which is closely related to the pathology evolution in the prophase of pancreas cancer, comprising the following steps of, (1) under the condition that the hybridization probe and a target sequence can form a steady hybrid complex, contacting the RNA to be detected in a substrate with the hybridization probe, to form a hybrid complex; (2) detecting the hybrid complex. The kit and the detecting method of the invention can detect the expression level of the Klotho gene at mRNA level and are earlier than medical imaging and present clinic biochemistry detecting indexes. The kit and the detecting method of the invention can realize real mRNA-level screening in precancerous periods, and meanwhile, the detecting method of the invention is simple and convenient and has low cost, which makes the method easy to be applied in county level hospitals.

Description

The horizontal hybridization in situ detection kit of mRNA and the detection method and the application of pancreas canceration KLOTHO gene in early stage

Technical field

The present invention relates to field of biological detection, more particularly, relate to cancer before the mRNA that becomes express the correlation detection technology that changes (pathology evolution process).

Background technology

According to the data that domestic and international authoritative institution provides, the newly-increased number 2,600,000 of the annual cancer of China, death toll nearly 2,100,000; The patient more than 700 ten thousand; The annual newly-increased cancer patients 8,000,000 in the whole world, death toll is near 8,000,000, and the patient has more than 8,400 ten thousand people approximately; To double to the above number of the year two thousand twenty, this is one group of fearful numeral.Cancer diagnosis and treatment cost is increasingly high; (poor area maybe be higher by cancer patients's year medical expense 200,000; Developed regions possibly exceed 200,000 far away), more than 700 ten thousand patients, annual cost is 1.4 trillion Renminbi; Deduction cost 35% is about 400,000,000,000, has every year 1000000000000 Renminbi to consume in vain approximately.And the cancer patients is most of can be dead soon after treatment.Therefore, existing clinical cancer diagnosis and treatment pattern must change, and innovative point of the present invention is to accomplish preventative examination in advance, in time gets involved preventative regulation and control and prophylactic treatment then, accomplishes preventiveing treatment of disease of gene level cancer.

An annual report has been done by eight tame units such as U.S. sanitary research institute in 2005, cancer research institute, Disease Control and Prevention Center; Anticancer Great War to initiation in 1972 is looked back; Report thinks that the mankind are failures in anticancer Great War; Conclusion is that cancer mortality does not reduce, and it is enumerated out and causes the Several Factors of anticancer Great War failure to be: 1. tumour cell heterogeneous (polymorphum); 2. tumor cell drug resistance; 3. cancer therapy drug mentality of designing imperfection (animal model designs not science) etc.Simultaneously, also propose to examine closely again the measure of existing diagnosis and treatment cancer in this report.

The inventor is in the middle discovery that studies for a long period of time, and the major reason that causes cancer mortality not fallen is to accomplish real early diagnosis.Come diagnosing cancer according to existing clinical medicine image (B ultrasonic, CT, zeugmatography etc.) and with other biochemistry (cancer antigen, CEACAMS, carbohydrate hormone, the cytolemma factor, the nucleus factor, cell streaming technology) index; All be that tumour forms the back diagnosis; The former will learn in a organized way and change or existing occupying lesion, latter's major part be tumour form the back secreted, discharge or the affinity tag of tumour.The clinical idea of tradition thinks that occupancy cancer piece is the diagnosis that belongs to early-stage cancer under 2 centimeters; This notion is worth conscientiously discussing, and it is rigorous inadequately that 2 centimeters early stage these of following cancer piece genus define science, analyzes from the cytology angle; 1 centimeter lump has 100,000,000 tumour cells approximately; Its three-dimensional cell stack number of 2 centimeters lump is far above 200,000,000 tumour cells, produces from canceration early stage to the mono-clonal cancer cells and forms 2 centimeters cancer piece, and its pathology evolution process is quite long; Possibly be (except the special case) more than 5 years or 10 years or even 10 years; What be difficult to confirm is in this pathology evolution process, and lump is unique spot of cancer and independent focus, and possible cancer cells is moved to other tissue or organ growth already.Clinical study confirms already, in case when forming lump, other cancer cells is moved to other position clonal growth through different approaches, in case behind the excision primary tumor, other organ recurrent foci or multiple cancer piece kitchen range successively form.Therefore; Whether define in early days with the cancerous swelling piece size below 2 centimeters clinically, rigorous inadequately (some case is when finding primary lesion; Find metastatic lesion simultaneously; Not in the content of our statement), at this moment be diagnosis in late period and treatment of late stage in fact, this is the true cause that causes cancer mortality not fallen.

Along with molecular biotechnology is perfect day by day, functional genomics, the deep expansion of research such as cancer genomics in order to seek more early stage diagnosing cancer, treatment cancer and preventing cancer, makes great progress.So far, we might do more accurate early screening and diagnosis on the one-level functional transcription product (mRNA level) of gene, preceding in canceration early stage or cancer cells formation (mono-clonal), just can accomplish early prediction and examination.The present invention adopts the nucleic acid hybridization in situ technology, selects many group clinical samples (cancer patient, high risk population, family members Shi Renqun, normal control), and check and analysis are carried out in the early warning of KLOTHO gene and carcinoma of the pancreas.

Scientist discovers that a kind of protein that can delay senility perhaps can be prevented and treated carcinoma of the pancreas.The protein of this Klotho of being called (Clotho) is a kind of natural hormone that brain and kidney produce.Find that through experiment Klotho protein has the effect that stops the mammary cancer differentiation, variation appears in this protein, can increase the probability that the women suffers from breast cancer greatly.The research of some other country shows that this protein also has the ability that stops liver cancer and uterus carcinoma cellular invasion.Carcinoma of the pancreas is that a kind of invasive is strong, diffusion is rapid, the high malignancy tumour, and sickness rate was ascendant trend year by year in recent years.Patient age is many in 40-60 year.In the U.S., carcinoma of the pancreas has been classified as the cause of the death of second largest digestive tract tumor.Some cities in China, sickness rate increased by 4 times in nearly 20 years.The obstructive jaundice of no pain and gall-bladder enlargement are the clinical diagnosis foundations of carcinoma of the pancreas, and therefore, early diagnosis and early treatment are the keys that improves and improve the carcinoma of the pancreas prognosis, early stage thoroughly radical cure tumour, and survival rate was greater than 20% in 5 years.Yet, the lack of specific performance clinically of early stage carcinoma of the pancreas, before jaundice did not occur, carcinoma of the pancreas was often out in the cold, caused easily and failed to pinpoint a disease in diagnosis or mistaken diagnosis.Though early stage vexed bloated sense of some upper abdomen of some patient or discomfort do not draw attention more.Because the carcinoma of the pancreas grade malignancy is high, very easily to shift, when waiting to occur the jaundice prescription on individual diagnosis, about 3/4 patient has belonged to late period, at present this cancer is not also had the efficacious therapy method.Experiment shows that the interior klotho gene expression amount of carcinoma of the pancreas patient's body reduces, and cancer can shift very soon.Use synthetic klotho albumen, the injection animal is after one or two week, and cancer cells has stopped diffusion, and tumour is also dwindled gradually.

Canceration has very important clinical diagnosis meaning in earlier stage as early screening pancreas with the mRNA of KLOTHO.The mRNA of the KLOTHO index that expression amount reduces in pancreas canceration early stage and canceration process is done in prostatitis canceration examination in early stage, and the recurrence of carcinoma of the pancreas art, is shifted early warning very important clinical meaning is also arranged.

The contriver is in long term studies; Drawn a kind of new concept, the clinical diagnosis and treatment pattern of cancer and other clinical major disease must change, and can not only stop present treating the disease affected (morbidity back diagnosis and treatment); Accomplish preventative diagnosis and treatment; Accomplish treating the disease affected, only in this way could reduce the M & M of major disease, reduce social cost and medical treatment cost.Therefore, the contriver innovates theoretical and technical in the horizontal kit for screening of mRNA and medicine of exploitation and production major disease.Particularly screen clinical samples (normal population, cancer high risk population, tumour patient); Broken through healthy tissues and tumor tissues consistency research and development thinking relatively, sought and developed the mRNA level that becomes before the cancer, developed closely related with cancer early gene physiopathology; And the extremely important target of clinical meaning; Tumour is clinically formed the preventative diagnosis and treatment that diagnosis and treatment pattern in back becomes tumour, striven for the time and the space of tumor diagnosis and treatment, reach preventing cancer.

At present the high flux gene chip technology is all adopted in the research of KLOTHO gene, and these methods are used for the scientific research aspect more, the incompatibility clinical application.Detection technique and test kit according to existing literature data KLOTHO gene mRNA level do not appear in the newspapers.

The inventor is in the requirement to the novelty invention; Designed different pieces of information example group (Pancreas cancer patients, high risk population, normal control people) example group; Detect with hybridization in situ technique; The result shows the low expression of above pancreatic cancer patient KLOTHO gene, and the high risk population has expression in various degree, and the normal people is a high expression level.The important symbol thing that shows KLOTHO gene pancreas canceration examination in early stage.

Hybridization in situ technique (in situ hybridization) combines molecular biology and cytochemistry technology, is probe with the nucleic acid molecule of mark, in the technology of histocyte in situ detection specific nucleic acid molecule.Its principle is to make the nucleic acid strand (being probe) that contains distinguished sequence, process mark; Under optimum conditions with histocyte in the complementary nucleic acid strand be that target nucleic acid is hybridized; With radioautograph or immunocytochemistry label probe is surveyed again, thereby shown special DNA or RNA molecule at cell in-situ.

The probe of in situ hybridization is molecule or sequence the unknown but the known nucleic acid molecule of molecule (though indeterminate this molecule full sequence of known array; But what target molecule known its is directed against), the kind of probe can be divided into dna probe, cDNA probe, cRNA probe and synthetic oligonucleotide probe again by the properties of nucleic acids difference.For the ease of spike, probe must be used certain means mark in addition, is beneficial to later detection.Affinity tag commonly used comprises radionuclide and non-radioactive marker's two big classes.Isotopic label commonly used has³H,³⁵S,¹²⁵I with³²P.Advantages such as susceptibility is high though isotopic label has, the back of the body end is comparatively clear because ri all can damage human and environment, have by the substituted trend of heterotope recently.At present the most frequently used in the heterotope affinity tag have three kinds of vitamin H, digoxin and resorcinolphthaleins.The method that detects these affinity tags all is a sensitive extremely.

According to used probe and to detect nucleic acid difference can be divided into DNA-DNA again, RNA-DNA, RNA-RNA hybridization.No matter but the hybridization of any form, all must be through five big processes, promptly histiocytic fixing, prehybridization, hybridization, flushing and demonstration.The present invention adopts the hybridization mode of RNA-RNA; Synthetic probe (mRNA) and the said target mrna that detects are the principles that adopts base complementrity (hybridization is complementary); Simultaneously through long-time research and observation; Start and termination place the result not influence (because mRNA sequence that contriver adopt all surpass 600bp more than) of residue to detecting.

In view of the diagnosis of cancer clinically (medical imaging and biochemical indicator thing all are the diagnosis after tumour forms) at present is the diagnosis in late period, treatment also is a treatment of late stage, the treatment pattern that causes mortality ratio not fallen.Original intention of the present invention is to want to change at present the diagnosis and treatment pattern of major disease clinically; Become preventative preventiveing treatment of disease from treating the disease affected; Reach preventative diagnosis and treatment; Present medical imaging means and numerous biochemical marker can't be detected become the mRNA level before the cancer and quantize changes technology, do the technological breakthrough of novelty, provide that to become the horizontal examination of mRNA before the cancer technological.Making has had a preceding technology that becomes the real early screening of mRNA level of new cancer clinically, for the diagnosis and treatment of clinical cancer are raced against time and the space.

Summary of the invention

The object of the invention at first provides a kind of hybridization in situ detection kit, and it comprises in situ hybridization detection probes and affinity tag.

Secondly, the present invention also will provide the mentioned reagent box to be used for pancreatic cell canceration examination in early stage and the recurrence of treatment back, to shift the relevant in situ hybridization detection method of early warning.

For realizing the object of the invention, technical scheme of the present invention is following:

The present invention at first provides a kind of hybridization in situ detection kit, and it comprises hybridization probe and affinity tag, wherein; Described hybridization probe is sequence shown in SEQ ID NO.1, is in the middle of the KLOTHO gene order one section, from 1000 to 1800bp; The sequence of KLOTHO gene is shown in SEQ ID NO.2; The nucleotide sequence length of gene is 5102bp, CDS:9 ... 3047bp, the number of being positioned at karyomit(e) 13q12^"If (gene order is oversize in the probe mark process; Surpass more than the 1000bp, we use the sequence of CDS and come designing probe, if the sequence of CDS also surpasses more than the 1000bp; Can adopt one section base sequence in centre of gene to come synthesising probing needle; Base sequence is no less than 500bp, will do sequential detection after probe is synthetic, and function is carried out the analysis of clinical meaning).

A preferred version of test kit of the present invention is that described affinity tag is selected from radioactive substance, chemoluminescence or substance that show color, vitamin H, metal king crab, resorcinolphthalein, enzyme and nano material.

A preferred version of test kit of the present invention is also to comprise hybridization solution.

A preferred version of test kit of the present invention is also to comprise toughener.

A preferred version of test kit of the present invention is also to comprise developer.

Canceration of the present invention KLOTHO gene screening in early stage test kit using value is, can be in the mRNA level, to pancreas canceration examination in early stage, and after the canceration or postoperative recurrence, transfer, diffusion generation early warning, further cooperate clinical treatment.

The present invention also provides a kind of detection method of KLOTHO gene hybridization in situ, may further comprise the steps:

(1) described in the above hybridization probe and target sequence can form under the condition of stablizing hybridization complex, and RNA to be measured in the substrate is contacted with hybridization probe, form hybridization complex; With

(2) detect said hybridization complex.

Detection method of the present invention, wherein preferably, the described condition of stablizing hybridization complex that forms is: the temperature of nucleic acid hybridization is 42 ℃; The time of nucleic acid hybridization is 16-24 hour.

Detection method of the present invention, wherein preferably, described substrate is selected people's blood leucocyte sample or other organ-tissue cell specimen for use.More preferably be that described blood preparation or other organ-tissue cell specimen are from Pancreas cancer patients, carcinoma of the pancreas high risk population, healthy subjects.

Detection method of the present invention, wherein preferably, other cancers such as described cancer high risk population, Pancreas cancer patients and mammary cancer, liver cancer.

Detection kit of the present invention is to adopt nucleic acid hybridization technique and groupization immunization method to combine; One-level functional transcription product mRNA with the KLOTHO gene is a detected object; Synthesising probing needle is the mRNA sequence of KLOTHO gene, and the substrate of detection is the expression amount of blood of human body sample white corpuscle or histiocytic RNA.The display packing of hybridization in situ technique can provide the sxemiquantitative or the quantitative expression deciding degree of KLOTHO gene.Above expression of gene amount is judged in colour developing according to hybridization back immunohistochemical methods, and normal people KLOTHO gene is low expresses, i.e. colour developing in a small amount, and the KLOTHO gene has apparent difference cancer patient and normal people, and this expression of gene amount is all higher than normal people expression amount.

The component of diagnostic kit of the present invention is by hybridization probe, hybridization solution, developer, compositions such as synergistic agent.The nucleic acid hybridization principle of this test kit is that the molecular biology insider all knows, and the concrete operations step is to carry out quantitative analysis, result's report under sample disposal, prehybridization, hybridization, immunohistochemical staining, the mirror, and wherein the concrete steps of hybridization comprise:

1). sample to be measured is put into reactive tank;

2). instrument discards liquid automatically, adds Digestive system automatically;

3). instrument discards liquid automatically, and the back is fixing automatically;

4). instrument discards liquid automatically, automatically prehybridization (42 ℃);

5). instrument discards liquid automatically, cleans automatically;

6). instrument discards liquid automatically, automatically hybridization (42 ℃);

7). instrument discards liquid automatically, cleans automatically;

8). instrument discards liquid automatically, and automatic and DIG antibody is cultivated (room temperature);

9). instrument discards liquid automatically, cleans colour developing automatically;

10). take out the mounting microscopy.

The scheme of a preferred embodiment of the present invention is: with the one-level functional transcription product mRNA synthetic nucleic probe of KLOTHO gene with digoxigenin labeled (cDNA of digoxigenin labeled, RNA and oligonucleotide probe; Not only probe has a biotin labeling advantage; Also having overcome biotin labeled probe is organized the endogenous vitamin H to do shortcomings such as sorrow in the crossover process in position); This hybridization probe and the leukocytic RNA nucleic acid to be measured of blood of human body are hybridized,, under light microscopic, observe existence and the location of mRNA again with the method colour developing of immunohistochemical methods; According to painted cell count, judge the expression amount of goal gene.

The inventive method is a nucleic acid hybridization in situ technology commonly used at present; This method is through detecting the KLOTHO gene expression amount in the substrate cell; Be used for confirming the canceration mRNA variable quantity in early stage, the prediction that whether canceration of early warning pancreas takes place and whether the Pancreas cancer patients postoperative recurs, shifts.Because KLOTHO gene high expression level in the normal people; If the KLOTHO gene expression amount is high; Explain that the risk of suffering from carcinoma of the pancreas is low, how the KLOTHO gene expression amount reduces the explanation canceration takes place, or the cancer patient postoperative recurs, shifts; Thereby obtain the early diagnosis information in the cancer pathology evolution process, help the doctor to get involved diagnosis and treatment early.A test kit can many person-portions use or person-portion use.

As stated, KLOTHO expression of gene amount reduces or zero expression when detecting, and then measurable experimenter is taken place for pancreas canceration pathologic process.

The present invention has following beneficial effect:

Clinical meaning of the present invention is the more early stage variation that detects KLOTHO gene mRNA expression amount in pancreas canceration generation and the pathology evolution process of following the tracks of, and the early warning carcinoma of the pancreas takes place, development trend.Diagnostic kit of the present invention and other detection and cancer markers clinically, and the medical imaging inspection has apparent difference.The present invention can be at the one-level function product mRNA of genetic transcription level detection KLOTHO gene unconventionality expression; Before the recurrence of occupancy carninomatosis kitchen range is not found in the medical imaging inspection; Before the cancer biochemical indicator does not produce unusually; Also do not form before the tumour, can accomplish the information acquisition of above abnormal gene expression early, predict for real early diagnosis of clinical cancer sufferer and treatment back transfer and relapse early.So just might implement early screening, early prevention, the early treatment of cancer, might from the source, thoroughly effect a radical cure the carcinoma of the pancreas foul disease.

In addition, characteristics highly sensitive, high specificity that test kit provided by the invention has, simultaneously, detection method of the present invention is convenient and simple for operation, can be widely used and promoted in Municipal Hospitals.

Description of drawings

Fig. 1 is a KLOTHO gene hybridization in situ techniqueflow chart of the present invention.

Fig. 2 is that pancreatic cancer patient KLOTHO expresses the reduction picture in the embodiment of the invention.

Fig. 3 is high risk population's picture.

Fig. 4 is that normal people KLOTHO expresses picture in the embodiment of the invention.

Embodiment

Below in conjunction with embodiment, content of the present invention is described more specifically.Should be appreciated that following embodiment is used for explanation and non-limiting content of the present invention, any pro forma change or accommodation will fall into protection scope of the present invention.

Embodiment 1

Prepare the in situ hybridization test kit of present embodiment according to ordinary method, this test kit comprises with the KLOTHO gene being hybridization probe, affinity tag, the specification sheets of testing goal gene design, wherein:

The probe mark thing of present embodiment is selected digoxin for use.

The test kit hybridization solution is formed:

Digestive system	100 μ L/pipe	1 pipe/box	Colourless transparent liquid
				Protection liquid	100 μ L/pipe	1 pipe/box	Colourless transparent liquid
Prehybridization solution	1300 μ L/ pipe	2 pipe/boxes	Colourless transparent liquid
				The justice hybridization solution	10 μ L/pipe	1 pipe/box	Colourless transparent liquid
The antisense hybridization solution	10 μ L/pipe	1 pipe/box	Colourless transparent liquid
				Confining liquid	1000 μ L/pipe	1 pipe/box	Colourless transparent liquid
The alkalinephosphatase enzyme antibody	1 μ L/pipe	1 pipe/box	Colourless transparent liquid
				Developer A	175 μ L/pipe	1 pipe/box	Yellow liquid
Developer B	320 μ L/pipe	1 pipe/box	Colourless transparent liquid
				The damping fluid I	The 90mL/bottle	1 bottle/box	Light yellow or colourless transparent liquid
The damping fluid II	The 80mL/bottle	1 bottle/box	Light yellow or colourless transparent liquid
				The damping fluid III	The 20mL/ bottle	3 bottle/boxes	Light yellow or colourless transparent liquid
The damping fluid IV	The 90mL/bottle	1 bottle/box	Light yellow or colourless transparent liquid
				Stationary liquid	The 90mL/bottle	1 bottle/box	Colourless transparent liquid
The positive control sample	6/box	?	?

The reagent preparation working concentration

1). 10 * damping fluid I is diluted to 1 * damping fluid I with tri-distilled water by 1:10;

2). 20 * damping fluid II is diluted to 2 * damping fluid II with tri-distilled water by 1:10;

Be diluted to 0.2 * damping fluid II by 1:100; Be diluted to 0.1 * damping fluid II by 1:200;

3). 10 * damping fluid III is diluted to 1 * damping fluid III with tri-distilled water by 1:10;

4) .10 * damping fluid IV with tri-distilled water by 1:10 be diluted to * the damping fluid IV (get 1#, 2#, each 10mL of 3#, add water to 100mL both can).

Embodiment 2

Use the implementation process of nucleic acid hybridization in situ detection method to each group blood preparation KLOTHO gene expression amount:

1). get two of samples to be measured;

2). in glass jar, add Digestive system (Digestive system 100 μ L add 1 * damping fluid I 99.9ml, are working concentration) 50 ml, 37 ℃ of water-bath preheating 10min put 16 slides into, handle 12 min,use 1 * damping fluid I to wash 5min again for 37 ℃;

3). (protection liquid 1ml adds 1 * damping fluid

to the protection liquid with 0.2%; 99ml is working concentration) wash 10min; Tri-distilled water is washed 5min (above process is all carried out at glass jar); Take out slide, let its seasoning;

4). slide is put into the box of preserving moisture, add prehybridization solution 25 μ L/ sheets (being added in the place of cell), covered, the lid box of tightly preserving moisture is placed in 42 ℃ of constant water bath box more than the 3h;

5). take out slide, discard deckglass, slide is put into glass jar, the ethanol with 70%, 90%, 95% is respectively washed 2min, takes out seasoning;

6). slide is put into the box of preserving moisture, and one adds just hybridization solution 25 μ L/ sheets, and another adds antisense hybridization solution 25 μ L/ sheets, and covered is covered the box of tightly preserving moisture, and is placed on 16-24h in 42 ℃ of constant water bath box;

7). take out slide, discard deckglass, slide is put into glass jar:

In 42 ℃ of constant water bath box, wash twice, each 15min with 2 * damping fluid II;

In 42 ℃ of constant water bath box, wash once each 15min with 0.2 * damping fluid II;

In 42 ℃ of constant water bath box, wash twice, each 15min with 0.1 * damping fluid II;

8). wash 30s with 1 * damping fluid III, take out slide, seasoning;

9). slide is put into the box of preserving moisture, add 0.5% confining liquid (the 1ml confining liquid adds5ml 1 * damping fluid III), 100 μ L/ sheets, cover the box of tightly preserving moisture, at room temperature act on 30min.(this step need not add deckglass);

10). take out slide, wash 30s with 1 * damping fluid III, seasoning;

11). slide is put into the box of preserving moisture; Add X-AP antibody (getting a pipe alkaline phosphatase enzyme antibody) 100 μ L/ sheets, cover the box of tightly preserving moisture and at room temperature act on 30min to wherein adding 1.8ml 1 * damping fluid III; Time can not be long, otherwise can produce false positive (this step need not add deckglass);

12). take out slide, wash 3 times, each 15min with 1 * damping fluid III;

13). wash 2min with 1 * damping fluid IV, add developer (developer A73.3 μ L, developer B157.5 μ L is added in30mL 1 * damping fluid IV, mixing), room temperature lucifuge 16h is to more than the 18h;

14). wash 5min with tri-distilled water, seasoning, (add with glycerine 10% 1 * damping fluid I mixing) mounting microscopy.

Nucleic acid hybridization in situ detection method of the present invention is used digoxigenin labeled with goal gene; Become the RNA nucleic probe; The RNA nucleic acid to be measured of probe and human leukocytes is hybridized,, therefore under light microscopic, observe existence and the location of mRNA again with the method colour developing of immunohistochemical methods; According to painted cell count, judge the expression amount of goal gene.

20 of carcinoma of the pancreas patients, 20 of high-risk and family members' histories, 20 of normal control groups.Take out all people's to be checked peripheral blood 3-5 milliliter (separation white corpuscle) and do in situ hybridization.The result representes that all cancer patients KLOTHO genetic expressions reduce, the shallow or dye-free of cell dyeing; High risk population and family members Shi Renyuan express and reduce, decimal dyeing; Normal control group KLOTHO genetic expression is high, and the most dyeing of cell concrete outcome is seen Fig. 2, Fig. 3, Fig. 4.

The carcinoma of the pancreas number	Expression amount %	High-risk number	Expression amount %	Normal number	Expression amount %
						1	?0	1	12	1	78
2	0	2	16	2	69
						3	2	3	13	3	66
4	0	4	18	4	80
						5	0	5	15	5	64
6	0	6	16	6	72
						7	0	7	12	7	70
8	0	8	13	8	82
						9	0	9	16	9	77
? 10	3	? 10	20	? 10	69
						? 12	0	? 12	12	? 12	86
? 13	4	? 13	15	? 13	90
						? 14	0	? 14	11	? 14	88
? 15	0	? 15	16	? 15	82
						? 16	0	? 16	13	? 16	76
? 17	2	? 17	15	? 17	75
						? 18	0	? 18	16	? 18	87
? 19	3	? 19	21	? 19	90
						? 20	0	? 20	20	? 20	78

。

Sequence table

 

SEQ ID NO:1 (probe sequence)

1000?atttattgat?ggtgactatc

1021?ccgagagcat?gaagaataac?ctttcatcta?ttctgcctga?ttttactgaa?tctgagaaaa

1081?agttcatcaa?aggaactgct?gacttttttg?ctctttgctt?tggacccacc?ttgagttttc

1141?aacttttgga?ccctcacatg?aagttccgcc?aattggaatc?tcccaacctg?aggcaactgc

1201?tttcctggat?tgaccttgaa?tttaaccatc?ctcaaatatt?tattgtggaa?aatggctggt

1261?ttgtctcagg?gaccaccaag?agagatgatg?ccaaatatat?gtattacctc?aaaaagttca

1321?tcatggaaac?cttaaaagcc?atcaagctgg?atggggtgga?tgtcatcggg?tataccgcat

1381?ggtccctcat?ggatggtttc?gagtggcaca?gaggttacag?catcaggcgt?ggactcttct

1441?atgttgactt?tctaagccag?gacaagatgt?tgttgccaaa?gtcttcagcc?ttgttctacc

1501?aaaagctgat?agagaaaaat?ggcttccctc?ctttacctga?aaatcagccc?ctagaaggga

1561?catttccctg?tgactttgct?tggggagttg?ttgacaacta?cattcaagta?gataccactc

1621?tgtctcagtt?taccgacctg?aatgtttacc?tgtgggatgt?ccaccacagt?aaaaggctta

1681?ttaaagtgga?tggggttgtg?accaagaaga?ggaaatccta?ctgtgttgac?tttgctgcca

1741?tccagcccca?gatcgcttta?ctccaggaaa?tgcacgttac?acattttcgc?ttctccctgg

 

The sequence of SEQ ID NO:2 KLOTHO gene and number: NM_004795.3

1?cgcgcagcat?gcccgccagc?gccccgccgc?gccgcccgcg?gccgccgccg?ccgtcgctgt

61?cgctgctgct?ggtgctgctg?ggcctgggcg?gccgccgcct?gcgtgcggag?ccgggcgacg

121?gcgcgcagac?ctgggcccgt?ttctcgcggc?ctcctgcccc?cgaggccgcg?ggcctcttcc

181?agggcacctt?ccccgacggc?ttcctctggg?ccgtgggcag?cgccgcctac?cagaccgagg

241?gcggctggca?gcagcacggc?aagggtgcgt?ccatctggga?tacgttcacc?caccaccccc

301?tggcaccccc?gggagactcc?cggaacgcca?gtctgccgtt?gggcgccccg?tcgccgctgc

361?agcccgccac?cggggacgta?gccagcgaca?gctacaacaa?cgtcttccgc?gacacggagg

421?cgctgcgcga?gctcggggtc?actcactacc?gcttctccat?ctcgtgggcg?cgagtgctcc

481?ccaatggcag?cgcgggcgtc?cccaaccgcg?aggggctgcg?ctactaccgg?cgcctgctgg

541?agcggctgcg?ggagctgggc?gtgcagcccg?tggtcaccct?gtaccactgg?gacctgcccc

601?agcgcctgca?ggacgcctac?ggcggctggg?ccaaccgcgc?cctggccgac?cacttcaggg

661?attacgcgga?gctctgcttc?cgccacttcg?gcggtcaggt?caagtactgg?atcaccatcg

721?acaaccccta?cgtggtggcc?tggcacggct?acgccaccgg?gcgcctggcc?cccggcatcc

781?ggggcagccc?gcggctcggg?tacctggtgg?cgcacaacct?cctcctggct?catgccaaag

841?tctggcatct?ctacaatact?tctttccgtc?ccactcaggg?aggtcaggtg?tccattgccc

901?taagctctca?ctggatcaat?cctcgaagaa?tgaccgacca?cagcatcaaa?gaatgtcaaa

961?aatctctgga?ctttgtacta?ggttggtttg?ccaaacccgt?atttattgat?ggtgactatc

1021?ccgagagcat?gaagaataac?ctttcatcta?ttctgcctga?ttttactgaa?tctgagaaaa

1081?agttcatcaa?aggaactgct?gacttttttg?ctctttgctt?tggacccacc?ttgagttttc

1141?aacttttgga?ccctcacatg?aagttccgcc?aattggaatc?tcccaacctg?aggcaactgc

1201?tttcctggat?tgaccttgaa?tttaaccatc?ctcaaatatt?tattgtggaa?aatggctggt

1261?ttgtctcagg?gaccaccaag?agagatgatg?ccaaatatat?gtattacctc?aaaaagttca

1321?tcatggaaac?cttaaaagcc?atcaagctgg?atggggtgga?tgtcatcggg?tataccgcat

1381?ggtccctcat?ggatggtttc?gagtggcaca?gaggttacag?catcaggcgt?ggactcttct

1441?atgttgactt?tctaagccag?gacaagatgt?tgttgccaaa?gtcttcagcc?ttgttctacc

1501?aaaagctgat?agagaaaaat?ggcttccctc?ctttacctga?aaatcagccc?ctagaaggga

1561?catttccctg?tgactttgct?tggggagttg?ttgacaacta?cattcaagta?gataccactc

1621?tgtctcagtt?taccgacctg?aatgtttacc?tgtgggatgt?ccaccacagt?aaaaggctta

1681?ttaaagtgga?tggggttgtg?accaagaaga?ggaaatccta?ctgtgttgac?tttgctgcca

1741?tccagcccca?gatcgcttta?ctccaggaaa?tgcacgttac?acattttcgc?ttctccctgg

1801?actgggccct?gattctccct?ctgggtaacc?agtcccaggt?gaaccacacc?atcctgcagt

1861?actatcgctg?catggccagc?gagcttgtcc?gtgtcaacat?caccccagtg?gtggccctgt

1921?ggcagcctat?ggccccgaac?caaggactgc?cgcgcctcct?ggccaggcag?ggcgcctggg

1981?agaaccccta?cactgccctg?gcctttgcag?agtatgcccg?actgtgcttt?caagagctcg

2041?gccatcacgt?caagctttgg?ataacgatga?atgagccgta?tacaaggaat?atgacataca

2101?gtgctggcca?caaccttctg?aaggcccatg?ccctggcttg?gcatgtgtac?aatgaaaagt

2161?ttaggcatgc?tcagaatggg?aaaatatcca?tagccttgca?ggctgattgg?atagaacctg

2221?cctgcccttt?ctcccaaaag?gacaaagagg?tggctgagag?agttttggaa?tttgacattg

2281?gctggctggc?tgagcccatt?ttcggctctg?gagattatcc?atgggtgatg?agggactggc

2341?tgaaccaaag?aaacaatttt?cttcttcctt?atttcactga?agatgaaaaa?aagctaatcc

2401?agggtacctt?tgactttttg?gctttaagcc?attataccac?catccttgta?gactcagaaa

2461?aagaagatcc?aataaaatac?aatgattacc?tagaagtgca?agaaatgacc?gacatcacgt

2521?ggctcaactc?ccccagtcag?gtggcggtag?tgccctgggg?gttgcgcaaa?gtgctgaact

2581?ggctgaagtt?caagtacgga?gacctcccca?tgtacataat?atccaatgga?atcgatgacg

2641?ggctgcatgc?tgaggacgac?cagctgaggg?tgtattatat?gcagaattac?ataaacgaag

2701?ctctcaaagc?ccacatactg?gatggtatca?atctttgcgg?atactttgct?tattcgttta

2761?acgaccgcac?agctccgagg?tttggcctct?atcgttatgc?tgcagatcag?tttgagccca

2821?aggcatccat?gaaacattac?aggaaaatta?ttgacagcaa?tggtttcccg?ggcccagaaa

2881?ctctggaaag?attttgtcca?gaagaattca?ccgtgtgtac?tgagtgcagt?ttttttcaca

2941?cccgaaagtc?tttactggct?ttcatagctt?ttctattttt?tgcttctatt?atttctctct

3001?cccttatatt?ttactactcg?aagaaaggca?gaagaagtta?caaatagttc?tgaacatttt

3061?tctattcatt?cattttgaaa?taattatgca?gacacatcag?ctgttaacca?tttgcacctc

3121?taagtgttgt?gaaactgtaa?atttcataca?tttgacttct?agaaaacatt?tttgtggctt

3181?atgacagagg?ttttgaaatg?ggcataggtg?atcgtaaaat?attgaataat?gcgaatagtg

3241?cctgaatttg?ttctcttttt?gggtgattaa?aaaactgaca?ggcactataa?tttctgtaac

3301?acactaacaa?aagcatgaaa?aataggaacc?acaccaatgc?aacatttgtg?cagaaatttg

3361?aatgacaaga?ttaggaatat?tttcttctgc?acccacttct?aaatttaatg?tttttctgga

3421?agtagtaatt?gcaagagttc?gaatagaaag?ttatgtacca?agtaaccatt?tctcagctgc

3481?cataataatg?cctagtggct?tcccctctgt?caaatctagt?ttcctatgga?aaagaagatg

3541?gcagatacag?gagagacgac?agagggtcct?aggctggaat?gttcctttcg?aaagcaatgc

3601?ttctatcaaa?tactagtatt?aatttatgta?tctggttaat?gacatacttg?gagagcaaat

3661?tatggaaatg?tgtattttat?atgatttttg?aggtcctgtc?taaaccctgt?gtccctgagg

3721?gatctgtctc?actggcatct?tgttgagggc?cttgcacata?ggaaactttt?gataagtatc

3781?tgcggaaaaa?caaacatgaa?tcctgtgata?ttgggctctt?caggaagcat?aaagcaattg

3841?tgaaatacag?tataccgcag?tggctctagg?tggaggaaag?gaggaaaaag?tgcttattat

3901?gtgcaacatt?atgattaatc?tgattataca?ccatttttga?gcagatcttg?gaatgaatga

3961?catgaccttt?ccctagagaa?taaggatgaa?ataatcactc?attctatgaa?cagtgacact

4021?actttctatt?ctttagctgt?actgtaattt?ctttgagttg?atagttttac?aaattcttaa

4081?taggttcaaa?agcaatctgg?tctgaataac?actggatttg?tttctgtgat?ctctgaggtc

4141?tattttatgt?ttttgctgct?acttctgtgg?aagtagcttt?gaactagttt?tactttgaac

4201?tttcacgctg?aaacatgcta?gtgatatcta?gaaagggcta?attaggtctc?atcctttaat

4261?gccccttaaa?taagtcttgc?tgattttcag?acagggaagt?ctctctatta?cactggagct

4321?gttttataga?taagtcaata?ttgtatcagg?caagataaac?caatgtcata?acaggcattg

4381?ccaacctcac?tgacacaggg?tcatagtgta?taataatata?ctgtactata?taatatatca

4441?tctttagagg?tatgattttt?tcatgaaaga?taagcttttg?gtaatattca?ttttaaagtg

4501?gacttattaa?aattggatgc?tagagaatca?agtttatttt?atgtatatat?ttttctgatt

4561?ataagagtaa?tatatgttca?ttgtaaaaat?ttttaaaaca?cagaaactat?atgcaaagaa

4621?aaaataaaaa?ttatctataa?tctcagaacc?cagaaatagc?cactattaac?atttcctacg

4681?tattttattt?tacatagatc?atattgtata?tagttagtat?ctttattaat?ttttattatg

4741?aaactttcct?ttgtcattat?tagtcttcaa?aagcatgatt?tttaatagtt?gttgagtatt

4801?ccaccacagg?aatgtatcac?aacttaaccg?ttcccgtttg?ttagactagt?ttcttattaa

4861?tgttgatgaa?tgttgtttaa?aaataatttt?gttgctacat?ttactttaat?ttccttgact

4921?gtaaagagaa?gtaattttgc?tccttgataa?agtattatat?taataataaa?tctgcctgca

4981?actttttgcc?ttctttcata?atcataaaaa?aa

 

Claims (10)

1. a hybridization in situ detection kit comprises hybridization probe and affinity tag, it is characterized in that, described hybridization probe is the sequence shown in the sequence table SEQ ID NO.1.

2. test kit as claimed in claim 1 is characterized in that, described affinity tag is selected from radioactive substance, chemoluminescence or substance that show color, vitamin H, metal king crab, resorcinolphthalein, enzyme and nano material.

3. test kit as claimed in claim 1 is characterized in that this test kit also comprises hybridization solution.

4. test kit as claimed in claim 1 is characterized in that this test kit also comprises synergistic agent.

5. test kit as claimed in claim 1 is characterized in that this test kit also comprises developer.

6. KLOTHO gene hybridization in situ detection method is characterized in that this method may further comprise the steps:

(1) can form under the condition of stablizing hybridization complex at described hybridization probe of claim 1 and target sequence, RNA to be measured in the substrate is contacted with hybridization probe, form hybridization complex; With

(2) detect said hybridization complex.

7. detection method as claimed in claim 6 is characterized in that, the described condition of stablizing hybridization complex that forms is: the temperature of nucleic acid hybridization is 42 ℃; The time of nucleic acid hybridization is 16-24 hour.

8. detection method as claimed in claim 6 is characterized in that, described substrate is selected people's blood leucocyte sample for use.

9. detection method as claimed in claim 8 is characterized in that, described blood leucocyte sample is selected from cancer, high risk population, normal people's sample.

10.KLOTHO gene detects the application in the carcinoma of the pancreas in situ hybridization test kit in preparation.

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