CN116949176A - Application of reagent for detecting FAS gene mutation site in preparation of pancreatic duct adenocarcinoma prognosis detection product - Google Patents

Abstract

The application relates to an application of a reagent for detecting FAS gene mutation in preparing pancreatic duct adenocarcinoma prognosis detection products; the mutation site is selected from g.8901119C > T or/and g.89021477G > A. Compared with the prior art, the application has the following beneficial effects: based on long-term clinical and massive case studies, the inventor of the present inventors found that mutations at two sites in the FAS gene (g.8901119c > T or/and g.89021477g > a) are closely related to prognosis of pancreatic ductal adenocarcinoma, and by detecting the mutation conditions of these two sites in the FAS gene, prognosis of patients after radical PDAC treatment can be predicted, including prediction of survival. The application can provide accurate guidance and clinical advice for prognosis and subsequent adjuvant treatment of pancreatic cancer patients.

Description

Application of reagent for detecting FAS gene mutation site in preparation of pancreatic duct adenocarcinoma prognosis detection product

Technical Field

The application relates to the technical field of biomedicine, in particular to application of a reagent for detecting FAS gene mutation sites in preparation of pancreatic duct adenocarcinoma prognosis detection products.

Background

Pancreatic ductal adenocarcinoma (pancreatic ductal adenocarcinoma, PDAC) accounts for more than 90% of pancreatic malignancies (pancreatic cancer), a tumor of the digestive system with high malignancy and poor prognosis, called "king in cancer", with a survival rate of less than 10% in 5 years. There is currently no effective treatment and means to predict disease progression. The method can evaluate the PDAC treatment effect, predict the disease progress and the prognosis situation in time and has great significance for the clinical treatment of PDAC and the social hygiene and economy.

The pancreatic duct adenocarcinoma has high malignancy degree and poor prognosis, and the traditional CT, nuclear magnetism, tumor marker CA19-9 and the like can not accurately know the disease progress and prognosis, so that the prognosis can not be predicted in advance, the difficulty is brought to clinical diagnosis and treatment, and the risk is increased.

In view of this, the present application has been made.

Disclosure of Invention

One of the purposes of the embodiment of the application comprises that a reagent for detecting the proper mutation site of the FAS gene is used for preparing a pancreatic ductal adenocarcinoma prognosis detection product, so that an effective means is provided for prognosis of pancreatic ductal adenocarcinoma.

In a first aspect of the application, there is provided the use of a reagent for detecting FAS gene mutation in the preparation of a pancreatic ductal adenocarcinoma prognostic assay product;

the mutation site is selected from g.8901119C > T or/and g.89021477G > A.

In some embodiments of the application, the reagent comprises a specific hybridization probe.

In some embodiments of the application, the specific hybridization probe targets an aRNA that binds to the FAS gene.

In some embodiments of the application, the specific hybridization probe that detects g.8901119C > T has a sequence as shown in SEQ ID No. 1.

In some embodiments of the application, the specific hybridization probe that detects g.89021477g > a has a sequence as shown in SEQ ID No. 2.

In some embodiments of the application, the sample detected is a tissue or a cell.

In a second aspect of the application there is provided a kit for pancreatic ductal adenocarcinoma prognosis, the kit comprising the reagents defined in the first aspect.

In some embodiments of the application, the kit comprises a detection chip on which detection probes are immobilized, the detection probes comprising specific hybridization probes as defined in the first aspect.

In some embodiments of the application, the kit further comprises one or more of sample RNA preservation reagents, RNA extraction reagents, RNA amplification reagents, nucleic acid dyes, and hybridization reagents.

In a third aspect of the present application, there is provided a method for detecting FAS gene for non-diagnostic purposes, the method comprising the steps of:

obtaining aRNA of a sample;

fluorescence labeling the aRNA;

and (3) carrying out hybridization reaction on the aRNA marked by fluorescence and the detection chip defined in the second aspect, detecting the fluorescence intensity of the detection chip after the hybridization reaction, and judging the mutation condition of the FAS gene of the sample according to the detection result.

In some embodiments of the present application, determining the mutation status of the FAS gene of the sample according to the detection result includes:

under the condition that fluorescence is not detected on a detection chip after hybridization reaction, the FAS gene of the sample does not exist g.8901119C > T and g.89021477G > A;

under the condition that fluorescence is detected on a detection chip after hybridization reaction, g.8901119C > T or/and g.89021477G > A exists in the FAS gene of the sample.

Compared with the prior art, the application has the following beneficial effects:

based on long-term clinical and massive case studies, the inventor of the present inventors found that mutations at two sites in the FAS gene (g.8901119c > T or/and g.89021477g > a) are closely related to prognosis of pancreatic ductal adenocarcinoma, and by detecting the mutation conditions of these two sites in the FAS gene, prognosis of patients after radical PDAC treatment can be predicted, including prediction of survival. The application can provide accurate guidance and clinical advice for prognosis and subsequent adjuvant treatment of pancreatic cancer patients.

Detailed Description

The present application will be described in further detail with reference to embodiments and examples. It should be understood that these embodiments and examples are provided solely for the purpose of illustrating the application and are not intended to limit the scope of the application in order that the present disclosure may be more thorough and complete. It will also be appreciated that the present application may be embodied in many different forms and is not limited to the embodiments and examples described herein, but may be modified or altered by persons skilled in the art without departing from the spirit of the application, and equivalents thereof are also intended to fall within the scope of the application. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the application, it being understood that the application may be practiced without one or more of these details.

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 application belongs. The terminology used herein in the description of the application is for the purpose of describing the embodiments and examples only and is not intended to be limiting of the application.

Terminology

Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:

the term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from the group consisting of "and/or", "and/or", it should be understood that, in the present application, the technical solutions include technical solutions that all use "logical and" connection, and also include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical scheme of "logical or" connection), and also include any and all combinations of A, B, C, D, i.e., any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical scheme of "logical and" connection).

The terms "plurality", "plural", "multiple", and the like in the present application refer to, unless otherwise specified, an index of 2 or more in number. For example, "one or more" means one kind or two or more kinds.

As used herein, "a combination thereof," "any combination thereof," and the like include all suitable combinations of any two or more of the listed items.

The "suitable" in the "suitable combination manner", "suitable manner", "any suitable manner" and the like herein refers to the fact that the technical scheme of the present application can be implemented, the technical problem of the present application is solved, and the technical effect expected by the present application is achieved.

Herein, "preferred", "better", "preferred" are merely to describe better embodiments or examples, and it should be understood that they do not limit the scope of the application.

In the present application, "further", "still further", "particularly" and the like are used for descriptive purposes to indicate differences in content but should not be construed as limiting the scope of the application.

In the present application, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.

In the present application, the terms "first", "second", "third", "fourth", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of a technical feature being indicated. Moreover, the terms "first," "second," "third," "fourth," and the like are used for non-exhaustive list description purposes only, and are not to be construed as limiting the number of closed forms.

In the application, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present application, a numerical range (i.e., a numerical range) is referred to, and optional numerical distributions are considered to be continuous within the numerical range and include two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range and each numerical value between the two numerical endpoints unless otherwise specified. Where a numerical range merely refers to integers within the numerical range, including both end integers of the numerical range, and each integer between the two ends, unless otherwise indicated, each integer is recited herein as directly, such as where t is an integer selected from 1 to 10, and where t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Further, when a plurality of range description features or characteristics are provided, these ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

The temperature parameter in the present application is not particularly limited, and may be a constant temperature treatment or may vary within a predetermined temperature range. It should be appreciated that the constant temperature process described allows the temperature to fluctuate within the accuracy of the instrument control. Allows for fluctuations within a range such as + -5 ℃, + -4 ℃, + -3 ℃, + -2 ℃, + -1 ℃.

In the present application,% (w/w) and wt% each represent weight percent,% (v/v) represents volume percent, and% (w/v) represents mass volume percent.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Unless otherwise indicated to the contrary by the intent and/or technical aspects of the present application, all references to which this application pertains are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in the present application, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated. In the case of the cited documents, examples and preferred modes of the cited relevant technical features are also incorporated into the present application by reference, but are not limited to being able to implement the present application. It should be understood that when a reference is made to the description of the application in conflict with the description, the application is modified in light of or adaptive to the description of the application.

Gene probe: the method is characterized in that a large number of probe molecules are immobilized on a support and hybridized with labeled sample molecules, the hybridization signal intensity of each probe molecule is detected, so that the number and sequence information of the sample molecules are obtained, and the batch detection of multiple genes is carried out.

Gene probes are applied to accurate gene information interpretation of tumor specimens at present, but no gene probes for evaluating and predicting PDAC disease prognosis conditions exist at present, so that clinicians cannot know the full-scale and prognosis conditions of PDAC disease from the disease sources of gene mutation. There is currently no genetic probe for pancreatic ductal adenocarcinoma or any pancreatic malignancy.

First aspect of the application

The application provides an application of a reagent for detecting FAS gene mutation in preparing pancreatic duct adenocarcinoma prognosis detection products;

the mutation site is selected from g.8901119C > T or/and g.89021477G > A.

In the present application, FAS gene mutation means a mutation occurring in a wild-type FAS gene.

It should be noted that, the above-mentioned mutation sites are all referred to the content recorded in the NCBI database, and those skilled in the art should understand that the mutation sites may be slightly different or changed due to the update of the database or the difference of the database, and the difference or change may be found by taking the content in the database as a standard, and the difference or change is also included in the protection scope of the present application. Moreover, it will be appreciated by those skilled in the art that the wild-type FAS gene in the human genome is the reference for the FAS gene in the present application, but when the wild-type FAS gene is present in other species, the sequence may be different, and the wild-type FAS gene in the species may be aligned with the wild-type FAS gene in the human genome to obtain the corresponding position in the wild-type FAS gene in the species.

It will be appreciated that the means of detection are not limited under conditions capable of detecting the FAS gene mutation, including, but not limited to, at least one of antibodies, probes, primers, and mass spectrometry detection reagents specific for at least one of the gene mutation or the polypeptide encoded thereby. For example, the presence or absence of such mutations in a test sample can be detected by the specific binding of an antibody that specifically recognizes the polypeptide to the polypeptide, i.e., by the interaction of a specific antibody with an antigen; the existence of the nucleic acid or the gene mutation can also be identified by designing a probe which specifically recognizes the gene mutation in advance and carrying out complementary pairing on the probe and the nucleic acid fragment where the nucleic acid or the gene mutation site is positioned; specific primers for amplifying the exons where the above-mentioned genetic mutations are located can also be designed, and then the presence or absence of the above-mentioned genetic mutations can be determined by gene amplification and sequencing; the presence or absence of the polypeptide expressed by the mutation can also be determined by mass spectrometry to detect the m/z of the polypeptide. At least one of the antibody, the probe, the primer and the mass spectrometry detection reagent according to the specific embodiment of the application can specifically and highly sensitively screen out the gene mutation or the polypeptide, thereby specifically and highly sensitively predicting the prognosis of pancreatic ductal adenocarcinoma.

Optionally, the reagent comprises a specific hybridization probe.

Alternatively, the specific hybridization probe targets an aRNA that binds to the FAS gene.

Alternatively, the specific hybridization probe for detecting g.8901119C > T has a sequence as shown in SEQ ID No. 1.

Alternatively, the specific hybridization probe for detecting g.89021477G > A has a sequence as shown in SEQ ID No. 2.

Alternatively, the sample detected is a tissue or a cell.

Second aspect of the application

The present application provides a pancreatic ductal adenocarcinoma prognosis kit comprising the reagents defined in the first aspect.

Optionally, the kit comprises a detection chip on which detection probes are immobilized, the detection probes comprising specific hybridization probes as defined in the first aspect.

Optionally, the kit further comprises one or more of sample RNA preservation reagents, RNA extraction reagents, RNA amplification reagents, nucleic acid dyes, and hybridization reagents.

Third aspect of the application

The application provides a detection method of FAS genes for non-diagnostic purposes, which comprises the following steps:

obtaining aRNA of a sample;

fluorescence labeling the aRNA;

and (3) carrying out hybridization reaction on the aRNA marked by fluorescence and the detection chip defined in the second aspect, detecting the fluorescence intensity of the detection chip after the hybridization reaction, and judging the mutation condition of the FAS gene of the sample according to the detection result.

Optionally, determining the mutation status of the FAS gene of the sample according to the detection result includes:

under the condition that fluorescence is not detected on a detection chip after hybridization reaction, the FAS gene of the sample does not exist g.8901119C > T and g.89021477G > A;

under the condition that fluorescence is detected on a detection chip after hybridization reaction, g.8901119C > T or/and g.89021477G > A exists in the FAS gene of the sample.

Embodiments of the present application will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to the guidelines given in the present application, and may be according to the experimental manual or conventional conditions in the art, the conditions suggested by the manufacturer, or the experimental methods known in the art.

In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.

Pancreatic Ductal Adenocarcinoma (PDAC) is a malignancy with high malignancy, poor prognosis, high recurrence-metastasis rate, and less than 10% of 5-year survival, and is called "king in cancer". At present, no effective treatment means exists clinically, and no method capable of specifically evaluating the disease progress and accurately predicting the prognosis of a patient exists. The research work of the application relates to tumor specimen sequencing and long-term postoperative follow-up visit are carried out on 315 patients treated by pancreatic cancer radical surgery in Beijing co-ordination hospitals in 2008 to 2015, and the FAS gene mutation is found to be positively correlated with the prognosis of a PDAC patient, the higher the FAS gene mutation level is, the worse the prognosis of the PDAC patient is, and the shorter the postoperative survival time is.

The application includes embodiments including:

1. in the research work, clinical test indexes and operation conditions of all patients undergoing the pancreatic cancer radical operation treatment in 2008-2015 are analyzed in detail, clinical baseline data (sex, age, disease course, symptoms, past history, personal history, family history, preoperative physical examination and the like) are screened, no obvious statistical difference exists in the preoperative test (blood convention, liver and kidney functions, blood coagulation, tumor markers (including CA 19-9) and the like), no obvious statistical difference exists in the preoperative examination (tumor size, position and the like), the operations are radical excision (including pancreas-duodenal excision, pancreas-tail-spleen excision and the like), the pathological cutting edges are all negative, the pathological diagnosis is pancreatic duct adenocarcinoma, and the postoperative radiotherapy and chemotherapy treatment scheme does not have obvious statistical difference in 315, and the application is common examples.

2. The 315 patients are followed up to obtain detailed and accurate prognosis data such as total postoperative survival time, disease-free survival time (no recurrence and metastasis), death time and the like.

3. The tumor samples of the 315 patients were subjected to whole genome sequencing analysis (sequencing depth 40×) and whole exome sequencing analysis (sequencing depth 200×) to obtain over 2.7 ten thousand genes with effective mutation. The applicant team then performed detailed, thorough, and detailed bioinformatic analyses:

(1) Mutation ratio sequencing and difference sequencing are respectively carried out on 2.7 ten thousand genes with effective mutation according to the radical post-operation survival time length of a patient, and 2414 genes with clinical significance and mutation statistics difference (within the top 10% of the ranking in the ratio sequencing or the difference sequencing) are screened according to the original clinical phenotype (clinical significance), mutation phenotype and mutation site of each gene.

(2) The above 2414 first screened important genes are subjected to bioinformatics analysis and signal path analysis by a bioinformatics analysis platform such as IPA, KEGG, GO, STRING, and 9 important signal paths are sorted and screened by enriching the mutation ratio sum and the difference sum of all genes in each signal path.

(3) And (3) carrying out total evaluation and scoring on the analysis results and clinical effects and signal path values of the genes in the aspects of pancreatic origin, tumor (tumor pathological characteristics such as adenocarcinoma, squamous carcinoma, adenosquamous carcinoma and the like), endocrine, immunity and the like, sorting according to the obtained scores, and screening to obtain 97 key genes.

(4) The 97 key genes are made into initial gene probes, 315 tumor specimens are subjected to sequence sequencing with the depth of 1000 x, and the obtained sequencing result is refined again, so that the analysis process is repeated.

(5) Finally, integrating information such as clinical characterization, gene functions, bioinformatics, basic medicine and the like, and finally finding out all factors such as the mutation of the FAS gene (g.8901119C > T and g.89021477G > A) to have a high-specificity prediction effect on the prognosis survival period of a patient after the radical PDAC treatment according to the biological effect of each gene, the positive and negative values of the correlation between the gene mutation and the survival period of the patient, the position and the connection effect of the gene mutation in a key signal path, the multiple ranking order of the actual mutation difference, the difference ranking order of the actual mutation difference and the like: as long as at least one of the two mutation sites of the FAS gene shows positive detection results, the survival time of the radical cure post-operation survival time does not exceed 24 months.

Of all 315 PDAC tumor specimens involved in sequencing, 22 patients detected FAS gene mutations (sensitivity 6.98%), with a total survival of 4 to 18 months, a median survival of 11 months, an ultrashort survival (postoperative survival < 12 months) of 12 patients (54.5%), and a short survival (postoperative survival < 15 months) of 20 patients (90.9% of specificity).

The FAS mutation sites in 22 patients were mainly focused on the following sites:

TABLE 1

Mutation	Cases	Survival(mo)
			C>T	15	6、7、7、9、10、11、11、12、12、12、13、13、15、16、18
G>A	7	5、5、8、11、11、12、14

4. Based on the clinical data and the biological informatics analysis detection results, the applicant team designs a FAS gene mutation detection probe which is a PDAC specific prognosis detection probe. The specific sequence is as follows:

TABLE 2

5. Gene probes were prepared from the above sequences, and tumor tissue specimens of PDAC patients who underwent radical surgery treatment were examined for 69 other patients (not included in the 315 patients and not statistically different from clinical baseline data), comprising the steps of:

(1) And immersing the pancreatic cancer tissue specimens collected in the operation and surrounding normal pancreatic tissues in an RNA Later for normal temperature preservation.

(2) Total mRNA was extracted using Trizol method: the tissue specimen pieces were placed in a 10ml centrifuge tube, added with about 10 volumes of Trizol, placed on ice, homogenized well and centrifuged briefly. The homogenate was then aspirated 5-10 times with a syringe 0.9mm in diameter until complete homogenization, and well lysed in Trizol reagent. Then adding 1/5 volume of chloroform into the homogenate, shaking and mixing thoroughly, standing on ice for 10min until the two phases are separated, centrifuging at maximum speed for 15min, sucking the supernatant to a new 1.5ml centrifuge tube, adding 70% ethanol with equal volume, and mixing repeatedly. Then, 700. Mu.l of the solution RW1 was added to the centrifugal purification column, and the mixture was centrifuged at maximum speed for 15s. The column was placed in a new collection tube and 500. Mu.l of RPE solution was added and centrifuged at maximum speed for 15s. Then, 500. Mu.l of RPE solution was added, the mixture was centrifuged at maximum speed for 2min, the filtrate was discarded, and the mixture was centrifuged at maximum speed for 1min, and the filtrate was discarded. The column was placed in a new sample collection tube, 100. Mu.l DEPC water was added, centrifuged at maximum speed for 1min, and the two filtrates were combined. Adding 2.5 times volume of absolute ethanol and 1/10 volume of 3M sodium acetate (pH 5.2, prepared by DEPC water), standing at room temperature for 30min or precipitating at-20deg.C for longer time, standing at 4deg.C for 13, 400rpm×15min, discarding supernatant, adding 1ml of 75% ethanol for resuspension, 7500rpm×5min, discarding supernatant, repeating once, and air drying at room temperature.

Adding proper amount of DEPC water to dissolve precipitate, and determining concentration and total amount of RNA sample, wherein when TE buffer/EB buffer is used as blank control and diluent, the A260/A280 ratio of RNA sample should be 1.8-2.0.

(3) RNA micro-amplification, and cDNA synthesis through reverse transcription: taking 0.5-2ug of total RNA, adding T7 Oligo (dT) primer and water without Nuclease-pollution, denaturing at 70 ℃ for 5min, cooling on ice for 5min, adding 10 x first strand synthesis buffer, ribonucleoase inhibitor, dNTP and reverse transcriptase mixed solution, and preserving heat at 42 ℃ for 2h. Mu.l of the cDNA sample was taken, and then, water free of nucleic acid-contamination, 10 Xsecond strand synthesis buffer, dNTP Mix, DNA Polymerase, RNase H were added thereto, and incubated at 16℃for 2 hours. The Cartridge was placed in a 2ml collection tube, 100. Mu.l cDNA Binding was added and allowed to stand at room temperature for 5min. After adding 250 mu l cDNA Binding Buffer to the obtained cDNA sample and shaking and mixing, the cDNA sample was added to the Cartridge, centrifuged at 10000 Xg for 1min, the filtrate was discarded, and the Filter Cartridge was returned to a 2ml recovery tube. Then 650 mu l cDNA Wash Buffer is added into the Filter Cartridge, 10000 Xg is centrifuged for 1min, the filtrate is discarded, and the Filter Cartridge is centrifuged for 1min, so that the Filter Cartridge is free of residual ethanol, and the Cartridge is transferred into a new collection tube. 50 μl of preheated Nuclear-Free Water was added, left at room temperature for 2min, centrifuged at 10000 Xg for 90s, and repeated.

(4) Amplified to aRNA and purified: 14. Mu.l of the cDNA sample was taken, and ATP, CTP, GTPmix, UTP solution, aaUTP, T7X Reaction Buffer, T7 Enzyme Mix were added thereto, incubated at 37℃for 14 hours, and RNase-free water was added thereto to adjust the volume to 100. Mu.l. Add 350. Mu. l aRNA Binding Buffer and mix well. Then 250 μl of absolute ethanol is added and mixed well. Then adding the mixture into the prepared Filter Cartridge, centrifuging the mixture for 1min at 10000 Xg, discarding the filtrate, and placing the Filter Cartridge into a 2ml recovery tube. Adding 650 mu l aRNA Wash Buffer to Filter Cartridge, centrifuging 10000 Xg for 1min, discarding the filtrate, centrifuging for 1min again, leaving Filter Cartridge free of residual ethanol, and transferring the Cartridge to a new collection tube. 50 μl of preheated Nuclear-Free Water was added, left at room temperature for 2min, centrifuged at 10000 Xg for 90s, and repeated. The aRNA concentration and yield were measured.

(5) Labeling with Cy3 fluorescent molecules: taking 15-20 mu g of aRNA, drying in vacuum, re-dissolving in 9 mu l of coupling Buffer, adding 11 mu l of mono-active Cy3 dye, mixing uniformly, and standing at room temperature in a dark place for 30min. 4.5. Mu.l of hydroxyamine was added, and the mixture was left to stand in the dark for 15 minutes after being mixed uniformly. Nuclease-free water was added to a volume of 30. Mu.l, 105. Mu. l aRNA Binding Buffer was added, mixed well, 75. Mu.l absolute ethanol was added, mixed well and transferred to aRNA Filter Cartridge purification column. Centrifugation was performed for 10000 g.times.1 min, after addition of Wash Buffer, re-centrifugation was performed, and then Nuclear-free water was added for 20. Mu.l, centrifugation was performed, and re-centrifugation was performed. The two products were combined and the total amount of recovered product was obtained by spectrophotometric measurement of A260/A280/A550/A650.

(6) Hybridization of the probe: taking 5 mug of the marked sample, vacuum-pumping, re-dissolving in about 3 mug of nucleic-free water, carrying out water bath for 5min at 95 ℃, adding 27 mug l Operon hybridization Buffer on ice for 1min, fully mixing, centrifuging, and carrying out hybridization reaction with the detection chip of the application, wherein the detection chip is a sample application method hybridization chip formed by combining FAS oligonucleotide probe arrays in table 2.

(7) Scanning and data processing: scanning was performed using a scanner (LuxScan, beijing bloo), after which the image was converted into a digital signal based on fluorescence intensity (GenePix), followed by data analysis and processing.

After correction and accounting of the data of the experimental group and the control group, 4 FAS gene mutation patients (2 of which are detected by the detection probes shown by SEQ ID No.1 and 2 of which are detected by the detection probes shown by SEQ ID No. 2) are detected, and the survival time after radical treatment is respectively 5 months, 11 months, 12 months and 13 months, which proves that the specificity of the FAS probe in the application for predicting the pancreatic cancer radical treatment short survival time (the survival time after operation is less than 15 months) reaches 100%, and fully shows that the FAS mutation probe can predict the survival condition of the patients after PDAC radical treatment: FAS mutations are positively correlated with short patient survival and poor prognosis following radical PDAC surgery.

In the early study, 97 key genes are obtained through screening, but through full analysis and screening of the study, other than FAS and other 31 core factors which are closely related to prognosis conditions after radical pancreatic cancer treatment, 65 genes are related to pancreatic cancer, such as TRAPPC13, RNF122, PPIL6, CXorf56 and the like, but the prediction effect of statistical difference cannot be generated on the prognosis judgment of pancreatic cancer. In particular, the present application has found that only the FAS gene can predict prognosis of pancreatic cancer by its presence or absence of one of the mutations in table 2. Furthermore, in the present application, no correlation of other sequence mutations in FAS gene with pancreatic cancer prognosis was found in the previous study.

In conclusion, through the probe detection of FAS gene mutation, the accurate prognosis situation prediction is carried out on patients with pancreatic duct adenocarcinoma, so that clinicians can comprehensively know the disease situation of the patients, assist in guiding the treatment decision of the clinicians, and play an important role in the treatment, prognosis evaluation and sanitation economics of the diseases.

The technical features of the above-described embodiments and examples may be combined in any suitable manner, and for brevity of description, all of the possible combinations of the technical features of the above-described embodiments and examples are not described, however, as long as there is no contradiction between the combinations of the technical features, they should be considered to be within the scope described in the present specification.

The above examples merely represent a few embodiments of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Further, it is understood that various changes and modifications of the present application may be made by those skilled in the art after reading the above teachings, and equivalents thereof are intended to fall within the scope of the present application. It should also be understood that, based on the technical solutions provided by the present application, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the patent of the application should therefore be determined with reference to the appended claims, which are to be construed as in accordance with the doctrines of claim interpretation.

Claims (10)

1. Application of reagent for detecting FAS gene mutation in preparing pancreatic duct adenocarcinoma prognosis detection product;

the mutation site is selected from g.8901119C > T or/and g.89021477G > A.

2. The use according to claim 1, wherein the reagent comprises a specific hybridization probe.

3. The use according to claim 2, wherein the specific hybridization probe targets an aRNA that binds to the FAS gene.

4. The use according to claim 3, wherein the specific hybridization probe for detecting g.8901119C > T has the sequence shown in SEQ ID No. 1.

5. The use according to claim 3, wherein the specific hybridization probe for detecting g.89021477g > a has the sequence shown in SEQ ID No. 2.

6. The use according to any one of claims 1 to 5, wherein the sample detected is a tissue or a cell.

7. A kit for the prognosis of pancreatic ductal adenocarcinoma, characterized in that it comprises a reagent as defined in any one of claims 1 to 6.

8. The pancreatic ductal adenocarcinoma prognosis kit according to claim 7, characterized in that it comprises a detection chip on which detection probes are immobilized, said detection probes comprising specific hybridization probes as defined in claim 4 or/and 5; optionally, the kit further comprises one or more of sample RNA preservation reagents, RNA extraction reagents, RNA amplification reagents, nucleic acid dyes, and hybridization reagents.

9. A method for detecting FAS genes for non-diagnostic purposes, comprising the steps of:

obtaining aRNA of a sample;

fluorescence labeling the aRNA;

carrying out hybridization reaction on the aRNA marked by fluorescence and the detection chip defined in claim 8 or 9, detecting the fluorescence intensity of the detection chip after the hybridization reaction, and judging the mutation condition of the FAS gene of the sample according to the detection result.

10. The method for detecting a FAS gene for non-diagnostic purposes according to claim 9, wherein determining the mutation of the FAS gene in the sample based on the detection result comprises:

under the condition that fluorescence is not detected on a detection chip after hybridization reaction, the FAS gene of the sample does not exist g.8901119C > T and g.89021477G > A;

under the condition that fluorescence is detected on a detection chip after hybridization reaction, g.8901119C > T or/and g.89021477G > A exists in the FAS gene of the sample.