CN109486954A - A kind of diagnosing cancer of liver kit and its application based on AFP mRNA detection - Google Patents
A kind of diagnosing cancer of liver kit and its application based on AFP mRNA detection Download PDFInfo
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- CN109486954A CN109486954A CN201811584271.0A CN201811584271A CN109486954A CN 109486954 A CN109486954 A CN 109486954A CN 201811584271 A CN201811584271 A CN 201811584271A CN 109486954 A CN109486954 A CN 109486954A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Abstract
The invention proposes a kind of diagnosing cancer of liver kit based on AFP mRNA detection and its applications, kit includes RNAscope hydrogen peroxide, RNAscope protease P lus, RNAscope target reparation reagent, 2.5 hypersensitivity detection kit of RNAscope, RNAscope cleaning buffer solution, wherein, 2.5 hypersensitivity detection kit of RNAscope includes signal amplifying system AMP1-AMP6 and DAB-A and DAB-B.The present invention improves the positive rate of AFP in hepatocellular carcinoma by paraffin organization in situ detection AFP mRNA, assists diagnosing cancer of liver.It replaces RNAscope hydrogen peroxide, RNAscope protease P lus, RNAscope target to repair reagent respectively in addition, 3% hydrogen peroxide, the gastric enzyme of 2.5g/L, citric acid are repaired liquid, testing cost can be substantially reduced.
Description
Technical field
The present invention relates to a kind of diagnosing cancer of liver kit based on AFP mRNA detection and its applications.
Background technique
Hepatocellular carcinoma is a kind of very high tumour of grade malignancy, Serum AFP diagnoses HCC all the time sensibility and
Specificity is all fine, but organizes the positive rate of AFP Protein Detection not high always.
AFP is the higher tumor associated antigen of Primary hepatic carcinoma cell specificity.Therefore detection peripheral blood in patients, chest and abdomen
Whether there is or not the expression of AFP mRNA in the samples such as water, puncture fluid, marrow and specimens from pri, can provide the diagnosing and treating of tumour
Strong evidence, can also transfer to tumour and recurrence and observation of curative effect help is provided.In the past to the inspection of AFP mRNA
It surveys using qualitative RT-PCR method, technically, although qualitative PCR is square from traditional ELISA of remolding sensitivity etc.
Method is significantly improved, but its specificity is relatively poor, and can not accurate quantification.
Summary of the invention
It is an object of the invention to propose it is a kind of based on AFP mRNA detection diagnosing cancer of liver kit and its application.
In order to solve the above technical problems, the present invention is achieved by the following scheme:
A kind of diagnosing cancer of liver kit based on AFP mRNA detection, the kit include:
RNAscope hydrogen peroxide, protease P lus, RNAscope target repair reagent (10 ×), 2.5 hypersensitivity of RNAscope
Detection kit (brown), RNAscope cleaning buffer solution (50 ×), wherein the 2.5 hypersensitivity detection reagent of RNAscope
Box includes signal amplifying system AMP1-AMP6 and DAB-A and DAB-B.
RNAscope Probe- Hs-AFP(ACD, the Cat No. designed by AFP nucleic acid sequence
427411), there are unique bis- " Z " type structures, it can be with tissue AFP mRNA specific binding.RNAscope kit (ACD)
Principle: the preposition amplification molecule of oligonucleotides (PreAMP) hybridizes with each pair of " ZZ " probe, signal amplification molecule (AMP) and label
Probe hybridizes with preposition amplification molecule, specific " ZZ " probe hybridization signal Cascaded amplification, is able to detect low abundance expression RNA
(1-20 copy/cell).
A kind of application of the diagnosing cancer of liver kit in diagnosing cancer of liver based on AFP mRNA detection, the application pass through paraffin
Tissue in situ detects AFP mRNA, improves the positive rate of AFP in hepatocellular carcinoma, assists liver cancer clinical diagnosis and antidiastole.For
The positive rate of tissue AFP is improved, attempts to specifically comprise the following steps: from the method for rna level detection tissue AFP
S1, the slice that the paraffin specimen of fixed embedding is cut into 4um, 65 DEG C roasting piece 1 hour；
S2, dewaxing: slice is put into fresh dimethylbenzene A 5 minutes, and fresh dimethylbenzene B 5 minutes, fresh dehydrated alcohol C 2 minutes,
Fresh dehydrated alcohol D 2 minutes；
S3, by 50 × cleaning buffer solution be made into 1 × it is stand-by, by 10 × pretreatment fluid be made into 1 × it is stand-by；
S4, slice instill pretreating reagent three and drip, and are incubated at room temperature 10 minutes, wash with distilled water 3 times, every time 2 minutes；
S5, sample is immersed in the 1 × pretreatment fluid to boil, slowly boiling 15 minutes, immediately move hot object slide stand
Into the staining jar for filling distilled water, distilled water cleaning 3 times, 2 minutes every time, fresh washes of absolute alcohol 3 times, 2 minutes every time,
It air-dries；
S6, immunohistochemistry stroke water blocking ring, 3 times wash with distilled water, every time 2 minutes；
3 drop pretreating reagents are added in S7, every sample, 30 minutes under the conditions of being placed in 40 DEG C of HYbZE hybrid heater；
S8, it will be preheated 10 minutes under the conditions of 40 DEG C of AFP mRNA probe, shake up and be placed on room temperature；
S9, slice 3 times wash with distilled water, 2 minutes every time, 3 drop AFP mRNA probes were added dropwise in each sample, and it is miscellaneous to be placed in HYbZE
It is incubated for 2 hours under the conditions of handing over 40 DEG C of furnace；
S10, it is rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes are every time, remove excess liq, and 3 drop AMP1 are added dropwise in each sample,
It is incubated for 30 minutes under the conditions of being placed in 40 DEG C of HYbZE hybrid heater；
S11, it is rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes are every time, remove excess liq, and 3 drop AMP2 are added dropwise in each sample,
It is incubated for 15 minutes under the conditions of being placed in 40 DEG C of HYbZE hybrid heater；
S12, it is rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes are every time, remove excess liq, and 3 drop AMP3 are added dropwise in each sample,
It is incubated for 30 minutes under the conditions of being placed in 40 DEG C of HYbZE hybrid heater；
S13, it is rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes are every time, remove excess liq, and 3 drop AMP4 are added dropwise in each sample,
It is incubated for 15 minutes under the conditions of being placed in 40 DEG C of HYbZE hybrid heater；
S14, it is rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes are every time, remove excess liq, and 3 drop AMP5 are added dropwise in each sample,
Incubation at room temperature 30 minutes；
S15, it is rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes are every time, remove excess liq, and 3 drop AMP6 are added dropwise in each sample,
Incubation at room temperature 15 minutes；
S16, it is rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes every time, removes excess liq, and 3 drop DAB-A are added dropwise in each sample
Isometric mixed liquor with DAB-B is incubated at room temperature 10 minutes；
S17, it redyes, is redyed 2 minutes with haematoxylin, flowing water rinses 2 minutes；
S18, indigo plant is returned, unsaturated carbonate lithium 1 minute, flowing water rinsed 10 minutes；
S19, suitable neutral gum mounting；
The brown signal in histotomy, the positive rate of computation organization AFP mRNA are observed under S20, ordinary optical microscope；
The positive rate of AFP mRNA, statistics in liver cancer, cirrhosis and a variety of benign livers slice that S21, statistics are selected in
The positive rate and specificity of liver cancer tissue AFP mRNA；
S22, the positive rate of result liver cancer tissue AFP mRNA are very high, and up to 65% or more, specificity is very well, it was demonstrated that detection AFP
MRNA is a kind of good hepatocellular carcinoma detection method.
The present invention has the beneficial effect that the detection of liver cancer tissue AFP all the time is always compared with prior art
Using the AFP albumen of AFP antibody test tissue, but the positive rate of liver cancer tissue AFP albumen is not high always (only 20% or so),
Liver cancer AFP positive rate is set to be increased to 65% or more using RNAscope technology detection tissue AFP mRNA, specificity is also very high.This
Invention improves the positive rate of liver cancer tissue AFP, meanwhile, testing cost is greatly reduced, the economy for reducing patient and family members is negative
Load.
Detailed description of the invention
Fig. 1 is RNAscope technical schematic diagram of the present invention.
Specific embodiment
The present invention is got information about to allow those skilled in the art to be more clear, the present invention will be made below further
Explanation.
The diagnosing cancer of liver kit based on AFP mRNA detection of the invention, including main agents it is as follows:
1.RNAscope hydrogen peroxide & protease P lus
2.RNAscope target repairs reagent (10 ×)
2.5 hypersensitivity detection kit (brown) of 3.RNAscope (including signal amplifying system AMP1-AMP6 and DAB-A with
DAB-B)
4.RNAscope cleaning buffer solution (50 ×)
The specific operation process of detection is as follows:
1. the paraffin specimen of fixed embedding to be cut into the slice of 4um, 65 DEG C roasting piece 1 hour.
2. dewaxing: slice is put into fresh dimethylbenzene A 5 minutes, and fresh dimethylbenzene B 5 minutes, fresh dehydrated alcohol C 2 divided
Clock, fresh dehydrated alcohol D 2 minutes.
3. by 50 × cleaning buffer solution be made into 1 ×, by 10 × pretreatment fluid be made into 1 ×.
It drips, is incubated at room temperature 10 minutes 4. slice instills pretreating reagent three.3 times wash with distilled water, every time 2 minutes.
5. sample is immersed in the 1 × pretreatment fluid to boil, slowly boiling 15 minutes, immediately by hot object slide stand
It moves in the staining jar for filling distilled water, distilled water cleaning 3 times, 2 minutes every time, fresh washes of absolute alcohol 3 times, 2 points every time
Clock air-dries.
6. immunohistochemistry stroke water blocking ring, 3 times wash with distilled water, every time 2 minutes.
7. 3 drop pretreating reagents are added in every sample, it is placed in HYbZE hybrid heater 40 DEG C × 30 minutes.
8. 40 DEG C of probe are preheated 10 minutes, shakes up and be placed on room temperature.
9. slice 3 times wash with distilled water, 2 minutes every time, 3 drop AFP mRNA probes were added dropwise in each sample, it is placed in HYbZE
40 DEG C of hybrid heater are incubated for 2 hours.
10. being rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes every time, removes excess liq, and 3 drops are added dropwise in each sample
Amp1 is placed in 40 DEG C of HYbZE hybrid heater and is incubated for 30 minutes.
11. being rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes every time, removes excess liq, and 3 drops are added dropwise in each sample
Amp2 is placed in 40 DEG C of HYbZE hybrid heater and is incubated for 15 minutes.
12. being rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes every time, removes excess liq, and 3 drops are added dropwise in each sample
Amp3 is placed in 40 DEG C of HYbZE hybrid heater and is incubated for 30 minutes.
13. being rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes every time, removes excess liq, and 3 drops are added dropwise in each sample
Amp4 is placed in 40 DEG C of HYbZE hybrid heater and is incubated for 15 minutes.
14. being rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes every time, removes excess liq, and 3 drops are added dropwise in each sample
Amp5 is incubated at room temperature 30 minutes.
15. being rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes every time, removes excess liq, and 3 drops are added dropwise in each sample
Amp6 is incubated at room temperature 15 minutes.
16. being rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes every time, removes excess liq, and 3 drops are added dropwise in each sample
DAB-A and DAB-B isometric mixed liquor are incubated at room temperature 10 minutes.
17. redying, redyed 2 minutes with haematoxylin, flowing water rinses 2 minutes.
18. returning indigo plant, unsaturated carbonate lithium 1 minute, flowing water was rinsed 10 minutes.
19. suitable neutral gum mounting.
20. observing the brown signal in histotomy, the positive of computation organization AFP mRNA under ordinary optical microscope
Rate.
21. the positive rate of AFP mRNA in the liver cancer that statistics is selected in, cirrhosis and a variety of benign livers slice,
Count the positive rate and specificity of liver cancer tissue AFP mRNA.
22. the positive rate of result liver cancer tissue AFP mRNA is very high, up to 65% or more, specificity is very well, it was demonstrated that detection AFP
MRNA is a kind of good hepatocellular carcinoma detection method.
Confirmatory experiment:
Firstly, we collect non-tumour hepatic tissue by 150 liver cancer fresh specimens and its corresponding cancer, carry out qRT-PCR and
RNAscope detection analyzes two kinds of technologies to the detection correlation of AFP mRNA expression, as a result confirms that RNAscope can
Represent the true horizon of tissue AFP mRNA.
Then, the pervious paraffin organization of 1000 many cases (including liver cancer, cirrhosis and a variety of benign livers) is collected, used
The expression of RNAscope technology in situ detection AFP mRNA.As a result positive rate and specificity of the AFP mRNA in liver cancer tissue
Very well, suitable with Serum AFP.
Finally, this detection method is applied to the sample of liver biopsy and operation patients that hospital newly accepts for medical treatment, obtained with
Identical result before.
The improvements of the present invention in the application:
1. we substitute 3% hydrogen peroxide of RNAscope hydrogen peroxide autogamy in actual experiment, as a result with autogamy hydrogen peroxide and
Two groups of experimental results of ACD company kit hydrogen peroxide are consistent.3% pair of the ACD company RNAscope hydrogen peroxide price than autogamy
Oxygen water is much higher, has saved cost by this method.
2. we substitute the gastric enzyme of the 2.5g/L of RNAscope protease P lus autogamy in actual experiment, as a result with certainly
Two groups of experimental results with gastric enzyme ratio ACD company kit protease P lus are consistent.ACD company RNAscope protease P lus valence
Lattice are more much higher than the gastric enzyme of autogamy, saved cost by this method.And the sample bad for certain fixations, ACD are public
The protease P lus of department, which will cause digestion, excessively makes cell dissolution, we are using the side for adjusting digestion time in actual experiment
Method can reduce cytolytic ratio well, to keep result more correct.
3. repairing the pH value of reagent by surveying RNAscope target, infer that its actual constituent is that citric acid repairs liquid.It is practical
Testing us and repairing fluid exchange is citric acid, and it is higher very than common citric acid reparation liquid that the target of ACD company repairs reagent price
It is more, it reduces costs in this way.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (3)
1. a kind of diagnosing cancer of liver kit based on AFP mRNA detection, which is characterized in that the kit includes:
RNAscope hydrogen peroxide, RNAscope protease P lus, RNAscope target repair reagent (10 ×), RNAscope 2.5
Hypersensitivity detection kit (brown), RNAscope cleaning buffer solution (50 ×), wherein 2.5 hypersensitivity of RNAscope
Detection kit includes signal amplifying system AMP1-AMP6 and DAB-A and DAB-B.
2. a kind of application of diagnosing cancer of liver kit based on AFP mRNA detection in diagnosing cancer of liver.
3. application as claimed in claim 2, which is characterized in that the application is mentioned by paraffin organization in situ detection AFP mRNA
The positive rate of AFP in high hepatocellular carcinoma assists liver cancer clinical diagnosis and antidiastole, in order to improve the positive rate of tissue AFP, from
The method that rna level detects tissue AFP, specifically comprises the following steps:
S1, the slice that the paraffin specimen of fixed embedding is cut into 4um, 65 DEG C roasting piece 1 hour；
S2, dewaxing: slice is put into fresh dimethylbenzene A 5 minutes, and fresh dimethylbenzene B 5 minutes, fresh dehydrated alcohol C 2 minutes,
Fresh dehydrated alcohol D 2 minutes；
S3, by 50 × cleaning buffer solution be made into 1 × it is stand-by, by 10 × pretreatment fluid be made into 1 × it is stand-by；
S4, slice instill pretreating reagent three and drip, and are incubated at room temperature 10 minutes, wash with distilled water 3 times, every time 2 minutes；
S5, sample is immersed in the 1 × pretreatment fluid to boil, slowly boiling 15 minutes, immediately move hot object slide stand
Into the staining jar for filling distilled water, distilled water cleaning 3 times, 2 minutes every time, fresh washes of absolute alcohol 3 times, 2 minutes every time,
It air-dries；
S6, immunohistochemistry stroke water blocking ring, 3 times wash with distilled water, every time 2 minutes；
3 drop pretreating reagents are added in S7, every sample, 30 minutes under the conditions of being placed in 40 DEG C of HYbZE hybrid heater；
S8, it will be preheated 10 minutes under the conditions of 40 DEG C of AFP mRNA probe, shake up and be placed on room temperature；
S9, slice 3 times wash with distilled water, 2 minutes every time, 3 drop AFP mRNA probes were added dropwise in each sample, and it is miscellaneous to be placed in HYbZE
It is incubated for 2 hours under the conditions of handing over 40 DEG C of furnace；
S10, it is rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes are every time, remove excess liq, and 3 drop AMP1 are added dropwise in each sample,
It is incubated for 30 minutes under the conditions of being placed in 40 DEG C of HYbZE hybrid heater；
S11, it is rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes are every time, remove excess liq, and 3 drop AMP2 are added dropwise in each sample,
It is incubated for 15 minutes under the conditions of being placed in 40 DEG C of HYbZE hybrid heater；
S12, it is rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes are every time, remove excess liq, and 3 drop AMP3 are added dropwise in each sample,
It is incubated for 30 minutes under the conditions of being placed in 40 DEG C of HYbZE hybrid heater；
S13, it is rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes are every time, remove excess liq, and 3 drop AMP4 are added dropwise in each sample,
It is incubated for 15 minutes under the conditions of being placed in 40 DEG C of HYbZE hybrid heater；
S14, it is rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes are every time, remove excess liq, and 3 drop AMP5 are added dropwise in each sample,
Incubation at room temperature 30 minutes；
S15, it is rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes are every time, remove excess liq, and 3 drop AMP6 are added dropwise in each sample,
Incubation at room temperature 15 minutes；
S16, it is rinsed at room temperature with cleaning buffer solution 2 times, 2 minutes every time, removes excess liq, and 3 drop DAB-A are added dropwise in each sample
Isometric mixed liquor with DAB-B is incubated at room temperature 10 minutes；
S17, it redyes, is redyed 2 minutes with haematoxylin, flowing water rinses 2 minutes；
S18, indigo plant is returned, unsaturated carbonate lithium 1 minute, flowing water rinsed 10 minutes；
S19, suitable neutral gum mounting；
The brown signal in histotomy, the positive rate of computation organization AFP mRNA are observed under S20, ordinary optical microscope；
The positive rate of AFP mRNA, statistics in liver cancer, cirrhosis and a variety of benign livers slice that S21, statistics are selected in
The positive rate and specificity of liver cancer tissue AFP mRNA；
S22, the positive rate of result liver cancer tissue AFP mRNA are very high, and up to 65% or more, specificity is very well, it was demonstrated that detection AFP
MRNA is a kind of good hepatocellular carcinoma detection method.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116769912A (en) * | 2023-06-09 | 2023-09-19 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Kit for detecting liver cancer AFP mRNA |
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| CN1410546A (en) * | 2002-11-12 | 2003-04-16 | 浙江大学医学院附属第二医院 | Real time fluorescence quantitative RT-PCR detection human alpha-fetoprotein reagent box |
| CN101429550A (en) * | 2007-11-30 | 2009-05-13 | 芮屈生物技术（上海）有限公司 | Synthetic detection kit for hybridization in situ and detection method |
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Patent Citations (3)
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| CN1410546A (en) * | 2002-11-12 | 2003-04-16 | 浙江大学医学院附属第二医院 | Real time fluorescence quantitative RT-PCR detection human alpha-fetoprotein reagent box |
| CN101429550A (en) * | 2007-11-30 | 2009-05-13 | 芮屈生物技术（上海）有限公司 | Synthetic detection kit for hybridization in situ and detection method |
| US20180251851A1 (en) * | 2015-09-10 | 2018-09-06 | Mathias HEIKENWALDER | Ectopic lymphoid structures as targets for liver cancer detection, risk prediction and therapy |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116769912A (en) * | 2023-06-09 | 2023-09-19 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Kit for detecting liver cancer AFP mRNA |
| CN116769912B (en) * | 2023-06-09 | 2024-03-19 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Kit for detecting liver cancer AFP mRNA |
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