CN107345236A - The specificity amplification primer and diagnosis for liver cancer kit of a kind of AMY2B mRNAs - Google Patents
The specificity amplification primer and diagnosis for liver cancer kit of a kind of AMY2B mRNAs Download PDFInfo
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- CN107345236A CN107345236A CN201610290859.XA CN201610290859A CN107345236A CN 107345236 A CN107345236 A CN 107345236A CN 201610290859 A CN201610290859 A CN 201610290859A CN 107345236 A CN107345236 A CN 107345236A
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Abstract
The present invention provides a kind of specificity amplification primer and diagnosis for liver cancer kit of AMY2B mRNAs, and the specificity amplification primer includes：Forward primer：5’‑ATCAACCTAGCCATCCAACCT‑3’；Reverse primer：5’‑AACCGAATTTTCAATTGTTTTCACA‑3’.The kit includes：(1) specificity amplification primer of AMY2B mRNAs claimed in claim 1；(2) PCR reactions enzyme and reagent, and/or the specific primer of the mRNA of reference gene mark.Using the biological tissue storehouse of large sample size, the validity of this kit is demonstrated, and prognosis of patients with feminine survival state is analyzed, it was demonstrated that the kit of the present invention can effectively predict the life cycle length of patient.
Description
Technical field
The present invention relates to biotechnology and medical domain, more particularly, to a kind of AMY2B courier
RNA specificity amplification primer, and the diagnosis for liver cancer kit including the specificity amplification primer.
Background technology
Hepatocellular carcinoma (hepatocellular carcinoma, HCC) is that liver is most common primary
Property malignant tumour, position is ranked third in the cancer associated cause of the death in the world.Liver cancer can be cured by generally acknowledging at present
Means are only performed the operation, including partial hepatectomy and liver transfer operation.And success rate of operation and postoperative tumour are answered
The clinical stages of hair rate and tumour, is closely related, and the size of gross tumor volume and the position of growth are to influence hand
The deciding factor of art difficulty and risk.Therefore find that liver cancer is to treat the key of liver cancer as early as possible.And mesh
It is preceding clinically to find that early liver cancer is highly difficult.On the one hand, early liver cancer atypical symptom, when going out
Middle and advanced stage is in more during the clinical manifestations such as existing pain, jaundice, dyshepatia.On the other hand, liver
Cancer examination means are deficient.Clinically the method currently used for examination liver cancer have imageological examination (ultrasonic wave,
CT, MRI) and alpha-fetoprotein (AFP) blood testing.But the sensitiveness of these means and special
Property is all unsatisfactory.Therefore, a kind of sensitiveness height, specific good and easy to operate inspection are invented
It is necessary to look into means.
The content of the invention
The purpose of the present invention is to be directed to above-mentioned clinical demand, there is provided a kind of spy of AMY2B mRNAs
Specific amplification primers and the diagnosis for liver cancer kit including the specificity amplification primer.
The present inventor has found that (cDNA sequence is SEQ ID to AMY2B genes under study for action
NO：1) late significantly lowered in liver cancer patient, with RT-PCR technology in early liver cancer patient specimen
Middle checking learns, AMY2B expression quantity also has part reduction liver cancer is early interim.Further analyze liver
Cancer patient's prognosis life cycle finds that low expression AMY2B patient survival significantly shortens.
Based on above-mentioned discovery, the first aspect of the present invention is to provide a kind of spy of AMY2B mRNAs
Specific amplification primers, the specificity amplification primer include：
Forward primer：5’-ATCAACCTAGCCATCCAACCT-3’(SEQ ID NO：2)；
Reverse primer：5’-AACCGAATTTTCAATTGTTTTCACA-3’(SEQ ID NO：
3)。
The second aspect of the present invention is to provide a kind of diagnosis for liver cancer kit, and the kit includes：
(1) specificity amplification primer of above-mentioned AMY2B mRNAs；
(2) PCR reactions enzyme and reagent, and/or the mRNA of reference gene mark are special
Property primer.
The reference gene mark is preferably HPRT.The selection of reference gene is based on international tumor research
Researchs of the authoritative magazine Journal of Cancer Research and Clinical Oncology in 2008
Article：Selection of reference genes for real-time PCR in human hepatocellular
carcinoma tissues.The author of this article is：Gao Q1,Wang XY,Fan J,Qiu SJ,Zhou J,
Shi YH,Xiao YS,Xu Y,Huang XW,Sun J。
The specific primer of reference gene HPRT mRNA can be conventional for verifying
HPRT specific primer, it is preferably：
Forward primer：5’-CCTGGCGTCGTGATTAGTGAT-3’(SEQ ID NO：4)；
Reverse primer：5’-AGACGTTCAGTCCTGTCCATAA-3’(SEQ ID NO：5).
According to the present invention, the PCR reactions are preferably used for the various normal of PCR reactions with enzyme and reagent
Reagent is advised, is included but is not limited to：Taq enzyme, 4 × dNTP mixed liquors, MgCl2Solution and deionized water.
Preferably, the kit also includes：Standard items AMY2B gene orders.
Preferably, the kit also includes：Reference substance hprt gene sequence.
Pass through the rich of the AMY2B that analyzes liver cancer patient tissue sample and normal structure mRNA
Degree, AMY2B abundance and the Pathological degree of liver cancer and prognosis existence highlights correlations are found, and developed
Go out to be easy to the diagnosis for liver cancer kit of clinical practice, molecular indexes are provided for the diagnosis of liver cancer
Support, and tutorial message is provided for clinical application.
The effect of the present invention is as follows：
(1) there is very high Sensitivity and Specificity using the QPCR detections for carrying out tumor markers,
And it is quantitative accurate, contribute to the auxiliary diagnosis of liver cancer, also provided for the biomarker of other diseases
Use for reference, while AMY2B expression quantity is clinically judges that patient's course of disease provides Informational support.
(2) the biological tissue storehouse of large sample size is utilized, demonstrates the validity of kit of the present invention, and
And prognosis of patients with feminine survival state is analyzed, it was demonstrated that kit of the present invention can be effectively
Predict the life cycle length of patient.For clinical detection patients ' recovery situation and formulate personalized therapy program
Provide help.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Brief description of the drawings
Exemplary embodiment of the invention is described in more detail in conjunction with the accompanying drawings.
Fig. 1 is mankind AMY2B genome.
Fig. 2 displays are using the kit of the present invention to AMY2B courier in liver cancer and normal liver sample
RNA amplification situation.
Fig. 3 displays detect the expression quantity situation of AMY2B in liver cancer sample using the kit of the present invention,
And analyze the pathogenesis of patient.Wherein, p represents the statistical test of difference, is examined using t.
Fig. 4 displays detect the expression quantity situation of AMY2B in liver cancer sample using the kit of the present invention,
And prognosis and the existence time limit of patient are analyzed on this basis.Wherein, when transverse axis represents patient's existence
Between (my god), p represents the statistical test of difference, is examined using Log Rank.
Embodiment
The development process of the diagnosis for liver cancer kit of the present invention includes：(1) systematic collection patient liver
Cancerous tissue and clinical data, and patient's prognosis situation is tracked for a long time；(2) study in liver cancer tissue
Associating between the abundance of AMY2B mRNAs and clinical indices and patient's prognosis existence；(3) build
Vertical mathematical modeling, the abundance of checking AMY2B mRNAs are used for the repeatability of clinical diagnosis；(4)
Develop the diagnosis for liver cancer kit based on AMY2B.
In above-mentioned steps (3), the abundance of AMY2B mRNAs is detected using QPCR,
Specifically operating procedure is：
(1) total serum IgE in liver cancer tissue sample is isolated and purified；
(2) by the total serum IgE reverse transcription of step (1) into cDNA；
(3) AMY2B and reference gene are subjected to augmentation detection on fluorescence real-time quantitative PCR instrument；
The specificity amplification primer of AMY2B mRNAs is：
Forward primer：5’-ATCAACCTAGCCATCCAACCT-3’(SEQ ID NO：2)；
Reverse primer：5’-AACCGAATTTTCAATTGTTTTCACA-3’(SEQ ID NO：
3)。
The specific primer of reference gene HPRT mRNA is：
Forward primer：5’-CCTGGCGTCGTGATTAGTGAT-3’(SEQ ID NO：4)；
Reverse primer：5’-AGACGTTCAGTCCTGTCCATAA-3’(SEQ ID NO：5).
Above-mentioned steps can be this area normal experiment step.
For example, the concrete operation step of step (1) includes：
A. tissue homogenate：Tissue specimen is ground to powdery in liquid nitrogen bath, adds appropriate TRIzol examinations
Agent, it is transferred in 1.5ml eppendorf pipes, places 5 minutes at room temperature.
B. 0.2ml chloroforms are added in every ml tissue grinder liquid, acutely concussion 15 seconds, stand at room temperature
3 minutes, centrifuge is put into afterwards, and 12000g is centrifuged 15 minutes.
C. supernatant is carefully pipetted to new pipe, is added the isopropanol of same volume, is overturned and mix.It is quiet at 4 DEG C
Put 15 minutes, 7500g centrifugations afterwards 15 minutes.
D. move and abandon supernatant, add the ethanol of 1ml 75%, 7500g is centrifuged 5 minutes.
E. it is air-dried RNA precipitate 10 minutes, is dissolved and precipitated with 20 μ l DEPC water.
F. UV spectrophotometer measuring OD260 and OD280 value are used.
The concrete operations of step (2) include：
The 1 μ g total serum IgEs that step (1) is extracted are dissolved in 14 μ l DEPC water, add 2 μ l 10 ×
Buffer solution, 2 μ l 10mM dNTP mixed liquors, 1 μ l oligo (dT) 18 primer (0.5 μ g/ul) and 1 μ l
Reverse transcriptase, slight concussion.42 DEG C are incubated 60 minutes, are placed in 95 DEG C 3 minutes.Cooled on ice it
Of short duration centrifugation afterwards, 380 μ l deionized waters are added, it is standby.
The concrete operations of step (3) include：
Using 12 μ l reaction systems, each sample sets 3 repeating pipes, to ensure the reliability of result.
SYBR Green PCRs system 6 μ l, forward and reverse each 1 μ l of primer, step (2) it is anti-
Answer the μ l of product 4.Amplification program is：94 DEG C, 5 minutes, (94 DEG C, 15 seconds；60 DEG C, 60 seconds) ×
40 circulations.In the enterprising performing PCR reaction of fluorescence real-time quantitative PCR instrument.
Fig. 2 is shown to be believed AMY2B in liver cancer and normal liver sample using the kit of the present invention
Make RNA amplification situation.Demonstrate the validity that the kit of the present invention detects to hepatoma sample.From
As a result as can be seen that the kit of the present invention can be believed with AMY2B in effective detection liver cancer tissue sample
Make RNA abundance, the relative quantity of AMY2B mRNAs can be calculated according to amplification curve.
In order to continue to assess value of the present invention in liver cancer clinical assistant diagnosis, inventor uses this reagent
369 hepatoma samples and 50 Carcinoma side normal tissue samples that box is collected to clinic are detected.Its
In, 50 cancer beside organism's samples are as a control group.
First than right kit the classification of testing result and liver cancer clinical pathology result.Such as Fig. 3
Shown, in liver cancer early stage patient, the abundance of AMY2B mRNAs is slightly lowered but unobvious；
And in later period of hepatocarcinoma patient, the abundance of AMY2B mRNAs reduces more than one times, and is uniting
Meter shows high conspicuousness on learning.This result is prompted, and monitors the rich of AMY2B mRNAs
Degree can be used for the disease state of development of auxiliary judgment hepatocarcinoma patient, for there is AMY2B courier
The patient that RNA abundance declines to pay a great deal of attention and active treatment.
Inventor also compares the testing result of this kit and the prognosis evaluation of hepatocarcinoma patient.Such as Fig. 4
Shown, patient is divided into AMY2B mRNAs Gao Shui by the monitoring result according to 369 hepatocarcinoma patients
Flat group and low-level group.It was found that the existence of AMY2B mRNAs high level group is expected significantly
Better than low-level group, the former 5 years survival rates are 1.6 times of the latter, and the former median survival time is
More than 2 times of the latter.The result is shown, is survived with prognosis of the kit of the present invention to hepatocarcinoma patient
Assessment has highly important reference value, and the existence of patient can be prompted to be expected.
Various embodiments of the present invention are described above, described above is exemplary, and exhaustive
Property, and it is also not necessarily limited to disclosed each embodiment.In the model without departing from illustrated each embodiment
Enclose and spirit in the case of, many modifications and changes for those skilled in the art
It will be apparent from.
Claims (7)
1. a kind of specificity amplification primer of AMY2B mRNAs, it is characterised in that this is special
Property amplimer includes：
Forward primer：5’-ATCAACCTAGCCATCCAACCT-3’；
Reverse primer：5’-AACCGAATTTTCAATTGTTTTCACA-3’.
2. a kind of diagnosis for liver cancer kit, it is characterised in that the kit includes：
(1) specificity amplification primer of AMY2B mRNAs claimed in claim 1；
(2) PCR reactions enzyme and reagent, and/or the mRNA of reference gene mark are special
Property primer.
3. kit according to claim 2, wherein, the kit also includes：
The reference gene mark is HPRT.
4. kit according to claim 3, wherein, reference gene mark HPRT letter
The specific primer for making RNA is：
Forward primer：5’-CCTGGCGTCGTGATTAGTGAT-3’；
Reverse primer：5’-AGACGTTCAGTCCTGTCCATAA-3’.
5. according to the kit described in any one in claim 2-4, wherein, the PCR reactions
Included with enzyme and reagent：Taq enzyme, 4 × dNTP mixed liquors, MgCl2Solution and deionized water.
6. according to the kit described in any one in claim 2-4, wherein, the kit is also
Including：Standard items AMY2B gene orders.
7. the kit according to claim 3 or 4, wherein, the kit also includes：It is right
According to product hprt gene sequence.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610290859.XA CN107345236A (en) | 2016-05-04 | 2016-05-04 | The specificity amplification primer and diagnosis for liver cancer kit of a kind of AMY2B mRNAs |
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| CN201610290859.XA CN107345236A (en) | 2016-05-04 | 2016-05-04 | The specificity amplification primer and diagnosis for liver cancer kit of a kind of AMY2B mRNAs |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020142981A1 (en) * | 2000-06-14 | 2002-10-03 | Horne Darci T. | Gene expression profiles in liver cancer |
| CN104969071A (en) * | 2012-11-30 | 2015-10-07 | 应用蛋白质组学公司 | Method for evaluation of presence of or risk of colon tumors |
-
2016
- 2016-05-04 CN CN201610290859.XA patent/CN107345236A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020142981A1 (en) * | 2000-06-14 | 2002-10-03 | Horne Darci T. | Gene expression profiles in liver cancer |
| CN104969071A (en) * | 2012-11-30 | 2015-10-07 | 应用蛋白质组学公司 | Method for evaluation of presence of or risk of colon tumors |
Non-Patent Citations (4)
| Title |
|---|
| GENBANK: "Homo sapiens amylase, alpha 2B (pancreatic) (AMY2B), mRNA，NCBI Reference Sequence: NM_020978.4", 《NCBI数据库》 * |
| IWAO KOYAMA ET AL.: "α-Amylase expressed in human liver is encoded by the AMY-2B gene identified in tumorous tissues", 《CLINICA CHIMICA ACTA》 * |
| QIANG GAO ET AL.: "Selection of reference genes for real-time PCR in human hepatocellular carcinoma tissues", 《JOURNAL OF CANCER RESEARCH AND CLINICAL ONCOLOGY》 * |
| 谢树高 等: "miR-145靶向调控DAB2对前列腺癌PC3细胞迁移和侵袭能力的影响", 《遗传》 * |
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Application publication date: 20171114 |