CN107815492A - A kind of detection method based on QPCR Diagnosis of Breast cancers - Google Patents
A kind of detection method based on QPCR Diagnosis of Breast cancers Download PDFInfo
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- CN107815492A CN107815492A CN201711298853.8A CN201711298853A CN107815492A CN 107815492 A CN107815492 A CN 107815492A CN 201711298853 A CN201711298853 A CN 201711298853A CN 107815492 A CN107815492 A CN 107815492A
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Abstract
The invention discloses a kind of detection method based on QPCR Diagnosis of Breast cancers, specifically comprise the following steps：Step 1. extracts serum 4 in tester's body and managed first, and often pipe is according to clinical criteria often pipe 4ml, and be put into incubator and stored with standby；Step 2. chooses 2 serology tubes and is separately added into primer into 2 test tubes, by shaking test tube and invisible spectro serum being instilled into detection ware, is observed by fluorescent quantitative detector；Step 3. chooses remaining 2 serology tubes, and serum dilution is instilled into test tube, the 10s of fully shaking 5；Serum after being shaken in step 3 is added dropwise on the slide of PCR instrument by step 4., and the long-chain non-coding RNA in serum and microRNA are detected by PCR instrument；Testing result in step 2 and step 4 is carried out data statistics by step 5.；The present invention relates to technical field of biological, and the detection method is simple to operate, and detecting the breast cancer correlating markings gene in human serum using qpcr methods effectively increases detection rates.
Description
Technical field
The present invention relates to technical field of biological, specially a kind of detection method based on QPCR Diagnosis of Breast cancers.
Background technology
Breast cancer is one of most common malignant tumour of women, and the incidence of disease accounts for the 7-10% of the various malignant tumours of whole body,
Women is only second to uterine cancer, it has also become threatens the Etiological of WomanHealth.Its morbidity is often relevant with heredity, and 40-60
Between year, women's incidence of disease before and after climacteric is higher.It is that one kind generally occurs in breast galandular epithelium tissue, has a strong impact on woman
Physically and mentally healthy even one of the most common malignant tumour of threat to life of female.Early detection, the early diagnosis of breast cancer, are to improve
The key of curative effect.
Tumour cell can reduce body's immunity, escape immunosurveillance by secretory immune inhibiting substances, such a to exempt from
Epidemic disease escape is to cause one of important mechanisms of tumor development.There are some researches prove the glucose and insulin of high concentration can show
Write the secretion of the increase some immunosuppressive substances of breast cancer tumor cells；The exception that generally existing is metabolized in tumour cell.Third
Pyruvate kinase M2 (PKM2) is the key enzyme of glycolysis, in tumour cell, PKM2 great expression, instead of original acetone
Acid kinase normal phenotype, before inside and outside experiment find that PKM2 overexpression can be remarkably reinforced Warburg effects, can not only
Energy necessary to enough be provided for growth of tumour cell, more tumour cell provide the raw material of synthesising biological macromolecular substances, so as to
Promote tumour growth.
The inspection of existing breast cancer is typically detected using some complicated Medical Devices such as color ultrasound to tester,
Not only testing cost is big for it, and increases medical personnel labour, and operation is more complicated, to medical personnel's technical requirements ratio
It is higher, it is impossible to meet user's request.
The content of the invention
The technical problem of solution
In view of the shortcomings of the prior art, the invention provides a kind of detection method based on QPCR Diagnosis of Breast cancers, solve
The existing problem to breast cancer detection operating difficulties.
Technical scheme
To realize object above, the present invention is achieved by the following technical programs：One kind is based on QPCR Diagnosis of Breast cancers
Detection method, specifically comprise the following steps：
Step 1. extracts serum 4 in tester's body and managed first, and often pipe is according to clinical criteria often pipe 4ml, and be put into incubator
Stored with standby；
Step 2. chooses 2 serology tubes and is separately added into primer into 2 test tubes, by shaking test tube and by test tube
Serum instill detection ware, observed by fluorescent quantitative detector；
Step 3. chooses remaining 2 serology tubes, and serum dilution is instilled into test tube, fully shaking 5-10s；
Serum after being shaken in step 3 is added dropwise on the slide of PCR instrument by step 4., and by PCR instrument in serum
Long-chain non-coding RNA and microRNA detected；
Testing result in step 2 and step 4 is carried out data statistics by step 5..
Preferably, the primer in the step 2 specifically includes one in miR-93-5p, miR-17-5p and miR-20-5p
The primer of kind or a variety of miRNA；Preferably include in serum miRNA two kinds in miR-93-5p, miR-17-5p and miR-20-5p
Or two or more miRNA primer；More preferably include miR-93-5p, miR-17-5p and miR-20- in serum miRNA
Tri- kinds of miRNA of 5p primer.
Preferably, the serum dilution in the step 3 is made up of following components content：Translate glycine 12-15g/L, essence
Propylhomoserin 10-14g/L, glutamine 2-6g/L, potassium phosphate 1-5g/L, Sodium Pyruvate 3-7g/L, sodium chloride 20-24g/L, magnesium chloride
13-17g/L, EDTA-2K0.2-0.8g/L, water 22.4-26.4.
Preferably, the blood in test tube is respectively dropped on detection ware and slide in the step 2 and step 4
It is added dropwise using rubber head dropper, is easy to control of the staff to amount.
Preferably, the serum dilution in the step 3 is turned into by following best composition content groups：Translate glycine 14g/L,
Arginine 12g/L, glutamine 4g/L, potassium phosphate 3g/L, Sodium Pyruvate 5g/L, sodium chloride 22g/L, magnesium chloride 15g/L,
EDTA-2K0.4g/L, water 24.4.
Beneficial effect
The invention provides a kind of detection method based on QPCR Diagnosis of Breast cancers.Possesses following beneficial effect：
The detection method is simple to operate, has tool using the breast cancer correlating markings gene in human serum is detected using qpcr methods
It has been improved the speed of detection；Joint-detection long-chain non-coding RNA H19, miR93-5P and miR17-5P, can not only improve inspection
Judgement can also be predicted to the migration of tumour by surveying the degree of accuracy of breast cancer, provide certain thinking for the treatment in later stage, accurately
Rate is higher, avoids causing testing result mistake because of human error.
Embodiment
The present invention provides a kind of technical scheme：A kind of detection method based on QPCR Diagnosis of Breast cancers, is specifically included as follows
Step：
Step 1. extracts serum 4 in tester's body and managed first, and often pipe is according to clinical criteria often pipe 4ml, and be put into incubator
Stored with standby；
Step 2. chooses 2 serology tubes and is separately added into primer into 2 test tubes, by shaking test tube and by test tube
Serum instill detection ware, observed by fluorescent quantitative detector；
Step 3. chooses remaining 2 serology tubes, and serum dilution is instilled into test tube, fully shaking 5-10s；
Serum after being shaken in step 3 is added dropwise on the slide of PCR instrument by step 4., and by PCR instrument in serum
Long-chain non-coding RNA and microRNA detected；
Testing result in step 2 and step 4 is carried out data statistics by step 5..
Present invention further optimization scheme, primer in the step 2 specifically comprising miR-93-5p, miR-17-5p and
The primer of one or more miRNA in miR-20-5p；Preferably include serum miRNA in miR-93-5p, miR-17-5p and
Two or more miRNA primer in miR-20-5p；More preferably include serum miRNA in miR-93-5p,
Tri- kinds of miRNA of miR-17-5p and miR-20-5p primer.
Present invention further optimization scheme, the serum dilution in the step 3 are made up of following components content：Translate sweet
Propylhomoserin 12-15g/L, arginine 10-14g/L, glutamine 2-6g/L, potassium phosphate 1-5g/L, Sodium Pyruvate 3-7g/L, sodium chloride
20-24g/L, magnesium chloride 13-17g/L, EDTA-2K0.2-0.8g/L, water 22.4-26.4.
Present invention further optimization scheme, the blood in test tube is respectively dropped into the step 2 and step 4
It is added dropwise on detection ware and slide using rubber head dropper, is easy to control of the staff to amount.
Present invention further optimization scheme, the serum dilution in the step 3 are made up of following best composition content
For：Translate glycine 14g/L, arginine 12g/L, glutamine 4g/L, potassium phosphate 3g/L, Sodium Pyruvate 5g/L, sodium chloride 22g/
L, magnesium chloride 15g/L, EDTA-2K0.4g/L, water 24.4.
It should be noted that herein, such as first and second or the like relational terms are used merely to a reality
Body or operation make a distinction with another entity or operation, and not necessarily require or imply and deposited between these entities or operation
In any this actual relation or order.Moreover, term " comprising ", "comprising" or its any other variant are intended to
Nonexcludability includes, so that process, method, article or equipment including a series of elements not only will including those
Element, but also the other element including being not expressly set out, or it is this process, method, article or equipment also to include
Intrinsic key element.In the absence of more restrictions.
Although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of changes, modification can be carried out to these embodiments, replace without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (5)
1. a kind of detection method based on QPCR Diagnosis of Breast cancers, it is characterised in that specifically comprise the following steps：
Step 1. extracts serum 4 in tester's body and managed first, and often pipe is according to clinical criteria often pipe 4ml, and be put into incubator progress
Storage is with standby；
Step 2. chooses 2 serology tubes and is simultaneously separately added into primer into 2 test tubes, by shaking test tube and by invisible spectro blood
It is clear to instill detection ware, observed by fluorescent quantitative detector；
Step 3. chooses remaining 2 serology tubes, and serum dilution is instilled into test tube, fully shaking 5-10s；
Serum after being shaken in step 3 is added dropwise on the slide of PCR instrument by step 4., and by PCR instrument to the length in serum
Chain non-coding RNA and microRNA are detected；
Testing result in step 2 and step 4 is carried out data statistics by step 5..
A kind of 2. detection method based on QPCR Diagnosis of Breast cancers according to claim 1, it is characterised in that：The step
Primer in 2 specifically includes the primer of one or more miRNA in miR-93-5p, miR-17-5p and miR-20-5p；It is preferred that
To include the primer of two or more miRNA in miR-93-5p, miR-17-5p and miR-20-5p in serum miRNA；Enter
One step is preferably to include the primer of tri- kinds of miRNA of miR-93-5p, miR-17-5p and miR-20-5p in serum miRNA.
A kind of 3. detection method based on QPCR Diagnosis of Breast cancers according to claim 1, it is characterised in that：The step
Serum dilution in 3 is made up of following components content：Translate glycine 12-15g/L, arginine 10-14g/L, glutamine 2-
6g/L, potassium phosphate 1-5g/L, Sodium Pyruvate 3-7g/L, sodium chloride 20-24g/L, magnesium chloride 13-17g/L, EDTA-2K 0.2-
0.8g/L, water 22.4-26.4.
A kind of 4. detection method based on QPCR Diagnosis of Breast cancers according to claim 1, it is characterised in that：It is described
The blood in test tube is respectively dropped on detection ware and slide in step 2 and step 4 and is added dropwise using rubber head dropper,
It is easy to control of the staff to amount.
A kind of 5. detection method based on QPCR Diagnosis of Breast cancers according to claim 3, it is characterised in that：The step
Serum dilution in 3 is turned into by following best composition content groups：Translate glycine 14g/L, arginine 12g/L, glutamine 4g/
L, potassium phosphate 3g/L, Sodium Pyruvate 5g/L, sodium chloride 22g/L, magnesium chloride 15g/L, EDTA-2K0.4g/L, water 24.4.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009015071A1 (en) * | 2007-07-23 | 2009-01-29 | Dharmacon, Inc. | Screening of micro-rna cluster inhibitor pools |
| CN101389770A (en) * | 2006-01-05 | 2009-03-18 | 俄亥俄州立大学研究基金会 | Microrna-based methods and compositions for the diagnosis, prognosis and treatment of solid cancers |
| CN106855575A (en) * | 2016-12-30 | 2017-06-16 | 广州华弘生物科技有限公司 | A kind of quick detection kit of breast cancer three |
| CN107076709A (en) * | 2014-09-03 | 2017-08-18 | 加利福尼亚大学董事会 | It is determined that the method for circulation RNA distribution profile |
| CN107429295A (en) * | 2015-03-09 | 2017-12-01 | 新加坡科技研究局 | The method of risk to suffer from breast cancer is determined by detecting the expression of Microrna (miRNA) |
-
2017
- 2017-12-08 CN CN201711298853.8A patent/CN107815492A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101389770A (en) * | 2006-01-05 | 2009-03-18 | 俄亥俄州立大学研究基金会 | Microrna-based methods and compositions for the diagnosis, prognosis and treatment of solid cancers |
| WO2009015071A1 (en) * | 2007-07-23 | 2009-01-29 | Dharmacon, Inc. | Screening of micro-rna cluster inhibitor pools |
| CN107076709A (en) * | 2014-09-03 | 2017-08-18 | 加利福尼亚大学董事会 | It is determined that the method for circulation RNA distribution profile |
| CN107429295A (en) * | 2015-03-09 | 2017-12-01 | 新加坡科技研究局 | The method of risk to suffer from breast cancer is determined by detecting the expression of Microrna (miRNA) |
| CN106855575A (en) * | 2016-12-30 | 2017-06-16 | 广州华弘生物科技有限公司 | A kind of quick detection kit of breast cancer three |
Non-Patent Citations (1)
| Title |
|---|
| 王川等: "miR-17-5p与乳腺癌侵袭及预后的相关性研究", 《福建医科大学学报》 * |
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Application publication date: 20180320 |