CN108085391A - A kind of RNA quality-control products for the lung cancer fusion detection kit for stablizing preservation - Google Patents
A kind of RNA quality-control products for the lung cancer fusion detection kit for stablizing preservation Download PDFInfo
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Abstract
The invention discloses a kind of RNA quality-control products for the lung cancer fusion detection kit for stablizing preservation, the RNA quality-control products include following components：Positive reference product, negative reference product and detection limit reference material.Contain corresponding fusion RNA, aurin front three acid ammonium salt, preservative and bestatin hydrochloride respectively in the positive reference product, negative reference product and detection limit reference material；The preservative is selected from glycine, ε polylysines or cysteine.RNA quality-control products provided by the invention can effectively prevent the degradation of free RNA, stable environment is provided for RNA, extend the holding time of RNA, have many advantages, such as that manufacturing cost is low, performance is stable and easy to maintain simultaneously, the factors such as personnel's operation, instrument state and kit validity can be effectively monitored, drastically increase the accuracy and reliability of detection kit.
Description
Technical field
The invention belongs to genetic test fields, and in particular to a kind of lung cancer fusion detection kit for stablizing preservation
RNA quality-control products.
Background technology
Lung cancer is the highest malignant tumour of morbidity and mortality in worldwide, seriously endangers human health.2012
Year, global cancer epidemiology statistics was shown, the annual cancer new cases about 14,100,000 in the whole world are about 8,200,000 dead,
Wherein, lung cancer morbidity is 182.5 ten thousand, and death about 159.0 ten thousand, rank first example.Lung cancer is divided into non-small cell lung cancer (non-
Small cell lung cancer, NSCLC) and Small Cell Lung Cancer, NSCLC account for 80% or so of whole lung cancer.Dye weight
Row and gene rearrangement are that common science of heredity changes in malignant tumour, and Soda in 2007 etc. has found spine in patients with lung cancer body for the first time
The fusion of 4 (EML4) gene of skin animal micro-pipe correlation albuminoid and anaplastic lymphoma kinase (ALK), the discovery is for the first time
Confirm that there are Gene Fusions in NSCLC.The ALK fusion gene oncogene new as one participates in the evolution of NSCLC,
Its incidence is about 5%, for ALK fusion gene targeted inhibition agent Gefitinib, Tarceva, gram azoles for Buddhist nun at present
It is widely used in NSCLC clinical treatments.It has also been found that ROS1, RET fusion in NSCLC.
Before targeted inhibition agent treatment lung cancer is used, fusion examination has become an important and necessary index.
US National synthesis cancer network clinical practice guideline, European Internal Medicine-Oncology association lung cancer common recognition and Chinese Advanced Non-Small Cell
Molecular targeted therapy for lung cancer Consensus of experts is clearly suggested：NSCLC patient receive treat before should first detect EGFR, ALK and
ROS1 genes determine corresponding therapeutic strategy according to gene appearance, and suggest being carried out at the same time three genes under condition license
Detection.
At present, detecting the method for the fusion of lung cancer driving mainly includes fluorescence in situ hybridization technique (FISH), is immunized
Histochemical method (IHC) and real-time RT-PCR (RT-PCR).A variety of lungs have been had been developed that based on the above method
Cancer fusion detection kit, but there is no economic, effective, stable and general lung cancer fusions on the market at present to detect
Kit quality-control product sale, and existing RNA Quality Controls category product have the shortcomings that more it is unstable, degradable and not easy to maintain,
And quality-control product is the important method and quality index of measurement and the performances such as a kit detection accuracy of assessment and stability.
The content of the invention
Based on this, it is an object of the present invention to overcome in place of above-mentioned the deficiencies in the prior art and providing one kind can stablize
The RNA quality-control products of the lung cancer fusion detection kit of preservation, low manufacture cost, performance are stable, easy to maintain, can be effectively
The factors such as monitoring personnel operation, instrument state and kit validity drastically increase the standard of lung cancer fusion detection
True property and reliability.
To achieve the above object, the technical solution taken of the present invention is：A kind of lung cancer fusion inspection for stablizing preservation
The RNA quality-control products of test agent box, the RNA quality-control products include following components：Positive reference product, negative reference product and detection limit ginseng
Examine product；
The positive reference product include following components：The lung cancer fusion RNA, aurin front three acid ammonium salt, preservative
With bestatin hydrochloride；
The negative reference product include following components：The non-lung cancer fusion RNA, aurin front three acid ammonium salt, anti-corrosion
Agent and bestatin hydrochloride；
The detection limit reference material includes following components：The lung cancer fusion RNA, the non-lung cancer fusion
RNA, aurin front three acid ammonium salt, preservative and bestatin hydrochloride, and the lung cancer fusion RNA accounts for the matter of total serum IgE
Amount is than being 1~5%；
The preservative is selected from glycine, epsilon-polylysine or cysteine.
Preferably, aurin front three acid ammonium salt, glycine in the positive reference product, negative reference product and detection limit reference material
Molar concentration with bestatin hydrochloride is：100~500 μM of aurin front three acid ammonium salt, 80~180mM of glycine and benzene fourth
20~60nM of inhibin hydrochloride.
Preferably, aurin front three acid ammonium salt concentration is in the positive reference product, negative reference product and detection limit reference material
200~400 μM, glycine concentration be 90~110mM, bestatin hydrochloride concentration is 30~50nM；It is it is highly preferred that described
Aurin front three acid ammonium salt concentration is 300 μM in positive reference product, negative reference product, detection limit reference material, glycine concentration is
100mM, bestatin hydrochloride concentration are 40nM.
Preferably, aurin front three acid ammonium salt concentration is in the positive reference product, negative reference product and detection limit reference material
100~500 μM, epsilon-polylysine concentration be 2~4mg/ml, bestatin hydrochloride concentration is 20~60nM.
Preferably, the positive reference product, negative reference product and detection limit reference material in aurin front three acid ammonium salt 200~
400 μM, epsilon-polylysine concentration be 2.5~3.5mg/ml, 30~50nM of bestatin hydrochloride；It is highly preferred that the sun
Property reference material, negative reference product and detection limit reference material in aurin front three acid ammonium salt concentration be 300 μM, epsilon-polylysine concentration is
3mg/ml and bestatin hydrochloride concentration are 40nM.
Preferably, aurin front three acid ammonium salt concentration is in the positive reference product, negative reference product and detection limit reference material
100~500 μM, semicystinol concentration be 5~10mM, bestatin hydrochloride concentration is 20~60nM.
Preferably, the positive reference product, negative reference product and detection limit reference material in aurin front three acid ammonium salt 200~
400 μM, semicystinol concentration be 5~10mM, 30~50nM of bestatin hydrochloride；It is highly preferred that the positive reference product,
Aurin front three acid ammonium salt concentration is 300 μM in negative reference product and detection limit reference material, semicystinol concentration is 7.5mM and benzene fourth
Inhibin hydrochloride concentration is 40nM.
Preferably, the positive reference product melt selected from ALK fusion gene reference material, ROS1 fusions reference material and RET
Close at least one of gene reference product；
The ALK fusion gene reference material includes following components：The ALK fusion gene RNA, aurin front three acid ammonium salt,
Preservative and bestatin hydrochloride；The ROS1 fusions reference material includes following components：The ROS1 fusions
RNA, aurin front three acid ammonium salt, preservative and bestatin hydrochloride；The RET fusions reference material is included with the following group
Point：The RET fusions RNA, aurin front three acid ammonium salt, preservative and bestatin hydrochloride.
Preferably, the negative reference product are selected from BCR-ABL fusions reference material, AML1-ETO fusion reference materials
At least one of with CBF β-MYH11 fusion reference materials；
The BCR-ABL fusions reference material includes following components：BCR-ABL fusions RNA, aurin tricarboxylic acid ammonium
Salt, preservative and bestatin hydrochloride；The AML1-ETO fusions reference material includes following components：AML1-ETO melts
Close gene RNA, aurin front three acid ammonium salt, preservative and bestatin hydrochloride；CBF β-MYH11 fusions the reference
Product include following components：AML1-ETO fusions RNA, aurin front three acid ammonium salt, preservative and bestatin hydrochloride.
Preferably, ALK fusion gene reference material is selected from ALK-EML4, ALK-KIF5B, ALK- in the positive reference product
KLC1, ALK-TFG, ALK-TPR, ALK-HIP1, ALK-STRN, ALK-DCTN1, ALK-SQSTM1, ALK-BIRC6 and ALK-
At least one of CLTC fusion reference materials；
The ROS1 fusions reference material be selected from ROS1-CD74, ROS1-SLC34A2, ROS1-GOPC, ROS1-
SDC4, ROS1-TPM3, ROS1-EZR, ROS1-LRIG3, ROS1-KDELR2, ROS1-LIMA1, ROS1-MSN and ROS1-
At least one of TMEM106B fusion reference materials；
The RET fusions reference material is selected from KIF5B-RET and RET-CCDC6 fusion reference materials.
Preferably, ALK fusion gene reference material is selected from ALK-EML4, ALK-KIF5B and ALK- in the positive reference product
TFG；
The ROS1 fusions reference material is selected from：ROS1-SLC34A2, ROS1-CD74, ROS1-EZR and ROS1-
SDC4；
The RET fusions reference material is selected from：RET-CCDC6.
Above-mentioned positive reference product, be inventor at work by being drawn after great amount of samples Analysis and Screening, type described above
Fusion as positive reference product, the Lung Cancer Types of most Chinese population can be covered, it is existing preferable with reference to effect
Fruit, and cost can be reduced.
It is another object of the present invention to provide the RNA of the lung cancer fusion detection kit for stablizing preservation
The preparation method of quality-control product.
To achieve the above object, the technical solution adopted by the present invention is a kind of lung cancer fusion inspection for stablizing preservation
The preparation method of the RNA quality-control products of test agent box, which is characterized in that comprise the following steps：
(1) preparation of positive reference product：1. the lung cancer fusion is obtained by PCR amplification；2. the lung cancer is melted
It closes gene and carrier carries out digestion, connection, transformed competence colibacillus cell, positive bacterium colony of the screening acquisition containing the lung cancer fusion
And sequence verification；3. the correct bacterium colony progress of sequencing result is supersonic induced, prepare pseudovirus solution；4. extract pseudovirus solution
RNA, add in aurin front three acid ammonium salt, preservative and bestatin hydrochloride to get；
(2) preparation of negative reference product：1. the non-lung cancer fusion is obtained by PCR amplification；2. by the non-lung
Cancer fusion and carrier carry out digestion, connection, transformed competence colibacillus cell, sun of the screening acquisition containing the non-lung cancer fusion
Property bacterium colony and sequence verification；The correct bacterium colony progress of sequencing result is supersonic induced, prepare pseudovirus solution；4. extract pseudovirus
The RNA of solution, add in aurin front three acid ammonium salt, preservative and bestatin hydrochloride to get；
(3) preparation of detection limit reference material：1. the lung cancer fusion RNA is prepared by step (1) the method；
2. the non-lung cancer fusion RNA is prepared by step (2) the method；3. by the lung cancer fusion RNA and non-
The lung cancer fusion RNA mixing so that the mass ratio that the lung cancer fusion RNA accounts for total serum IgE is 1~5%, adds in gold
Smart front three acid ammonium salt, preservative and bestatin hydrochloride to get.
It is prepared by the RNA quality-control products the present invention also provides the lung cancer fusion detection kit for stablizing preservation
Purposes in lung cancer fusion detection kit.
Present inventor has found, aurin front three acid ammonium salt, glycine and bestatin salt are added in RNA quality-control products
Hydrochlorate can effectively protect the RNA in quality-control product, prevent its degradation, extend the shelf-life of quality-control product.
Compared with the prior art, beneficial effects of the present invention are：(1) protected in RNA quality-control products provided by the invention containing RNA
Ingredient is protected, each component and dosage are that inventor is got by substantial amounts of research and analysis, effectively can prevent RNA from degrading, make it
It is more stable, it is more easy to maintain, extend the term of validity of product；(2) the RNA matter of lung cancer fusion detection kit provided by the invention
The Stability and veracity of control product energy effective monitoring kit solves the problems, such as kit quality, performance evaluation, ensures lung cancer
The accuracy and reliability of fusion examination, while there is simple and direct, convenient, use cost is low etc.；(3) present invention provides
Lung cancer fusion detection kit RNA quality-control products on the premise of detection kit validity is ensured, can monitoring personnel
The factors such as operation and instrument state further ensure the accuracy of testing result.
Description of the drawings
Fig. 1 is RNA segments after the ALK-EML4 fusion RNA reference materials of experimental group 3 preserve 2 weeks under the conditions of -80 DEG C
Size detection figure.
Fig. 2 is that RNA segments are big after the ALK-EML4 fusion RNA reference materials of experimental group 3 preserve 2 weeks under the conditions of 37 DEG C
Small detection figure.
Fig. 3 is RNA segments after the ALK-EML4 fusion RNA reference materials of control group 1 preserve 2 weeks under the conditions of -80 DEG C
Size detection figure.
Fig. 4 is that RNA segments are big after the ALK-EML4 fusion RNA reference materials of control group 2 preserve 2 weeks under the conditions of 37 DEG C
Small detection figure.
Specific embodiment
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment to the present invention
It is described further.
Embodiment 1
A kind of embodiment of the RNA quality-control products of lung cancer fusion detection kit of the present invention, includes positive reference product, the moon
Property reference material and detection limit reference material.
The positive reference product include ALK fusion gene reference material, ROS1 fusions reference material and RET fusions ginseng
Examine product.The ALK fusion gene reference material include ALK-EML4, ALK-KIF5B, ALK-KLC1, ALK-TFG, ALK-TPR,
ALK-HIP1, ALK-STRN, ALK-DCTN1, ALK-SQSTM1, ALK-BIRC6 and ALK-CLTC fusion reference material；It is described
ROS1 fusions reference material include ROS1-CD74, ROS1-SLC34A2, ROS1-GOPC, ROS1-SDC4, ROS1-TPM3,
ROS1-EZR, ROS1-LRIG3, ROS1-KDELR2, ROS1-LIMA1, ROS1-MSN and ROS1-TMEM106B fusion are joined
Examine product；The RET fusions reference material includes KIF5B-RET and RET-CCDC6 fusion reference materials.The fusion
Reference material contains corresponding fusion RNA, 100 μM of aurin front three acid ammonium salt, glycine 80mM and bestatin salt respectively
Hydrochlorate 20nM.
The negative reference product include BCR-ABL fusions reference material, AML1-ETO fusions reference material and CBF β-
MYH11 fusion reference materials.The fusion reference material is respectively containing corresponding fusion RNA, aurin tricarboxylic acid ammonium
Salinity is 100 μM, epsilon-polylysine concentration is 2mg/ml, bestatin hydrochloride concentration is 20nM.
The detection limit reference material includes ALK-EML4, ALK-KIF5B, ALK-KLC1, ALK-TFG, ALK-TPR, ALK-
HIP1、ALK-STRN、ALK-DCTN1、ALK-SQSTM1、ALK-BIRC6、ALK-CLTC、ROS1-CD74、ROS1-
SLC34A2、、ROS1-GOPC、ROS1-SDC4、ROS1-TPM3、ROS1-EZR、ROS1-LRIG3、ROS1-KDELR2、ROS1-
LIMA1, ROS1-MSN, ROS1-TMEM106B, KIF5B-RET and RET-CCDC6 fusion detection limit reference material；It is described to melt
It closes genetic test limit reference material and includes corresponding fusion RNA, BCR-ABL fusion RNA, AML1-ETO fusion respectively
RNA, CBF β-MYH11 fusions RNA, aurin front three acid ammonium salt concentration are 100 μM, the suppression of semicystinol concentration 5mM, benzene fourth
The plain hydrochloride concentration of system is 20nM, and the fusion RNA accounts for the mass ratio of total serum IgE as 2%.
Embodiment 2
A kind of embodiment of the RNA quality-control products of lung cancer fusion detection kit of the present invention, includes positive reference product, the moon
Property reference material and detection limit reference material.
The positive reference product include ALK fusion gene reference material, ROS1 fusions reference material and RET fusions ginseng
Examine product.The ALK fusion gene reference material includes ALK-EML4, ALK-KIF5B, ALK-KLC1 and ALK-BIRC6 fusion
Reference material；The ROS1 fusions reference material is referred to comprising ROS1-MSN, ROS1-EZR and ROS1-TMEM106B fusion
Product；The RET fusions reference material includes KIF5B-RET fusion reference materials.The fusion reference material contains respectively
There are corresponding fusion RNA, 500 μM of aurin front three acid ammonium salt, glycine 180mM and bestatin hydrochloride 60nM.
The negative reference product include AML1-ETO fusions reference material and CBF β-MYH11 fusion reference materials.Institute
It states fusion reference material and contains corresponding fusion RNA, aurin front three acid ammonium salt concentration respectively for 500 μM, ε-poly- bad ammonia
Acid concentration is 4mg/ml, bestatin hydrochloride concentration is 60nM.
It is described detection limit reference material include ALK-EML4, ALK-KIF5B, ALK-KLC1, ALK-BIRC6, ROS1-MSN,
ROS1-EZR, ROS1-TMEM106B and KIF5B-RET fusion detection limit reference material；The fusion detection limit reference
Product are respectively comprising corresponding fusion RNA, AML1-ETO fusion RNA, CBF β-MYH11 fusions RNA, aurin front three
Acid ammonium salt concentration is 500 μM, semicystinol concentration 10mM, bestatin hydrochloride concentration are 60nM, and the fusion base
Because the RNA mass ratioes for accounting for total serum IgE are 1%.
Embodiment 3
A kind of embodiment of the RNA quality-control products of lung cancer fusion detection kit of the present invention, includes positive reference product, the moon
Property reference material and detection limit reference material.
The positive reference product include ALK fusion gene reference material and ROS1 fusion reference materials.The ALK merges base
Because reference material include ALK-EML4, ALK-KIF5B, ALK-KLC1, ALK-TFG, ALK-TPR, ALK-HIP1, ALK-STRN,
ALK-DCTN1, ALK-SQSTM1, ALK-BIRC6 and ALK-CLTC fusion reference material；The ROS1 fusions reference material
Comprising ROS1-CD74, ROS1-SLC34A2, ROS1-GOPC, ROS1-SDC4, ROS1-TPM3, ROS1-EZR, ROS1-
LRIG3, ROS1-KDELR2, ROS1-LIMA1, ROS1-MSN and ROS1-TMEM106B fusion reference material；The fusion
Gene reference product inhibit respectively containing corresponding fusion RNA, 400 μM of aurin front three acid ammonium salt, glycine 110mM and benzene fourth
Plain hydrochloride 35nM.
The negative reference product include BCR-ABL fusions reference material, AML1-ETO fusions reference material and CBF β-
MYH11 fusion reference materials.The fusion reference material is respectively containing corresponding fusion RNA, aurin tricarboxylic acid ammonium
Salinity is 400 μM, epsilon-polylysine concentration is 2.5mg/ml, bestatin hydrochloride concentration is 35nM.
The detection limit reference material includes ALK-EML4, ALK-KIF5B, ALK-KLC1, ALK-TFG, ALK-TPR, ALK-
HIP1、ALK-STRN、ALK-DCTN1、ALK-SQSTM1、ALK-BIRC6、ALK-CLTC、ROS1-CD74、ROS1-
SLC34A2、、ROS1-GOPC、ROS1-SDC4、ROS1-TPM3、ROS1-EZR、ROS1-LRIG3、ROS1-KDELR2、ROS1-
LIMA1, ROS1-MSN and ROS1-TMEM106B detection limit reference material；The fusion detection limit reference material includes phase respectively
Answer fusion RNA, BCR-ABL fusion RNA, AML1-ETO fusion RNA, CBF β-MYH11 fusions RNA, gold
Smart front three acid ammonium salt concentration is 400 μM, semicystinol concentration 8mM, bestatin hydrochloride concentration are 35nM, and described is melted
It closes gene RNA and accounts for the mass ratio of total serum IgE as 5%.
Embodiment 4
A kind of embodiment of the RNA quality-control products of lung cancer fusion detection kit of the present invention, includes positive reference product, the moon
Property reference material and detection limit reference material.
The positive reference product include ALK fusion gene reference material.The ALK fusion gene reference material includes ALK-
EML4、ALK-KIF5B、ALK-KLC1、ALK-TFG、ALK-TPR、ALK-HIP1、ALK-STRN、ALK-DCTN1、ALK-
SQSTM1, ALK-BIRC6 and ALK-CLTC fusion reference material.The fusion reference material contains corresponding fusion
RNA, 300 μM of aurin front three acid ammonium salt, glycine 100mM and bestatin hydrochloride 40nM.
The negative reference product include BCR-ABL fusions reference material, AML1-ETO fusions reference material and CBF β-
MYH11 fusion reference materials.It is dense that the fusion reference material contains corresponding fusion RNA, aurin front three acid ammonium salt
Spend for 300 μM, epsilon-polylysine concentration is 3mg/ml and bestatin hydrochloride concentration is 40nM.
The detection limit reference material includes ALK-EML4, ALK-KIF5B, ALK-KLC1, ALK-TFG, ALK-TPR, ALK-
HIP1, ALK-STRN, ALK-DCTN1, ALK-SQSTM1, ALK-BIRC6 and ALK-CLTC detection limit reference material；The fusion base
Because detection limit reference material is dense comprising corresponding fusion RNA, CBF β-MYH11 fusions RNA, aurin front three acid ammonium salt respectively
It is 40nM to spend for 300 μM, semicystinol concentration 7.5mM, bestatin hydrochloride concentration, and the fusion RNA is accounted for
The mass ratio of total serum IgE is 3%.
Embodiment 5
Lung base cancer fusion of the present invention is because of a kind of embodiment of the RNA quality-control product preparation methods of detection kit, including following
Step：
(1) preparation of positive reference product：1. the preparation of purpose lung cancer fusion：According to purpose lung cancer in ncbi database
Fusion gene mRNA sequence, design covering fracture point sequence, synthesizes purpose lung cancer fusion.It is merged using containing purposeful lung cancer
The DNA of gene order carries out PCR amplification for template, and primer sequence is as shown in table 1.By PCR product into row agarose gel electrophoresis,
Glue is cut to the band containing purposeful lung cancer fusion segment, purpose lung cancer fusion base is carried out with Ago-Gel QIAquick Gel Extraction Kit
The recycling of cause.2. plamid vector construction and the verification of the fusion of lung cancer containing purpose：Respectively with restriction enzyme to purpose lung
Cancer fusion segment and carrier carry out double digestion, and digestion products are detected into row agarose gel electrophoresis, and gel extraction.It will
Digestion products are attached, and obtain connection product, then by connection product transformed competence colibacillus cell, are coated in LB culture medium flat plates
On, 37 DEG C are inverted culture, select the positive bacterium colony of single white.The DNA of the positive bacterium colony of extraction, carries out PCR verifications, PCR is verified
Sequence verification is carried out for the bacterium colony of the purpose lung cancer fusion positive.3. the preparation of purpose lung cancer fusion pseudovirus solution：
The single positive bacterium colony confirmed through sequencing is seeded in LB fluid nutrient mediums, after culture, centrifugation abandons supernatant, stays precipitation.Add
Enter sonication buffer (50mmol/L Tris-HCl, pH 8.0；2mmol/L EDTA；Volume fraction is 20% glycerine；
100mmol/L NaCl；Volume fraction is 0.1% Triton X-100), ice-bath ultrasonic process is carried out, ultrasound condition is：
350W, interval 5s, ultrasonic 5s, 30 Xun Huans.Then ultrasonic product in 6000rpm is centrifuged 10 minutes, collects supernatant, be vacation
Viral solution.4. prepared by quality-control product：DNaseI is added in pseudovirus solution, the DNA in digestion solution.It is carried using Trizol
Follow the example of the RNA of extraction pseudovirus solution, then add in aurin front three acid ammonium salt, glycine and bestatin hydrochloric acid to get.
(2) preparation of negative reference product：1. the preparation of non-purpose lung cancer fusion：According to non-purpose in ncbi database
Lung cancer fusion gene mRNA sequence, design covering fracture point sequence, synthesizes non-purpose lung cancer fusion.Using containing non-purpose
The DNA of lung cancer fusion gene sequence carries out PCR amplification for template, and primer sequence is as shown in table 1.PCR product is subjected to agarose
Gel electrophoresis is cut glue to the band for containing non-purpose lung cancer fusion segment, is carried out with Ago-Gel QIAquick Gel Extraction Kit non-
The recycling of purpose lung cancer fusion.2. plamid vector construction and verification containing non-purpose lung cancer fusion：Respectively with limitation
Property restriction endonuclease double digestion is carried out to non-purpose lung cancer fusion segment and carrier, by digestion products into row agarose gel electrophoresis
Detection, and gel extraction.Digestion products are attached, obtain connection product, then by connection product transformed competence colibacillus cell,
It is coated on LB culture medium flat plates, 37 DEG C are inverted culture, select the positive bacterium colony of single white.The DNA of the positive bacterium colony of extraction, carries out
PCR verifies that the bacterium colony that PCR is verified as to the non-purpose lung cancer fusion positive carries out sequence verification.3. non-purpose lung cancer fusion
The preparation of gene pseudovirus solution：The single positive bacterium colony confirmed through sequencing is seeded in LB fluid nutrient mediums, after culture,
Centrifugation, abandons supernatant, stays precipitation.Add in sonication buffer (50mmol/L Tris-HCl, pH 8.0；2mmol/L EDTA；Volume
Fraction is 20% glycerine；100mmol/L NaCl；Volume fraction is 0.1% Triton X-100), it carries out at ice-bath ultrasonic
Reason, ultrasound condition are：350W, interval 5s, ultrasonic 5s, 30 Xun Huans.Then ultrasonic product is centrifuged 10 minutes in 6000rpm,
Supernatant is collected, is pseudovirus solution.4. prepared by quality-control product：DNaseI is added in pseudovirus solution, in digestion solution
DNA.Using the RNA of Trizol extraction methods extraction pseudovirus solution, aurin front three acid ammonium salt, epsilon-polylysine and benzene are then added in
Fourth inhibin hydrochloric acid to get.
(3) preparation of detection limit reference material：1. purpose lung cancer fusion RNA is prepared by step (1) the method；
2. non-purpose lung cancer fusion RNA is prepared by step (2) the method；3. by purpose lung cancer fusion RNA and non-
Purpose lung cancer fusion RNA is mixed so that the mass ratio that purpose lung cancer fusion RNA accounts for total serum IgE is 2%, adds in aurin
Front three acid ammonium salt, cysteine and bestatin hydrochloride to get.
1 primer sequence of table
Embodiment 6
The present embodiment studies the preservation effect for inventing RNA in the RNA quality-control products.
1st, experimental design
By taking ALK-EML4 fusions reference material in positive reference product as an example, RNA is in difference in research quality-control product of the present invention
Preservation effect under preservation condition.Experimental group 1~6 and control group 1~2 be set, the ALK-EML4 fusion bases in experimental group 1~6
Because reference material respectively containing ALK-EML4 fusions RNA, aurin front three acid ammonium salt, preservative (glycine, epsilon-polylysine,
Cysteine) and bestatin hydrochloride, the ALK-EML4 fusion reference materials in control group 1~2 contain ALK- respectively
EML4 fusion RNA and DEPC water, it is specific as shown in table 1：
1 experimental design of table
2nd, experimental method
The pseudovirus solution containing ALK-EML4 fusions is prepared by 5 the method for embodiment and extracts pseudovirus
The RNA of solution is equally divided into 8 groups, adds in aurin front three acid ammonium salt, glycine and benzene fourth into RNA solution by the design of table 1
Each group ALK-EML4 fusion reference materials are prepared in inhibin hydrochloride or DEPC water.Experiment starts preceding to each group ALK-
EML4 fusions reference material carries out RNA concentration mensurations, and records.Then each group ALK-EML4 fusions reference material is preserved
Each group ALK-EML4 fusions are measured respectively under the conditions of corresponding, after 2 weeks by 2100 biological analysers of Agilent to join
The RNA clip sizes and concentration of product are examined, the stability of each group ALK-EML4 fusion reference materials and degradation situation are detected with this.
Wherein, detecting the RNA segments of 3100-3200bp, to illustrate that ALK-EML4 fusion reference materials do not have degradable, while profit
The degradation rate of ALK-EML4 fusion reference materials is calculated with the following formula：Degradation rate %=is (before experiment after RNA concentration-experiment
RNA concentration)/experiment before RNA concentration × 100%.
3rd, experimental result
After 2 weeks, each group ALK-EML4 fusion reference materials are detected, experimental group 3, experimental group 6,1 and of control group
The RNA clip sizes testing result of control group 2 is respectively as shown in Fig. 1,2,3 and 4, and the results are shown in Table 2 for specific experiment：
2 experimental result of table
From above-mentioned experimental result, aurin tricarboxylic acid is included in ALK-EML4 fusions reference material provided by the invention
Ammonium salt, preservative and bestatin hydrochloride, it is notable in -80 DEG C (experimental groups 1~3) and 37 DEG C of (experimental group 4~6) Shi Junneng
The stability of RNA is improved, can effectively prevent degradations of the RNA during preservation, wherein, the ALK- in experimental group 3 and experimental group 6
EML4 fusions reference material is best to the preservation effect of RNA.Under identical preservation condition, RNA in 1~2 reference material of control group
Degradation rate be significantly higher than ALK-EML4 fusions reference material of the present invention.Especially under 37 DEG C of preservation conditions, in control group 2
RNA solution it is degradable after 2 weeks (degradation rate 100%), and in ALK-EML4 fusions reference material of the present invention RNA degrade
Rate (experimental group 4~6) below 30%.RNA in the ALK-EML4 fusion reference materials of the embodiment of the present invention 3 is within 2 weeks
Do not occur degradation situation, according to medical instrument accelerated aging tests determine basic principle and the correlation technique of the term of validity,
The term of validity is 6 months 3 years.The preservation of RNA in other types positive reference product of the present invention, negative reference product and detection limit reference material
Effect is similar to the present invention, and specific data are omitted.
Embodiment 7
The present embodiment is by taking the RNA quality-control products of the lung cancer fusion detection kit in embodiment 1 as an example, described in research
The accuracy of RNA quality-control products.
1st, detection method
RNA quality-control products are analyzed using high throughput targeting PCR sequencing PCR, evaluate the accuracy of the RNA quality-control products.
2nd, testing result
Testing result is as shown in table 3：
3 RNA quality-control product accuracy testing results of table
From above-mentioned experimental result, the fusion in positive reference product, negative reference product and detection limit reference material is equal
It can accurately detect, and fusion type complies fully with.It is 100% to illustrate reference material accuracy rate provided by the invention.To implementing
The testing result of RNA quality-control products is same as Example 1 in example 2~4, and accuracy rate is 100%, and detailed data is omitted.
Embodiment 8
The present embodiment is by taking the RNA quality-control products of the lung cancer fusion detection kit in embodiment 1 as an example, described in research
The stability of RNA quality-control products.
1st, detection method
It is analyzed using the RNA quality-control products of three set different production batch of the high-throughput targeting PCR sequencing PCR to randomly selecting.
2nd, testing result
Testing result is as shown in table 4：
The RNA quality-control product Detection of Stability results of 4 three sets of different batches of table
From the above results, the testing result for the three sets of RNA quality-control products randomly selected is accurate and fusion type is complete
It is complete consistent, illustrate the testing result indifference between three sets of RNA quality-control products, and testing result is completely correct.Due to the three of detection
Set RNA quality-control products are randomly selected, and can be deduced that the testing result between other RNA quality-control products is also consistent, be illustrated this hair
The RNA quality-control product performances of bright offer are very stable.And three sets of repeated reference material precision meet the coefficient of variation (CV, %) and are not more than
15.0.Same as Example 1 to the testing result of RNA quality-control products in embodiment 2~4, detailed data is omitted.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, those of ordinary skill in the art should
Understand, technical scheme can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention
And scope.
Claims (10)
- A kind of 1. RNA quality-control products for the lung cancer fusion detection kit for stablizing preservation, which is characterized in that the RNA matter Control product include following components：Positive reference product, negative reference product and detection limit reference material；The positive reference product include following components：The lung cancer fusion RNA, aurin front three acid ammonium salt, preservative and benzene Fourth inhibin hydrochloride；The negative reference product include following components：The non-lung cancer fusion RNA, aurin front three acid ammonium salt, preservative and Bestatin hydrochloride；The detection limit reference material includes following components：The lung cancer fusion RNA, the non-lung cancer fusion RNA, gold Smart front three acid ammonium salt, preservative and bestatin hydrochloride, and the lung cancer fusion RNA accounts for the mass ratio of total serum IgE as 1 ~5%；The preservative is selected from glycine, epsilon-polylysine or cysteine.
- 2. the RNA quality-control products of the lung cancer fusion detection kit according to claim 1 for stablizing preservation, feature It is, in the positive reference product, negative reference product and detection limit reference material, aurin front three acid ammonium salt, glycine and the suppression of benzene fourth The molar concentration of the plain hydrochloride of system is respectively：100~500 μM of aurin front three acid ammonium salt, 80~180mM of glycine and benzene fourth inhibit Plain 20~60nM of hydrochloride.
- 3. the RNA quality-control products of the lung cancer fusion detection kit according to claim 1 for stablizing preservation, feature It is, in the positive reference product, negative reference product and detection limit reference material, aurin front three acid ammonium salt concentration is 100~500 μ M, epsilon-polylysine concentration is 2~4mg/ml, bestatin hydrochloride concentration is 20~60nM.
- 4. the RNA quality-control products of the lung cancer fusion detection kit according to claim 1 for stablizing preservation, feature Be, the positive reference product, negative reference product and detection limit reference material in aurin front three acid ammonium salt concentration for 100~500 μM, Semicystinol concentration is 5~10mM, bestatin hydrochloride concentration is 20~60nM.
- 5. the RNA Quality Controls of the lung cancer fusion detection kit of preservation can be stablized according to Claims 1 to 4 any one of them Product, which is characterized in that the positive reference product are selected from ALK fusion gene reference material, ROS1 fusions reference material and RET fusions At least one of gene reference product；The ALK fusion gene reference material includes following components：The ALK fusion gene RNA, aurin front three acid ammonium salt, anti-corrosion Agent and bestatin hydrochloride；The ROS1 fusions reference material includes following components：The ROS1 fusions RNA, Aurin front three acid ammonium salt, preservative and bestatin hydrochloride；The RET fusions reference material includes following components：Institute State RET fusions RNA, aurin front three acid ammonium salt, preservative and bestatin hydrochloride.
- 6. the RNA Quality Controls of the lung cancer fusion detection kit of preservation can be stablized according to Claims 1 to 4 any one of them Product, which is characterized in that the negative reference product are selected from BCR-ABL fusions reference material, AML1-ETO fusion reference materials At least one of with CBF β-MYH11 fusion reference materials；The BCR-ABL fusions reference material includes following components：BCR-ABL fusions RNA, aurin front three acid ammonium salt, Preservative and bestatin hydrochloride；The AML1-ETO fusions reference material includes following components：AML1-ETO is merged Gene RNA, aurin front three acid ammonium salt, preservative and bestatin hydrochloride；CBF β-MYH11 fusion the reference materials Include following components：AML1-ETO fusions RNA, aurin front three acid ammonium salt, preservative and bestatin hydrochloride.
- 7. the RNA quality-control products of the lung cancer fusion detection kit according to claim 5 for stablizing preservation, feature It is, ALK fusion gene reference material is selected from ALK-EML4, ALK-KIF5B, ALK-KLC1, ALK- in the positive reference product TFG, ALK-TPR, ALK-HIP1, ALK-STRN, ALK-DCTN1, ALK-SQSTM1, ALK-BIRC6 and ALK-CLTC merge base Because of at least one of reference material；The ROS1 fusions reference material be selected from ROS1-CD74, ROS1-SLC34A2, ROS1-GOPC, ROS1-SDC4, ROS1-TPM3, ROS1-EZR, ROS1-LRIG3, ROS1-KDELR2, ROS1-LIMA1, ROS1-MSN and ROS1-TMEM106B At least one of fusion reference material；The RET fusions reference material is selected from KIF5B-RET and RET-CCDC6 fusion reference materials.
- 8. the RNA quality-control products of the lung cancer fusion detection kit according to claim 7 for stablizing preservation, feature It is, ALK fusion gene reference material is selected from ALK-EML4, ALK-KIF5B and ALK-TFG in the positive reference product；The ROS1 fusions reference material is selected from：ROS1-SLC34A2, ROS1-CD74, ROS1-EZR and ROS1-SDC4；The RET fusions reference material is selected from：RET-CCDC6.
- 9. the RNA Quality Controls of the lung cancer fusion detection kit of preservation can be stablized according to Claims 1 to 4 any one of them The preparation method of product, which is characterized in that comprise the following steps：(1) preparation of positive reference product：1. the lung cancer fusion is obtained by PCR amplification；2. the lung cancer is merged into base Cause and carrier carry out digestion, connection, and transformed competence colibacillus cell screens positive bacterium colony of the acquisition containing the lung cancer fusion and simultaneously surveys Sequence is verified；3. the correct bacterium colony progress of sequencing result is supersonic induced, prepare pseudovirus solution；4. extract pseudovirus solution RNA, add in aurin front three acid ammonium salt, preservative and bestatin hydrochloride to get；(2) preparation of negative reference product：1. the non-lung cancer fusion is obtained by PCR amplification；2. the non-lung cancer is melted It closes gene and carrier carries out digestion, connection, transformed competence colibacillus cell, positive bacteria of the screening acquisition containing the non-lung cancer fusion Fall simultaneously sequence verification；The correct bacterium colony progress of sequencing result is supersonic induced, prepare pseudovirus solution；4. extract pseudovirus solution RNA, add in aurin front three acid ammonium salt, preservative and bestatin hydrochloride to get；(3) preparation of detection limit reference material：1. the lung cancer fusion RNA is prepared by step (1) the method；2. it presses The non-lung cancer fusion RNA is prepared in step (2) the method；3. by the lung cancer fusion RNA and non-described Lung cancer fusion RNA is mixed so that the mass ratio that the lung cancer fusion RNA accounts for total serum IgE is 1~5%, adds in aurin three Formic acid ammonium salt, preservative and bestatin hydrochloride to get.
- 10. the RNA matter of the lung cancer fusion detection kit of preservation can be stablized according to Claims 1 to 4 any one of them Purposes of the control product in lung cancer fusion detection kit is prepared.
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