CN107529556A - A kind of method that Brc2 hypotypes in PML RAR alpha fusion genes are identified using ddPCR - Google Patents
A kind of method that Brc2 hypotypes in PML RAR alpha fusion genes are identified using ddPCR Download PDFInfo
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Abstract
The invention discloses a kind of method that Brc2 hypotypes in PML RAR alpha fusion genes are identified using ddPCR, this method includes the RNA in extraction blood and carries out QC assessments, in addition to RNA reverse transcriptions cDNA and carries out QC assessments.DdPCR of the present invention detection PML RARA fusion bcr2 hypotypes up to 0.01% abundance, the 0.1% of remote super RT PCR, reproducible, non-false positive；In low AF（0.01%）In the case of, it is proposed that the increase of sample input amount, or repeat multiple operation repetitives.Due to the high sensitivity of the technology, therefore the early stage that can be applied to disease is made a definite diagnosis, dynamic monitoring and preventing and treating are recurred.The present invention is directed to common Brc2 types, design detection fusion probes, and primer is expanded for fusion both sides sequence；Using RARA genes as internal reference, Ref probes are designed, primer only expands RARA genes.
Description
Technical field
The invention belongs to gene identification method technical field, particularly relates to a kind of using ddPCR identifications PML-
The method of Brc2 hypotypes in RAR alpha fusion genes.
Background technology
PML-RAR alpha fusion genes are by t (15:17)(q22:Q21) transposition is formed, and is common in the white blood of acute progranulocyte
Sick (APL).RAR α and PML fusion have following characteristics：RAR α gene breaks site is located at intron 2；And PML bases
Because there is three regions to take part in t (15：17) transposition, i.e. introne 6 (Brc1, account for 55%), exon 6 (brc2, accounts for
5%), 40%) introne 3 (brc3, accounts for.PML-RAR alpha fusion genes can be divided into three kinds of Asias according to the position of broken site
Type：As shown in Figure 1, elongated (L-type, Brc1), anomaly (V-type, brc2) and short (S types, brc3).The treatment of leukaemia at present
Relatively effectively, its patient is extended life cycle, but later stage follow-up finds that recurrence rate is higher in the several years, and possible cause is to adopt at present
With RT-PCR detection techniques, its detection sensitivity is only 0.1%.Limitation based on the detection technique, this project, which uses, more increases
Sensitive ddPCR methods are detected, it is intended to have directive significance to clinic, its sensitivity is up to 0.01%.
The content of the invention
The present invention is in order to overcome the shortcomings of the prior art, there is provided one kind has highly sensitive to be identified using ddPCR
The method of Brc2 hypotypes in PML-RAR alpha fusion genes.
The present invention is achieved by the following technical solutions：One kind is using in ddPCR identification PML-RAR alpha fusion genes
The method of Brc2 hypotypes, this method include the RNA in extraction blood and carry out QC assessments, and specific operation process is as follows：(1) extract
RNA：Blood plasma is divided in 1.5ml EP pipes, often pipe dispenses 300 μ l；(2) cracked using the cell carried in Tiangeng kit
Liquid CL, 750 μ l are added into blood, mixing of turning upside down, stand 1min；10000rpm centrifuges 1min；Supernatant is abandoned, stays precipitation；
700 μ l CL are added into blood, is centrifuged under equal conditions, abandons supernatant, stay precipitation；(3) 150 μ l buffer PKD are added,
Piping and druming mixes；Then 10 μ l Proteinase Ks are added, piping and druming mixes；Mix is not configured, need to individually be sequentially added；(4) sample is in constant temperature
3min on ice is immediately placed in after 56 DEG C of instrument, rotating speed 350rpm, 15min；(5) 13400rpm centrifuges 15min, transfer supernatant 160 μ l
Into 1.5ml EP pipes；(6) supernatant is placed in 80 DEG C of incubations 15min, 13400rpm on thermostat and quickly centrifuges 30s；(7) add
320 μ l buffer RLT, piping and druming mix, and add the ethanol of 1000 μ l 96~100%, are vortexed and mix；(8) 700 μ are shifted every time
L samples 13400rpm centrifugation 15s, abandon filtrate into RNeasy MinElute centrifugal columns, repeat this step until by all samples
This is crossed post and finished；(9) plus 350 μ l buffer FRN are to centrifugal column, 13400rpm centrifugation 15s, abandon filtrate；(10) mix is matched somebody with somebody:10
μ l DNaseI and 70 μ l buffer RDD, piping and druming mix, are added on the film in centrifugal column, are placed in 20~30 DEG C of temperature of room temperature
The lower 15min of degree；(11) plus 500 μ l buffer FRN are to centrifugal column, 13400rpm centrifugation 15s, retain filtrate；Change one it is new
2ml collecting pipes, filtrate is rejoined into centrifugal column, 13400rpm centrifugation 15s, abandons filtrate；(12) 500 μ l buffer are added
RPE 13400rpm centrifugation 15s, abandons filtrate, is repeated once to centrifugal column；(13) centrifugal column is placed in new 2ml collecting pipes,
Lid is opened, 13400rpm centrifugations 5min；(14) centrifugal column is placed in new 1.5ml EP pipes, adds 25 μ l RNase-
Free water close lid, stand 1min at a temperature of 15~25 DEG C of room temperature on the filter membrane in centrifugal column center；13400rpm from
Heart 1min；(15) RNA of extraction carries out reverse transcription or frozen in -80 DEG C immediately；(16) RNA QC are assessed：Use Nanodrop pairs
RNA is carried out quantitative and is referred to A260/280 data, or carries out RNA quality evaluations using labchip/2100, assesses RIN values；
This method also includes RNA reverse transcriptions cDNA and carries out QC assessments, and specific operation process is as follows：
(1) RNA reverse transcriptions cDNA, reverse transcription primer are combined with template ribonucleic acid, after PCR EPs (end of program), are positioned on ice,
No less than 1min；HELA need to only put into 1ul, i.e. 10ng as control.
(2) prepare RT reaction mixtures and carry out RT incubations：7ul mix are added into the 13ul of previous step, piping and druming is mixed
It is even, total system 20ul is reacted, reverse transcription reaction is carried out on PCR, storage extremely -20 DEG C of preservations after the completion of reaction.
(3) cDNA QC are assessed：The HELA control primer carried using kit, enter performing PCR reaction, run glue and test
Demonstrate,prove band, the performance of Quality Control reverse transcription reaction.
DdPCR systems：
DdPCR programs：
*Use a heated lid set to 105℃and set the samole volume to 40μl.
The beneficial effects of the invention are as follows：As shown in Fig. 2 the conception that the present invention detects is as follows：The present invention is for common
Brc2 types (account for 5%), and design detection fusion probes (FAM), primer is expanded for fusion both sides sequence；By RARA
Gene only expands RARA genes as internal reference, design Ref probes (HEX), primer.The present invention is using ddPCR identification PML-RAR α
Have in fusion the advantages of the method for Brc2 hypotypes：1st, ddPCR detects PML-RARA and merges bcr2 hypotypes up to 0.01%
Abundance, the 0.1% of remote super RT-PCR, reproducible, non-false positive；2nd, in the case of low AF (0.01%), it is proposed that sample is thrown
Enter amount increase, or repeat multiple operation repetitives.Due to the high sensitivity of the technology, thus the early stage that can be applied to disease make a definite diagnosis,
Dynamic monitoring and preventing and treating are recurred.
Brief description of the drawings
Fig. 1 is PML-RAR α configuration pictures of the present invention；
Fig. 2 is brc2 configuration pictures of the present invention；
Fig. 3 is cDNA of the present invention QC result figures：
Fig. 4 is ddPCR of the present invention result column diagram.
Embodiment
Below in conjunction with the drawings and specific embodiments, the invention will be further described.
A kind of method that Brc2 hypotypes in PML-RAR alpha fusion genes are identified using ddPCR, this method include extraction blood
In RNA and carry out QC assessments, specific operation process is as follows：
(1) RNA is extracted, blood plasma is divided in 1.5ml EP pipes, often pipe dispenses 300 μ l；
(2) using the cell pyrolysis liquid CL carried in Tiangeng kit, 750 μ l are added into blood, mixing of turning upside down,
Stand 1min；10000rpm centrifuges 1min；Supernatant is abandoned, stays precipitation；700 μ l CL are added into blood, under equal conditions from
The heart, supernatant is abandoned, stay precipitation；
(3) 150 μ l buffer PKD are added, piping and druming mixes；Then 10 μ l Proteinase Ks are added, piping and druming mixes；Do not configure
Mix, need to individually it sequentially add；
(4) sample is immediately placed in 3min on ice after 56 DEG C of thermostat, rotating speed 350rpm, 15min；
(5) 13400rpm centrifuges 15min, shifts in μ l to the 1.5ml EP pipes of supernatant 160；
(6) supernatant is placed in 80 DEG C of incubations 15min, 13400rpm on thermostat and quickly centrifuges 30s；
(7) 320 μ l buffer RLT are added, piping and druming mixes, and adds the ethanol of 1000 μ l 96~100%, is vortexed and mixes；
(8) 700 μ l samples of transfer into RNeasy MinElute centrifugal columns, 13400rpm centrifugation 15s, abandon filter every time
Liquid, repeat this step and finished until all samples are crossed into post；
(9) plus 350 μ l buffer FRN are to centrifugal column, 13400rpm centrifugation 15s, abandon filtrate；
(10) mix is matched somebody with somebody:10 μ l DNaseI and 70 μ l buffer RDD, piping and druming mix, the film being added into centrifugal column
On, it is placed in 15min at a temperature of 20~30 DEG C of room temperature；
(11) plus 500 μ l buffer FRN are to centrifugal column, 13400rpm centrifugation 15s, retain filtrate；Change a new 2ml
Collecting pipe, filtrate is rejoined into centrifugal column, 13400rpm centrifugation 15s, abandons filtrate；
(12) 500 μ l buffer RPE are added to centrifugal column, 13400rpm centrifugation 15s, filtrate is abandoned, is repeated once；
(13) centrifugal column is placed in new 2ml collecting pipes, lid is opened, 13400rpm centrifugations 5min；
(14) centrifugal column is placed in new 1.5ml EP pipes, adds 25 μ l RNase-free water into centrifugal column
On the filter membrane of centre, lid is closed, 1min is stood at a temperature of 15~25 DEG C of room temperature；13400rpm centrifuges 1min；
(15) RNA of extraction carries out reverse transcription or frozen in -80 DEG C immediately；
(16) RNA QC are assessed：RNA is carried out using Nanodrop quantitative and refer to A260/280 data, or used
Labchip/2100 carries out RNA quality evaluations, assesses RIN values.
This method also includes RNA reverse transcriptions cDNA and carries out QC assessments, and specific operation process is as follows：
(1) RNA reverse transcriptions cDNA, reverse transcription primer are combined with template ribonucleic acid；After PCR EPs (end of program), it is positioned on ice,
No less than 1min；HELA need to only put into 1ul, i.e. 10ng as control；
(2) prepare RT reaction mixtures and carry out RT incubations；7ul mix are added into the 13ul of previous step, piping and druming is mixed
It is even, total system 20ul is reacted, reverse transcription reaction is carried out on PCR, storage extremely -20 DEG C of preservations after the completion of reaction；
(3) cDNA QC are assessed：The HELA control primer carried using kit, enter performing PCR reaction, run glue
Verify band, the performance of Quality Control reverse transcription reaction.
Embodiment：Sample：Blood sample and NB4- simulations AF 0.01%；
Table one：RNA extracts result --- Nanodrop
| Sample names | Concentration (ng/ul) | Total amount (ug) | A260/280 | A260/230 |
| WBC | 105 | 3ug | 2.07 | 1.10 |
| NB4 | 790.2 | 15ug | 2.11 | 1.98 |
CDNA QC results are as shown in figure 3, ddPCR result is as shown in Figure 4.
First row：Without false sun detection, postive numbers are 0.Secondary series：K562 pattern detections, high abundance sample.3rd
Arrange to the 5th row：For 0.05% sample, its input amount gradually increases AF, and positive detections number also increases.6th row are to the
Eight row：For 0.01% sample, its input amount gradually increases AF, and positive detections number also increases.
Finally it should be noted that above content is merely illustrative of the technical solution of the present invention, rather than the present invention is protected
The limitation of scope, the simple modification or equivalent substitution that one of ordinary skill in the art is carried out to technical scheme,
All without departing from the spirit and scope of technical solution of the present invention.
Claims (1)
- A kind of 1. method that Brc2 hypotypes in PML-RAR alpha fusion genes are identified using ddPCR, it is characterised in that：First, this method includes the RNA in extraction blood and carries out QC assessments, and specific operation process is as follows：（1）RNA is extracted, blood plasma is divided in 1.5ml EP pipes, often pipe dispenses 300 μ l；（2）Using the cell pyrolysis liquid CL carried in Tiangeng kit, 750 μ l are added into blood, mixing of turning upside down, are stood 1min；10000rpm centrifuges 1min；Supernatant is abandoned, stays precipitation；700 μ l CL are added into blood, centrifuges, abandons under equal conditions Supernatant, stay precipitation；（3）150 μ l buffer PKD are added, piping and druming mixes；Then 10 μ l Proteinase Ks are added, piping and druming mixes；Mix is not configured, Need to individually it sequentially add；（4）Sample is immediately placed in 3min on ice after 56 DEG C of thermostat, rotating speed 350rpm, 15min；（5）13400rpm centrifuges 15min, shifts in μ l to the 1.5ml EP pipes of supernatant 160；（6）Supernatant is placed in 80 DEG C of incubations 15min, 13400rpm on thermostat and quickly centrifuges 30s；（7）320 μ l buffer RLT are added, piping and druming mixes, and adds the ethanol of 1000 μ l 96~100%, is vortexed and mixes；（8）700 μ l samples of transfer 13400rpm centrifugation 15s, are abandoned filtrate, repeated into RNeasy MinElute centrifugal columns every time This step finishes until all samples are crossed into post；（9）350 μ l buffer FRN are added 13400rpm centrifugation 15s, to abandon filtrate to centrifugal column；（10）With mix:10 μ l DNaseI and 70 μ l buffer RDD, piping and druming mix, are added on the film in centrifugal column, put The 15min at a temperature of 20~30 DEG C of room temperature；（11）500 μ l buffer FRN are added 13400 rpm centrifugation 15s, to retain filtrate to centrifugal column；A new 2ml is changed to receive Collector, filtrate is rejoined into centrifugal column, 13400rpm centrifugation 15s, abandons filtrate；（12）500 μ l buffer RPE are added to centrifugal column, 13400rpm centrifugation 15s, filtrate is abandoned, is repeated once；（13）Centrifugal column is placed in new 2ml collecting pipes, lid is opened, 13400rpm centrifugations 5min；（14）Centrifugal column is placed in new 1.5ml EP pipes, adds 25 μ l RNase-free water to centrifugal column center On filter membrane, lid is closed, 1min is stood at a temperature of 15~25 DEG C of room temperature；13400rpm centrifuges 1min；（15）The RNA of extraction carries out reverse transcription or frozen in -80 DEG C immediately；（16）RNA QC are assessed：RNA is carried out using Nanodrop quantitative and refer to A260/280 data, or used Labchip/2100 carry out RNA quality evaluations, assess RIN values；2nd, this method also includes RNA reverse transcriptions cDNA and carries out QC assessments, and specific operation process is as follows：（1）RNA reverse transcription cDNA, reverse transcription primer are combined with template ribonucleic acid：After PCR EPs (end of program), it is positioned on ice, much In 1 min；HELA need to only put into 1ul, i.e. 10ng as control；（2）Prepare RT reaction mixtures and carry out RT incubations：7ul mix are added into the 13ul of previous step, piping and druming mixes, instead Total system 20ul is answered, reverse transcription reaction is carried out on PCR, storage extremely -20 DEG C of preservations after the completion of reaction；（3）CDNA QC are assessed：The HELA control primer carried using kit, enter performing PCR reaction, run glue checking bar Band, the performance of Quality Control reverse transcription reaction.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112626208A (en) * | 2020-12-16 | 2021-04-09 | 中山大学达安基因股份有限公司 | Kit and method for quantitative detection of PML-RARA fusion gene by digital PCR |
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| CN102719540A (en) * | 2012-06-12 | 2012-10-10 | 福州艾迪康医学检验所有限公司 | Detection kit for detecting relative expression level of PML-RAR (alpha) fusion gene by use of fluorescent quantitative PCR (polymerase chain reaction) technology |
| CN105229175A (en) * | 2013-03-15 | 2016-01-06 | 雅培分子公司 | For increasing and measuring the method for RNA fusion gene variant, the method distinguishing them and relevant primer, probe and test kit |
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| CN112626208A (en) * | 2020-12-16 | 2021-04-09 | 中山大学达安基因股份有限公司 | Kit and method for quantitative detection of PML-RARA fusion gene by digital PCR |
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