Disclosure of Invention
The invention provides a primer group and a probe group for detecting the digital PCR chimerism rate and application thereof, which aim to solve at least one technical problem.
In order to solve the above problems, as an aspect of the present invention, there is provided a primer set for detecting digital PCR chimerism rate, wherein based on Indel site data, 14 Indel sites with high individual recognition rate for asian population are selected based on asian population, and a specific primer is designed for each site, including the following 14 primer pairs:
preferably, the forward and reverse primers are located at least 10bp before and after the del/indel position, respectively.
The invention also provides a detection probe set for the digital PCR chimerism rate, which is characterized in that 14 Indel sites aiming at the high individual recognition rate of Asian population are screened out based on the Indel site data and the Asian population, and a specific probe set is designed aiming at each site, wherein the detection probe set comprises the following 14 probe pairs:
preferably, the forward and reverse primers are located at least 10bp before and after the del/indel position, respectively.
The invention also provides application of the primer group for detecting the digital PCR chimerism rate and the probe group for detecting the digital PCR chimerism rate in preparing a chimerism detection reagent.
The primer group and the detection method provided by the invention and the chimerism rate result obtained by the primer group and the detection method can be applied to a patient after hematopoietic stem cell transplantation, and have the characteristics of higher coverage rate, high sensitivity and wide linear range.
Detailed Description
The following detailed description of embodiments of the invention, but the invention can be practiced in many different ways, as defined and covered by the claims.
The invention discloses a primer and probe combination based on digital PCR chimeric detection and a method for chimeric detection by using the primer and probe combination.
Based on Indel site data, 14 Indel sites with high individual recognition rate for Asian population are screened out based on the Asian population, and specific primer and probe combinations are further designed for each site. The primer group consists of 28 primers, and the probe group consists of 28 primers. The primer group and the detection method provided by the invention and the chimerism rate result obtained by the primer group and the detection method can be applied to a patient after hematopoietic stem cell transplantation, and have the characteristics of higher coverage rate, high sensitivity and wide linear range.
The primer group consists of 28 primers, the probe group consists of 28 primers, the forward and reverse primers are respectively located at least 10bp positions before and after the del/indel position, each pair of primers can stably amplify a del/indel product, the MGB probe group respectively designs a probe (+) and a probe (-) aiming at the del/indel position and can accurately react with the del/indel amplification product, the dual control of a detection site is realized by applying a method of combining the specific primers and the MGB probe, the specificity of the detection site and the accuracy of the result are ensured, the specificity is good, and the sensitivity is high.
Step 1, extracting DNA of a sample to be detected, diluting the DNA to about 40 ng/mu L by using RNase-Free ddH2O, mixing uniformly and centrifuging for later use.
And 2, the concentrations of the primer mother liquor and the probe mother liquor are both 100uM, the concentration of each primer in the working solution is 9uM, and the concentration of the probe is 9 uM. The working solution preparation scheme is as follows: 9ul and 6ul of each primer probe stock solution in each tube were taken, and finally 100 ul of M ltraPure ™ DNase/RNase-Free Distilled Water (Invitrogen, cat. No. 10977-015) was used for complementation.
Step 3, preparing a chip set of the acute digital PCR instrument, uniformly mixing reagents, centrifuging for a short time for standby, preparing 8 connecting pipes according to a digital PCR reaction table, numbering, and preparing a digital PCR reaction system according to the following table:
and 4, oscillating the 8-connection pipe for short-time centrifugation for later use.
And 5, adding 75ul of wrapping oil into corresponding hole positions in the digital chip, transferring 20ul of reaction liquid in the 8-way tube to a circular hole of the chip, and adding liquid-phase oil into the circular hole (so that the oil covers the surface of the reaction liquid and the bubbles are not added) while paying attention to the process that no bubbles are added.
And 6, covering a soft rubber cover on the machine, and finishing the sample adding in the step 10 within 10min as much as possible.
And 7, running a program on the sharp digital PCR instrument (the position of the chip needs to be correctly selected according to the actual placement condition):
step 8, off-line analysis
Opening the PCR analyzer, clicking "move out", putting the amplified chip together with the chip rack into the analyzer, clicking "move in" touch screen to select numbers 1-16, inputting the number in "plate name", selecting "mutation detection" in "experiment type", reserving FAM and HEX for the fluorescence channel of "target", clicking "add", and clicking "scan sample". At which time the instrument begins to analyze the chip results. The data were copied after the analysis was completed as follows: and inserting a USB flash disk into the USB interface, clicking 'settings' on the analysis interface, clicking 'data export', finding a file name, and clicking 'export'.
The first embodiment is as follows: design and Synthesis of primers
InDel site screening
The InDel locus is screened by adopting the following principle:
1. the length difference (the number of inserted or deleted bases) of the InDel allele fragments is more than 3bp and less than 30 bp;
2. located on 22 human autosomes;
3. the spacing between InDel sites on the same chromosome is more than 5 Mb;
4. minimum Allele Frequency (MAF) between 0.30 and 0.50 (based on the east asian population);
5. the total number of the sites is not less than 30, and the accumulated individual identification rate reaches more than 0.9999;
6. no linkage is formed on the same chromosome, and the distribution in the population conforms to Hardy-Weinberg genetic balance;
7. to avoid the InDel sites being associated with certain specific disease phenotypes, it is preferred that polymorphic sites be located in the intron regions.
By adopting the principle, primary screening is carried out on InDel sites with stronger polymorphism through websites shown by http:// www.ensemb1.org/index. html and http:// www.ncbi.nlm.nih.gov/projects/SNP, and finally 14 InDel sites are selected, wherein the sites are respectively as follows: n1-1(rs2308010), N1-2(rs34855933), N1-3(rs34855933), N2-1(rs3042783), N5-4(rs1610932), N21-1(rs16663), N9-1(rs2308112), N11-1(rs2307696), N13-1(rs3038530), N14-1(rs2067204), N16-2(rs2067204), N7-2(rs 1041618), N8-1(rs3081400) and N22-1(rs6481), wherein the number rs in brackets indicates the number of the site in dbSNP database.
EXAMPLE two primer and Probe specificity test
5 insertion homozygotes, deletion homozygotes and heterozygotes are respectively selected for carrying out digital PCR for detecting the specificity of the primer and the probe.
1. Digital PCR amplification system
A 20uL digital PCR amplification system was used: 10uL 2 × dPCR Mix reaction; luL 9uM primer; luL 6uM probe; 8uL 40ng/uL DNA.
2. Digital PCR reaction conditions
The following fluorescent PCR reaction conditions were used: pre-denaturation at 95 ℃ for 10min, then annealing and extension at 95 ℃ for 30 s-55 ℃ for 1min, and carrying out 39 cycles in total, denaturation at 98 ℃ for 10min, and extension at 20 ℃ for 2 min.
3. Opening the PCR analyzer, clicking "move out", putting the amplified chip together with the chip rack into the analyzer, clicking "move in" touch screen to select numbers 1-16, inputting the number in "plate name", selecting "mutation detection" in "experiment type", reserving FAM and HEX for the fluorescence channel of "target", clicking "add", and clicking "scan sample". At which time the instrument begins to analyze the chip results. The data were copied after the analysis was completed as follows: and inserting a USB flash disk into the USB interface, clicking 'settings' on the analysis interface, clicking 'data export', finding a file name, and clicking 'export'.
4. Specificity determination
The state of each genetic marker insertion/deletion is judged through a signal value, and if FAM, HEX, double negative and double positive groups are obvious, the specificity of the primer and the probe is good. Taking the Nl-2(+) insertion genetic marker as an example, FAM signal values are distributed between 4000-7000, HEX signal values are distributed between 3000-5000 and double negativity are below 2000, indicating that the specificity of the primer and the probe is good.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.