A kind of detection method for the chimeric rate being based only upon chimera
阅读说明：本技术 一种仅基于嵌合体的嵌合率的检测方法 (A kind of detection method for the chimeric rate being based only upon chimera ) 是由 彭海 于 2019-07-19 设计创作，主要内容包括：本发明涉及一种仅基于嵌合体的嵌合率的检测方法,属于生物检测领域。所述检测方法包括：利用引物组通过多重PCR扩增嵌合体的基因组DNA的检测位点,获得多重PCR扩增产物；对所述多重PCR扩增产物进行高通量测序,获得测序片段；根据所述测序片段获得所述检测位点的真实多核苷酸多态性的等位基因型；利用所述真实多核苷酸多态性的等位基因型判定所述嵌合体是否被污染；若所述嵌合体未被污染,则计算所述嵌合体的嵌合率。当嵌合体样本中存在任何情况的污染类型时,嵌合体的多核苷酸多态性的等位基因型的数目可能超过没有污染的最大值,即4个,从而判定其存在污染,实现污染的全面质量控制。(The present invention relates to a kind of detection methods of chimeric rate for being based only upon chimera, belong to field of biological detection.The detection method includes: the detection site using primer sets by the genomic DNA of multiplexed PCR amplification chimera, obtains multiplex PCR amplification product；High-flux sequence is carried out to the multiplex PCR amplification product, obtains sequencing fragment；The allelotype of the true polynucleotides polymorphism of the detection site is obtained according to the sequencing fragment；Determine whether the chimera is contaminated using the allelotype of the true polynucleotides polymorphism；If the chimera is not contaminated, the chimeric rate of the chimera is calculated.When, there are when the pollution type of any situation, the number of the allelotype of the polynucleotides polymorphism of chimera can exceed that the maximum value not polluted, i.e., 4 in chimera sample, therefore, it is determined that it realizes the overall quality control of pollution in the presence of pollution.)
1. a kind of detection method for the chimeric rate for being based only upon chimera, which is characterized in that the detection method includes:
Using primer sets by the detection site of the genomic DNA of multiplexed PCR amplification chimera, multiplex PCR amplification product is obtained, The primer sets include forward primer and reverse primer；
High-flux sequence is carried out to the multiplex PCR amplification product, obtains sequencing fragment；
The allelotype of the true polynucleotides polymorphism of the detection site is obtained according to the sequencing fragment, it is described true The determination method of the allelotype of polynucleotides polymorphism are as follows:
The sequencing fragment is compared onto reference genome, the allelotype of potential polynucleotides polymorphism is obtained, it is described The allelotype of potential polynucleotides polymorphism be the sequencing fragment on from the base sequence different with reference to genome Combination,
It calculatesWherein, niFor the equipotential base for determining i-th of potential polynucleotides polymorphism Because type is true decision threshold, k is coefficient of determination, and k >=1, NjFor the support jth in the sequencing fragment, observed The number of the sequencing fragment of the allelotype of the kind potential polynucleotides polymorphism, e are detection error rate, mijIt is i-th kind The different base number between the allelotype of potential polynucleotides polymorphism described in jth kind,
When the number of the sequencing fragment of the allelotype of i-th kind of potential polynucleotides polymorphism of the support observed Ni≥niWhen, determine that the allelotype of potential polynucleotides polymorphism described in jth kind is true, and by the potential multicore Allelotype of the allelotype of nucleotide polymorphism as the true polynucleotides polymorphism；
Determine whether the chimera is contaminated using the allelotype of the true polynucleotides polymorphism；
If the chimera is not contaminated, the chimeric rate of the chimera is calculated.
2. detection method according to claim 1, which is characterized in that the detection site includes multiple mononucleotide polymorphics The base position of property.
3. detection method according to claim 2, which is characterized in that the sequence of the primer sets is respectively as in sequence table Shown in SEQ ID NO:1~SEQ ID NO:200.
4. detection method according to claim 1, which is characterized in that the detection site is as follows:
* starting point and * terminal refer to the base positions on the reference genome for the people that version number is hg19.
5. detection method according to claim 1, which is characterized in that the allele of the true polynucleotides polymorphism The allelotype of type and the potential polynucleotides polymorphism is the group of the allelotype of multiple single nucleotide polymorphism It closes.
6. detection method according to claim 1, which is characterized in that k=10.
7. detection method according to claim 1, which is characterized in that the whether contaminated method of judgement chimera Are as follows:
The quantity of the detection site of the quantity greater than 4 of the allelotype of the true polynucleotides polymorphism is counted, And it is denoted as M；
As M >=1, then determine that the chimera is contaminated；
As M=0, then determine that the chimera is not contaminated.
8. detection method according to claim 7, which is characterized in that the method for the chimeric rate for calculating the chimera Are as follows:
It, will be in the detection site in the chimera, supporting i-th of detection when determining that the chimera is not contaminated The descending arrangement of number of the sequencing fragment of the allelotype of the true polynucleotides polymorphism, ranking front three The number of the sequencing fragment be denoted as N respectivelyi1、Ni2And Ni3If only have in i-th of detection site 1 it is described true more The allelotype of nucleotide polymorphisms, then provide Ni2=Ni3=0, if only have in i-th of detection site 2 it is described true The allelotype of real polynucleotides polymorphism, then provide Ni3=0；
The ratio r of the support sequencing fragment of the receptor allele type of i-th of detection site is calculated as followsi:
WhenWhen, thenWherein, NiFor the number summation of the sequencing fragment of i-th of detection site；
WhenWhen, then
Be denoted as it is all be 0 riValue median be r；
R value is substituted into equation of linear regression, obtains the chimeric rate R of the chimera.
9. detection method according to claim 8, which is characterized in that the method for obtaining the equation of linear regression are as follows:
The sample for taking chimera known to chimeric rate, as training sample, the chimeric rate of the training sample is R '；
Calculate the r value of the training sample；
Using r value as independent variable, R ' value is dependent variable, calculates and obtains the equation of linear regression.
10. detection method according to claim 9, which is characterized in that the equation of linear regression is R=2.338r- 0.0167。
Technical field
The present invention relates to field of biological detection, the in particular to detection method of chimera.
Background technique
In the experiment of the bone-marrow transplantation of research animal and people, the chimeric rate of chimera is whether instruction donor bone marrow plays function The important reference indicator of energy.In recent years, the detection method of the chimeric rate of chimera includes red cell antigens method, karyotype Method, human leucocyte antigen typing's method and short tandem repeat (Short tandem repeat, STR) method.Wherein, STR method It is most extensive with obtaining, but since STR method is in PCR (Polymerase Chain Reaction, polymerase chain reaction) amplification procedure In, phenomena such as there are slippages, the sensitivity that this will lead to detection is very low.
A kind of existing method for detecting chimera, this method is using primer sets to the genome of donor, receptor and chimera DNA carries out multiplexed PCR amplification, obtains amplified production, amplified production is detected by high-flux sequence, and parting acquisition is more A (25~96) SNP site, then the chimeric rate of each SNP site is calculated separately, take the flat of the chimeric rate of all SNP sites Chimeric rate of the mean value as sample to be tested (chimera).Above-mentioned primer sets include Tag primer, pass through label in sequencing result It can recognize the cross contamination situation of sample to be tested, to improve the sensitivity of detection.
The method of the existing detection chimera when detecting, needs to use between the adjacent sample to be tested in same batch Primer sets with different Tag primers carry out multiplexed PCR amplification, for avoiding between adjacent sample to be tested in PCR reaction reagent There is a situation where cross contaminations in process for preparation and during PCR amplification.In addition to this, the pollution for influencing testing result is also wrapped The pollution between non-adjacent sample to be tested, the pollution before the preparation of PCR reaction reagent and the Aerosol Pollution in air are included, this is What above-mentioned detection method was not avoided that, it thus will lead to testing result inaccuracy.
Summary of the invention
In order to solve problems in the prior art, the embodiment of the invention provides a kind of inspections of chimeric rate for being based only upon chimera Survey method.The technical solution is as follows:
The embodiment of the invention provides a kind of detection method of chimeric rate for being based only upon chimera, the detection method packets It includes:
Using primer sets by the detection site of the genomic DNA of multiplexed PCR amplification chimera, multiplexed PCR amplification is obtained Product, the primer sets include forward primer and reverse primer；
High-flux sequence is carried out to the multiplex PCR amplification product, obtains sequencing fragment；
The allelotype of the true polynucleotides polymorphism of the detection site is obtained according to the sequencing fragment, it is described The determination method of the allelotype of true polynucleotides polymorphism are as follows:
The sequencing fragment is compared onto reference genome, the allelotype of potential polynucleotides polymorphism is obtained, The allelotype of the potential polynucleotides polymorphism be the sequencing fragment on from the base different with reference to genome The combination of sequence,
It calculatesWherein, niTo determine i-th of potential polynucleotides polymorphism Allelotype is true decision threshold, and k is coefficient of determination, and k >=1, NjFor the branch in the sequencing fragment, observed The number of the sequencing fragment of the allelotype of potential polynucleotides polymorphism described in jth kind is held, e is detection error rate, mijFor Different base number between the allelotype of potential polynucleotides polymorphism described in i-th kind and jth kind,
When the sequencing fragment of the allelotype of i-th kind of potential polynucleotides polymorphism of the support observed Number Ni≥niWhen, determine that the allelotype of potential polynucleotides polymorphism described in jth kind is true, and will be described potential Allelotype of the allelotype of polynucleotides polymorphism as the true polynucleotides polymorphism；
Determine whether the chimera is contaminated using the allelotype of the true polynucleotides polymorphism；
If the chimera is not contaminated, the chimeric rate of the chimera is calculated.
Specifically, the detection site includes the base position of multiple single nucleotide polymorphism.
Specifically, the sequence of the primer sets is respectively as shown in SEQ ID NO:1~SEQ ID NO:200 in sequence table.
Specifically, the detection site is as follows:
* starting point and * terminal refer to the base positions on the reference genome for the people that version number is hg19.
Specifically, the allelotype of the true polynucleotides polymorphism and the potential polynucleotides polymorphism etc. Position genotype is the combination of the allelotype of multiple single nucleotide polymorphism.
Specifically, which is characterized in that k=10.
Specifically, the whether contaminated method of judgement chimera are as follows:
Count the number of the detection site of the quantity greater than 4 of the allelotype of the true polynucleotides polymorphism Amount, and it is denoted as M；
As M >=1, then determine that the chimera is contaminated；
As M=0, then determine that the chimera is not contaminated.
Further, the method for the chimeric rate for calculating the chimera are as follows:
When determining that the chimera is not contaminated, by the chimera, the detection site of i-th of detection is supported In the true polynucleotides polymorphism allelotype the sequencing fragment the descending arrangement of number, before ranking The number of three sequencing fragments is denoted as N respectivelyi1、Ni2And Ni3If only have in i-th of detection site 1 it is described true The allelotype of real polynucleotides polymorphism, then provide Ni2=Ni3=0, if only having 2 institutes in i-th of detection site The allelotype for stating true polynucleotides polymorphism, then provide Ni3=0；
The ratio of the support sequencing fragment of the receptor allele type of i-th of detection site is calculated as follows Example ri:
WhenWhen, thenWherein, NiFor the number summation of the sequencing fragment of i-th of detection site；
WhenWhen, then
Be denoted as it is all be 0 riValue median be r；
R value is substituted into equation of linear regression, obtains the chimeric rate R of the chimera.
Still further, the method for obtaining the equation of linear regression are as follows:
The sample for taking chimera known to chimeric rate, as training sample, the chimeric rate of the training sample is R '；
Calculate the r value of the training sample；
Using r value as independent variable, R ' value is dependent variable, calculates and obtains the equation of linear regression.
Further, the equation of linear regression is R=2.338r-0.0167.
The embodiment of the invention provides a kind of detection methods of chimeric rate for being based only upon chimera, deposit when in chimera sample In the pollution type of any situation, the number of the allelotype of the polynucleotides polymorphism of chimera can be can exceed that without dirt The maximum value of dye, i.e., 4 realize the overall quality control of pollution therefore, it is determined that it has pollution.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, embodiment of the present invention will be made into one below Step ground detailed description.The operating process or working specification for being not specified or being described in detail in the embodiment of the present invention are that common molecular is raw Operation known to object technical staff.The reagent or biomaterial being not specified in the embodiment of the present invention are available on the market Common agents or biomaterial are known to common molecular biology technical staff, and can buy on the market.