CN112458155A

CN112458155A - Polyploid detection method and system based on time-of-flight mass spectrum

Info

Publication number: CN112458155A
Application number: CN202011374801.6A
Authority: CN
Inventors: 蒋馥蔓
Original assignee: Shenzhen Jingke Gene Technology Co ltd; South China University of Technology SCUT
Current assignee: Shenzhen Jingke Gene Technology Co ltd; South China University of Technology SCUT
Priority date: 2020-11-30
Filing date: 2020-11-30
Publication date: 2021-03-09
Anticipated expiration: 2040-11-30
Also published as: CN112458155B

Abstract

The invention discloses a method and a system for detecting polyploidy based on a time-of-flight mass spectrum, wherein the risk of sample polyploidy is calculated based on the ratio value of the maximum base peak height value and the second maximum base peak height value of a heterozygous locus by selecting SNP loci with MAF of 0.3-0.7 on a chromosome and determining the corresponding peak height value by using the time-of-flight mass spectrum. Some embodiments of the invention have the advantages of simple and convenient operation, rapidness, high flux, lower detection cost and good accuracy. In some embodiments of the invention, no additional reference is needed, and the detection result can be determined only by a single sample, so that the defect that the reference sample is difficult to obtain is overcome.

Description

Polyploid detection method and system based on time-of-flight mass spectrum

Technical Field

The invention relates to the field of biological detection, in particular to polyploid detection and a polyploid detection system, and specifically relates to a polyploid detection method and a polyploid detection system based on a time-of-flight mass spectrum.

Background

Diploid refers to an individual organism having two chromosome sets in a somatic cell, and polyploid refers to an individual having three or more chromosome sets in a somatic cell. The normal person is diploid, polyploidy refers to that the content of one or more chromosomes is increased, and the chromosome has 3 times or more of genetic material, and the excessive genetic material can cause abnormal and disordered functions of the body, genetic abnormality and even no survival. Clinically, the abortion, repeated abortion and the like with unknown reasons are possibly caused by the reasons, so that the polyploidy detection is recommended for patients with related family history besides the conventional reasons.

Currently, methods conventionally used clinically for detecting polyploids are karyotype detection and specific STR method as gold standard and comparative genomic hybridization (aCGH) method. Karyotyping is a gold standard, but it relies on cell culture and microscopic observation, rather than direct detection of DNA changes based on DNA material, and there are cases of cell culture failure and time consuming comparisons. The specific STR method is a DNA sequence formed by using 2-6 base pairs as core units and repeatedly connecting in series, and the change of the repeated units and the repeated times of the series connection forms the genetic polymorphism of the STR locus. The STR method is to design a fluorescent primer to amplify a specific STR sequence, then carry out capillary electrophoresis, express the amplified quantity of products through the fluorescent peak area, and calculate the dosage change of alleles to diagnose the abnormal number of chromosomes. STR methods involve a limited number of STR sites and are costly, which is extremely difficult to reduce due to the need for fluorescent primers and flux limitations. The aCGH method is characterized in that different fluorescein is marked on a sample to be detected and a control sample, and then co-hybridization is carried out on a chip to detect the DNA copy number change of a genome of the sample to be detected relative to the genome of the control sample, so that the deletion or amplification of the genome DNA in the whole chromosome group can be visually shown. The aCGH method has the advantages of complex detection steps, low flux and higher cost.

The detection principle of the flight time mass spectrum is that laser with certain intensity is used for irradiating a cocrystallized film formed by a sample and a matrix, the matrix absorbs energy from the laser, the sample is desorbed, charge transfer occurs between the matrix and the sample to ionize sample molecules, the ionized sample is accelerated to fly through a flight pipeline under the action of an electric field, the detection is distinguished according to different flight times reaching a detector (the mass-to-charge ratio of the ions is in direct proportion to the flight time), and the relative molecular mass of the sample molecules can be obtained. Based on this principle, a gene detection method based on time-of-flight mass spectrometry has been developed, in which a gene to be detected is amplified by using a specific primer, and then the gene is determined by a difference in molecular weight. There is no literature showing that time-of-flight mass spectrometry can be used for polyploid detection.

The development of a low-cost and quick multi-time detection method and system has very practical significance.

Disclosure of Invention

The invention aims to overcome at least one defect of the prior art and provides a polyploid detection method and system based on time-of-flight mass spectrometry.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided:

a method for polyploid detection for non-diagnostic purposes comprising:

1) selecting SNP loci with the minimum allele frequency MAF of 0.3-0.7, preferably 0.4-0.6, and more preferably about 0.5 on a chromosome, and designing a primer group for amplifying conserved regions of upstream and downstream sequences of the SNP loci;

2) amplifying a sample conserved region sequence by using the primer group, further processing an amplification product, then performing spotting on a computer, and performing time-of-flight mass spectrometry;

3) acquiring a corresponding peak height value read by a mass spectrometer at each SNP site of a sample, wherein each SNP site has a corresponding peak height value of 4 basic groups;

4) determining the total number N of sample genome heterozygous sites: the selection standard of the heterozygous locus is that 2 peak heights in 4 base peak heights are higher than 1, the other 2 base peak heights are smaller than 1 and close to 0, and N is not lower than 5;

5) calculating the ratio value of the maximum base peak height value and the second maximum base peak height value of each heterozygous site for each heterozygous site selected by the sample, and counting the number n of heterozygous sites in the sample, wherein the ratio value of heterozygous sites is more than 1.5;

6) calculating a Ratio value of the sample, wherein the Ratio value is N/N, and determining the polyploidy risk of the sample according to the Ratio value, wherein:

the Ratio value is more than or equal to 0.8, and the triploid is judged to have high risk;

when the Ratio value is 0.4-0.6, determining that the risk is quadruple high;

judging the diploid when the Ratio value is less than or equal to 0.3;

and (3) the Ratio value is more than 0.3 and less than 0.4, or the Ratio value is more than 0.6 and less than 0.8, and the triploid/tetraploid chimera is judged.

The biallelic SNP locus with the MAF of 0.3-0.7, preferably 0.4-0.6, and more preferably about 0.5 is used, so that the heterozygous locus of the SNP locus can be ensured to have a higher probability in the sample, and the subsequent further determination of the polyploidy risk of the sample is facilitated.

The larger the number of SNPs is, the more beneficial the total number N of the genomic heterozygous sites in the sample is to be ensured to be not less than 5. Preferably, the number of the SNPs is selected so that the theoretical probability that the total number N of the genomic heterozygous sites is lower than 5 is lower than 0.001, so that the probability that the total number N of the genomic heterozygous sites in the sample is lower than 5 can be almost ignored, and the defect that the total number N of the genomic heterozygous sites is lower than 5 to supplement experiments is avoided.

In order to detect polyploidy conditions of all chromosomes more comprehensively, the number of SNP sites is determined according to the difference of samples to be detected and the purpose to be detected. In order to comprehensively and accurately detect the multiple cases of all chromosomes, aiming at an animal sample with sex chromosomes, the number of the selected SNP sites is not less than (the logarithm of the autosomes +2 sex chromosomes), and at least one SNP site is ensured to be selected on each chromosome. Aiming at a plant sample without sex chromosomes, the number of the selected SNP sites is not lower than the logarithm of the autosomes. If only polyploidy of a specific chromosome needs to be detected, corresponding SNP sites can be selected from the chromosome only.

In some examples, the sample to be tested is from human, and in order to comprehensively detect polyploidy of each chromosome, the total number of SNPs selected from each chromosome is not less than 24, namely, at least one SNP site of each of 22 autosomes + X chromosome + Y chromosome. Based on clinical experience, if it is determined that 4 chromosomes are polyploid, the whole body can be basically judged to be polyploid, and the selected SNP sites are preferably dispersed on more than 4 chromosomes in view of reducing the detection amount.

In some examples, the conserved regions are located within 200bp each upstream and downstream of the SNP site. In particular, the conserved regions should not have repeating regions.

SNP sites are not particularly required. In some examples, the includes rs9934438, rs1042713, rs9934438, rs11881222, rs1801280, rs72554664, rs776746, rs1065852, rs4244285, rs1799930, rs16947, rs12979860, rs9923231, rs1799931, rs8099917, rs1799931, rs1041983, rs1801280, rs20417, rs1135840, rs4244285, rs4149056, rs1057910, rs 1208.

In a second aspect of the present invention, there is provided:

a polyploid detection system comprising:

gene amplification apparatus: a conserved region for amplifying sequences upstream and downstream of the SNP locus with the minimum allele frequency MAF of 0.3-0.7, preferably 0.4-0.6, on the chromosome;

time-of-flight mass spectrometry detection device: processing the amplified product and carrying out flight time mass spectrum detection to determine the corresponding peak height value of the SNP site, wherein each SNP site has 4 corresponding base peak height values;

a data analysis device:

determining the total number N of sample genome heterozygous sites: the selection standard of the heterozygous locus is that 2 peak heights in 4 base peak heights are higher than 1, the other 2 base peak heights are smaller than 1 and close to 0, and N is not lower than 5;

calculating the ratio value of the maximum base peak height value and the second maximum base peak height value of each heterozygous site for each heterozygous site selected by the sample, and counting the number n of heterozygous sites in the sample, wherein the ratio value of heterozygous sites is more than 1.5;

calculating a Ratio value of the sample, wherein the Ratio value is N/N, and determining the polyploidy risk of the sample according to the Ratio value, wherein:

when the Ratio value is 0.4-0.6, determining that the risk is quadruple high;

judging the diploid when the Ratio value is less than or equal to 0.3;

the Ratio value is more than 0.3 and less than 0.4, or the Ratio value is more than 0.6 and less than 0.8, and the triploid/tetraploid chimera is judged;

a result output device: used for outputting the result obtained by the data analysis device.

In some examples, the conserved regions are located within 200bp each upstream and downstream of the SNP site.

In some examples, the SNP site includes rs9934438, rs1042713, rs9934438, rs11881222, rs1801280, rs72554664, rs776746, rs1065852, rs4244285, rs1799930, rs16947, rs12979860, rs9923231, rs1799931, rs8099917, rs1799931, rs1041983, rs1801280, rs20417, rs1135840, rs4244285, rs4149056, rs1057910, rs 1208.

In some examples, the amplification reaction system of the gene amplification device is as follows:

taking 0.5 mu M of forward and reverse primers of each SNP locus to prepare a multiplex PCR primer mixture,

in some examples, the amplification reaction conditions of the gene amplification device are as follows:

in some examples, the amplification products are subjected to an SAP reaction and desalting prior to time-of-flight mass spectrometry detection.

In some examples, the SAP reaction conditions include:

SAP reaction system proportion:

SAP reaction procedure:

extension reaction system:

extension reaction procedure:

the invention has the beneficial effects that:

some embodiments of the invention have the advantages of simple and convenient operation, rapidness, high flux, lower detection cost and good accuracy.

In some embodiments of the invention, no additional reference is needed, and the detection result can be determined only by a single sample, so that the defect that the reference sample is difficult to obtain is overcome.

Detailed Description

Part of the technical principles of the invention are as follows:

taking the triploid test as an example, if a sample is triploid, and if the composition of a normal diploid heterozygous site is AT as seen from a genomic heterozygous site, then if it is triploid, the T peak height will be about 2 times the a peak height assuming the composition is ATT; assuming the composition is AAT, the peak height of a will be twice that of T. Regardless of the composition, and without regard to the particular base combination, we can measure whether this site is triploid by dividing the maximum peak height by the next largest peak height. If there are enough points to justify it as a triploid, then this sample is represented as a triploid. For normal diploids, the maximum peak height is substantially equal to 1 over the minimum peak height.

The technical scheme of the invention is further explained by combining the examples. It should be noted that the implementation is only for illustrating the technical solution, and is not to be considered as limiting the technical solution itself.

1. Selection site

24 points with a minimum allele frequency MAF of around 0.5 were selected from chromosomes 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 22, X.

2. Design of primers

And selecting 200bp sequences of upstream and downstream by taking the selection site as a center to design the primer.

3. Selecting a sample

7 clinical samples were selected for the experiment (experimental procedure was according to the flight mass spectrometer procedure).

PCR amplification

1) 0.5. mu.M of each primer was used to prepare a PCR primer mixture containing forward and reverse primers for each SNP site in a multiplex PCR reaction. The PCR mix was prepared using a new 2ml centrifuge tube according to the following table, placed at 4 ℃ and the DNA sample was carefully not added to the PCR mix.

2) The DNA samples were diluted to 10 ng/. mu.l, 1. mu.l was added to the 384 well plates, and then 4. mu.l of PCR suspension was added to each well containing the sample, after which the plates were sealed with a membrane, vortexed and centrifuged.

Put on a PCR instrument to perform the following procedures:

SAP reaction

SAP Mixed solution preparation was performed using 1 new 1.5ml centrifuge tube according to the following table

Add 2. mu.l of the SAP mixture prepared in this step to each well of the sample plate obtained in the previous step, seal the plate with a membrane after completion, vortex, shake, and centrifuge instantaneously.

Put on a PCR instrument to perform the following procedures:

6. extension reaction

The extension primer mixture was prepared in advance, and 1 new 1.5ml centrifuge tube was used to prepare the extension mixture according to the following table.

And adding 2 mu l of the iPLEX mixed solution prepared in the step into each hole of the sample plate obtained in the previous step, sealing the plate by using a membrane after the completion, and performing vortex oscillation and instantaneous centrifugation.

Put on a PCR instrument to perform the following procedures:

7. desalination treatment

Clean Resin (Resin) was spread flat on 384/6mg of sample plate and air dried for a minimum of 10 minutes. Then, 16ul of water was added to each well of the sample plate having the sample. After completion, the plates were sealed with a membrane, vortexed and centrifuged. Then, 6mg of clean Resin (Resin): the sample plate is gently inverted in the air, placed on the sample plate with the resin placed, and then the sample plate is inverted with the sample plate to allow the resin to fall into the wells. The plates were sealed with a membrane and placed on a rotator and shaken upside down for 15 minutes. Plates were centrifuged for 5 minutes at 3200g (2000 rpm of standard plate centrifuge).

8. Spotting is carried out

A volume test was performed with a spotting machine to find out the appropriate spotting speed. However, the spotting speed of the 3-spot standard should be as low as 90 mm/sec. Volume testing was performed with authentic samples on the plate, followed by MassARRAY^TMThe Nanodispenser RS1000/Fusio (RS1000/Fusio spotting instrument) or a self-contained spotting instrument spots the sample onto a SpectroCHIP (chip).

MALDI-TOF MS matrix-assisted laser desorption ionization-time of flight mass spectrometry

The chip was placed in MassARRAY type workbench MA4 or Compact for mass spectrometry, and the parameters of FlexControl and SpectroAcquire were set to iPLEX XCold (iPLEX. par).

10. Data analysis

1) After the mass spectrum detection is finished, extracting partial data sequenced by the mass spectrum, and the method comprises the following steps:

the first column is the sample ID value, the second column is the base case of the heterozygous site, the third column is the rs number of the corresponding site, the fourth column is the well number, the fifth column is the base peak height, the sixth column is the base peak height with the shortest flight time, the seventh column is the base peak height with the second shortest flight time, the eighth column is the base peak height with the third shortest flight time, and the ninth column is the base peak height with the fourth flight time.

2) The SNP site information and sequencing results of the samples are as follows

The ratio of the maximum peak height divided by the second largest value, and the position of the chromosome corresponding to the matching rs number, need to be calculated: the data for 7 samples are as follows:

3) result determination and consistency determination

And judging the chromosome ploidy of the sample to be detected according to the method threshold value and comparing the results of clinical chromosome karyotypes to determine whether the results judged by the technical method are correct.

Sample numbering	n:	N：	Ratio value	Risk prediction	Whether the result is correct or not
						01	2	7	0.29	Diploid body	Correction of
02	11	13	0.85	Triploid	Correction of
						03	5	9	0.56	Tetraploid	Correction of
04	1	5	0.20	Diploid body	Correction of
						05	1	9	0.11	Diploid body	Correction of
06	6	10	0.60	Tetraploid	Correction of
						07	1	8	0.13	Diploid body	Correction of

In the table, n is the number of heterozygous sites with a ratio value of 1.5 or more; n is the total number of heterozygous sites; the Ratio value is N/N.

And (4) conclusion: the prediction results of the 7 samples to be detected are correct and accord with the clinical detection results.

The experimental data show that some examples of the invention have simple and convenient operation, high speed, high flux and lower detection cost; the detection result can be determined only by a single sample, so that the defect that a reference sample is difficult to obtain is avoided; meanwhile, the method has good accuracy.

Claims

1. A method for polyploid detection for non-diagnostic purposes comprising:

selecting SNP loci with the minimum allele frequency MAF of 0.3-0.7, preferably 0.4-0.6 on a chromosome, and designing a primer group for amplifying conserved regions of upstream and downstream sequences of the SNP loci;

amplifying a sample conserved region sequence by using the primer group, further processing an amplification product, then performing spotting on a computer, and performing time-of-flight mass spectrometry;

acquiring a corresponding peak height value read by a mass spectrometer at each SNP site of a sample, wherein each SNP site has a corresponding peak height value of 4 basic groups;

when the Ratio value is 0.4-0.6, determining that the risk is quadruple high;

judging the diploid when the Ratio value is less than or equal to 0.3;

2. The detection method according to claim 1, characterized in that: the number of the selected SNPs is not less than 24.

3. The detection method according to claim 1 or 2, characterized in that: the conserved regions are located within 200bp of each upstream and downstream of the SNP locus.

4. A polyploid detection system comprising:

a data analysis device:

when the Ratio value is 0.4-0.6, determining that the risk is quadruple high;

judging the diploid when the Ratio value is less than or equal to 0.3;

5. The system of claim 4, wherein: the conserved regions are located within 200bp of each upstream and downstream of the SNP locus.

6. The system of claim 4, wherein: the SNP sites comprise rs9934438, rs1042713, rs9934438, rs11881222, rs1801280, rs72554664, rs776746, rs1065852, rs4244285, rs1799930, rs16947, rs12979860, rs9923231, rs1799931, rs8099917, rs1799931, rs1041983, rs1801280, rs20417, rs1135840, rs4244285, rs4149056, rs1057910 and rs 1208.

7. The system of claim 4, wherein: the amplification reaction system of the gene amplification device comprises the following components in percentage by weight: taking 0.5 mu M of forward and reverse primers of each SNP locus to prepare a multiplex PCR primer mixture,

8. the system according to claim 4 or 7, characterized in that: the amplification reaction conditions of the gene amplification device are as follows:

。

9. the system of claim 4, wherein: before the detection of the time-of-flight mass spectrum, SAP reaction and desalting treatment are carried out on the amplification product.

10. The system of claim 9, wherein: the SAP reaction conditions include:

SAP reaction system proportion:

SAP reaction procedure:

extension reaction system:

extension reaction procedure:

。