CN113337597A

CN113337597A - Detection kit for pregnancy vitamin A deficiency risk assessment and application method

Info

Publication number: CN113337597A
Application number: CN202110748964.4A
Authority: CN
Inventors: 叶辉铭; 李娟�; 苏志英; 吴琦嫦; 曹佳莉; 邹悦
Original assignee: Xiamen Maternity & Child Healthcare Hospital (xiamen City Family Planning Service Center)
Current assignee: Xiamen Maternal And Child Health Hospital Xiamen Eugenics Service Center Xiamen University Affiliated Women's And Children's Hospital Xiamen Linqiaozhi Women's And Children's Hospital
Priority date: 2021-07-02
Filing date: 2021-07-02
Publication date: 2021-09-03
Anticipated expiration: 2041-07-02
Also published as: CN113337597B

Abstract

A detection kit for pregnancy vitamin A deficiency risk assessment and an application method relate to molecular biology. The primer group for screening pregnancy vitamin A deficiency risk prediction is used for amplifying single nucleotide polymorphism sites of vitamin A metabolic genes; the length of the primer is 18-30 bp. Adding SNP sequence specific extension primers into a product amplified by multiple PCR, extending 1 base on an SNP locus, extending different bases of different genotypes, exciting by using instantaneous nanosecond (10-9 s) strong laser, separating in a non-electric field drift region according to the mass-to-charge ratio of the product, and distinguishing different genotypes according to different flying time of the product in a vacuum tubule and different arrival time of the product in the vacuum tubule.

Description

Detection kit for pregnancy vitamin A deficiency risk assessment and application method

Technical Field

The present invention relates to molecular biology, in particular to

MALDI-TOF System (time flight mass spectrometry biochip System) is used for pregnant vitamin A deficiency risk assessment detection kit and application method of pregnant vitamin A.

Background

Vitamin A (VA) is a group of fat-soluble vitamins with all-trans retinol bioactivity, mainly including retinol, retinoic acid, retinal and esters, retinol is the main form of vitamin a transport in the body; retinoic acid and retinal are the active forms of vitamin a that exert biological effects in vivo; retinyl esters are the major storage form of vitamin a in the body. The physiological functions of vitamin A mainly include maintaining the functions of skin epithelial cells, vision and reproductive functions and lung functions, and are closely related to the development and functions of the central nervous system.

2011 statistics show that nearly 980 ten thousand pregnant women suffer from nyctalopia, and nearly 1910 ten thousand pregnant women (with the highest proportion of africa and southeast asia) have too low serum retinol concentration. According to 2016 statistical data of the world health organization, the pregnant women with global vitamin A deficiency are up to 15.3 percent. Researches show that the health influence of vitamin A deficiency on women in gestation period is more serious than that of other people, the vitamin A deficiency of pregnant women can cause the vitamin A deficiency of infants, and the vitamin A deficiency of the pregnant women is related to anemia of the pregnant women, premature fetal birth, delayed fetal intrauterine development, low birth weight, fetal deformity, delayed infant growth, death of newborn and infant, death of pregnant women and the like in many researches. Excess vitamin A can also cause central nervous system development malformation and heart malformation, animal experiments prove that the excess vitamin A has teratogenic toxicity to embryos, and a series of birth defects of the embryos caused by the reduction of pregnant women in pregnancy or in the early pregnancy or the wrong use of retinoic acid drugs are reported in people.

There are many proteins that mediate vitamin a transport and function, including serum retinol binding protein, cellular retinol binding protein, retinol egg phosphatidyltransferase, STRA6, and the like. Researches show that related gene polymorphism in the vitamin A metabolic process possibly influences the intake and absorption of vitamin A, so that a method for realizing individualized and accurate supplement of vitamin A in pregnancy through screening of vitamin A gene polymorphism is needed in the field.

Disclosure of Invention

The first purpose of the invention is to provide a primer set for screening pregnancy vitamin A deficiency risk prediction.

A second object of the invention is to provide a test kit for screening a prediction of the risk of vitamin a deficiency during pregnancy.

The third purpose of the invention is to solve the problem that the sequencing of gene polymorphism in the existing pregnancy vitamin A deficiency risk prediction product is expensive

MALDI-TOF System (time-of-flight mass spectrometry biochip System) method for detecting vitamin A in pregnancy and screening pregnancy vitamin A deficiency risk by using the detection kit.

The primer group for screening pregnancy vitamin A deficiency risk prediction is used for amplifying Single Nucleotide Polymorphism (SNP) sites of vitamin A metabolic genes; the length of the primer is 18-30 bp.

The single nucleotide polymorphism sites comprise rs12724719, rs5755368, rs3798709, rs2791952, rs2501175, rs10991408, rs10882272, rs12265684, rs6564851, rs7196470, rs8043708, rs1247620, rs12591551, rs12139131, rs3758538, rs2241057 and rs 10882273.

The primer group is shown as SEQ ID NO. 1-51.

An assay kit for screening for a prediction of risk of vitamin a deficiency during pregnancy comprising:

(1) a primer set as described above;

(2) the reaction solution for multiplex PCR comprises the following components:

a method for screening pregnancy vitamin a deficiency risk by using the detection kit, comprising the following steps:

1) collecting a blood sample of a pregnant woman, and extracting genome DNA;

2) carrying out PCR amplification on a target fragment of the genome DNA by adopting a primer group, wherein the PCR amplification comprises at least one gene fragment amplification, at least one product alkaline phosphatase treatment and at least one single-base primer extension reaction amplification to obtain an amplification product mixed solution;

3) purifying the amplification product mixed solution by resin to obtain an amplification product;

4) detecting and analyzing the amplification product by adopting MassARRAY Analyzer Comac mass spectrum detection and outputting the result.

The sample comprises human peripheral blood.

The product amplified by multiple PCR is added with SNP sequence specific extension primers, 1 base is extended on an SNP locus, different bases are extended by different genotypes, then the product is excited by instantaneous nanosecond (10-9 s) strong laser, the product is separated according to the mass-to-charge ratio in a non-electric field drift region, and different genotypes are distinguished according to different qualities of the extended bases and different flying time of the extended bases in a vacuum tubule to reach a detector.

Compared with the prior art, the invention has the following outstanding advantages:

1) ultra-high sensitivity: the lower detection limit was 0.4 ng.

2) And (3) ultrahigh flexibility: the method has the advantages that multiple PCR is automatically designed, common primers are synthesized, a universal kit and a chip can be used for several times, PCR conditions do not need to be optimized, new detection can be quickly established under standard conditions, and only two days are needed from design to result.

3) High quality data: and (3) fully automatically analyzing data, completing a genotyping report, and giving the mass spectrum to obtain a sample state and result reliability analysis.

4) High PCR multiplicity and conversion to power: better results can be obtained by 10-36 PCR, and the maximum can reach 40 PCR; > 95% of SNP sites can be analyzed with this platform.

5) High accuracy: the INDELs and the transposition fusion genes can be effectively detected by designing the primers in a forward and reverse direction, and the accuracy rate is more than 99.7%.

6) High sample throughput: the number of samples that can be processed per day is more than 3,000, 100,000 genotypes.

7) High cost performance: and 10-40 SNP sites are simultaneously measured, fluorescent markers are not needed, and the cost of consumables and the sample consumption are low.

8) Low DNA sample size and quality requirements: only 10ng of DNA was required for each PCR reaction

The invention provides a primer group, a kit and a method for detecting vitamin A metabolism related gene mutation, which can detect vitamin A metabolism related gene polymorphism.

Drawings

FIG. 1 is a mass spectrometric cluster diagram of different bases at point rs 12724719.

FIG. 2 is a mass spectrometric clustering diagram of different bases at the locus rs 5755368.

FIG. 3 is a mass spectrometric clustering diagram of different bases at position rs 3798709.

FIG. 4 is a diagram of mass spectrometric clustering of different bases at position rs 2791952.

FIG. 5 is a mass spectrometric clustering diagram of different bases at site rs 2501175.

FIG. 6 is a mass spectrometric clustering diagram of different bases at locus rs 10991408.

Figure 7 is a mass spectrometric clustering plot of different bases at point rs 10882272.

FIG. 8 is a mass spectrometric clustering plot of different bases at point rs 12265684.

FIG. 9 is a mass spectrometric cluster diagram of different bases at site rs 6564851.

FIG. 10 is a mass spectrometric clustering plot of different bases at position rs 7196470.

FIG. 11 is a mass spectrometric clustering plot of different bases at site rs 8043708.

FIG. 12 is a mass spectrometric clustering plot of different bases at locus rs 1247620.

FIG. 13 is a mass spectrometric clustering plot of different bases at locus rs 12591551.

FIG. 14 is a mass spectrometric clustering plot of different bases at site rs 12139131.

FIG. 15 is a mass spectrometric clustering plot of different bases at locus rs 3758538.

FIG. 16 is a plot of mass spectra clustering of different bases at position rs 2241057.

FIG. 17 is a mass spectrometric clustering plot of different bases at site rs 10882273.

FIG. 18 is a basic flow chart of time-of-flight mass spectrometry.

Detailed Description

The following examples will further illustrate the present invention with reference to the accompanying drawings.

Example 1

Design of primer composition

The invention selects the optimized selection of 17 SNP site systems from ALDH1A2 gene, CRABBP 2 gene, CYP26B1 gene, BCO1 gene, RBP4 gene, ABCA1 gene, ELOVL2 gene, CXCL8 gene, ISX gene, RPE65 gene, PKD1L2 gene and SOD2 gene, designs a PCR primer composition for amplifying DNA fragments containing 17 SNP sites on different genes, wherein the length range of the primer is 20-35bp at most, the length of PCR amplification products is 100-200 bp, the GC content is 40-60% and the primer is prevented from generating hairpin structures and continuous arrangement of more than 5 purine or pyrimidine nucleotides. In addition, because the mass spectrometry adopts a multiplex PCR method, and the number of primers in the same reaction system is large, the complementary occurrence between the primers needs to be paid particular attention, the 3' end of the SNP extension primer is absolutely prevented from being complementary with more than 3 bases of other primers, otherwise, the non-specific extension occurs, and the detection result is greatly interfered. In addition, the designed primer has no obvious homology with other sequences in the genome, so that the wrong detection result is prevented. The 5 ' ends of the front primer and the rear primer are additionally provided with adaptor sequences of 10bp in length ACGTTGGATG commonly called as ' protective bases ', the sequences of the protective bases enable the molecular weight of the PCR primers to be increased, and the PCR primers remaining in the reaction can be prevented from entering a mass spectrum detection window so as to avoid interference with the detection effect. The design principle for the single-base extension primer is as follows: the first base of the primer amplification is the base to be detected, and a hairpin structure and a repeated nucleotide sequence are avoided.

The genes related to the vitamin A metabolism detection of the invention are ABCA1(rs2791952, rs10991408), ALDH1A2(rs12591551), BCO1(rs6564851, rs7196470), CRABP2(rs12724719), CXCL8(rs1247620), CYP26B1(rs2241057), ELOVL2(rs3798709), ISX (rs5755368), PKD1L2(rs8043708), RBP4(rs10882272, rs 65684, rs3758538, rs10882273), RPE65(rs12139131), SOD2(rs2501175)

The primer group is shown as SEQ ID NO. 1-51.

Example 2

The use of the kit of the present invention is described in detail below with reference to example 1, and example 2 is carried out under the technical premise of the present invention, and detailed embodiments and specific procedures are given.

S1: DNA extraction:

1. sample treatment: adding 2 times volume of Buffer TBP into 200ul of whole blood of pregnant woman, mixing well, standing at room temperature for 1min until erythrocytes are completely lysed. Centrifuge at 8,000rpm for 1min and discard the supernatant. The pellet was resuspended in 500. mu.l TE Buffer, centrifuged at 8,000rpm for 1min, the supernatant discarded, the pellet was washed once with TE Buffer until white, and 200. mu.l PBS solution was added.

2. Add 20. mu.l of Proteinase K and mix well. Then 200. mu.l of Buffer DL was added, mixed by shaking, and then heated in a 56 ℃ water bath for 10 min. The mixed solution becomes clear and transparent, and the cracking is complete.

3. Add 200. mu.l of absolute ethanol to the tube and mix well by inversion.

4. Putting the adsorption column into a collecting tube, adding the solution and the semitransparent fibrous suspended matters into the adsorption column by a liquid transfer device, standing for 2min, centrifuging at 10,000rpm at room temperature for 1min, and pouring off waste liquid in the collecting tube.

5. The column was returned to the collection tube, 500. mu.l of GW Solution was added to the column, and the column was centrifuged at 10,000rpm for 30 seconds to discard the waste liquid.

6. The column was returned to the collection tube, and 700. mu.l of Wash Solution was added to the column, and the column was centrifuged at 10,000rpm for 30 seconds to discard the waste.

7. Repeat step 6 once.

8. The column was replaced in the collection tube and centrifuged at 12,000rpm for 2min at room temperature to remove the remaining Wash Solution. The cover of the adsorption column is opened and placed at room temperature for a plurality of minutes to thoroughly dry the Wash Solution remained in the adsorption material,

9. the adsorption column was taken out, and placed in a new 1.5ml centrifuge tube, 50. mu.l of CE Buffer was added thereto, and left to stand for 3min, and centrifuged at 12,000rpm at room temperature for 2min, and the DNA solution was collected.

S2: and (3) DNA quality detection:

1. detecting by taking 5 mu l of DNA solution 1% agarose and 1X TAE buffer solution electrophoresis (voltage of 120-180V), wherein a single band indicates that the DNA is complete and not degraded, and an obvious band indicates that the concentration can meet the PCR requirement;

2. and detecting the concentration and the purity by a spectrophotometer, taking 1 mul to detect the OD value, wherein the OD260/280 is 1.7-2.0, which shows that the DNA quality is better, and the DNA is polluted by protein less than 1.7 and RNA more than 2.0. :

s3: primer design

Designing primer software, designing PCR amplification primers and single base extension primers of the to-be-detected sites by using Genotyping Tools and Massarray Assay Design software of Sequenom. The specific amplification primer sequences and single base extension primer sequences are shown in Table 1.

TABLE 1 primer set sequences

1. The length of the primer is 18-30 bp, and the PCR product is 80-200 bp;

2. the Tm value is 55-65 ℃, and the annealing temperature is about 60 ℃;

3. GC content is 40-70%;

4. particular care should be taken to avoid primer dimer and the presence of non-specific amplification.

S4 PCR amplification

1. Using multiplex PCR, the reaction was carried out in 384-well plates, and the total volume of each reaction system was 5. mu.l, as follows:

PCR reaction conditions:

s5: alkaline phosphatase treatment of PCR products

After the PCR reaction, the PCR product was treated with SAP (Shamp alikaine phosphonase shrimp alkaline phosphatase), dNTPs in the reaction were removed, and SAP reaction solutions (384 samples as an example) were prepared in the order of Table 1

Table 2 SAP reaction liquid composition.

Note: the above 384-well reaction solution had a 4% excess.

1. A row of 12 tubes was taken, and the SAP reaction was dispensed 66. mu.l per well, briefly centrifuged, and then dispensed into a 384-well PCR reaction plate with a 10. mu.l line gun, 2. mu.l per well, sealed and centrifuged.

2. The 384-well plate to which the SAP reaction solution had been added was placed in a PCR instrument, and a reaction program named "SAP" was run.

3. SAP procedure: at 37 ℃ for 40 min; 5min at 85 ℃; 4 ℃ and forever.

4. At the end of the reaction, the 384-well plate is removed and centrifuged briefly for use.

S6: extension reaction

The iPLex reaction reagent was prepared as in Table 2.

Table 3 iPlex reaction reagents.

Note: the above 384-well reaction solution had a 4% excess.

1. Taking a row of 12 tubes, subpackaging the iPlex reaction solution with 66 mu l per well, centrifuging briefly, subpackaging with 10 mu l row gun to 384-well PCR reaction plates, adding 2 mu l per well, sealing the membrane, and centrifuging.

2. The 384-well plate to which the iPlex reaction solution had been added was placed in a PCR apparatus, and a reaction program named "extension" was run.

3. At the end of the reaction, the 384-well plate is removed and centrifuged briefly for use.

S7: purification of the product

1. 6mg of the resin was placed on a 384-hole resin scraper, and the resin was uniformly covered, scraped of excess resin, and left to stand for 20 min.

2. The 384 well plate after the reaction was centrifuged at 1000rpm for 1min, 25. mu.l of deionized water was added to each well, the plate was inverted on the resin plate (note fixed and not displaceable), and then the plate was placed upside down and the resin was dropped into the 384 well plate by tapping, and the membrane was sealed.

3. The 384-well plate is turned over for 20min by taking the long axis of the 384-well plate as the axis, and centrifuged at 3500rpm for 5min for standby.

S8: mass spectrometric detection

1. The nanodispenner SpectroCHIP chip was spotted, and the assay samples were transferred from 384-well reaction plates to a MassARRAY SpectroCHIP chip with a surface-coated substrate.

2. Mass spectrometric detection of MassARRAY Analyzer Comac

3. And transferring the sample to a SpectroCHIP chip, and then putting the chip into a mass spectrometer for detection, wherein each detection point only needs 3-5 s, and full-automatic analysis is carried out.

4. TYPER software analyzes the experimental results to obtain typing data (see FIGS. 1-17).

Example 3

In order to verify that the kit can accurately detect the 17 SNP site polymorphisms of 12 genes, 3 samples to be detected, which are detected by using the kit disclosed by the invention, are subjected to one-generation sequencing verification at the same time.

The first-generation sequencing result is consistent with the detection result of the kit related to the patent of the invention (see table 3), which shows that the patent of the invention can accurately detect the polymorphism of the corresponding SNP site.

TABLE 4 comparison of results of two detection methods

^*The patent: detecting results by using the kit provided by the invention;

^#generation: and (5) sequencing results of one generation.

The invention is as described

MALDI-TOF System (time of flight mass spectrometry biochip System) is a genotyping detection System developed and produced exclusively by Sequenom, Inc., USA, and is also the only device for directly performing SNP genotyping detection by adopting the principle of time of flight mass spectrometry at present. The main characteristics are as follows: adding SNP sequence specific extension primer to the product amplified by multiple PCR, extending 1 base on SNP site, extending different bases with different genotypes, and then using instantaneous nanosecond (10)^-9s) strong laser excitation, separating according to the mass-to-charge ratio in a non-electric field drift region, and distinguishing different genotypes according to different masses of extended bases and different flying time of the extended bases in a vacuum small tube to reach a detector, wherein the basic flow is shown in figure 18.

Sequence listing

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Claims

1. A primer group for screening pregnancy vitamin A deficiency risk prediction, which is characterized in that the primer group is used for amplifying a single nucleotide polymorphism site of a vitamin A metabolic gene; the length of the primer is 18-30 bp.

2. The primer set for screening pregnancy vitamin a deficiency risk prediction as claimed in claim 1, wherein the single nucleotide polymorphism sites comprise rs12724719, rs5755368, rs3798709, rs2791952, rs2501175, rs10991408, rs10882272, rs12265684, rs6564851, rs7196470, rs8043708, rs1247620, rs12591551, rs12139131, rs3758538, rs2241057, rs 10882273.

3. The primer group for screening prediction of pregnancy vitamin A deficiency risk according to claim 1, wherein the primer group is shown as SEQ ID No. 1-51.

4. An assay kit for screening for a prediction of risk of vitamin a deficiency during pregnancy comprising:

(1) the primer set of claim 1;

(2) the reaction solution for multiplex PCR comprises the following components:

5. a method for screening pregnancy vitamin a deficiency risk using the test kit of claim 4, comprising the steps of:

1) collecting a blood sample of a pregnant woman, and extracting genome DNA;

6. The method of claim 5, wherein said sample comprises peripheral blood of a human.