CN116606926A - Digital PCR (polymerase chain reaction) kit for noninvasively detecting fetal chromosomal aneuploidy - Google Patents
Digital PCR (polymerase chain reaction) kit for noninvasively detecting fetal chromosomal aneuploidy Download PDFInfo
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- 238000007847 digital PCR Methods 0.000 title claims abstract description 31
- 230000001605 fetal effect Effects 0.000 title claims abstract description 18
- 208000036878 aneuploidy Diseases 0.000 title claims abstract description 12
- 231100001075 aneuploidy Toxicity 0.000 title claims abstract description 12
- 230000002759 chromosomal effect Effects 0.000 title claims abstract description 12
- 238000003752 polymerase chain reaction Methods 0.000 title description 11
- 239000000523 sample Substances 0.000 claims abstract description 41
- 210000000349 chromosome Anatomy 0.000 claims abstract description 40
- 238000001514 detection method Methods 0.000 claims abstract description 31
- 238000000137 annealing Methods 0.000 claims abstract description 8
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- 239000004094 surface-active agent Substances 0.000 claims description 6
- 108010006785 Taq Polymerase Proteins 0.000 claims description 5
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- 238000006243 chemical reaction Methods 0.000 description 6
- 210000005259 peripheral blood Anatomy 0.000 description 5
- 239000011886 peripheral blood Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 201000006360 Edwards syndrome Diseases 0.000 description 4
- 201000009928 Patau syndrome Diseases 0.000 description 4
- 206010044686 Trisomy 13 Diseases 0.000 description 4
- 208000006284 Trisomy 13 Syndrome Diseases 0.000 description 4
- 208000007159 Trisomy 18 Syndrome Diseases 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 206010053884 trisomy 18 Diseases 0.000 description 4
- 239000012807 PCR reagent Substances 0.000 description 3
- 238000007481 next generation sequencing Methods 0.000 description 3
- 201000003738 orofaciodigital syndrome VIII Diseases 0.000 description 3
- 238000009609 prenatal screening Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 208000032170 Congenital Abnormalities Diseases 0.000 description 2
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- 230000015572 biosynthetic process Effects 0.000 description 2
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- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
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- 208000031404 Chromosome Aberrations Diseases 0.000 description 1
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- 210000004381 amniotic fluid Anatomy 0.000 description 1
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- 238000003149 assay kit Methods 0.000 description 1
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- 210000003917 human chromosome Anatomy 0.000 description 1
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Abstract
The application relates to a digital PCR kit for noninvasively detecting fetal chromosomal aneuploidies, which comprises one or more primers designed for 21, 18 and 13 chromosomes respectively, wherein the primers meet the following conditions: (1) all primer annealing temperatures are 60+/-1 ℃; (2) the length of all the primers is 17-25 bp; (3) the amplified sequence of the primer is less than or equal to 100bp; one or more probes designed for the 21, 18, 13 chromosomes respectively, the probes satisfying the following conditions: (1) all probe annealing temperatures are 70+/-2 ℃; (2) all probes have lengths of 25-35 bp; (3) The 21 chromosome probe was fluorescently labeled with FAM-BHQ1, the 18 chromosome was fluorescently labeled with VIC-BHQ1, and the 13 chromosome was fluorescently labeled with Cy5-BHQ 2. The kit has the advantages of high detection speed, high flux, high accuracy, low cost and the like.
Description
Technical Field
The application relates to the field of detection, and in particular relates to a digital PCR kit for noninvasively detecting fetal chromosomal aneuploidies.
Background
Birth defects of newborns are a wide variety of diseases, wherein chromosomal abnormalities are a relatively harmful and common type of birth defects of newborns, such as Down syndrome (T21), edwards syndrome (T18) and Patau syndrome (T13), with morbidity of 1:600-1:800, 1:6,000, 1:10,000 respectively; the birth number of Down's infants per year in China is about 26,600, and the factors such as food safety deterioration of living environment and the like are in an ascending trend along with the improvement of the growth age. At present, there is no effective treatment method for Down syndrome, and the prevention is mainly carried out by means of prenatal detection, and the Down syndrome causes great mental and economic burden on families and society.
Serology is currently used to indirectly assess the risk of developing down's disease in a fetus, and pregnant women who are "positive" (i.e., at high risk) in serological tests typically do further amniotic fluid puncture tests to confirm whether a Tang Shai fetus is present. The former, although noninvasive, has very poor sensitivity and specificity, while the latter, although accurate (about 99%), must be sampled by invasive methods, which can result in yields of up to 1%. The noninvasive fetal prenatal screening, which is emerging in recent 2-3 years and directly quantitatively determines the free fetal DNA in the peripheral blood of the pregnant woman by using a molecular diagnosis technology, has the advantages of noninvasive property and high accuracy (99%), and is certainly widely popularized in clinic.
From the university of hong Kong, 1997, LOYM et al reported that fetal free DNA (Cff DNA) could be repeatedly and stably detected in the peripheral blood of pregnant women, opening the way for noninvasive prenatal testing (Noninvasive Prenatal Diagnostics, NIPD). Based on a second generation gene sequencing (Next-Generation Sequencing, NGS) method in China, noninvasive prenatal screening work is carried out, and the detection accuracy rate can reach 99.9%. The core idea of the second generation gene sequencing is sequencing-while-synthesis (sequencing by synthesis), which has higher flux, but high price, requires trained special personnel to prepare libraries and analyze bioinformatics data, has longer time, restricts the noninvasive gene detection based on NGS to be directly used in clinical laboratory of hospitals, and has low clinical popularization rate at present.
The digital PCR is a nucleic acid quantitative analysis technology which is rapidly developed in the later 2000 s, and the core principle is that the traditional 1-relatively-large-volume (50-100 mu L) PCR reaction is subdivided into 2-1000 ten thousand micro-volume units (nano-scale or smaller) in a reaction container, each micro-volume unit does not contain or theoretically only contains a nucleic acid molecule to be detected, after PCR amplification, each micro-volume unit is detected, the micro-volume unit with fluorescent signals is interpreted as 1, the micro-volume unit without fluorescent signals is interpreted as 0 (therefore called as digital PCR), and finally, according to the statistical Poisson distribution principle and the proportion of positive results, the analysis software can directly calculate the concentration or copy number of the target molecule to be checked. The technology realizes absolute quantification in a true sense, and the accuracy, the accuracy and the sensitivity of the result are better. The microdroplet digital PCR technology is particularly suitable for accurately quantifying low-abundance targets in a sample (such as fetal free DNA targets in peripheral blood of a pregnant woman), and is therefore very suitable for noninvasive fetal prenatal screening.
Disclosure of Invention
In order to solve the technical problems, the application aims to provide a digital PCR detection kit for noninvasively detecting Down syndrome (T21), edwards syndrome (T18) and Patau syndrome (T13) with high detection speed, high flux, low cost and accurate detection result.
The application provides a digital PCR kit for noninvasively detecting fetal chromosomal aneuploidy, which comprises the following components:
one or more primers designed for the 21, 18 and 13 chromosomes respectively meet the following conditions: (1) all primer annealing temperatures are 60+/-1 ℃; (2) the length of all the primers is 18-30 bp; (3) the amplified sequence of the primer is less than or equal to 100bp;
one or more probes designed for the 21, 18, 13 chromosomes respectively, the probes satisfying the following conditions: (1) all probe annealing temperatures are 70+/-2 ℃; (2) all probes have lengths of 25-35 bp; (3) The 21 chromosome probe was fluorescently labeled with FAM-BHQ1, the 18 chromosome was fluorescently labeled with VIC-BHQ1, and the 13 chromosome was fluorescently labeled with Cy5-BHQ 2.
In order to improve the accuracy of the detection result, primer pairs synthesized by 21, 18 and 13 chromosomes respectively are designed, wherein the number of the primer pairs is 1-20, 5-20 is optional, and 10 pairs are optional; the number of the probe strips synthesized by 21, 18 and 13 chromosomes respectively is designed to be between 1 and 20, optionally between 5 and 20, and optionally 10. The adoption of the multiple primer probe to detect the target can solve the problem of low target concentration of clinical samples.
The length of free DNA is 50-300bp, main peak is about 150bp, in order to amplify fragments as many as possible, the size of amplified fragments of the designed primer is 50-300bp, optionally 50-150bp, and further 70-90 bp.
Further, the primer and the probe are obtained by the following steps:
(1) Designing a plurality of pairs of primers and probes aiming at specific sequences of 13, 18 and 21 chromosomes, wherein the 13 chromosomes are fluorescently labeled with Cy5-BHQ2, the 18 chromosomes are fluorescently labeled with VIC-BHQ1, and the 21 chromosome probes are fluorescently labeled with FAM-BHQ 1;
(2) Extracting DNA from the plasma of pregnant women with normal fetuses as a digital PCR amplification template, adding the single pair of primer probes in the step (1), preparing a digital PCR reagent for detection, and taking the primer probes with normal copy number results for standby;
(3) And (3) carrying out combined pairing on the normal primer probes in the step (2), taking the pregnant woman plasma extracted DNA with the normal fetus as a digital PCR amplification template, preparing a digital PCR reagent for detection, and obtaining the primer probes with normal copy number results.
Further, the primer probe is selected from: SEQ ID NO1-SEQ ID NO 90.
Further, the kit also comprises a digital PCR testAssay reagents comprising Taq DNA polymerase, glycerol, dNTPs, KCl, mgCl 2， Surfactants, and the like.
Further, in each reaction, taq DNA polymerase is 1-4IU, glycerol concentration is 1-5%, dNTPs is 0.2-0.6 mM, KCl is 40-60 mM, mgCl 2 1 to 5mM, and the concentration of the surfactant is 0.1 to 1%.
Further, the surfactant is one or more of Tween-20, tween-40, tween-60, tween-80, triton X-100, triton X-200, NP-10, NP-20, and NP-40.
After digital PCR amplification, statistical PCR results were counted with chromosome copy numbers of 13, 18, 21 as N 13 、N 18 、N 21 Calculating the ratio of 3 by two respectively by taking the copy numbers of chromosome 13, 18 and 21 as reference, and marking as R 21/13 、R 21/18 、R 18/13 Counting the three Z-score (Z-score) 21/13 、Z-score 21/18 、Z-score 18/13 ) If Z-score is less than 1.96, the chromosome is normal; if Z-score is more than 1.96 and less than or equal to 3, the sample is suspected to be positive and needs to be retested and confirmed; if Z-score > 3, the chromosome is abnormal.
The detection method of the kit comprises the following steps:
(1) Extracting maternal plasma DNA with normal or abnormal chromosomes as a digital PCR detection template;
(2) Preparing a digital PCR reagent containing a primer, and adding the template in the step (1);
(3) Generating water-in-oil droplets with a droplet generation apparatus;
(4) Performing conventional PCR amplification;
(5) The data is detected and read.
Further, the PCR reaction procedure was:
after digital PCR amplification, statistical PCR results were counted with chromosome copy numbers of 13, 18, 21 as N 13 、N 18 、N 21 With chromosome 13, 18, 21 copy numbers as referencesCalculating the ratio of 3 to R 21/13 、R 21/18 、R 18/13 Counting the three Z-score (Z-score) 21/13 、Z-score 21/18 、Z-score 18/13 ) If Z-score is less than 1.96, the chromosome is normal; if Z-score is more than 1.96 and less than or equal to 3, the sample is suspected to be positive and needs to be retested and confirmed; if Z-score > 3, the chromosome is abnormal.
The kit for noninvasively screening the trisomy 21, the trisomy 18 and the trisomy 13 solves the problem of low target concentration of clinical samples through a tubular multiplex detection design and combines a unique innovative detection result judgment standard, so that the false positive rate is controlled within an acceptable range while the false negative result is reduced, and finally the kit overcomes the problems of small sample volume (namely low DNA target concentration) of pregnant women, low ratio of fetal DNA (cffDNA) and the like which are common in NIPT detection and seriously influences the detection sensitivity, and effectively solves the problems. The kit has the advantages of high detection speed, high flux, high accuracy, low cost and the like, and can be used as an effective means for clinically screening Down syndrome, edwards syndrome and Patau syndrome.
Drawings
FIG. 1 shows the results of 21 chromosome sequence single digit PCR.
FIG. 2 shows the results of 18 chromosome sequence single digit PCR.
FIG. 3 shows the results of 13 chromosome sequence single digit PCR.
FIG. 4 shows the results of multiplex digital PCR for chromosome sequences 21, 18, and 13.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present application, the present application will be further described with reference to the following examples, which are obviously only a part of the examples of the present application and are not intended to limit the scope of the present application.
Example 1 design and verification of primer probes
The NCBI genome database is used for searching genes on human chromosomes 13, 18 and 21, the primer and the probe are designed by using oligo7, the annealing temperature of the primer is controlled to be 60+/-1 ℃, the annealing temperature of the probe is controlled to be 70+/-2 ℃, the amplified sequence is controlled to be less than 90bp, blast comparison is carried out on the amplified sequence, and a highly conserved sequence is selected and delivered to a gene synthesis company for synthesizing the primer and the probe.
The synthesized primer probe was formulated for single and multiplex digital PCR validation (FIGS. 1, 2, 3, 4) to finally obtain the appropriate sequences in Table 1.
TABLE 1 primer probe sequences
One embodiment of the application may select SEQ ID NO:1-10, seq ID NO:40-50, seq ID NO:70-80, adding digital PCR detection reagent including Taq DNA polymerase, glycerol, dNTPs, KCl, mgCl 2， And (3) preparing the surfactant into a detection kit.
In each reaction, taq DNA polymerase was 3IU, glycerol concentration was 3%, dNTPs was 0.4mM, KCl was 50mM, mgCl 2 4mM, surfactant Tween-80, at a concentration of 0.6%.
Example 2 detection of clinical samples
A baseline is established by using 20 trisomy negative diagnosis samples, and 15 pregnant woman peripheral blood samples with the pregnancy period of 12-20 weeks are tested, wherein the specific experimental steps are as follows:
(1) Taking 5ml of a peripheral blood sample of a pregnant woman, centrifuging, taking a supernatant, and extracting DNA for later use by using a free DNA extraction kit (example 1);
(2) Preparing 12.5 mu L of digital PCR Mix, adding 12.5 mu L of the extracted product in the step (1), uniformly mixing, and generating microdroplets;
(3) Placing the mixture on a PCR instrument for reaction, wherein the PCR reaction program is as follows:
(4) The data were analyzed and the results are shown in Table 2.
TABLE 2.15 clinical sample detection results
The final detection of clinical samples verifies that the detection sensitivity of the kit is 100% and the specificity is 95.14%. One measure of the quality of the assay is Receiver Operating Characteristic Curve (ROC) and Area Under the Curve (AUC), the AUC of the assay kit being 0.992.
Claims (7)
1. A digital PCR kit for noninvasive detection of fetal chromosomal aneuploidies, the kit comprising the following components:
one or more primers designed for the 21, 18, 13 chromosomes respectively, the primers satisfying the following conditions: (1) all primer annealing temperatures are 60+/-1 ℃; (2) the length of all the primers is 18-30 bp; (3) the amplified sequence of the primer is less than or equal to 100bp;
one or more probes designed for the 21, 18, 13 chromosomes respectively, which probes fulfil the following conditions: (1) all probe annealing temperatures are 70+/-2 ℃; (2) all probes have lengths of 25-35 bp; (3) The 21 chromosome probe was fluorescently labeled with FAM-BHQ1, the 18 chromosome was fluorescently labeled with VIC-BHQ1, and the 13 chromosome was fluorescently labeled with Cy5-BHQ 2.
2. The digital PCR kit for noninvasive detection of fetal chromosomal aneuploidies according to claim 1 wherein the number of probes for 21, 18, 13 chromosomes is 1-20, respectively.
3. The digital PCR kit for noninvasive detection of fetal chromosomal aneuploidies according to claim 2 wherein the number of probes for 21, 18, 13 chromosomes is 10, respectively.
4. The digital PCR kit for non-invasive detection of fetal chromosomal aneuploidies according to claim 1 wherein the primer amplified fragment size is 50-300bp.
5. The digital PCR kit for noninvasive detection of fetal chromosomal aneuploidies of claim 1, wherein the kit components further comprise Taq DNA polymerase, glycerol, dNTPs, KCl, mgCl 2 And a surfactant.
6. The digital PCR kit for noninvasive detection of fetal chromosomal aneuploidies according to claim 1, wherein the kit counts the digital PCR results after digital PCR amplification, the 13, 18, 21 chromosome copy numbers are counted as N13, N18, N21, the ratio of 3 is calculated by taking the chromosome 13, 18, 21 copy numbers as references, and the ratio is counted as R21/13, R21/18, R18/13, and the Z-score of the three is counted, and if the Z-score is less than 1.96, the chromosome is normal; if Z-score is more than 1.96 and less than or equal to 3, the sample is suspected to be positive and needs to be retested and confirmed; if Z-score > 3, the chromosome is abnormal.
7. The digital PCR kit for the non-invasive detection of fetal chromosomal aneuploidies according to claim 1 wherein said probe is selected from the group consisting of SEQ ID NO1-SEQ ID NO 90.
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