TWI576431B - Screening platform for checking aneuploidy in human embryos by quantitative polymerase chain reaction (qpcr) and the assembly thereof - Google Patents

Description

Detection of human embryos by real-time quantitative polymerase chain reaction (qPCR) Aneuploid screening platform and its set

The present invention relates to preimplantation genetic screening, in particular to the detection of embryo aneuploidy by real-time quantitative polymerase chain reaction (qPCR). Check the platform and the set.

On day 3, cleavage stage embryo biopsy (Day-3 cleavage stage embryo biopsy) and fluorescence in situ hybridization (FISH) test is the current acceptance of women with advanced maternal age. A high preimplantation genetic screening (PGS) program. The hypothesis of the aforementioned genetic screening program is based on the fact that older maternal susceptible embryos exhibit aneuploidy, which leads to embryo implantation failure or the production of chromosomal abnormalities in the fetus, so the aforementioned genetic screening program can improve pregnancy success rate. And the production of a normal fetus (reproductive outcome). However, many randomized controlled trials (RCTs) have confirmed that the aforementioned genetic screening protocol (FISH) is less effective than other PGS techniques that have not been widely adopted, and has not only failed to effectively improve pregnancy success. Rate, more detrimental to the live birth rate of older women (live-birth rate). Therefore, other researchers have proposed different genetic screening programs, including biopsy at different embryonic development stages and different embryo transfer timings, such as trophectoderm biopsy on day 5/6 of embryonic development. Vs cleavage-stage blastomere biopsy on day 3 of embryonic development, and frozen embryos implanted periodically with fresh embryos vs. periodic implants to detect abnormal copy number It also replaces the fluorescence detection technique that can only selectively detect a few chromosomes (FISH detection is mainly limited by the choice of fluorescent species).

There is growing evidence that blastomere biopsy on day 3 cleavage stage embryos do impair the implantation potential, whereas vegetative ectoderm on day 5/6 embryos There is no embryo damage in the biopsy. In addition, the use of more advanced genetic testing techniques may screen for embryos that are more suitable for implantation. Related technologies include array-based comparative genomic hybridization (aCGH), single nucleotide polymorphism array (SNP array), qPCR, and next generation sequencing (NGS, next generation). Sequencing). Among these technologies, NGS technology has considerable research value in data analysis in the field of bioinformatics, but its cost is high, thus reducing the popularity of its clinical application; currently the most feasible tool is qPCR and Array-based technologies, including SNP array and aCGH. It is currently known that mosaicism is a common phenomenon in early human embryo development, so how to screen embryos with optimal implantation potential becomes a very important topic. Among them, qPCR has a lower false-positive rate than aCGH, therefore, qPCR is a preferred PGS scheme.

The commercially available aCGH PGS platform is available on the market, due to its easy to use and easy-to-use system, for artificial reproduction centers (IVF centers) that do not have a genetic testing laboratory. ), high acceptability. However, due to the aforementioned qPCR The PGS results are quite convincing, so it is necessary for researchers in the same field to further confirm the feasibility of qPCR to replace or replace the aCGH platform.

The object of the present invention is to provide a screening platform for detecting embryo aneuploidy by using real-time quantitative polymerase chain reaction (qPCR) and a probe thereof, which mainly adopts the 5th/6th day embryo. Embryo, selectively amplify chromosomal markers by qPCR technology to detect aneuploidy, and modify the probe with LNA (locked nucleic acid) technology; thereby providing a rapid screening and false A PGS platform with a low positive rate, and the probe of the present invention has the technical efficacy of detecting at least 5 chromosomes.

To achieve the foregoing objective, the present invention provides a screening platform for detecting aneuploidy of an embryo using a real-time quantitative polymerase chain reaction (qPCR), which adopts a 5/6 day embryo stage. Embryos and selectively amplify chromosomal markers via a two-color qPCR to detect aneuploidy; wherein the screening platform includes at least 2 probes and 1 set for amplification of chromosome 1 The reference locus and the complex array are used to amplify the outer primer sets and the inner primer sets of the target loci of the target chromosome. The needle contains at least three LNA-modified nucleotides, and the probe recognition region sequences on the chromosome are located on the exon sequence, and the probe is accurately identified by the conservation of the recognition region sequence. And combined with the chromosomal probe recognition region sequence to improve the detection stability; in addition, one of the specific primer pairs is located in the intron sequence to ensure the amplified gene sequence Begin genome sequence of deoxyribonucleic acid (genomic DNA) as a template, RNA interference can be avoided message sequence (precursor mRNA) and improve the accuracy of detection.

The screening platform of the present invention, wherein the probe for the control locus sequence is selected from the probe of the sequence 5'-CCTGGCCACCG-3' (sequence list: SEQ ID NO: 1); The needle is selected from the probe of the sequence 5'-GGGCAGCAGC-3' (sequence list: SEQ ID NO: 2), and the two locked nucleic acid (LNA) modified probes are respectively labeled with two different fluorescences for quantification. The number of amplified loci of the target locus and the control locus; and the control locus is the specific sequence on the first chromosome, which defines the range of the plural specific primer pairs within a particular sequence range.

Wherein, the control locus is taken as FAM20B, and there are two sets of primer pairs (1FAM20B-F/R-out, 1FAM20B-F/R-in), wherein the control locus is outside the primer pair (1FAM20B-F/ The sequence range of R-out) is >chr1:179040981+179041348, and the sequence range of the primer pair in the control locus (1FAM20B-F/R-in) is >chr1:179041176+179041262.

The screening platform of the present invention, wherein the target locus can be at least a chromosome of the 13th, 18th, 21st, Xth, and Yth, respectively, or other chromosomes, and the target locus is defined within a specific sequence range. The sequence range of a particular primer pair.

The screening platform of the present invention, wherein the fluorescent nucleic acid (LNA) modified fluorescent marker comprises FAMTM and HEXTM respectively connected to the 5'-end of the two probes, and the 3'-end of the two probes is linked to the BHQTM As a fluorescent sign.

The present invention also provides a kit for detecting aneuploidy of human embryos, which is based on the aforementioned screening platform to provide a fast and accurate aneuploid detection kit.

The technical effect of the present invention is that the proposed aneuploid screening platform and probe are a novel preimplantation genetic screening (PGS) scheme proposed by the inventor of the present invention. Through the aforementioned screening platform and probe, it is possible to quickly and accurately detect whether the number of embryonic chromosome sets is aneuploid, so that the tested euploid embryos have a better continuous pregnancy rate (ongoing pregnancies), about 53.8. % shows that the solution proposed by the present invention is clinically feasible. And the aforementioned screening and probes can be widely used for complex dyeing The color-screened PGS can be extended to 24 chromosomes (refer to chromosome 1-22, X, Y) for comprehensive screening.

A secondary object of the present invention is that the present invention can be quickly diagnosed, and a euploid embryo can be diagnosed in about four hours, thereby performing a fresh embryo transplantation operation.

The present invention is directed to the above-described objects, the screening platform and the probes thereof, and the embodiments are described in detail with reference to the drawings. The objects, features and other advantages of the present invention are believed to be Understand.

1A and 1B are schematic diagrams showing the first amplification process of the aneuploid of the embryo by the instant quantitative polymerase chain reaction (qPCR) of the present invention.

Figure 1C is a graph of temperature versus time in a first amplification run of the present invention for detecting aneuploidy of an embryo using an instant quantitative polymerase chain reaction (qPCR).

2 is a schematic diagram showing the probe adhesion and the second amplification process of the aneuploid of the embryo by the instantaneous quantitative polymerase chain reaction (qPCR) of the present invention.

The following describes a specific embodiment of the present invention for the detection of aneuploidy of embryos and probes thereof by real-time quantitative polymerase chain reaction (qPCR).

The present invention provides a platform for aneuploidy detection of all 24 chromosomes by real-time quantitative polymerase chain reaction (qPCR), wherein a specific control locus and a probe of a target locus are defined by the present inventors, and Repaired by locked nucleic acid technology (LNA) Decoration, the first amplification of the locus sequence by the appropriate plural primer pair, the sequence of the locus after the first amplification, and the other plurality of primer pairs can be bonded to the probe, and then the second amplification is performed. The fluorescent marker of the probe is released and effectively detected, forming a detection platform for real-time quantitative polymerase chain reaction (qPCR) detection of fluorescent signals.

As shown in FIG. 1A to FIG. 2, a schematic flow chart of the amplification principle of the screening platform for detecting aneuploidy of human embryos by qPCR and a graph of the relationship between the warm end and the time are shown. The process comprises the following steps: first amplification: as shown in FIG. 1A and FIG. 1B, which uses a pair of control loci and a primer pair outside the target locus to perform amplification of the locus sequence on the embryonic cell; 2, which is to bind the primer pair and the probe in the control locus and the target locus simultaneously on the locus sequence after the first amplification; the second amplification and release of fluorescence: FIG. 2 As shown, the primers in the control locus and the target locus are re-amplified on the locus sequence after the first amplification, and the original is bound to the sequence of the first amplified post-occupation locus. The probe is destroyed, and the fluorescence is released, so that the fluorescent marker HEXTM and FAMTM of the probe are separated from the fluorescent marker BHQ at the 3' end respectively, and the fluorescent markers HEXTM and FAMTM can emit fluorescence; The steps of bonding, second amplification, and release of fluorescence are used as quantification by real-time quantitative polymerase chain reaction (qPCR).

The present inventors performed preimplantation genetic screening (PGS) using an instant quantitative polymerase chain reaction (qPCR) assay. The present invention initially targets only the 13th, 18th, 21st, and Xth and Y chromosomes of aneuploidy, and selects the first chromosome as a control set for control, and uses all chromosomes, Including: 22 body chromosomes and X, Y chromosomes, all can be the target set of detected aneuploid.

As shown in Table 1 above, the present invention is a two-color real-time quantitative polymerase chain reaction (qPCR), the screening system comprising: targeting the first chromosome having the least trisomy in the human body as a control chromosome, and targeting the control chromosome The designed control locus sequence probe is selected from the probe of the sequence 5'-CCTGGCCACCG-3' (SEQ ID NO: 1 of the Sequence Listing); the other chromosome is the target chromosome, and the target designed for the target chromosome The locus sequence probe is selected from the probes of the sequence 5'-GGGCAGCAGC-3' (SEQ ID NO: 2 of the Sequence Listing), which are each modified with two different fluorescent markers for nucleic acid (LNA) modification.

In the aforementioned primer sets, it can be divided into an outer primer set and an inner primer set, wherein, as shown in Table 1, the control locus of the control group is introduced. For example, in FAM20B, there are two pairs of primers, 1FAM20B-F/R-out and 1FAM20B-F/R-in, which bind to the aforementioned control locus sequence probe (SEQ ID NO: 1), wherein the control gene The sequence range of the extra-primer pair (1FAM20B-F/R-out) is >chr1:179040981+179041348, and the sequence range of the primer pair (1FAM20B-F/R-in) in the control locus is >chr1:179041176+179041262 .

As shown in Table 1, the chromosome of the target group is taken as the 21st chromosome. When the target loci are URB1, TIAM1, TRAPPC10, and PRDM15, there are 21URB1-F/R-out, 21URB1-F/R-in, and 21TIAM1. -F/R-out, 21TIAM1-F/R-in, 21TRAPPC10-F/R-out, 21TRAPPC10-F/R -in, 21PRDM15-F/R-out, 21PRDM15-F/R-in, a total of eight pairs of primer pairs binding to the aforementioned target locus sequence probe (SEQ ID NO: 2), wherein the target locus is other than URB1 The sequence range of the primer pair (21URB1-F/R-out) is >chr21:33706393-33706556, and the sequence range of the target locus in the URB1 primer pair (21URB1-F/R-in) is >chr21:33706443- 33706522; the target locus is the sequence range of the TIAM1 extra-primer pair (21 TIAM1-F/R-out) > chr21:32582430-32582602, and the target locus is the primer pair within TIAM1 (21 TIAM1-F/R-in The sequence range is >chr21:32582484-32582588; the target locus is the sequence of the TRAPPC10 extra-primer pair (21 TRAPPC10-F/R-out) >chr21:45457611+45457826, and the target locus is within TRAPPC10 The sequence range of the primer pair (21 TRAPPC10-F/R-in) is >chr21:45457699+45457783; the target locus is the sequence range of the primer pair (21 PRDM15-F/R-out) of PRDM15>chr21: 43279112-43279292, the sequence of the target locus in the primer set of PRDM15 (21 PRDM15-F/R-in) is >chr21:43279123-43279253.

As shown in Table 1, the chromosome of the target group is exemplified by chromosome 18, and when the target loci are MBD1, ZNF236, and CTDP1, there are 18MBD1-F/R-out, 18MBD1-F/R-in, and 18ZNF236-F. /R-out, 18ZNF236-F/R-in, 18CTDP1-F/R-out, 18CTDP1-F/R-in, a total of six pairs of primer pairs binding to the aforementioned target locus sequence probe (SEQ ID NO: 2) Wherein, the target locus is the sequence of the MBD1 extra-primer pair (18MBD1-F/R-out) > chr18: 47800159-47800294, and the target locus is the primer pair of MBD1 (18MBD1-F/R-in) The sequence range is >chr18:47800188-47800275; the target locus is ZNF236 and the sequence of the primer pair (18ZNF236-F/R-out) is >chr18:74592149+74592311, and the target locus is the primer pair of ZNF236. The sequence range of (18 ZNF236-F/R-in) is >chr18:74592157+74592265; the sequence locus of the target locus is CTDP1 (18 CTDP1-F/R-out). The sequence range is >chr18:77513629+ 77513793, the target locus is within the CTDP1 primer pair (18 CTDP1-F/R-in) has a sequence range of >chr18:77513666+77513758.

As shown in Table 1, the chromosome of the target group is exemplified by chromosome 13 which has 13MTMR6-F/R-out, 13MTMR6-F/R-in, 13PCDH8-F when the target locus is MTMR6, PCDH8 and IPO5. /R-out, 13PCDH8-F/R-in-1, 13PCDH8-F/R-in-2, 13IPO5-F/R-out, 13IPO5-F/R-in, a total of seven pairs and the aforementioned target locus sequence A pair of primers (SEQ ID NO: 2) that binds, wherein the target locus is the sequence of the MTMR6 primer pair (13 MTMR6-F/R-out) > chr13: 25823301-25823552, and the target locus is MTMR6 The sequence range of the primer pair (13 MTMR6-F/R-in) is >chr13:25823445-25823508; the target locus is the sequence range of the primer pair (13 PCDH8-F/R-out) of PCDH8 > Chr13:53419845-53420030, the target locus is the sequence of the primer pair (13 PCDH8-F/R-in-1, 13PCDH8-F/R-in-2) in PCDH8. The sequence range is >chr13:53419866-53419995 And >chr13:53419922-53419995; the target locus is IPO5 and the sequence of the primer pair (13 IPO5-F/R-out) is >chr13:98655080+98655268, and the target locus is the primer pair of IPO5 (13 IPO5) The sequence range of -F/R-in) is >chr13:98655149+98 655224.

As shown in Table 1, the target group chromosome is exemplified by chromosome Y. When the target locus is ZFY and KDM5D, there are YZFY-F/R-in, YZFY-F/R-in, and YKDM5D-F/R-out. , YKDM5D-F/R-in a total of four pairs of primer pairs binding to the aforementioned target locus sequence probe (SEQ ID NO: 2), wherein the target locus is a ZFY-introducing pair (YZFY-F/R-out The sequence range is >chrY:2844794+2844938, and the sequence range of the target locus is ZFY (YZFY-E/R-in) is >chrY: 2844826+2844897; the target locus is KDM5D. The sequence range of (YKDM5D-F/R-out) is >chrY: 21871539-21871720, and the sequence range of the target locus is KDM5D (YKDM5D-F/R-in) is >chrY: 21871564-21871641 .

As shown in Table 1, the chromosome of the target group is exemplified by chromosome X. When the target loci are ZFX, HUWE1, EFNB1, and XPNPEP2, there are XZFX-F/R-out, XZFX-F/R-in, and XHUWE1-F. /R-out, XHUWE1-F/R-in, XEFNB1-F/R-out, XEFNB1-F/R-in, XXPNPEP2-F/R-out, XXPNPEP2-F/R-in, a total of eight pairs of primers that bind to the aforementioned target locus sequence probe (SEQ ID NO: 2), wherein the target locus is a primer pair other than ZFX ( The sequence range of X ZFX-F/R-out) is >chrX:24226376+24226585, and the sequence range of the primer locus (X ZFX-F/R-in) of the target locus is >chrX:24226424+24226508 The sequence of the target locus is HUWE1 (X HUWE1-F/R-out), the sequence range is >chrX:53573627-53573781, and the target locus is the primer pair of HUWE1 (X HUWE1-F/R-in) The sequence range is >chrX:53573688-53573767; the target locus is EFNB1 and the sequence range of the primer pair (X EFNB1-F/R-out) is >chrX:68060064+68060217, and the target locus is the primer of EFNB1. The sequence range of (X EFNB1-F/R-in) is >chrX:68060099+68060172; the target locus is XPNPEP2 and the sequence range of the primer pair (X XPNPEP2-F/R-out) is >chrX:128873141 +128873289, the sequence range of the primer locus (X XPNPEP2-F/R-in) of the target locus is XPNPEP2 is >chrX: 128873173 + 128873264.

In addition to the adhesion and detection of the aforementioned chromosomes, the target locus sequence probe of the present invention can perform the same detection operation for other chromosomes.

The control locus sequence probe and the target locus sequence probe of the present invention contain LNA modified nucleotides; wherein the 5' end of the control locus sequence probe is labeled with HEXTM fluorescent label, the target locus sequence probe The 5' end is labeled with FAMTM fluorescent label (IDT, Integrated DNA Technologies, Iowa, USA/USA, custom nucleic acid supplier), and the 3' end of the control locus sequence probe and the target locus sequence probe are The fluorescent marker BHQ is suppressed.

<110> Chen Ming

<120> Screening platform and set of aneuploidy for human embryos using real-time quantitative polymerase chain reaction (qPCR)

<160> 2

<210> 1

<211> 11

<212> DNA

<213> Artificial sequence

<400> 1

<210> 2

<211> 10

<212> DNA

<213> Artificial sequence

<400> 2

Claims (11)

A screening platform for detecting aneuploidy of an embryo by real-time quantitative polymerase chain reaction (qPCR), which selectively amplifies a chromosomal locus via a two-color qPCR to detect aneuploidy; wherein the screening platform includes a a control locus sequence probe, a target locus sequence probe, a plurality of specific primer pairs for a control locus and a target locus, the control locus being a gene sequence inside the first chromosome, the first chromosome The designed control locus sequence probe is the probe of SEQ ID NO: 1 of the sequence 5'-CCTGGCCACCG-3'; the target locus is selected from the gene sequence of 24 chromosomes, selected from the chromosome 24 design The target locus sequence probe is selected from the probe of SEQ ID NO: 2 of the sequence 5'-GGGCAGCAGC-3', and the control locus sequence probe and the target locus sequence probe contain LNA modified nucleotides. The screening platform according to claim 1, wherein the control locus is FAM20B, and the sequence range of the control locus exogenous pair is >chr1:179040981+179041348, and the sequence of the primer pair in the control locus The range is >chr1:179041176+179041262. For example, in the screening platform described in claim 1, the target locus is a gene sequence inside the 21st chromosome, and the target locus is selected from URB1, TIAM1, TRAPPC10, and PRDM15, wherein the target locus is URB1. The sequence range of the primer pair is >chr21:33706393-33706556, the sequence range of the primer pair of the target locus is URB1 is >chr21:33706443-33706522; the target locus is the sequence range of the primer pair of TIAM1> Chr21:32582430-32582602, the sequence range of the primer locus of TIAM1 is >chr21:32582484-32582588; the target locus is the sequence range of the primer pair of TRAPPC10>chr21:45457611+45457826, target locus The sequence range of the primer pair within TRAPPC10 is >chr21:45457699+45457783; the target locus is the sequence range of the primer pair of PRDM15 >chr21:43279112-43279292, the sequence range of the primer pair within the target locus of PRDM15 is >chr21:43279123-43279253. As for the screening platform described in claim 1, the target locus is a gene sequence within the 18th chromosome, and the target locus is selected from the group consisting of MBD1, ZNF236, and CTDP1, wherein the target locus is other than MBD1. The sequence range of the primer pair is >chr18:47800159-47800294, the sequence range of the primer pair in the target locus is >chr18:47800188-47800275; the target locus is the sequence range of the primer pair of ZNF236>chr18: 74592149+74592311, the target locus is within the sequence of ZNF236. The sequence range is >chr18:74592157+74592265; the target locus is CTDP1. The sequence range of the primer pair is >chr18:77513629+77513793, and the target locus is CTDP1. The sequence range of the primer pair is >chr18:77513666+77513758. For example, in the screening platform described in claim 1, the target locus is a gene sequence inside the 13th chromosome, and the target locus is selected from the group consisting of MTMR6, PCDH8 and IPO5, wherein the target locus is other than MTMR6. The sequence range of the primer pair is >chr13:25823301-25823552, the sequence range of the primer pair in the target locus is >chr13:25823445-25823508; the target locus is the sequence range of the primer pair of PCDH8>chr13: 53419845-53420030, the sequence range of the primer locus in the target locus is PCDH8 is >chr13:53419866-53419995 and >chr13:53419922-53419995; the target locus is IPO5 and the sequence range of the primer pair is >chr13: 98655080+98655268, the target locus is within the sequence of the primer pair within IPO5 is >chr13: 98655149+98655224. For example, in the screening platform described in claim 1, the target locus is a gene sequence inside the Y chromosome, and the target locus is selected from ZFY and KDM5D, wherein the target locus is a pair of ZFY primer pairs. The sequence range is >chrY: 2844794+2844938, and the target locus is The sequence range of the primer pair in ZFY is >chrY: 2844826+2844897; the target locus is KDM5D and the sequence range of the primer pair is >chrY: 21871539-21871720, and the target locus is the sequence range of the primer pair within KDM5D. Is >chrY: 21871564-21871641. For example, in the screening platform described in claim 1, the target locus is a gene sequence inside the X chromosome, and the target locus is selected from ZFX, HUWE1, EFNB1, and XPNPEP2, wherein the target locus is ZFX. The sequence range of the primer pair is >chrX:24226376+24226585, and the sequence range of the primer pair of the target locus is >chrX:24226424+24226508; the target locus is the sequence range of the primer pair of HUWE1> chrX:53573627-53573781, the target locus is the sequence of the primer pair of HUWE1 is >chrX:53573688-53573767; the target locus is the sequence range of the primer pair of EFNB1>chrX:68060064+68060217, target locus The sequence range of the primer pair in EFNB1 is >chrX:68060099+68060172; the target locus is XPNPEP2, and the sequence range of the primer pair is >chrX:128873141+128873289, and the target locus is the sequence range of the primer pair within XPNPEP2. The system is >chrX: 128873173+128873264. The screening platform according to claim 1, wherein the LNA modified nucleotide comprises a fluorescent label of FAM and HEX, and is respectively connected to the control locus sequence probe and the target locus sequence. 5' end of the probe. The screening platform according to claim 8, wherein the control locus sequence probe has a 5' end labeled HEX, and a 3' end is ligated with a fluorescent label BHQ, the target locus sequence probe. The 5' end is labeled with FAM and its 3' end is terminated with a fluorescent label BHQ. A kit for detecting aneuploidy of a human embryo, comprising the screening platform of any one of claims 1 to 9. A method for detecting aneuploidy of human embryos according to the screening platform described in claim 1 of the patent application, comprising: first amplification: using a control locus and a primer pair outside the target locus, targeting the embryo The cell is subjected to amplification of the locus sequence; binding: simultaneously binding the primer pair and the probe within the control locus and the target locus on the locus sequence after the first amplification, wherein the sequence 5'-CCTGGCCACCG-3 'SEQ ID NO: 1 is a probe for the control locus, and SEQ ID NO: 2 of the sequence 5'-GGGCAGCAGC-3' is a probe for the locus of interest; the second amplification and release of fluorescence: The control locus and the primer in the target locus re-amplify the sequence of the locus after the first amplification, and destroy the probe that binds to the first amplified post locus sequence. And release the fluorescent light, so that the fluorescent label HEX and FAM of the 5' end of the probe are separated from the fluorescent label BHQ of the 3' end, respectively, and the fluorescent label HEX and FAM can emit fluorescence; repeated bonding, second Steps of amplifying and releasing fluorescence for immediate quantification Polymerase chain reaction (qPCR) quantification.