A STR fluorescent labeling multiplex amplification system, kit and application

Abstract

The present invention discloses a STR fluorescence-labeled multiplex amplification system, which includes 6 STR loci on human chromosome 15, 12 STR loci on human chromosome 20, and corresponding 36 primer pairs, and each primer pair carries a fluorescent dye label. The present invention can serve as an effective supplement to existing kits, can significantly reduce the false positive rate, effectively improve the accuracy and reliability of detection, provide an important basis for prenatal diagnosis and genetic disease detection, and has important clinical application value and broad market prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular genetics, and particularly to a STR fluorescence labeling multiplex amplification system, a kit and an application thereof. Background Art

[0002] UPD (uniparental disomy) refers to the replacement of a chromosomal region / segment from one parent by a homologous part of the other, or both homologous chromosomes of an individual coming from the same parent. The occurrence mechanisms of UPD include trisomic rescue, monosomic duplication, gametic complementation, and somatic mitotic abnormalities, etc. According to the origin of homologous chromosomes, UPD can be divided into maternal UPD (matUPD) and paternal UPD (patUPD). UPD is associated with a variety of diseases. Due to abnormal expression of imprinted genes, UPD involving chromosomes 6, 7, 11, 14, 15, and 20 can lead to definite imprinting diseases. Other chromosomal UPDs are relatively rare, and there are currently no reports of definite related imprinting diseases.

[0003] Maternal UPD of chromosome 15 can cause Prader-Willi syndrome (PWS), also known as Prader-Willi syndrome, which is a rare congenital disease. Maternal uniparental disomy of chromosome 15 is one of the important pathogenic mechanisms of PWS, accounting for about 20-30% of PWS cases. It is mainly due to the nondisjunction of oocytes during meiosis I, resulting in the lack of paternal gene expression in the genetic imprinted 15q11.2-q13 region, while the maternal genes SNURF, SNRPN, UBE3A, and ATP10C are overexpressed. The clinical manifestations of PWS are neonatal hypotonia and poor sucking, inability to grow normally, developmental delay and / or intellectual impairment, childhood obesity, short stature, hypogonadism and abnormal behavior. Paternal UPD of chromosome 15 can lead to Angelman syndrome (AS), also known as Angelman syndrome, which is caused by paternal uniparental disomy of chromosome 15. This pathogenic mechanism accounts for about 3-7% of AS cases. Due to the non-disjunction of chromatids during oocyte meiosis II or early cleavage, paternal uniparental disomy in the genetic imprinted 15q11.2-q13 region occurs, and maternal genes are not expressed. Clinical manifestations include severe intellectual impairment, aphasia, ataxia, microcephaly, epileptic seizures, and a cheerful personality with paroxysmal laughter. Maternal UPD20 can cause Mulchandani-Bhoj-Conlin syndrome, which is characterized by intrauterine and postnatal maldevelopment and prominent feeding difficulties, and failure to thrive. Most patients do not have dysmorphic features, congenital malformations, or major developmental delays. Paternal UPD20 causes pseudohypoparathyroidism type 1b (PHP1B), which is characterized by renal resistance to parathyroid hormone, manifested as hypocalcemia, hyperphosphatemia, and abnormally high parathyroid hormone levels. This condition is usually caused by the loss of the DMR of the GNAS locus located on 20q13 or the loss of the STX gene (a remote control element for methylation of the GNAS locus), which results in the loss of expression of the maternal Gs-α isoform in kidney tissue.

[0004] DNA-based polymorphic markers are classic methods for studying UPD, and STR sequence markers are the most commonly used. STR (Short Tandem Repeat), also known as microsatellite DNA, is usually a segment of DNA repeat sequence composed of 2-6 base units in the genome. Due to the high variability of the number of core unit repeats among individuals, STR markers are very abundant throughout the genome. Many STR loci have very high heterozygosity, which reflects the differences in allele frequencies in the population and is very suitable for multiplex polymerase chain reaction. In addition to the proband's DNA sample, samples from both parents are usually required to describe the parental origin of the detected STR alleles. Traditional UPD detection methods include DNA methylation analysis, fluorescence in situ hybridization combined with chromosome karyotyping, chromosome microarray, gene sequencing, etc. These methods are also used for UPD detection, but these operations are relatively cumbersome and the costs are relatively high. The STR multiplex amplification technology is time-saving and simple to operate, with low costs, and is suitable as a commonly used means for UPD detection. Summary of the Invention

[0005] In view of the fact that there are relatively few STR loci on chromosomes 15 and 20 currently, and the false positive rate of existing kits is relatively high, the present invention provides an STR fluorescence-labeled multiplex amplification system, kit and application, which can provide STR loci with relatively high polymorphism and good heterozygosity for accurately judging the uniparental disomy of chromosomes 15 and 20 and giving a hint of uniparental diploidy.

[0006] To achieve the above object, the embodiments of the present invention adopt the following technical solutions:

[0007] An STR fluorescence-labeled multiplex amplification system includes 6 STR loci on human chromosome 15: D15S1232, GTTTT001, D15S1513, D15S231, CHLC.ATC3C11, D15S1233; 12 STR loci on human chromosome 20: 20A-P1, D20S164, 20A-P3, D20S430, D20S158, D20S443, D20S466, 20B-P2, D20S902, D20S481, D20S601, D20S206; 36 primer pairs corresponding to the above STR loci; and each primer pair carries a fluorescent dye label.

[0008] Preferably,

[0009] The upstream and downstream primer sequences for amplifying D15S1232 are shown as SEQ ID NO.1 and SEQ ID NO.2; the upstream and downstream primer sequences for amplifying GTTTT001 are shown as SEQ ID NO.3 and SEQ ID NO.4; the upstream and downstream primer sequences for amplifying D15S1513 are shown as SEQ ID NO.5 and SEQ ID NO.6; the upstream and downstream primer sequences for amplifying D15S231 are shown as SEQ ID NO.7 and SEQ ID NO.8; the upstream and downstream primer sequences for amplifying CHLC.ATC3C11 are shown as SEQ ID NO.9 and SEQ ID NO.10; the upstream and downstream primer sequences for amplifying D15S1233 are shown as SEQ ID NO.11 and SEQ ID NO.12.

[0010] The upstream and downstream primer sequences for amplifying 20A-P1 are shown as SEQ ID NO.13 and SEQ ID NO.14; the upstream and downstream primer sequences for amplifying D20S164 are shown as SEQ ID NO.15 and SEQ ID NO.16; the upstream and downstream primer sequences for amplifying 20A-P3 are shown as SEQ ID NO.17 and SEQ ID NO.18; the upstream and downstream primer sequences for amplifying D20S430 are shown as SEQ ID NO.19 and SEQ ID NO.20; the upstream and downstream primer sequences for amplifying D20S158 are shown as SEQ ID NO.21 and SEQ ID NO.22; the upstream and downstream primer sequences for amplifying D20S443 are shown as SEQ ID NO.23 and SEQ ID NO.24; the upstream and downstream primer sequences for amplifying D20S466 are shown as SEQ ID NO.25 and SEQ ID NO.26; the upstream and downstream primer sequences for amplifying 20B-P2 are shown as SEQ ID NO.27 and SEQ ID NO.28; the upstream and downstream primer sequences for amplifying D20S902 are shown as SEQ ID NO.29 and SEQ ID NO.30; the upstream and downstream primer sequences for amplifying D20S481 are shown as SEQ ID NO.31 and SEQ ID NO.32; the upstream and downstream primer sequences for amplifying D20S601 are shown as SEQ ID NO.33 and SEQ ID NO.34; the upstream and downstream primer sequences for amplifying D20S206 are shown as SEQ ID NO.35 and SEQ ID NO.36.

[0011] Preferably, the fluorescence labeling groups of the primers are as follows:

[0012] Group 1: D15S1232, GTTTT001, D15S1513, 20A-P1, D20S164, 20A-P3, D20S466, 20B-P2;

[0013] The second group: D15S231, CHLC.ATC3C11, D15S1233, D20S430, D20S158, D20S443, D20S902, D20S481, D20S601, D20S206;

[0014] The third group: internal standard primers used in the multiplex amplification system.

[0015] Preferably, in the fluorescence-labeled groups, the fluorophores for each group are respectively: the first group is labeled with FAM fluorescence; the second group is labeled with HEX fluorescence; the third group is labeled with ORG fluorescence.

[0016] Preferably, the concentrations of the primer pairs corresponding to the STR loci in the amplification detection system are as follows: D15S1232 is 0.3 - 0.5 μM; GTTTT001 is 0.3 - 0.5 μM; D15S1513 is 0.9 - 1.0 μM; 20A-P1 is 0.1 - 0.2 μM; D20S164 is 0.1 - 0.2 μM; 20A-P3 is 0.1 - 0.2 μM; D20S466 is 0.1 - 0.2 μM; 20B-P2 is 0.2 - 0.3 μM;

[0017] D15S231 is 0.4 - 0.5 μM; CHLC.ATC3C11 is 0.8 - 0.9 μM; D15S1233 is 0.9 - 1.0 μM; D20S430 is 0.2 - 0.3 μM; D20S158 is 0.1 - 0.2 μM; D20S443 is 1.2 - 1.3 μM; D20S902 is 0.1 - 0.2 μM; D20S481 is 0.2 - 0.3 μM; D20S601 is 0.2 - 0.3 μM; D20S206 is 0.5 - 0.6 μM.

[0018] Preferably, the amplification system includes 7.5 μl of PCR reaction solution, 2.5 μl of primer mixture, 5 - 20 ng of template DNA, and nuclease-free pure water is added to 25 μl.

[0019] Preferably, the amplification program of the amplification system is pre-denaturation at 95°C for 5 min, 1 cycle; denaturation, annealing and extension at 94°C for 10 sec; 59°C for 90 sec, 25 cycles; final extension at 72°C for 20 min; incubation at 4°C.

[0020] Preferably, the template DNA of the STR fluorescence-labeled multiplex amplification system is from one or more of human blood, blood stain, semen, seminal stain, saliva, saliva stain, amniotic fluid, cord blood or abortion tissue.

[0021] In a second aspect, a kit includes the above STR fluorescence-labeled multiplex amplification system.

[0022] An application for detecting UPD of human chromosomes 15 and 20 uses the above STR fluorescence-labeled multiplex amplification system or the above kit to detect DNA.

[0023] Advantages of the implementation of the present invention: The STR fluorescence-labeled multiplex amplification system of the present invention screens out 18 STR loci with high polymorphism and individual recognition rate. The primers of these loci are divided into three groups by fluorescence labeling. Combined with the molecular weight internal standard, multi-color fluorescence detection can be achieved, significantly improving the detection efficiency and accuracy. In addition, the STR loci of the present invention can be used as an effective supplement to existing kits, which can significantly reduce the false positive rate, effectively improve the accuracy and reliability of detection. According to the positive judgment value standard, the uniparental disomy of different chromosomes can be accurately judged, providing an important basis for prenatal diagnosis and genetic disease detection, and having important clinical application value and broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0025] Figure 1 It is the amplification map of 6 STR loci on chromosome 15 according to the present invention;

[0026] Figure 2 It is the amplification map of 6 STR loci of the A system (paternal UPD) on chromosome 20 according to the present invention;

[0027] Figure 3 It is the amplification map of 6 STR loci of the B system (maternal UPD) on chromosome 20 according to the present invention;

[0028] Figure 4 It is the locus arrangement map of chromosome 15 according to the present invention;

[0029] Figure 5 It is the locus arrangement map of the A system (paternal UPD) on chromosome 20 according to the present invention;

[0030] Figure 6 It is the locus arrangement map of the B system (maternal UPD) on chromosome 20 according to the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0032] Embodiment 1

[0033] The STR fluorescent labeling multiplex amplification system provided in this embodiment includes 6 STR loci on human chromosome 15, 12 STR loci on human chromosome 20, and 36 corresponding primer pairs, and each primer pair carries a fluorescent dye label.

[0034] The 6 STR loci on human chromosome 15 are D15S1232, GTTTT001, D15S1513, D15S231, CHLC.ATC3C11, D15S1233; the 12 STR loci on chromosome 20 are 20A-P1, D20S164, 20A-P3, D20S430, D20S158, D20S443, D20S466, 20B-P2, D20S902, D20S481, D20S601, D20S206.

[0035] Among them, GTTTT001, CHLC.ATC3C11, 20A-P1, 20A-P3, D20S466, 20B-P2 are self-named.

[0036] The specific positions of the 6 STR loci located on chromosome 15 are shown in Table 1:

[0037] Table 1

[0038]

[0039] The specific positions of the 12 STR loci located on chromosome 20 are shown in Table 2:

[0040] Table 2

[0041]

[0042] Note: Sorted according to the physical positions of each locus on each chromosome. The position information is from GRCH38.

[0043] The primer sequences of the 6 STR loci located on chromosome 15 are shown in Table 3:

[0044] Table 3

[0045]

[0046] The primer sequences of the 12 STR loci located on chromosome 20 are shown in Table 4 as follows:

[0047] Table 4

[0048]

[0049] The primers can be divided into two groups, and the two groups are respectively labeled with different fluorescent labels:

[0050] The first group: For D15S1232, GTTTT001, D15S1513, 20A-P1, D20S164, 20A-P3, D20S466, 20B-P2, one primer at the 5'-end is labeled with FAM fluorescent label;

[0051] The second group: For D15S231, CHLC.ATC3C11, D15S1233, D20S430, D20S158, D20S443, D20S902, D20S481, D20S601, D20S206, one primer at the 5'-end is labeled with HEX fluorescent label;

[0052] The internal standard primer used in the multiplex amplification system has an ORG fluorescent label at the 5'-end.

[0053] In the Y-STR fluorescent label multiplex amplification detection system of this embodiment, the STR multiplex amplification system includes 7.5 μl of PCR reaction solution (containing DNA polymerase and PCR buffer), 2.5 μl of primer mixture, and 1 μl (5 - 20 ng) of template DNA, and nuclease-free pure water is added to make up to 25 μl.

[0054] The amplification program of the STR multiplex amplification system is pre-denaturation at 95 °C for 5 min, 1 cycle; denaturation annealing extension at 94 °C for 10 sec; 59 °C for 90 sec, 25 cycles; final extension at 72 °C for 20 min; incubation at 4 °C.

[0055] The amplification products of the STR multiplex amplification system are detected by capillary electrophoresis using a genetic analyzer, such as ABI3500 Dx, 3730XL. The detection results are analyzed by GeneMapper® ID-X software to obtain the amplification map and the fragment sizes of the products at each STR locus, and the status of the sample to be detected is determined through the results.

[0056] The STR multiplex amplification system and kit of the present invention are applicable to the extraction of DNA samples, and the DNA samples are extracted from, but not limited to, human blood, blood stains, semen, sperm stains, saliva, saliva stains, amniotic fluid, cord blood, and abortion tissues. The DNA extracted from the blood of both parents and amniotic fluid, cord blood, and abortion tissues is used for detection, and the best results can be obtained by comparing and interpreting the results.

[0057] Currently, there are few STR loci available for detection before and after the onset segments of UPD on chromosomes 15 and 20. There are only 4 STR loci in the existing kits for detecting UPD15. The present invention has screened out 18 STR loci that can be used to detect UPD on human chromosomes 15 and 20. Some of these loci have no unified naming yet. Among the 18 loci, 17 STR loci have relatively high heterozygosity, polymorphism, and individual discrimination power. The 17 STR loci are: D15S1232, GTTTT001, D15S1513, CHLC.ATC3C11, D15S1233, 20A-P1, D20S164, 20A-P3, D20S430, D20S158, D20S443, D20S466, 20B-P2, D20S902, D20S481, D20S601, D20S206.

[0058] The statistical results of the genetic data of the 17 STR loci are shown in Table 5, where:

[0059] Hobs: Observed heterozygosity;

[0060] HExp: Expected heterozygosity;

[0061] DP: Individual discrimination ability;

[0062] PIC: Polymorphism information content;

[0063] PE: Probability of excluding non-fathers.

[0064] Table 5

[0065]

[0066] The existing kits for detecting UPD of human chromosome 15 using STR multiplex amplification technology cover fewer loci, have insufficient locus polymorphisms, and there are problems with locus mutations. With the widespread clinical application of uniparental disomy kits, the phenomenon of false positives in the determination of duos often occurs. After triple verification, there are still about 5% of suspected positive results that need to be further clarified. The STR loci provided by the present invention can be used as an effective supplement to existing kits. By detecting and statistically analyzing 565 family samples of the Han population, before and after using the additional detection system, the false positive rate of UPD of chromosome 15 changed from 2.48% to 0, and the false positive rate of UPD of chromosome 20 was 0. Using the existing kits in combination with the STR multiplex amplification system of the present invention can accurately determine the final sample results.

[0067] The STR multiplex amplification system and kits provided by the present invention are applicable to a variety of types of test materials, including but not limited to DNA samples extracted from blood, blood spots, semen, sperm stains, saliva, saliva stains, amniotic fluid, cord blood, and abortion tissues. Using the kits provided by the present invention to detect the DNA samples of one or both parents and the DNA sample of the fetus, and analyzing and interpreting the sample results can provide key tips for fetal UPD15 and UPD20-related diseases during prenatal diagnosis.

[0068] Example 2

[0069] STR loci were screened from positions 1 Mb before and after the maternally and paternally derived UPD disease segments of chromosome 15 at 15q11.2-q13, the maternally derived UPD disease segment of chromosome 20 at 20q11.1-13.2, and the paternally derived UPD disease segment of chromosome 20 at 20q13.32. The loci were sourced from genome.ucsc.edu. The selected STR loci contain simple repeats of 3-5 bases and do not overlap with the loci covered by existing UPD detection kits.

[0070] Using Primer Premier 5 software, primers were designed for amplification before and after the core repeat regions of the selected STR loci. When amplifying a single locus, it was required that the amplification product be a single band without non-specific amplification products. After the primers were determined, 368 DNA samples extracted from the blood of the Chinese Han population were tested, and the locus heterozygosity, individual polymorphism, and individual recognition ability were calculated. Combining the amplification situation and locus efficiency, finally 6 STR loci were selected on chromosome 15: D15S1232, GTTTT001, D15S1513, D15S231, CHLC.ATC3C11, D15S1233, and the chromosomal segment where they are located is 15q12 - 15q14; 12 STR loci were selected on chromosome 20: 20A - P1, D20S164, 20A - P3, D20S430, D20S158, D20S443, D20S466, 20B - P2, D20S902, D20S481, D20S601, D20S206, and the chromosomal segments where they are located are 20q11.1 - 20q13.2 and 20q13.31 - 20q13.32. The specific positions of the STR loci on the chromosome are shown in Table 6:

[0071] Table 6

[0072]

[0073] The lengths of the amplification products of the 18 STR loci in the present invention are 100 - 550 bp. According to the lengths of the amplification product fragments of each locus, loci with different amplification product lengths were labeled with the same fluorescence, and primers for loci with similar fragment lengths were labeled with two different colors of fluorescence to ensure that multiple loci could be amplified simultaneously in one tube in the same reaction system. The primers of the 18 loci in the present invention were labeled with two different colors of fluorescence, FAM and HEX respectively.

[0074] FAM fluorescence labeling: One primer of each of D15S1232, GTTTT001, D15S1513, 20A - P1, D20S164, 20A - P3, D20S466, 20B - P2 has a FAM fluorescence label at the 5' end;

[0075] HEX fluorescence labeling: One primer of each of D15S231, CHLC.ATC3C11, D15S1233, D20S430, D20S158, D20S443, D20S902, D20S481, D20S601, D20S206 has a HEX fluorescence label at the 5' end.

[0076] The present invention includes three STR multiplex amplification systems, namely, the multiplex amplification system for detecting maternal and paternal UPD of chromosome 15, which includes 6 STR loci: D15S1232, GTTTT001, D15S1513, D15S231, CHLC.ATC3C11, D15S1233; the multiplex amplification system for detecting maternal UPD of chromosome 20, which includes 6 STR loci: 20A-P1, D20S164, 20A-P3, D20S430, D20S158, D20S443; the multiplex amplification system for detecting paternal UPD of chromosome 20, which includes 6 STR loci: D20S466, 20B-P2, D20S902, D20S481, D20S601, D20S206.

[0077] Example 3

[0078] 1. DNA samples

[0079] DNA of the blood of the father and mother and amniotic fluid or aborted tissue extracted by the magnetic bead method. A total of 3 DNA samples of the father, mother and fetus are considered as one family.

[0080] 2. Primers

[0081] The primers for the 6 STR loci in the multiplex amplification system for detecting maternal and paternal UPD15 are mixed into one primer Mix. The primers for the 6 STR loci in the multiplex amplification system for detecting maternal UPD20 are mixed into one primer Mix. The primers for the 6 STR loci in the multiplex amplification system for detecting paternal UPD20 are mixed into one primer Mix. The amplification product of each locus is amplified by two primers, one of which is labeled with FAM or HEX fluorophore at the 5' end. The primer information is shown in Table 7:

[0082] Table 7

[0083]

[0084]

[0085] 3. PCR amplification

[0086] Prepare the multiplex amplification reaction system. Mix 7.5 μl of PCR reaction solution, 2.5 μl of primer mixture and 14 μl of nuclease-free pure water to form a mixture. Add 1 μl (5 - 20 ng) of template DNA, mix well and centrifuge, then use the ABI® PCR System ProFlex thermal cycler for amplification. The amplification program is as follows: pre-denaturation at 95°C for 5 min, 1 cycle; denaturation, annealing and extension at 94°C for 10 sec; 59°C for 90 sec, 25 cycles; final extension at 72°C for 20 min; hold at 4°C.

[0087] 4. Electrophoresis Detection

[0088] Add 2 μl of spectral calibration reagent to 100 μl of deionized formamide, mix well by shaking, dispense 10 μl into each well, denature at 95 °C for 3 min, immediately cool on ice for 3 min, perform spectral calibration electrophoresis, and select "J6" for Dye Set. Add 20 μl of molecular weight internal standard to 1 ml of formamide, mix well and dispense into a 96-well plate, 9 μl / well, then add 1 μl of PCR amplification product, centrifuge and place on an ABI3500Dx genetic sequencer for detection.

[0089] 5. Data Analysis

[0090] Import the original data. In the File menu on the main page, select Add sample to project, find the sample file and select it, click add to list, then click add, and the sample file will be displayed in the Project window; select the analysis parameters and define the analysis method, panel, and size standard. Browse the original data of the sample electrophoresis, select the file name of a certain sample, and select "raw data" in the "sample" menu. Move the tracking line so that the cursor stops on the right side of the primer peak (before the first orange internal standard peak), and use the value displayed on the X-axis at the lower left corner of the window at this time as the starting point in the analysis parameters of the analysis method; click the green analysis button, a save project dialog box will appear, name it and save it, and the software will start processing the data. After the analysis is completed, analysis completed will be displayed at the lower left corner. Analyze the data obtained by GeneMapper® ID-X software and generate a map, that is Figure 1-3 。

[0091] As Figure 1-3 shown, by comparing and analyzing the test results of the father, mother, and fetal DNA samples, within a certain test section, if there are double peaks at one or more loci in the fetus and corresponding sized fragments can be found in the father and mother samples respectively, it represents that the sample is a normal sample; if all loci in the fetus are single peaks and corresponding sized fragments can be found in the mother sample, it represents that the sample is maternal isodisomy; if the fragment sizes of all loci in the fetus are exactly the same as those of the mother and there are double peaks at one or more loci, it represents that the sample is maternal heterodisomy; if all loci in the fetus are single peaks and corresponding sized fragments can be found in the father sample, it represents that the sample is paternal isodisomy; if the fragment sizes of all loci in the fetus are exactly the same as those of the father and there are double peaks at one or more loci, it represents that the sample is paternal heterodisomy.

[0092] 6. Experimental Results

[0093] Table 8-10 respectively lists the test results of 1 normal fetal sample, 1 sample of maternal isodisomy of chromosome 15, and 1 sample of paternal isodisomy of chromosome 15. By jointly analyzing these results with the test results of the existing UPD15 detection kit, the final sample results can be accurately determined;

[0094] Table 8

[0095]

[0096] Table 9

[0097]

[0098] Table 10

[0099]

[0100] Advantages of the implementation of the present invention: The STR fluorescence-labeled multiplex amplification system of the present invention screens out 18 STR loci with high polymorphism and individual recognition rate. The primers of these loci are divided into three groups by fluorescence labeling. Combining with the molecular weight internal standard, multi-color fluorescence detection can be achieved, significantly improving the detection efficiency and accuracy. In addition, the STR loci of the present invention can be used as an effective supplement to the existing kits, which can significantly reduce the false positive rate, effectively improve the accuracy and reliability of the detection, provide an important basis for prenatal diagnosis and genetic disease detection, and have important clinical application value and broad market prospects.

[0101] The above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A STR fluorescent labeling composite amplification system, characterized in that: include: 18 primer pairs designed for 6 STR loci on human chromosome 15 and 12 STR loci on human chromosome 20, wherein the 6 STR loci on human chromosome 15 are: D15S1232, GTTTT001, D15S1513, D15S231, CHLC.ATC3C11, D15S1233; the 12 STR loci on human chromosome 20 are: 20A-P1, D20S164, 20A-P3, D20S430, D20S158, D20S443, D20S466, 20B-P2, D20S902, D20S481, D20S601, D20S206; Each primer pair carries a fluorescent dye marker; the upstream and downstream primer sequences for amplifying the D15S1232 are shown in SEQ ID NO.1 and SEQ ID NO.2; the upstream and downstream primer sequences for amplifying the GTTTT001 are shown in SEQ ID NO.3 and SEQ ID NO.4; the upstream and downstream primer sequences for amplifying the D15S1513 are shown in SEQ ID NO.5 and SEQ ID NO.6; the upstream and downstream primer sequences for amplifying the D15S231 are shown in SEQ ID NO.7 and SEQ ID NO.8; the upstream and downstream primer sequences for amplifying the CHLC.ATC3C11 are shown in SEQ ID NO.9 and SEQ ID NO.10; the upstream and downstream primer sequences for amplifying the D15S1233 are shown in SEQ ID NO.11 and SEQ ID NO.12; The upstream and downstream primer sequences for amplifying the 20A-P1 are shown in SEQ ID NO.13 and SEQ ID NO.14; the upstream and downstream primer sequences for amplifying the D20S164 are shown in SEQ ID NO.15 and SEQ ID NO.16; the upstream and downstream primer sequences for amplifying the 20A-P3 are shown in SEQ ID NO.17 and SEQ ID NO.18; the upstream and downstream primer sequences for amplifying the D20S430 are shown in SEQ ID NO.19 and SEQ ID NO.20; the upstream and downstream primer sequences for amplifying the D20S158 are shown in SEQ ID NO.21 and SEQ ID NO.22; the upstream and downstream primer sequences for amplifying the D20S443 are shown in SEQ ID NO.23 and SEQ ID NO.24; the upstream and downstream primer sequences for amplifying the D20S466 are shown in SEQ ID NO.25 and SEQ ID NO.26; the upstream and downstream primer sequences for amplifying the 20B-P2 are shown in SEQ ID NO.27 and SEQ ID NO. ID NO.28; the upstream and downstream primer sequences for amplifying the D20S902 are shown in SEQ ID NO.29 and SEQ ID NO.30; the upstream and downstream primer sequences for amplifying the D20S481 are shown in SEQ ID NO.31 and SEQ ID NO.32; the upstream and downstream primer sequences for amplifying the D20S601 are shown in SEQ ID NO.33 and SEQ ID NO.34; the upstream and downstream primer sequences for amplifying the D20S206 are shown in SEQ ID NO.35 and SEQ ID NO.36; The fluorescent labels of the primers are grouped as follows: Group 1: D15S1232, GTTTT001, D15S1513, 20A-P1, D20S164, 20A-P3, D20S466, 20B-P2; The second group: D15S231, CHLC.ATC3C11, D15S1233, D20S430, D20S158, D20S443, D20S902, D20S481, D20S601, D20S206; The third group: the internal standard primers used in the composite amplification system.

2. The STR fluorescent labeling composite amplification system according to claim 1, characterized in that: In the fluorescent marker groups, the fluorescent markers of each group are: the first group is marked with FAM fluorescent marker; the second group is marked with HEX fluorescent marker; and the third group is marked with ORG fluorescent marker.

3. The STR fluorescent labeling composite amplification system according to claim 1, characterized in that: The primer pair concentration corresponding to the D15S1232 is 0.3-0.5 μM; the primer pair concentration corresponding to the GTTTT001 is 0.3-0.5 μM; the primer pair concentration corresponding to the D15S1513 is 0.9-1.0 μM; the primer pair concentration corresponding to the 20A-P1 is 0.1-0.2 μM; the primer pair concentration corresponding to the D20S164 is 0.1-0.2 μM; the primer pair concentration corresponding to the 20A-P3 is 0.1-0.2 μM; the primer pair concentration corresponding to the D20S466 is 0.1-0.2 μM; the primer pair concentration corresponding to the 20B-P2 is 0.2-0.3 μM; The primer pair concentration corresponding to the D15S231 is 0.4-0.5 μM; the primer pair concentration corresponding to the CHLC.ATC3C11 is 0.8-0.9 μM; the primer pair concentration corresponding to the D15S1233 is 0.9-1.0 μM; the primer pair concentration corresponding to the D20S430 is 0.2-0.3 μM; the primer pair concentration corresponding to the D20S158 is 0.1-0.2 μM; the primer pair concentration corresponding to the D20S443 is 1.2-1.3 μM; the primer pair concentration corresponding to the D20S902 is 0.1-0.2 μM; the primer pair concentration corresponding to the D20S481 is 0.2-0.3 μM; the primer pair concentration corresponding to the D20S601 is 0.2-0.3 μM; the primer pair concentration corresponding to the D20S206 is 0.5-0.6 μM.

4. The STR fluorescent labeling composite amplification system according to claim 1, characterized in that: The amplification system includes 7.5 μl of PCR reaction solution, 2.5 μl of primer mixture, 5-20 ng of template DNA, and nuclease-free pure water is added to 25 μl.

5. The STR fluorescent labeling composite amplification system according to claim 1, characterized in that: The amplification program of the amplification system is pre-denaturation at 95°C for 5 minutes, 1 cycle; denaturation annealing extension at 94°C for 10 seconds; 59°C for 90 seconds, 25 cycles; final extension at 72°C for 20 minutes; and insulation at 4°C.

6. The STR fluorescent labeling composite amplification system according to claim 1, characterized in that: The template DNA of the STR fluorescent labeling composite amplification system comes from one or more of human blood, blood spots, semen, sperm spots, saliva, saliva spots, amniotic fluid, umbilical cord blood or abortion tissue.

7. A kit comprising the STR fluorescent labeling composite amplification system according to any one of claims 1 to 6.

Images