CN116837081A - YSTR reagent, system and application for rapid identification of biological individuals - Google Patents
YSTR reagent, system and application for rapid identification of biological individuals Download PDFInfo
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- CN116837081A CN116837081A CN202310011135.7A CN202310011135A CN116837081A CN 116837081 A CN116837081 A CN 116837081A CN 202310011135 A CN202310011135 A CN 202310011135A CN 116837081 A CN116837081 A CN 116837081A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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Abstract
The application discloses a reagent, a system and application for rapidly identifying biological individuals. Belongs to the field of biotechnology. The application aims to solve the problem of providing a reagent, a system and application for rapidly identifying biological individuals, in particular to a YSTR microfluidic chip amplification detection system. The application discloses the use of a product for detecting the gene sequences of DYS460, YGATAH4, DYS390, DYS19, DYS449, DYS481, DYF387S1a, DYF387S1b, DYS576, DYS635, DYS385a, DYS385b, DYS627, DYS438, DYS391, DYS393, DYS389I, DYS570, DYS389II, DYS392, DYS518, DYS437, DYS456, DYS439, DYS448, DYS533 and DYS458 in any of the following: a1 Identifying or assisting a biological individual; a2 Preparing a product that identifies or assists the biological subject; a3 STR typing; a4 Preparing STR parting products; a5 Amplification of the YSTR microfluidic chip; a6 Preparing a YSTR microfluidic chip amplification product. The amplification system has high detection sensitivity and accurate result, and can be used for male component identification and family investigation.
Description
Technical Field
The application relates to a YSTR reagent, a system and application for rapidly identifying biological individuals in the technical field of molecular biology.
Technical Field
The PCR-STR typing technology can be used for identifying individuals of the biological evidence at the crime scene, so that the suspects can be identified or excluded. Conventional STR typing processes include DNA extraction, PCR amplification, electrophoretic separation, and typing profile analysis. The whole detection process generally needs 6-8 hours, and is long in time consumption, multiple in steps, complex in operation, high in requirements on professional skills of operators and easy to cause sample pollution risks. At present, some serious and sudden cases often need to obtain test results in a short time to provide directions for case investigation. The conventional DNA typing detection method cannot meet the public security combat demand, and innovation is needed to be realized in the direction of quick and convenient integration on site.
The microfluidic chip technology integrates basic operation units of sample preparation, reaction, separation, detection and the like in biological, chemical and medical analysis processes onto a micron-scale chip, and automatically completes the whole analysis process. Compared with the traditional analysis method, the method has the advantages of small volume, less consumption of samples and reagents, easiness in realizing automation of operation flow, full integration and the like. At present, many scholars have developed many researches on rapid DNA inspection based on microfluidic chip technology, such as microfluidic chip PCR technology, microfluidic chip electrophoresis technology, integrated chip technology, and the like. On the basis, a series of DNA rapid inspection instruments are also developed and produced successively and applied, and STR rapid detection reagents matched with the instruments are also an indispensable part for realizing integrated rapid detection. At present, several types of foreign rapid inspection instruments mainly comprise: 200; single channel->ID and ANDE TM 6C, the above devices are all matched with an STR kit for individual identification, and no report has been made on the use of a Y-STR system for rapid detection devices.
Disclosure of Invention
The application solves the problem of how to provide a reagent, a system and application for rapidly identifying biological individuals, in particular to a YSTR microfluidic chip amplification detection system.
Based on the above problems, the present application provides the following applications.
Use of a substance for detecting loci which are the following 27 loci:
the DYS460 locus, the YGATAH4 locus, the DYS390 locus, the DYS19 locus, the DYS449 locus, the DYS481 locus, the DYF387S1a locus, the DYF387S1b locus, the DYS576 locus, the DYS635 locus, the DYS385a locus, the DYS385b locus, the DYS627 locus, the DYS438 locus, the DYS391 locus, the DYS393 locus, the DYS389I locus, the DYS570 locus, the DYS389II locus, the DYS392 locus, the DYS518 locus, the DYS437 locus, the DYS456 locus, the DYS439 locus, the DYS448 locus, the DYS533 locus and the DYS458 locus.
The application is any one of the following:
a1 Detecting the use of a substance at a locus in identifying or assisting an individual of a living being;
A2 Use of a substance that detects a locus in the preparation of a product that identifies or assists in the biological individual;
a3 Detecting the use of a locus species in STR typing;
a4 Detecting the use of a locus material in the preparation of an STR typing product;
a5 Detecting the application of the locus substance in YSTR microfluidic chip amplification;
a6 The application of a substance for detecting a locus in preparing a YSTR microfluidic chip amplification product.
In the present application, the DYF387S1a locus and the DYF387S1b locus are double copy loci.
In the present application, the DYS385a gene locus and the DYS385b gene locus are double copy gene loci.
In the present application, the STR is Y-STR.
In the present application, a locus (locus) is the position occupied by a gene on a chromosome. At the molecular level, is a DNA sequence with genetic effects. In the sense that a pair of chromosomes can be thought of as two parallel lines, a given location on a chromosome is better than a point or segment of the corresponding location on the two parallel lines, called a locus.
STR loci, also known as "short tandem repeats", "microsatellite DNA". STR is an abbreviation for english short tandem repeat. A repeat sequence having a repeat unit length of 2 to 6 base pairs. Is the most widely used genetic marker of length polymorphism in forensic material science, and the length polymorphism is derived from individual difference of copy number of repeated units.
Y-STR is a judicial gene detection technique for determining whether a person contains a particular Y chromosome fragment, and thus knowing whether the person belongs to a particular family.
The DYS460 locus is as GenBank: MK990453.1 (18-JUN-2019) shows the DYS390 locus as Gen Bank: MZ325913.1 (29-JUN-2021), DYS19 locus such as GenBank: MT607266.1 (30-JUN-2020) as shown, DYS449 locus as shown in GenBank: BV679975.1 (04-JUN-2009) as shown, DYS481 locus as shown in GenBank: MW074021.1 (28-OCT-2020) as shown, DYF387S1a locus and DYF387S1b locus as shown in GenBank: MZ 908776.1 (16-NOV-2021) as shown, DYS576 locus as shown in GenBank: MW392186.1 (17-JAN-2021) as shown, DYS635 locus as shown in GenBank: MW074056.1 (28-OCT-2020) as shown, DYS385a locus as shown in GenBank: MK990450.2 (01-OCT-2021) as shown, DYS627 locus as shown in GenBank: MH814953.1 (09-JAN-2019) as shown, DYS438 locus as shown in GenBank: MZ325916.1 (29-JGen-2021) as shown, DYS 20235 locus as shown in GenBank: MW392186.1 (17-JAN-2021) as shown, DYS 20235 locus as shown in GenBank: MW074056.1 (28-JAN-9) as shown, DYS 074056.1-JA-9) as shown, DYS385 locus as shown in GenBank (35-JA-9) as shown in GenBank: 35-9-JA-9 as shown, MZ-9 as shown in Gen-9, MZ-9 as shown in DYS 393-990450.2 (9) as shown in Gen-9) as shown, MYS 393-9 locus as shown in Gen-9 (9) as shown, as shown in GenP-9 as shown, 35, and 35, and, as, 9, and, 9, and, 9, 9, the DYS456 locus is shown as GenBank: OK330029.1 (16-NOV-2021), the DYS 439 locus is shown as GenBank: MW073964.1 (28-OCT-2020), the DYS448 locus is shown as GenBank: MZ325917.1 (29-JUN-2021), the DYS533 locus is shown as GenBank: MW074029.1 (28-OCT-2020) and the DYS458 locus is shown as GenBank: MT607307.1 (23-DEC-2021).
In the above application, the substance is any one of the following:
d1 An in vitro nucleic acid amplification primer composition that specifically amplifies the locus;
d2 An in vitro nucleic acid amplification reagent comprising D1) the in vitro nucleic acid amplification primer composition;
d3 A kit comprising D1) the in vitro nucleic acid amplification primer composition or D2) the in vitro nucleic acid amplification reagent.
In the above application, the in vitro nucleic acid amplification primer composition consists of 27 primer pairs for the 27 loci, the 27 primer pairs are composed of a primer pair DYS460-FR for detecting a DYS460 locus, a primer pair YGATAAH 4-FR for detecting a YGATAAH 4 locus, a primer pair DYS390-FR for detecting a DYS390 locus, a primer pair DYS19-FR for detecting a DYS19 locus, a primer pair DYS449-FR for detecting a DYS449 locus, a primer pair DYS481-FR for detecting a DYS481 locus, a primer pair DYF387S1a locus and a primer pair DYF387S1ab-FR for detecting a DYS 387S1b locus, a primer pair DYS576-FR for detecting a DYS576 locus, a primer pair DYS635-FR for detecting a DYS385 locus and a primer pair DYS385ab-FR for detecting a stand, a primer pair DYS627-FR for detecting a stand, and a primer pair DYS438-FR for detecting a DYS385b locus the primer pair for detecting the DYS391 locus comprises a primer pair for detecting the DYS391-FR, a primer pair for detecting the DYS393 locus, a primer pair for detecting the DYS389I locus, a primer pair for detecting the DYS570 locus, a primer pair for detecting the DYS389II locus, a primer pair for detecting the DYS392 locus, a primer pair for detecting the DYS518-FR, a primer pair for detecting the DYS437 locus, a primer pair for detecting the DYS456-FR, a primer pair for detecting the DYS439-FR, a primer pair for detecting the DYS448 locus, a primer pair for detecting the DYS533-FR and a primer pair for detecting the DYS458 locus;
The primer pair for detecting DYS460 locus is two single-stranded DNA of which the nucleotide sequence is sequence 1 and sequence 2, the primer pair for detecting YGATAAH 4 locus is two single-stranded DNA of which the nucleotide sequence is sequence 3 and sequence 4, the primer pair for detecting DYS390 locus is two single-stranded DNA of which the nucleotide sequence is sequence 5 and sequence 6, the primer pair for detecting DYS19 locus is two single-stranded DNA of which the nucleotide sequence is sequence 7 and sequence 8, the primer pair for detecting DYS449 locus is two single-stranded DNA of which the nucleotide sequence is sequence 9 and sequence 10, the primer pair for detecting DYS481 locus is two single-stranded DNA of which the nucleotide sequence is sequence 11 and sequence 12, the primer pair for detecting DYF387S1a locus and DYF387S1b locus is two single-stranded DNA of which the nucleotide sequence 13 and sequence 14, the primer pair for detecting DYS576 locus is two single-stranded DNA of which the nucleotide sequence 15 and sequence 16, the primer pair for detecting DYS635 locus is two single-stranded DNA of which the nucleotide sequence is sequence 17 and sequence 18, the primer pair for detecting DYS385a locus and detecting DYS385b is two single-stranded DNA of which the nucleotide sequence is sequence 19 and sequence 20, the primer pair for detecting DYS627 locus is two single-stranded DNA of which the nucleotide sequence is sequence 21 and sequence 22, the primer pair for detecting DYS438 locus is two single-stranded DNA of which the nucleotide sequence is sequence 23 and sequence 24, the primer pair for detecting DYS391 locus is two single-stranded DNA of which the nucleotide sequence is sequence 25 and sequence 26, the primer pair for detecting DYS393 locus is two single-stranded DNA of which the nucleotide sequence is sequence 27 and sequence 28, the primer pair for detecting DYS389I locus is two single-stranded DNA of which the nucleotide sequence is sequence 29 and sequence 30, the primer pair for detecting DYS570 locus is two single-stranded DNA of which the nucleotide sequence is sequence 31 and sequence 32, the primer pair for detecting DYS389II locus is two single-stranded DNA of which the nucleotide sequence is sequence 33 and sequence 34, the primer pair for detecting DYS392 locus is two single-stranded DNA of which the nucleotide sequence is sequence 35 and sequence 36, the primer pair for detecting DYS518 locus is two single-stranded DNA of which the nucleotide sequence is sequence 37 and sequence 38, the primer pair for detecting DYS437 locus is two single-stranded DNA of which the nucleotide sequence is sequence 39 and sequence 40, the primer pair for detecting DYS456 locus is two single-stranded DNA of which the nucleotide sequence is sequence 41 and sequence 42, the primer pair for detecting DYS439 locus is two single-stranded DNA of which the nucleotide sequence 43 and sequence 44, the primer pair for detecting DYS448 locus is two single-stranded DNA of which the nucleotide sequence 45 and sequence 46, and the primer pair for detecting DYS437 locus is single-stranded DNA of which the nucleotide sequence 48 and the nucleotide sequence 48 is single-stranded DNA of which the nucleotide sequence 48 and the primer pair for detecting DYS533 is sequence 50.
In the above application, the composition of the in vitro nucleic acid amplification primer composition is as follows:
77 parts of DYS460-FR,128 parts of YGATAH4-FR,340 parts of DYS390-FR,381 parts of DYS19-FR,305 parts of DYS449-FR,104 parts of DYS481-FR,97 parts of DYS 387S1ab-FR,115 parts of DYS576-FR,170 parts of DYS635-FR,305 parts of DYS385ab-FR,67 parts of DYS627-FR,400 parts of DYS438-FR,429 parts of DYS391-FR,177 parts of DYS393-FR,114 parts of DYS389I-FR,132 parts of DYS570-FR,333 parts of DYS389II-FR,333 parts of DYS392-FR,162 parts of DYS518-FR,333 parts of DYS437-FR,146 parts of DYS456-FR,109 parts of DYS439-FR,171 parts of DYS448-FR,333 parts of DYS 533-FR; the parts are parts by weight.
The concentrations of the 27 primer pairs in the in vitro nucleic acid amplification reaction system are as follows: the YGATAAH-FR is 15mM, the YGATAAH-4-FR is 26mM, the DYS390-FR is 68mM, the DYS19-FR is 76mM, the DYS449-FR is 61mM, the DYS481-FR is 21mM, the DYF387S1ab-FR is 19mM, the DYS576-FR is 23mM, the DYS635-FR is 34mM, the DYS385ab-FR is 61mM, the DYS627-FR is 13mM, the DYS438-FR is 80mM, the DYS391-FR is 86mM, the DYS393-FR is 35mM, the DYS389I-FR is 23mM, the DYS570-FR is 26mM, the DYS389II-FR is 67mM, the DYS392-FR is 67mM, the DYS518-FR is 32mM, the DYS635-FR is 34mM, the DYS438-FR is 67mM, the DYS533-FR is 30mM, the DYS448-FR is 67mM, the DYS-FR is 30mM, the DYS-FR is 48 mM, and the DYS-FR is 67 mM.
The concentrations of the 27 primer pairs in the in vitro nucleic acid amplification reaction system are as follows:
in the above application, one single-stranded DNA or both single-stranded DNAs of each of the 27 primer pairs is labeled with a nucleic acid label.
In the above, the nucleic acid labels may be radioisotope labels and non-radioactive labels.
In the above, the nucleic acid label may be a radioactive phosphate, hapten or a fluorophore label.
In the above application, the nucleic acid label is a fluorescent label.
In the above, the 5' -end of one single-stranded DNA of the two single-stranded DNAs of each of the 27 primer pairs is labeled with a fluorescent group.
In the above, all fluorescent labels are labeled at the 5' end of the forward primer F of the detection locus primer pair. Namely the 5' -end of DYS460-F, YGATAH4-F, DYS390-F, DYS19-F, DYS449-F, DYS481-F, DYF387S1ab-F, DYS576-F, DYS635-F, DYS385ab-F, DYS627-F, DYS438-F, DYS391-F, DYS393-F, DYS389I-F, DYS570-F, DYS389II-F, DYS392-F, DYS518-F, DYS437-F, DYS456-F, DYS439-F, DYS448-F, DYS533-F or DYS 458-F.
In the above application, the 27 primer pairs label 4 fluorophores, the primer pair for detecting the DYS460 locus, the primer pair for detecting the YGATAH4 locus, the primer pair for detecting the DYS390 locus, the primer pair for detecting the DYS19 locus, the primer pair for detecting the DYS449 locus, the primer pair for detecting the DYS481 locus, and the primer pair for detecting the DYF387S1a locus and the DYF387S1b locus label fluorophores named fluorophore a; a primer pair for detecting the DYS576 locus, a primer pair for detecting the DYS635 locus, a primer pair for detecting the DYS385a locus and the DYS385B locus, a primer pair for detecting the DYS627 locus, a primer pair for detecting the DYS438 locus, and a primer pair for detecting the DYS391 locus labeled with a fluorophore designated as fluorophore B; a primer pair for detecting the DYS393 locus, a primer pair for detecting the DYS389I locus, a primer pair for detecting the DYS570 locus, a primer pair for detecting the DYS389II locus, a primer pair for detecting the DYS392 locus, and a primer pair for detecting the DYS518 locus label a fluorophore designated as fluorophore C; a primer pair for detecting the DYS437 locus, a primer pair for detecting the DYS456 locus, a primer pair for detecting the DYS439 locus, a primer pair for detecting the DYS448 locus, a primer pair for detecting the DYS533 locus, and a primer pair for detecting the DYS458 locus labeled with a fluorophore designated as fluorophore D;
The fluorescent group A, the fluorescent group B and the fluorescent group C and the fluorescent group D are different compounds.
In the above, the wavelengths of the emitted light of the fluorescent group a, the fluorescent group B, the fluorescent group C and the fluorescent group B are different, and different colors are generated under the excitation of the excitation light.
In the above application, the fluorescent group is selected from at least one of FAM, VIC, HEX, TRT, CY, CY5, ROX, JOE, FITC, TET, NED, TAMRA, LC RED640, LC RED705, quasar705, or Texas RED.
Primer pairs for detecting the DYS460 locus, primer pairs for detecting the YGATAH4 locus, primer pairs for detecting the DYS390 locus, primer pairs for detecting the DYS19 locus, primer pairs for detecting the DYS449 locus, primer pairs for detecting the DYS481 locus, primer pairs for detecting the DYF387S1a locus and primer pairs for detecting the DYF387S1b locus label FAM fluorescent markers; a primer pair for detecting the DYS576 locus, a primer pair for detecting the DYS635 locus, a primer pair for detecting the DYS385a locus and the DYS385b locus, a primer pair for detecting the DYS627 locus, a primer pair for detecting the DYS438 locus, and a primer pair for detecting the DYS391 locus labeled HEX fluorescent markers; a primer pair for detecting the DYS393 locus, a primer pair for detecting the DYS389I locus, a primer pair for detecting the DYS570 locus, a primer pair for detecting the DYS389II locus, a primer pair for detecting the DYS392 locus, and a primer pair for detecting the DYS518 locus label TAMRA fluorescent markers; the primer pair for detecting the DYS437 locus, the primer pair for detecting the DYS456 locus, the primer pair for detecting the DYS439 locus, the primer pair for detecting the DYS448 locus, the primer pair for detecting the DYS533 locus and the primer pair for detecting the DYS458 locus are labeled with ROX fluorescent markers.
In order to solve the problems, the application also provides a product.
The product contains the substance for detecting loci described above, and has at least one of the following uses:
g1 For identifying or assisting a biological individual;
g2 For use in the preparation of a product for identifying or assisting biological subjects;
g3 For STR typing;
g4 For preparing STR parting products;
g5 For amplification of a YSTR microfluidic chip;
g6 Used for preparing YSTR microfluidic chip amplification products.
In order to solve the problems, the application also provides a biological individual identification method.
The biological individual identification method comprises the steps of using genome DNA of a sample to be detected as a template in an in-vitro nucleic acid amplification system, performing in-vitro nucleic acid amplification by using the substance for detecting the genetic locus, the in-vitro nucleic acid amplification primer composition or the product, and performing biological individual identification according to the in-vitro nucleic acid amplification product.
The in vitro nucleic acid amplification may be performed in a microfluidic chip.
In the above, the in vitro nucleic acid amplification may be PCR.
The content of the in vitro nucleic acid amplification primer composition in the in vitro nucleic acid amplification system meets the following conditions: primer combination: DYS460-FR 15mM, YGATAAH4-FR 26mM, DYS390-FR 68mM, DYS19-FR 76mM, DYS449-FR 61mM, DYS481-FR 21mM, DYS 387ab-FR 19mM, DYS576-FR 23mM, DYS635-FR 34mM, DYS385ab-FR 61mM, DYS627-FR 13mM, DYS438-FR 80mM, DYS391-FR 86mM, DYS393-FR 35mM, DYS389I-FR 23mM, DYS570-FR 26mM, DYS389II-FR 67mM, DYS392-FR 67mM, DYS518-FR 32mM, DYS437-FR 67mM, DYS456-FR 29mM, DYS448-FR 34mM, DYS533-FR 67mM, and DYS458-FR 30mM.
In the above, the final concentrations of the two primers in each primer pair are the same in an in vitro nucleic acid amplification system.
In the above, the amplification reagents include RTyper27 Primer and RTyper27 PCR Mix.
The RTyper27 Primer comprises the 27 pairs of primers, wherein the final concentration of each Primer pair in the RTyper27 Primer is DYS460-FR (77 mM), YGATAAH 4-FR (128 mM), DYS390-FR (340 mM), DYS19-FR (381 mM), DYS449-FR (305 mM), DYS481-FR (104 mM), DYF387S1ab-FR (97 mM), DYS576-FR (115 mM), DYS635-FR (170 mM), DYS385ab-FR (305 mM), DYS627-FR (67 mM), DYS438-FR (400 mM), DYS391-FR (429 mM), DYS393-FR (177 mM), DYS389I-FR (114 mM), DYS570-FR (132 mM), DYS389II-FR (333 mM), DYS392-FR (333 mM), DYS518-FR (162 mM), S-FR (333 mM), DYS 385-FR (146 mM), DYS533-FR (149 mM), and DYS533-FR (533 mM) in the same Primer pair system.
The RTyper27 PCR Mix was purchased from Xinhai biotechnology Co., ltd (cat. NH 9365).
The RTyper27 PCR Mix may be an optimized RTyper27 PCR Mix.
The optimized RTyper27 PCR Mix: the RTyper27 PCR Mix further comprises Taq enzyme with a final concentration of 4U/10. Mu.L and MgCl with a final concentration of 0.2. Mu.M/10. Mu.L 2 。
The preparation method of the optimized RTyper27PCR Mix comprises the following steps: RTyper27PCR Mix (available from New sea Biotechnology Co., ltd., st. No. NH 9365) was added with Taq enzyme and MgCl2 to give a final concentration of Taq enzyme of 4U/10. Mu.L, mgCl 2 The final concentration was 0.2. Mu.M/10. Mu.L.
The Taq enzyme may be a hot start Taq enzyme.
The optimized RTyper27PCR Mix may also contain 2% -4% glycerol by mass volume.
The mass volume ratio of 2% -4% of glycerin can be 2%, 2.5%, 3%,3.3%, 3.5% or 4%.
The RTyper27PCRThe composition of Mix is as follows: taq enzyme, magnesium chloride, dNTPs, potassium chloride, BSA and Tris-HCL buffer solution are started at a hot state.
The RTyper27PCR Mix can be directly used, or the RTyper27PCR Mix can be prepared into RTyper27PCR Mix freeze-dried powder for transportation or storage.
In the above, the RTyper27PCR Mix lyophilized powder can be prepared according to the method comprising the following steps: uniformly mixing the freeze-dried auxiliary material liquid I with the RTyper27PCR Mix according to the volume ratio of 1:1, and freeze-drying to obtain RTyper27PCR Mix freeze-dried powder;
in the above, the freeze-dried auxiliary material liquid I contains 22% of trehalose by mass and 10% of dextran by mass. The freeze-dried auxiliary material liquid I consists of 22% of trehalose by mass fraction, 10% of glucan by mass fraction and water.
The freeze-dried auxiliary material liquid I can also contain 2-4% of glycerol by mass and volume.
The mass volume ratio of 2% -4% of glycerin can be 2%, 2.5%, 3%,3.3%, 3.5% or 4%.
The preparation method of the freeze-dried auxiliary liquid I comprises the following steps:
the lyophilized auxiliary solution I was prepared by weighing 0.22g of trehalose and 0.1g of dextran with a balance to a volume of 1mL with water.
In the above, the RTyper27Primer is prepared from the following raw materials: RTyper27Primer, 1×TTE.
The RTyper27Primer can be directly used or prepared into RTyper27Primer freeze-dried powder for transportation or storage.
In the above, the RTyper27Primer lyophilized powder can be prepared according to the method comprising the following steps:
mixing trehalose solution with 15% mass fraction and mannitol solution with 15% mass fraction according to the volume ratio of 1:8, mixing to obtain freeze-dried auxiliary material liquid II; and then uniformly mixing the freeze-dried auxiliary material liquid II and the RTyper27Primer according to the volume ratio of 1:1, and freeze-drying to obtain the RTyper27Primer freeze-dried powder.
The solvent of trehalose with mass fraction of 15% and mannitol with mass fraction of 15% can be water.
The preparation method of the freeze-dried auxiliary liquid II is as follows:
Weighing 0.15g mannitol to 1ml by a balance to obtain mannitol; weighing 0.15g of trehalose to a volume of 1ml by using a balance to obtain a trehalose solution; mixing the mannitol solution and the trehalose solution according to the volume ratio of 8:1 to form the freeze-dried auxiliary material liquid II.
The freeze-drying method of the RTyper27 Primer freeze-dried powder comprises the following steps:
uniformly mixing the freeze-dried auxiliary material liquid II and the primer mixed liquid according to the volume ratio of 1:1, and freeze-drying to obtain a primer mixed liquid freeze-drying reagent;
the primer mixture contains 13.3% of trehalose by mass and 1.67% of dextran by mass.
In the above, the conditions of the freeze-drying are as follows:
pre-freezing: -55 ℃,40min; sublimation drying: 240min at-45 ℃,360min at-35 ℃ and 240min at-25 ℃; and (5) analysis and drying: 20 ℃ for 360min.
In the above, the sample to be tested is derived from epithelial cells or blood cells of the throat, tonsil or oral cavity, or peripheral blood.
The throat, tonsil, oral epithelial cells or blood cell epithelial cells may be collected with a throat swab.
The peripheral blood may be from fingertip blood.
The fingertip blood is preserved by a blood card.
The RTyper27 PCR Mix was purchased from Xinhai biotechnology Co., ltd (cat. NH 9365).
In the application, the whole set of in-vitro nucleic acid amplification operation can be controlled by a microfluidic chip amplification technology.
Sample lysis can be performed by repeatedly pumping 20 times, extracting the feed solution to the amplification chip, and lysing for 10min at 95 ℃.
The amplification conditions can be that the amplification is started after the completion of the cleavage, and the thermal cycle parameter is 95 ℃ for 1min;95℃10s,59℃1min,72℃20s,28 cycles; and at 60℃for 10min.
The electrophoresis separation can be that the PCR amplification product is discharged, electrophoresis is started, the sample injection voltage is 480V, and the sample injection time is 180s; separation voltage 4600V, separation time 3600s; electrophoresis temperature was 59 ℃.
Data was collected and results analyzed using the companion software BioStrGenotyping v2.0
In the present application, the locus is derived from any one of the following:
h1 A primates;
h2 Ape subgenera;
h3 A human family;
h4 A) humans;
h5 Intellectual planting.
Advantageous effects
The application discloses a reagent, a system and application for rapidly identifying biological individuals. An amplification detection system suitable for YSTR microfluidic chips is disclosed. And the primer mixed solution and the amplification premix in the system are prepared into freeze-dried pellets, and the freeze-dried pellets have full appearance, smoother and non-atrophic surface, uniform color and good porosity. And dissolving the freeze-dried pellets and then using the freeze-dried pellets for amplification detection of the YSTR microfluidic chip. The results show that: the oral swab sample is subjected to full-integration detection, and compared with the conventional laboratory PCR-CE platform result, the full-integration detection result is completely typed, has no allele deletion and is completely consistent with the conventional result. And (3) carrying out full-integration detection on the oral swab and the blood card, wherein the allele peak height value is more than or equal to 200RFU (RFU value refers to relative fluorescence intensity) is taken as an analysis threshold value, and the sample can obtain complete Y-STR typing without allele loss. The allele peak height is more than or equal to 200RFU as a standard, a DNA standard 2800M is used as a template, and a RTyperY27 liquid reagent detection system and a freeze-drying reagent detection system can obtain complete typing; the peak heights of individual alleles in the freeze-dried reagent typing graph are lower (RFU values) compared with the liquid reagent detection system, but the typing judgment is not affected; in the whole, the RTyperY27 chip detection system, namely the freeze-drying reagent detection system, has good amplification effect and meets the experimental requirements. Therefore, the YSTR microfluidic chip amplification detection system provided by the application can keep the characteristics of high detection sensitivity and accurate result of the amplification system when facing various samples, and can be used for identifying or excluding suspects, male component identification and family investigation.
Drawings
FIG. 1 is a RTYperY27 site layout.
FIG. 2 is a RTyperY27 reagent; a: before lyophilization; b: after lyophilization, red was RTyperY27 Primer and white was RTyperY27 PCR Mix.
FIG. 3 is a graph comparing the typing of RTyperlY 27 lyophilized reagent detection system and liquid reagent detection system.
Fig. 4 (a) shows the detection result of a certain oral swab on a conventional laboratory PCR-CE platform (B) the detection result of the same oral swab on the whole integration of the rapid tester.
Fig. 5 is a full-integration test result of the oral swab sample.
Fig. 6 shows the result of the blood card sample full-integrated test.
FIG. 7 shows the amplification results of a PCR Mix (available from Xinhai Biotechnology Co., ltd., product number NH 9365) using RTyper27 on a chip.
FIG. 8 shows the result of amplification of the optimized RTyper27 PCR Mix on the chip
FIG. 9 shows the lyophilization results (MIX 1, MIX2, MIX3 in order from left to right) of RTyper Y27 PCR MIX1 to MIX3 using lyophilization flow 1 (FIG. A) and flow 2 (FIG. B).
FIG. 10 shows the lyophilization results of RTyper Y27 Primer using lyophilization protocol 1 (panel A) and protocol 2 (panel B).
Fig. 11 is a diagram of the fully integrated detection of three adjuvant MIX lyophilized according to procedure 1.
FIG. 12 is a graph of the total integrated detection of 2800M after lyophilization in procedure 2 for three MIX formulations.
FIG. 13 is a comparison of RTypery27 lyophilized reagent and liquid reagent detection typing.
Detailed Description
Materials and methods
Sample:
oral swab: provided by the present laboratory volunteers.
Blood card: provided by the present laboratory volunteers.
DNA standard 2800M: purchased from Promega corporation, USA (initial mass concentration 10 ng/. Mu.L).
RTyper27 PCR Mix was purchased from Xinhai biotechnology Co., ltd (cat. NH 9365).
Major reagents and instrumentation:
2.5 XS 10 PCR Mix: purchased from the new sea biotechnology Co., ltd.
Sample direct-expansion treatment fluid: purchased from the new sea biotechnology Co., ltd.
Bo Yi kang Pilot3-6E vacuum freeze drier: purchased from beijing bojikang laboratory instruments ltd; quick TargSeq full-integrated DNA on-site rapid tester: provided by beijing boao biotechnology limited.
Fully integrated chip card box: provided by beijing boao biotechnology limited.
Example 1 construction of primers involving RTyperY27 chip amplification System
1. Primer design and Synthesis
The 27Y chromosome short tandem repeat (Y-STRs) loci selected in this study were the DYS460 locus, the YGATAAH 4 locus, the DYS390 locus, the DYS19 locus, the DYS449 locus, the DYS481 locus, the DYF387S1a locus, the DYF387S1b locus, the DYS576 locus, the DYS635 locus, the DYS385a locus, the DYS385b locus, the DYS627 locus, the DYS438 locus, the DYS391 locus, the DYS393 locus, the DYS389I locus, the DYS570 locus, the DYS389II locus, the DYS392 locus, the DYS518 locus, the DYS437 locus, the DYS456 locus, the DYS439 locus, the DYS448 locus, the DYS533 locus and the DYS458 locus. The kit comprises 19 core loci (GB/T41009-2021) specified in national criminal investigation information professional application system Y-STR DNA database construction and management guidelines (hereinafter referred to simply as library construction guidelines), 5Y-STR loci selected from 15 preferred loci recommended by the library construction guidelines, and FAM, HEX, TAMRA, ROX fluorescent markers. All primer syntheses were provided by the soviet sea biotechnology Co., ltd, specific information is shown in Table 1, and fluorescent labels and site arrangements are shown in FIG. 1. The 27 loci are those described in Table 1.
The RTyper 27 chip detection system of the present invention detects 27Y STR loci. The amplification primers (i.e., in vitro nucleic acid amplification primers, the length of the amplified fragment ranges from 31 bp to 90 bp) were designed and optimized according to each locus, and the in vitro nucleic acid amplification primers were labeled with nucleic acid markers, specifically FAM, HEX, TAMRA, ROX, and all fluorescence markers were labeled at the 5' end of the forward primer F at each locus.
The method comprises the following steps:
the Primer composition (the name RgyperY 27 Primer) for detecting the above 27 loci was prepared from DYS460-FR (the nucleotide sequences are respectively two single-stranded DNAs of sequence 1 and sequence 2), YGATAAH 4-FR (the nucleotide sequences are respectively two single-stranded DNAs of sequence 3 and sequence 4), DYS390-FR (the nucleotide sequences are respectively two single-stranded DNAs of sequence 5 and sequence 6), DYS19-FR (the nucleotide sequences are respectively two single-stranded DNAs of sequence 7 and sequence 8), DYS449-FR (the nucleotide sequences are respectively two single-stranded DNAs of sequence 9 and sequence 10), DYS481-FR (the nucleotide sequences are respectively two single-stranded DNAs of sequence 11 and sequence 12), DYF387S 1-FR (the nucleotide sequences are respectively two single-stranded DNAs of sequence 13 and sequence 14), DYS576-FR (the nucleotide sequences are respectively two single-stranded DNAs of sequence 15 and sequence 16), DYS635-FR (the nucleotide sequences are respectively two single-stranded DNAs of sequence 17 and sequence 18), DYS 385-FR (the nucleotide sequences are respectively two single-stranded DNAs of sequence 24 and single-stranded DNAs of DYF 27-FR (the nucleotide sequences are respectively single-stranded DNAs of sequence 24) and DYF 38-FR (the nucleotide sequences are respectively two single-stranded DNAs of sequence 24 and two single-stranded DNAs of sequence 24) and DYF 38-FR (respectively) and DYF 38-FR (the nucleotide sequences are respectively) and single-stranded DNAs of sequence 20) DYS570-FR (the nucleotide sequence is two single-stranded DNA of sequence 31 and sequence 32, respectively), DYS389II-FR (the nucleotide sequence is two single-stranded DNA of sequence 33 and sequence 34, respectively), DYS392-FR (the nucleotide sequence is two single-stranded DNA of sequence 35 and sequence 36, respectively), DYS518-FR (the nucleotide sequence is two single-stranded DNA of sequence 37 and sequence 38, respectively), DYS437-FR (the nucleotide sequence is two single-stranded DNA of sequence 39 and sequence 40, respectively), DYS456-FR (the nucleotide sequence is two single-stranded DNA of sequence 41 and sequence 42, respectively), DYS439-FR (the nucleotide sequence is two single-stranded DNA of sequence 43 and sequence 44, respectively), DYS448-FR (the nucleotide sequence is two single-stranded DNA of sequence 45 and sequence 46, respectively), DYS533-FR (the nucleotide sequence is two single-stranded DNA of sequence 47 and sequence 48, respectively) and DYS458-F (the nucleotide sequence is two single-stranded DNA of sequence 49 and sequence 50, respectively) R. The RTyper Y27Primer was dissolved in TE buffer to obtain a RTyper Y27Primer solution.
Among the primer pairs for detecting DYF387S1a locus and DYF387S1ab locus are DYF387S1ab-FR (the nucleotide sequences are two single-stranded DNAs of sequence 13 and sequence 14, respectively), and DYS576-FR (the nucleotide sequences are two single-stranded DNAs of sequence 15 and sequence 16, respectively).
The primer pair for detecting the DYS385a locus and the DYS385b locus was DYS385ab-FR (the nucleotide sequences are two single-stranded DNA of sequence 19 and sequence 20, respectively).
TABLE 1 primer information Table
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2. Construction of RTyper27 chip amplification detection system
1. RTyper27 liquid amplification reagent
The RTyper27 liquid amplification reagent consisted of RTyper27 PCR Mix (product number NH9356, new sea Biotechnology Co., ltd.) and RTyper27 Primer. RTyper27 Primer consisted of 25 Primer pairs and TE (solvent TE) in Table 1, each Primer pair having a final concentration of DYS460-FR (77 mM), YGATAAH 4-FR (128 mM), DYS390-FR (340 mM), DYS19-FR (381 mM), DYS449-FR (305 mM), DYS481-FR (104 mM), DYF387S1ab-FR (97 mM), DYS576-FR (115 mM), DYS635-FR (170 mM), DYS385ab-FR (305 mM), DYS627-FR (67 mM), DYS438-FR (400 mM), DYS391-FR (429 mM), DYS393-FR (177 mM), DYS389I-FR (114 mM), DYS570-FR (132 mM), DYS389II-FR (333 mM), DYS392-FR (333 mM), S518-FR (162 mM), DYS 385-FR (146 mM), DYS448 mM), DYS (171-FR (533 mM), and DYS 109mM (533 mM), respectively. The final concentrations of the two primers in each Primer pair were identical in the RTyper27 Primer. RTyper27 Primer solution was obtained.
RTyper27 PCR Mix was optimized from RTyper27 PCR Mix (available from new sea biotechnology, inc., su) under the designation NH9356, optimized RTyper27 PCR Mix: RTyper27 PCR Mix (product number NH9356, new Biotechnology Co., ltd.) further contained Taq enzyme at a final concentration of 4U/10. Mu.L and MgCl2 at a final concentration of 0.2. Mu.M/10. Mu.L.
The preparation method of the optimized RTyper27 PCR Mix comprises the following steps: RTyper27 PCR Mix (available from New Biotechnology Co., ltd., product No. NH 9365) was added with Taq enzyme and MgCl2 so that the final concentration of Taq enzyme was 4U/10. Mu.L and the final concentration of MgCl2 was 0.2. Mu.M/10. Mu.L. The optimized RTyper27 PCR Mix is subsequently referred to as RTyper27 PCR Mix or optimized RTyper27 PCR Mix.
2. RTyperY27 lyophilized formulation
1) Freeze-dried component
The RTyper Y27 liquid amplification reagent comprises two parts of RTyper Y27 PCR Mix and RTyper Y27 Primer, and the liquid amplification reagent is subjected to chip storage in a vacuum freeze drying (abbreviated as freeze drying) mode.
Mixing trehalose, glucose and deionized water to obtain a mixed solution containing 22 mass percent of trehalose and 10 mass percent of dextran, obtaining a freeze-drying auxiliary material I, and mixing the freeze-drying auxiliary material with RTyper Y27 PCR Mix according to a volume ratio of 1:1 to obtain RTyperiY 27 PCR Mix freeze-dried mixed solution.
Mannitol and deionized water are mixed to obtain a mannitol solution containing 15 percent (mass fraction), trehalose and deionized water are mixed to obtain a trehalose solution containing 15 percent (mass fraction), and the mannitol solution and the trehalose solution are subjected to a volume ratio of 8:1, mixing to obtain a freeze-dried auxiliary material II, and mixing the freeze-dried auxiliary material with RTyperY27 Primer solution according to the volume ratio of 1:1 to obtain RTyperY27 Primer freeze-dried mixed solution.
2) Preparation of lyophilized pellets
A 10cm multiplied by 10cm metal plate is selected as a freeze-drying reagent carrying container, the metal plate is pre-frozen in liquid nitrogen for 30min before freeze-drying is started, a 12 mu L pipettor is used for suspending after the metal plate is taken out, RTyper Y27PCR Mix freeze-drying mixed solution or RTyper Y27 Primer freeze-drying mixed solution is respectively dripped on the metal plate, and the reagent is coagulated into spheres after touching the plate. The metal plate carrying the reagent was placed in a vacuum freeze dryer (Beijing Bo Yikang laboratory instruments Co., ltd.) and a lyophilization process flow was set, as shown in Table 2, and lyophilization was started with a vacuum of < 60pa throughout the lyophilization process.
Table 2 lyophilization process flow
3) Shaping effect
And respectively freeze-drying the RTyper Y27PCR Mix freeze-drying mixture and the RTyper Y27 Primer freeze-drying mixture according to the method to obtain a RTyper Y27 Primer freeze-drying agent (namely RTyper Y27 Primer freeze-drying pellets) and a RTyper Y27PCR Mix freeze-drying agent (namely RTyper Y27PCR Mix freeze-drying pellets). In order to ensure the activity of the freeze-drying reagent, the damage to protein substances such as enzymes and the like in the freeze-drying process is reduced by adding a freeze-drying protective agent. Meanwhile, in order to ensure that the freeze-dried reagent has a better framework structure and is convenient to transfer, excipients are often added to ensure that the freeze-dried reagent has certain hardness and toughness. FIG. 2 shows a comparison of RTypery27 reagent before and after lyophilization, A: before lyophilization; b: after lyophilization, red was RTyperY27 Primer and white was RTyperY27PCR Mix. The freeze-dried pellets can be seen to be full in appearance, smoother and non-atrophic in surface, uniform in color and luster and good in porosity.
EXAMPLE 2 application of RTyper27 amplification reagents
1. Experimental sample and method
1. Experimental sample
The test subjects provided oral swabs and blood card samples as test samples.
The preparation method of the two kinds of samples is as follows:
oral swab sample: volunteers scrape 8 times on the left and right of the inner wall of the mouth with sampling swabs, respectively;
blood card sample: taking 2 drops of finger tip blood of a volunteer, wherein the diameter of each drop is about 1cm, dripping the drops into a marking ring of a blood sampling card, collecting the samples, and drying in the shade for later use.
DNA standard 2800M available from Promega corporation, USA, (initial mass concentration 10 ng/. Mu.L)
2. Experimental method
1) Preparing a fully integrated chip card box:
embedding an amplification freeze-drying reagent in an amplification chip sample processing pool, wherein the amplification freeze-drying reagent comprises 4 RTyper Y27PCR Mix freeze-drying pellets and 2 RTyper Y27 Primer freeze-drying pellets; simultaneously, 6 mu L of formamide and AS 500 internal standard mixed solution (volume ratio is 2:1) are buried under an electrophoresis chip sample injection hole groove, 55 mu L of 5 xTTE is placed in each buffer solution hole of the chip, and the buffer solution holes are packaged by aluminum foil.
2) Sample adding:
200 mu L of direct amplification treatment liquid I (NH 9866 of Suzhou New Biotechnology Co., ltd.) is added into a 1.5mL centrifuge tube, 1 oral swab sample or a round blood card/saliva card 4 piece with the diameter of 2mm is put into the centrifuge tube, shaking is carried out for 5 to 10 times, oral epithelial cells or blood cells are eluted, then 24 mu L of the direct amplification treatment liquid I is mixed with 36 mu L of direct amplification treatment liquid II (NH 9867 of Suzhou New Biotechnology Co., ltd.) and added into an amplification chip sample treatment pool for re-dissolving and amplifying freeze-drying reagent (4 RTyperiy 27PCR Mix freeze-dried pellets and 2 RTyperiy 27 Primer freeze-dried pellets which are prepared by freeze-drying in units of 12 mu L).
The concentrations of the 27 primer pairs in the in vitro nucleic acid amplification reaction system were: DYS460-FR 15mM, YGATAAH4-FR 26mM, DYS390-FR 68mM, DYS19-FR 76mM, DYS449-FR 61mM, DYS481-FR 21mM, DYS 387ab-FR 19mM, DYS576-FR 23mM, DYS635-FR 34mM, DYS385ab-FR 61mM, DYS627-FR 13mM, DYS438-FR 80mM, DYS391-FR 86mM, DYS393-FR 35mM, DYS389I-FR 23mM, DYS570-FR 26mM, DYS389II-FR 67mM, DYS392-FR 67mM, DYS518-FR 32mM, DYS437-FR 67mM, DYS456-FR 29mM, DYS448-FR 34mM, DYS533-FR 67mM, and DYS458-FR 30mM.
3) Full integration detection:
inserting a fully integrated chip cartridge into a QuickTargSeqThe DNA on-site rapid tester sample injection bin inputs sample information, a preset program is selected, and after an operation key is clicked, the instrument automatically completes all processes of sample splitting, PCR amplification and electrophoresis separation. The main process and important parameters of each process of the full integration are as follows:
sample lysis: repeatedly pumping for 20 times, extracting the feed liquid to an amplification chip, and cracking for 10min at 95 ℃.
And (3) PCR amplification: starting amplification after completion of cleavage, wherein the thermal cycle parameter is 95 ℃ for 1min;95℃10s,59℃1min,72℃20s,28 cycles; and at 60℃for 10min.
And (3) electrophoresis separation: the PCR amplification product is discharged, electrophoresis is started, the sample injection voltage is 480V, and the sample injection time is 180s; separation voltage 4600V, separation time 3600s; electrophoresis temperature was 59 ℃.
Data was collected and the results were analyzed using the companion software BioStrGenotyping v 2.0.
2. Performance index validation
1. Freeze-drying reagent amplification effect
The amplification effect of the RTyperY27 chip system after lyophilization was tested using 10 ng/. Mu.L of DNA standard 2800M and compared to its liquid reagents.
Lyophilizing the reagent complex solution: the volume was 60. Mu.L, which included 36. Mu.L of the direct amplification treatment liquid II or deionized and sterilized water, 1. Mu.L of 2800M, 23. Mu.L of deionized and sterilized water.
Liquid reagent: a volume of 60. Mu.L was included of 24. Mu.L RTyperY27 PCR Mix (2U hot start Taq enzyme, 1.5mM magnesium chloride, 1mM dNTPs, 25mM potassium chloride, 0.5% BSA, 20mM Tris-HCl buffer), 12. Mu.L RTyperY27 Primer Mix, 1. Mu.L 2800M, 23. Mu.L deionized sterile water.
Full integration test operation the full integration test procedure described above is referred to.
The result shows that the complete typing can be obtained by using the allele peak height of more than or equal to 200RFU as a standard, using a DNA standard 2800M as a template, and using both a RTyperY27 liquid reagent detection system and a freeze-drying reagent detection system, wherein the typing diagram is shown in figure 3. The individual allele peak heights in the lyophilized reagent typing pattern were lower (RFU values) compared to the liquid reagent detection system, but did not affect the typing judgment. In the whole, the RTyperY27 chip detection system, namely the freeze-dried reagent detection system has good amplification effect, and meets the experimental requirement, namely the spot detection
2. Accuracy detection
The full-integration detection is carried out by taking 30 volunteer oral swab samples, and the result is compared with the result of a conventional laboratory PCR-CE platform to verify the accuracy of the RTyper Y27 chip amplification system on a rapid detector. The results showed that the full-integrated detection of 30 samples was completely typed, without allele loss, and completely consistent with the conventional results (results as in FIG. 4)
3. Adaptability of checking material
And carrying out full-integrated detection on different detection materials, including an oral swab and a blood card sample, so as to verify the detection capability of the RTyperY27 chip amplification system on different types of detection materials. The typing results are shown in figures 5 and 6, the allele peak height value is more than or equal to 200RFU as an analysis threshold, and the complete Y-STR typing can be obtained for all 2 samples without allele loss.
Conclusion: the research establishes a reagent system and a preparation method for a fully integrated micro-fluidic chip system. The RTyper Y27 chip detection system is constructed in a freeze-drying mode, can be stored in a microfluidic chip card box for a long time, can detect oral swab and blood card samples, completes the whole processes of sample splitting, PCR amplification and electrophoresis separation within 2 hours, and meets the urgent requirements of public security actual combat site and rapid detection.
Optimization of the comparative example 1 RTyper27 PCR Mix System
Liquid reagent RTyper27 PCR Mix, available from Xinhai Biotechnology Co., ltd., suzhou (cat. NH 9365).
1. The purchased RTyper27 PCR Mix reagent was amplified on a microfluidic chip by the method of example 2 one without any optimization, and the result was poor (only 6 loci had amplification product peaks) and a large number of allele losses occurred, and the peak heights were unbalanced, as shown in fig. 7.
2.RTyper27 PCR Mix the hot start Taq enzyme and MgCl2 are added to make the final concentration of Taq enzyme be 4U/10 mu L and the final concentration of MgCl2 be 0.2 mu M/10 mu L, so as to obtain the optimized RTyper27 PCR Mix, and the optimized RTyper27 PCR Mix is amplified by the microfluidic chip without any optimization through the steps of the method 1 in the example 2, and the amplification result on the chip is as shown in the following figure 8, except that the allele peak value of the individual loci is lower, but no allele loss exists, so that the complete Y-STR typing graph can be obtained.
Comparative example 2 selection of the lyophilization conditions for reagents
1. Experimental method
The RTyper Y27 liquid amplification reagent comprises two parts of RTyper Y27 PCR Mix and RTyper Y27 Primer, and the liquid amplification reagent is subjected to chip storage by a vacuum freeze drying (short for freeze drying) mode
A 10cm multiplied by 10cm metal plate is selected as a freeze-drying reagent carrying container, the metal plate is pre-frozen in liquid nitrogen for 30min before freeze-drying is started, a 12 mu L pipettor is used for suspending after the metal plate is taken out, a reagent is dripped on the metal plate in a hanging manner, and the reagent is coagulated into a sphere after touching the plate. The metal plate carrying the reagent is put into a vacuum freeze dryer (Beijing Bo Yikang laboratory instrument Co., ltd.) and a freeze-drying process flow is set, and freeze-drying is started, wherein the vacuum degree is less than 60pa in the whole freeze-drying process. And respectively comparing the freeze-drying auxiliary material proportion of the RTyper Y27PCR Mix and the RTyper Y27Primer with the freeze-drying process flow in parallel, and selecting the optimal one according to the freeze-dried form, the storage time and the amplification effect.
(1) Selection of lyophilization procedure
Two lyophilization program flows are preliminarily set, the specific steps are shown in table 3,RTyperY27 PCR Mix and RTyper Y27Prim er, the same lyophilization program is used for lyophilization, after lyophilization, the lyophilized amplification reagent is pre-buried in a full-integration card box, and 10ng 2800 is subjected to full-integration detection.
Table 3 selection of lyophilization procedure
(2) RTyper Y27PCR Mix and RTyper Y27Primer freeze-drying auxiliary material proportion
Three RTyper Y27PCR Mix auxiliary material ratios and one RTyper Y27Primer auxiliary material ratio are preliminarily set, the specific ratios are shown in table 4, lyophilization is carried out by using lyophilization processes 1 and 2 (the specific process is shown in table 3), after lyophilization is finished, the lyophilization molding state is observed, the lyophilization pellets are pre-buried in a full-integration card box, and 10ng 2800 is subjected to full-integration detection. RTyper Y27PCR Mix reagent the optimized RTyper Y27PCR Mix prepared in example 1 and RTyper Y27Primer prepared in example 1.
Table 4 lyophilized reagent adjuvant formulation
2. Experimental results
The RTyper Y27 PCR Mix and RTyper Y27 Primer were lyophilized to loose pellet form (as shown in fig. 9 and 10) using different lyophilization adjuvant ratios and lyophilization procedures, and the whole integration test results of the lyophilized RTyper Y27 PCR Mix and RTyper Y27 Pr timer were as shown in fig. 11 and 12 by the method of step two in example 2, and partial locus allele loss (red circle) was found using Mix2 of lyophilization procedure 1 and Mix1-Mix3 of lyophilization procedure 2, and Mix1 overall allele peak value and balance of lyophilization procedure 1 were better than Mix2, so Mix1 and lyophilization procedure 1 were selected as the optimal lyophilization adjuvant ratios and lyophilization procedure conditions for the study.
The results of full-integration detection of lyophilized reagents and liquid reagents (i.e., prior to lyophilization) using 2800M under this condition are shown in fig. 13, where the peak heights of individual alleles in the lyophilized reagent typing graph are reduced, but the typing judgment is not affected, which also indicates that the liquid reagents have less influence on the experimental results of the chip after lyophilization.
Screening of Glycerol content in comparative example 3RTyperY27 PCR Mix
In order to achieve long-term storage on the chip, it is necessary to observe whether the morphology of the lyophilized pellet changes after a period of time. The glycerol content has a large influence on storage, so that the glycerol content in the PCR Mix is optimized.
3.1 Experimental methods
Setting a glycerol content gradient in RTyper Y27 PCR Mix (optimized RTyper Y27 PCR Mix), sequentially taking 2%,2.5%,3%,3.3%,3.5% and 4% of the glycerol content gradient as mass volume ratio, adopting the MIX1 as a freeze-drying auxiliary material according to the experimental result, freeze-drying by using the freeze-drying process 1, collecting the freeze-dried amplification reagent in a 1.5mLEP tube after freeze-drying, sealing and storing the obtained product in a 4 ℃ tube by using an aluminum foil bag, observing the storage state of the obtained product for 7 days, observing the form of the obtained product, filling the freeze-dried pellets in a full-integration card box according to the corresponding proportion, respectively carrying out full-integration detection on 10ng 2800M, and calculating the peak height average value of the obtained product.
3.2 experimental results
RTyper Y27 PCR Mix containing different glycerol concentrations can be freeze-dried according to the standard freeze-drying procedure, but freeze-dried pellets with glycerol content of more than or equal to 3% have smaller volume compared with freeze-dried pellets with lower glycerol content, and after 7 days of storage, the pellets with reduced volume, viscosity and enzyme content of 3.5% and 4% are completely condensed into a group and cannot be stored and transferred to a full-integration cartridge, so that the freeze-dried pellets with the RTyper Y27 PCR Mix content of 2%,2.5%,3%,3.3% glycerol and the Y27 Primer Mix freeze-dried pellets prepared according to the mixture ratio of Table 4 can be fully integrated according to the method to obtain the full-integration detection, and the peak height average value of the full-integration cartridge is shown in Table 5. From the peak height values, the glycerol content of 2.5% and 3.3% was found to be better, and the glycerol content added to the RTypery27 PCR Mix was finally selected to be 2.5% in consideration of the morphology and storage condition of the pellets after lyophilization.
TABLE 5 MIX peak height means for different enzyme contents
3.3 success Rate and typing accuracy of RTyper27 reagent chip System
The chip total integration test, statistical success rate and typing accuracy were carried out on 115 parts of blood card using the RTyper27 freeze-dried reagent system (namely, RTyper27 PCR Mix freeze-dried pellets with 2.5% glycerol content prepared in 3.1 in example 3 and RTyper27 Primer freeze-dried pellets prepared in Table 4 according to the freeze-drying process 1), and the results are shown in Table 6.
Table 6 success rate and typing accuracy statistics
( And (3) injection: success rate refers to the ratio of samples with allele detection rate of more than or equal to 80% to all test samples; typing accuracy refers to the successful result of typing, the ratio of the alleles to the total alleles that are accurately typed by software )
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (10)
1. Use of a substance for detecting a locus, characterized in that: the loci are the following 27 loci:
the DYS460 locus, the YGATAH4 locus, the DYS390 locus, the DYS19 locus, the DYS449 locus, the DYS481 locus, the DYF387S1a locus, the DYF387S1b locus, the DYS576 locus, the DYS635 locus, the DYS385a locus, the DYS385b locus, the DYS627 locus, the DYS438 locus, the DYS391 locus, the DYS393 locus, the DYS389I locus, the DYS570 locus, the DYS389II locus, the DYS392 locus, the DYS518 locus, the DYS437 locus, the DYS456 locus, the DYS439 locus, the DYS448 locus, the DYS533 locus and the DYS458 locus.
The application is any one of the following:
a1 Detecting the use of a substance at a locus in identifying or assisting an individual of a living being;
a2 Use of a substance that detects a locus in the preparation of a product that identifies or assists in the biological individual;
a3 Detecting the use of a locus species in STR typing;
a4 Detecting the use of a locus material in the preparation of an STR typing product;
a5 Detecting the application of the locus substance in YSTR microfluidic chip amplification;
a6 The application of a substance for detecting a locus in preparing a YSTR microfluidic chip amplification product.
2. The use according to claim 1, wherein the substance is any one of the following:
d1 An in vitro nucleic acid amplification primer composition that specifically amplifies the locus;
d2 An in vitro nucleic acid amplification reagent comprising D1) the in vitro nucleic acid amplification primer composition;
d3 A kit comprising D1) the in vitro nucleic acid amplification primer composition or D2) the in vitro nucleic acid amplification reagent.
3. The use according to claim 2, wherein said in vitro nucleic acid amplification primer composition consists of 27 primer pairs for said 27 loci, the 27 primer pairs are composed of a primer pair DYS460-FR for detecting a DYS460 locus, a primer pair YGATAAH 4-FR for detecting a YGATAAH 4 locus, a primer pair DYS390-FR for detecting a DYS390 locus, a primer pair DYS19-FR for detecting a DYS19 locus, a primer pair DYS449-FR for detecting a DYS449 locus, a primer pair DYS481-FR for detecting a DYS481 locus, a primer pair DYF387S1a locus and a primer pair DYF387S1ab-FR for detecting a DYS 387S1b locus, a primer pair DYS576-FR for detecting a DYS576 locus, a primer pair DYS635-FR for detecting a DYS385 locus, a primer pair DYS385ab-FR for detecting a locus and a primer pair DYS627-FR for detecting a DYS385b locus, a primer pair DYS627-FR for detecting a gene locus, and a primer pair DYS438-FR for detecting a gene locus the primer pair for detecting the DYS391 locus comprises a primer pair for detecting the DYS391-FR, a primer pair for detecting the DYS393 locus, a primer pair for detecting the DYS389I locus, a primer pair for detecting the DYS570 locus, a primer pair for detecting the DYS389II locus, a primer pair for detecting the DYS392 locus, a primer pair for detecting the DYS518-FR, a primer pair for detecting the DYS437 locus, a primer pair for detecting the DYS456-FR, a primer pair for detecting the DYS439-FR, a primer pair for detecting the DYS448 locus, a primer pair for detecting the DYS533-FR and a primer pair for detecting the DYS458 locus;
The primer pair for detecting DYS460 locus is two single-stranded DNA of which the nucleotide sequence is sequence 1 and sequence 2, the primer pair for detecting YGATAAH 4 locus is two single-stranded DNA of which the nucleotide sequence is sequence 3 and sequence 4, the primer pair for detecting DYS390 locus is two single-stranded DNA of which the nucleotide sequence is sequence 5 and sequence 6, the primer pair for detecting DYS19 locus is two single-stranded DNA of which the nucleotide sequence is sequence 7 and sequence 8, the primer pair for detecting DYS449 locus is two single-stranded DNA of which the nucleotide sequence is sequence 9 and sequence 10, the primer pair for detecting DYS481 locus is two single-stranded DNA of which the nucleotide sequence is sequence 11 and sequence 12, the primer pair for detecting DYF387S1a locus and the primer pair for detecting DYF387S1b locus is two single-stranded DNA of which the nucleotide sequence 13 and sequence 14, the primer pair for detecting DYS449 locus is two single-stranded DNA of which the nucleotide sequence 15 and sequence 16, the primer pair for detecting DYS635 locus is two single-stranded DNA of which the nucleotide sequence is sequence 17 and sequence 18, the primer pair for detecting DYS385a locus and detecting DYS385b locus is two single-stranded DNA of which the nucleotide sequence is sequence 19 and sequence 20, the primer pair for detecting DYS627 locus is two single-stranded DNA of which the nucleotide sequence is sequence 21 and sequence 22, the primer pair for detecting DYS438 locus is two single-stranded DNA of which the nucleotide sequence is sequence 23 and sequence 24, the primer pair for detecting DYS391 locus is two single-stranded DNA of which the nucleotide sequence is sequence 25 and sequence 26, the primer pair for detecting DYS393 locus is two single-stranded DNA of which the nucleotide sequence is sequence 27 and sequence 28, the primer pair for detecting DYS389I locus is two single-stranded DNA of which the nucleotide sequence is sequence 29 and sequence 30, the primer pair for detecting DYS570 locus is two single-stranded DNA of which the nucleotide sequence is sequence 31 and sequence 32, the primer pair for detecting DYS389II locus is two single-stranded DNA of which the nucleotide sequence is sequence 33 and sequence 34, the primer pair for detecting DYS392 locus is two single-stranded DNA of which the nucleotide sequence is sequence 35 and sequence 36, the primer pair for detecting DYS518 locus is two single-stranded DNA of which the nucleotide sequence is sequence 37 and sequence 38, the primer pair for detecting DYS437 locus is two single-stranded DNA of which the nucleotide sequence is sequence 39 and sequence 40, the primer pair for detecting DYS456 locus is two single-stranded DNA of which the nucleotide sequence is sequence 41 and sequence 42, the primer pair for detecting DYS439 locus is two single-stranded DNA of which the nucleotide sequence 43 and sequence 44, the primer pair for detecting DYS448 locus is two single-stranded DNA of which the nucleotide sequence 45 and sequence 46, and the primer pair for detecting DYS437 locus is single-stranded DNA of which the nucleotide sequence 48 and the nucleotide sequence 48 is single-stranded DNA of which the nucleotide sequence 48 and the primer pair for detecting DYS533 is sequence 50.
4. The use according to claim 3, wherein the composition of the in vitro nucleic acid amplification primer composition is as follows:
77 parts of DYS460-FR,128 parts of YGATAH4-FR,340 parts of DYS390-FR,381 parts of DYS19-FR,305 parts of DYS449-FR,104 parts of DYS481-FR,97 parts of DYS 387S1ab-FR,115 parts of DYS576-FR,170 parts of DYS635-FR,305 parts of DYS385ab-FR,67 parts of DYS627-FR,400 parts of DYS438-FR,429 parts of DYS391-FR,177 parts of DYS393-FR,114 parts of DYS389I-FR,132 parts of DYS570-FR,333 parts of DYS389II-FR,333 parts of DYS392-FR,162 parts of DYS518-FR,333 parts of DYS437-FR,146 parts of DYS456-FR,109 parts of DYS439-FR,171 parts of DYS448-FR,333 parts of DYS 533-FR; the parts are parts by weight.
5. The use according to claim 3 or 4, wherein one or both of the two single stranded DNA of each of the 27 primer pairs is labeled with a nucleic acid marker.
6. The use according to claim 5, wherein the nucleic acid label is a fluorescent label.
7. The use of claim 6, wherein the 27 primer pairs label 4 fluorophores, the primer pair for detecting the DYS460 locus, the primer pair for detecting the YGATAH4 locus, the primer pair for detecting the DYS390 locus, the primer pair for detecting the DYS19 locus, the primer pair for detecting the DYS449 locus, the primer pair for detecting the DYS481 locus, the primer pair for detecting the DYF387S1a locus, and the primer pair for detecting the DYF387S1b locus label fluorophores designated as fluorophores a; a primer pair for detecting the DYS576 locus, a primer pair for detecting the DYS635 locus, a primer pair for detecting the DYS385a locus and the DYS385B locus, a primer pair for detecting the DYS627 locus, a primer pair for detecting the DYS438 locus, and a primer pair for detecting the DYS391 locus labeled with a fluorophore designated as fluorophore B; a primer pair for detecting the DYS393 locus, a primer pair for detecting the DYS389I locus, a primer pair for detecting the DYS570 locus, a primer pair for detecting the DYS389II locus, a primer pair for detecting the DYS392 locus, and a primer pair for detecting the DYS518 locus label a fluorophore designated as fluorophore C; a primer pair for detecting the DYS437 locus, a primer pair for detecting the DYS456 locus, a primer pair for detecting the DYS439 locus, a primer pair for detecting the DYS448 locus, a primer pair for detecting the DYS533 locus, and a primer pair for detecting the DYS458 locus labeled with a fluorophore designated as fluorophore D;
The fluorescent group A, the fluorescent group B and the fluorescent group C and the fluorescent group D are different compounds.
8. The use according to claim 7 or claim 7, wherein the fluorophore is selected from at least one of FAM, VIC, HEX, TRT, CY, CY5, ROX, JOE, FITC, TET, NED, TAMRA, LCRED640, LCRED705, quasar705 or Texas Red.
9. A product comprising a substance for detecting a locus according to any one of claims 1 to 8, the product having at least one of the following uses:
g1 For identifying or assisting a biological individual;
g2 For use in the preparation of a product for identifying or assisting biological subjects;
g3 For STR typing;
g4 For preparing STR parting products;
g5 For amplification of a YSTR microfluidic chip;
g6 Used for preparing YSTR microfluidic chip amplification products.
10. The method for identifying biological individuals is characterized by comprising the steps of using genomic DNA of a sample to be detected as a template in an in-vitro nucleic acid amplification system, performing in-vitro nucleic acid amplification by using the substance for detecting loci, the in-vitro nucleic acid amplification primer composition or the product according to any one of claims 1-8, and identifying biological individuals according to the in-vitro nucleic acid amplification product.
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