CN111705138A - Kit for detecting STR locus and detection method thereof - Google Patents
Kit for detecting STR locus and detection method thereof Download PDFInfo
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- 238000001514 detection method Methods 0.000 title abstract description 24
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 15
- 108020004414 DNA Proteins 0.000 claims description 101
- 102000053602 DNA Human genes 0.000 claims description 90
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 90
- 230000003321 amplification Effects 0.000 claims description 25
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 25
- 239000000047 product Substances 0.000 claims description 20
- 239000012634 fragment Substances 0.000 claims description 18
- 238000006243 chemical reaction Methods 0.000 claims description 17
- 239000000243 solution Substances 0.000 claims description 17
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 claims description 15
- 229940069446 magnesium acetate Drugs 0.000 claims description 15
- 235000011285 magnesium acetate Nutrition 0.000 claims description 15
- 239000011654 magnesium acetate Substances 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 15
- 108090000790 Enzymes Proteins 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 10
- 239000012264 purified product Substances 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000005251 capillar electrophoresis Methods 0.000 claims description 5
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- 229910052751 metal Inorganic materials 0.000 claims description 5
- 239000002184 metal Substances 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 108091092878 Microsatellite Proteins 0.000 abstract 4
- 108010091086 Recombinases Proteins 0.000 description 2
- 102000018120 Recombinases Human genes 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000004543 DNA replication Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011901 isothermal amplification Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The invention relates to a kit for detecting STR locus and a detection method thereof, comprising the following steps: the method comprises the following steps: preparing a primer group; step two: preparing a reagent by the primer group in the step one and a standard reference single product to obtain a standby reagent, and the step three: and preparing a kit by using the spare reagent in the step two. The invention belongs to the technical field of biology, and particularly relates to a kit for detecting STR (short tandem repeat) loci and a detection method thereof, which are used for solving the technical problems of long time consumption, complex operation, low sensitivity and the like in the prior art when the STR loci are identified for typing.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit for detecting STR loci and a detection method thereof.
Background
Since the successful solution of paternity testing cases involving immigrants in 1985 by DNA fingerprinting technology, DNA analysis technology has been widely used in criminal litigation. More and more DNA genetic markers with good polymorphism have been discovered in 20 years, and the analysis technology is more sensitive, rapid, simple, convenient and accurate. To date, DNA laboratories have been established and used by over 120 countries of the world to address cadaveric assertions in criminal cases, civil disputes, and large catastrophic events. The STR locus typing technology in the DNA used at present provides effective scientific evidence for case detection and judgment. However, the method has higher requirements on personnel, cost, instruments and environment, and the first-line detection popularization is difficult, so that the establishment of a quick, sensitive, simple, convenient, economic and easily-developed detection method is particularly important; the recombinase isothermal amplification (RPA) technology can get rid of the traditional PCR instrument and can obtain accurate detection results in a short time on site. RPA is based on recombinase polymerase mediated amplification principle, simulates the enzyme reaction process of in vivo DNA replication, amplifies the DNA template, and can complete the amplification reaction within 30 minutes at a constant temperature of about 37 ℃. And the accurate size of the gene fragment is obtained by combining gel electrophoresis separation with high resolution at the later stage to judge the genotype. The method can be used for amplifying the field trace samples in time for the multiple STR sites, and has great application value for forensic field detection. In view of the above, the present invention is particularly proposed.
Disclosure of Invention
Aiming at the situation and overcoming the defects of the prior art, the invention provides a kit for detecting STR loci and a detection method thereof, so as to relieve the technical problems of long time consumption, complex operation, low sensitivity and the like when the STR loci are identified in the prior art.
In order to achieve the above object, the present invention provides a kit for detecting STR loci, which is prepared by the following steps:
the method comprises the following steps: preparing a primer group;
step two: preparing a reagent by the primer group in the step one and a standard reference single product to obtain a standby reagent;
step three: and preparing a kit by using the spare reagent in the step two.
Further, the primer group in the first step includes a single-stranded DNA molecule shown in SEQ ID NO.1 to a single-stranded DNA molecule shown in SEQ ID NO.8, the name of the single-stranded DNA molecule shown in SEQ ID NO.1 is vWA-F, and the sequence of the single-stranded DNA molecule shown in SEQ ID NO.1 is GCCCTAGTGGATGATAAGAATAATCAGTATGTG; the name of the single-stranded DNA molecule shown by SEQ ID NO.2 is vWA-R, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.2 is GGACAGATGATAAATACATAGGATGGATGG; the name of the single-stranded DNA molecule shown by SEQ ID NO.3 is D8S1179-F, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.3 is ATTGCAACTTATATGTATTTTTGTATTTCATG; the name of the single-stranded DNA molecule shown by SEQ ID NO.4 is D8S1179-R, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.4 is TTACCAAATTGTGTTCATGAGTATAGTTTC; the name of the single-stranded DNA molecule shown by SEQ ID NO.5 is TPOX-F, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.5 is GACTGGCACAGAACAGGCACTTAGGGAACC; the name of the single-stranded DNA molecule shown by SEQ ID NO.6 is TPOX-R, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.6 is AAACGTGAGGTTGACTCTACTGTCCTGGG; the name of the single-stranded DNA molecule shown by SEQ ID NO.7 is FGA-F, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.7 is TTGAACTCACAGATTAAACTGTAACCAA; the name of the single-stranded DNA molecule shown by SEQ ID NO.8 is FGA-R, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.8 is GCTGGATATGCTGTACTTTTTCTATGAC.
Further, the final concentration of the primer group in the first step is 0.1-0.2 umol/L.
Further, the reagent in the second step includes a reference standard substance composed of 22 bands, the size of the band is from 60bp to 600bp, the fragment interval of the band is 20bp between 60bp and 200bp, the fragment interval of the band is 25bp between 200bp and 500bp, the fragment interval of the band is 50bp between 500bp and 600bp, that is, the band size is 60bp, 80bp, 100bp, 120bp, 140bp, 160bp, 180bp, 200bp, 225bp, 250bp, 275bp, 300bp, 325bp, 350bp, 375bp, 400bp, 425bp, 450bp, 475bp, 500bp, 550bp and 600bp, and the fluorescence intensity of the band of 100bp is twice that of other bands to simplify size distribution.
Further, the kit in the third step comprises a freeze-dried enzyme powder tube, a rehydration buffer solution, a magnesium acetate solution and a standby reagent, wherein the concentration of the magnesium acetate solution is 260-280 mmol/L.
Further, a detection method of the kit for detecting STR loci is as follows:
(1) taking genome DNA of a sample to be detected as a template, adopting the kit in the third step, adding 28.5-29.5 uL of rehydration buffer solution into 0.2mL of reaction tube containing freeze-dried enzyme powder, dissolving to obtain a mixture, adding 5.7-5.9 uL of the mixture into another 0.2mL of new tube, adding 0.5-1 uL of deionized water and 0.1-0.2 uL of primer group, and finally adding 0.3-0.5 uL of magnesium acetate solution to obtain an RPA amplification system;
(2) fully and uniformly mixing the RPA amplification system, and placing the mixture on a metal bath or a water bath kettle at the temperature of 35-37 ℃ for reaction for 20-30 min to obtain an RPA amplification product;
(3) after the reaction is finished, purifying the amplification product by using a PCR product purification kit and recovering the purified product to obtain a purified product;
(4) the band size was detected using capillary electrophoresis and the site type was calculated.
The invention with the structure has the following beneficial effects: the scheme is that a kit for detecting STR locus and its detection method, aiming at STR locus sequence, designing specific RPA amplification primer group, and establishing STR parting RPA detection method based on the primer group, which can carry out qualitative detection to STR locus, and tests prove that: the RPA amplification primer group has good specificity, high sensitivity and short detection time, does not need a PCR instrument, has the sensitivity equivalent to that of a common PCR method, but obviously reduces the cost of experiment time, the simplicity and convenience of operation and the cost of reagents, and can be used for the amplification of forensic field samples and the detection of trace samples. The reagent or kit containing the primer group can specifically detect and identify STR typing, and the RPA detection method of STR typing established based on the primer group is sensitive, accurate, simple, convenient and quick, can realize the quick detection of STR locus typing and locus typing comparison, is not limited by fields and reaction equipment, and can realize the accurate and quick detection of field trace samples.
Drawings
FIG. 1 is a diagram of the calculated locus typing results of the kit for detecting STR loci and the detection method thereof of the present invention.
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1, a kit for detecting STR loci, which is prepared by the steps of:
the method comprises the following steps: preparing a primer group;
step two: preparing a reagent by the primer group in the step one and a standard reference single product to obtain a standby reagent;
step three: and preparing a kit by using the spare reagent in the step two.
Further, the primer group in the first step includes a single-stranded DNA molecule shown in SEQ ID NO.1 to a single-stranded DNA molecule shown in SEQ ID NO.8, the name of the single-stranded DNA molecule shown in SEQ ID NO.1 is vWA-F, and the sequence of the single-stranded DNA molecule shown in SEQ ID NO.1 is GCCCTAGTGGATGATAAGAATAATCAGTATGTG; the name of the single-stranded DNA molecule shown by SEQ ID NO.2 is vWA-R, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.2 is GGACAGATGATAAATACATAGGATGGATGG; the name of the single-stranded DNA molecule shown by SEQ ID NO.3 is D8S1179-F, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.3 is ATTGCAACTTATATGTATTTTTGTATTTCATG; the name of the single-stranded DNA molecule shown by SEQ ID NO.4 is D8S1179-R, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.4 is TTACCAAATTGTGTTCATGAGTATAGTTTC; the name of the single-stranded DNA molecule shown by SEQ ID NO.5 is TPOX-F, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.5 is GACTGGCACAGAACAGGCACTTAGGGAACC; the name of the single-stranded DNA molecule shown by SEQ ID NO.6 is TPOX-R, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.6 is AAACGTGAGGTTGACTCTACTGTCCTGGG; the name of the single-stranded DNA molecule shown by SEQ ID NO.7 is FGA-F, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.7 is TTGAACTCACAGATTAAACTGTAACCAA; the name of the single-stranded DNA molecule shown by SEQ ID NO.8 is FGA-R, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.8 is GCTGGATATGCTGTACTTTTTCTATGAC.
Further, the final concentration of the primer sets in the first step is 0.1 umol/L.
Further, the reagent in the second step includes a reference standard substance composed of 22 bands, the size of the band is from 60bp to 600bp, the fragment interval of the band is 20bp between 60bp and 200bp, the fragment interval of the band is 25bp between 200bp and 500bp, the fragment interval of the band is 50bp between 500bp and 600bp, that is, the band size is 60bp, 80bp, 100bp, 120bp, 140bp, 160bp, 180bp, 200bp, 225bp, 250bp, 275bp, 300bp, 325bp, 350bp, 375bp, 400bp, 425bp, 450bp, 475bp, 500bp, 550bp and 600bp, and the fluorescence intensity of the band of 100bp is twice that of other bands to simplify size distribution.
Further, the kit in the third step comprises a freeze-dried enzyme powder tube, a rehydration buffer solution, a magnesium acetate solution and a standby reagent, wherein the concentration of the magnesium acetate solution is 260 mmol/L.
Further, a detection method of the kit for detecting STR loci is as follows:
(1) taking genome DNA of a sample to be detected as a template, adopting the kit in the third step, adding 28.5uL of rehydration buffer solution into 0.2mL of reaction tube containing freeze-dried enzyme powder, dissolving to obtain a mixture, adding 5.7 mixtures into another 0.2mL of new tube, adding 0.5uL of deionized water and 0.1uL of primer group, and finally adding 0.3uL of magnesium acetate solution to obtain an RPA amplification system;
(2) fully and uniformly mixing the RPA amplification system, and placing the mixture on a metal bath or a water bath kettle at the temperature of 35 ℃ for reaction for 20min to obtain an RPA amplification product;
(3) after the reaction is finished, purifying the amplification product by using a PCR product purification kit and recovering the purified product to obtain a purified product;
(4) the band size was detected using capillary electrophoresis and the site type was calculated.
Example 2, a kit for detecting STR loci, which is prepared by the steps of:
the method comprises the following steps: preparing a primer group;
step two: preparing a reagent by the primer group in the step one and a standard reference single product to obtain a standby reagent;
step three: and preparing a kit by using the spare reagent in the step two.
Further, the primer group in the first step includes a single-stranded DNA molecule shown in SEQ ID NO.1 to a single-stranded DNA molecule shown in SEQ ID NO.8, the name of the single-stranded DNA molecule shown in SEQ ID NO.1 is vWA-F, and the sequence of the single-stranded DNA molecule shown in SEQ ID NO.1 is GCCCTAGTGGATGATAAGAATAATCAGTATGTG; the name of the single-stranded DNA molecule shown by SEQ ID NO.2 is vWA-R, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.2 is GGACAGATGATAAATACATAGGATGGATGG; the name of the single-stranded DNA molecule shown by SEQ ID NO.3 is D8S1179-F, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.3 is ATTGCAACTTATATGTATTTTTGTATTTCATG; the name of the single-stranded DNA molecule shown by SEQ ID NO.4 is D8S1179-R, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.4 is TTACCAAATTGTGTTCATGAGTATAGTTTC; the name of the single-stranded DNA molecule shown by SEQ ID NO.5 is TPOX-F, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.5 is GACTGGCACAGAACAGGCACTTAGGGAACC; the name of the single-stranded DNA molecule shown by SEQ ID NO.6 is TPOX-R, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.6 is AAACGTGAGGTTGACTCTACTGTCCTGGG; the name of the single-stranded DNA molecule shown by SEQ ID NO.7 is FGA-F, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.7 is TTGAACTCACAGATTAAACTGTAACCAA; the name of the single-stranded DNA molecule shown by SEQ ID NO.8 is FGA-R, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.8 is GCTGGATATGCTGTACTTTTTCTATGAC.
Further, the final concentration of the primer sets in the first step is 0.15 umol/L.
Further, the reagent in the second step includes a reference standard substance composed of 22 bands, the size of the band is from 60bp to 600bp, the fragment interval of the band is 20bp between 60bp and 200bp, the fragment interval of the band is 25bp between 200bp and 500bp, the fragment interval of the band is 50bp between 500bp and 600bp, that is, the band size is 60bp, 80bp, 100bp, 120bp, 140bp, 160bp, 180bp, 200bp, 225bp, 250bp, 275bp, 300bp, 325bp, 350bp, 375bp, 400bp, 425bp, 450bp, 475bp, 500bp, 550bp and 600bp, and the fluorescence intensity of the band of 100bp is twice that of other bands to simplify size distribution.
Further, the kit in the third step comprises a freeze-dried enzyme powder tube, a rehydration buffer solution, a magnesium acetate solution and a standby reagent, wherein the concentration of the magnesium acetate solution is 270 mmol/L.
Further, a detection method of the kit for detecting STR loci is as follows:
(1) taking the genome DNA of a sample to be detected as a template, adopting the kit in the third step, adding 29uL of rehydration buffer solution into 0.2mL of reaction tube containing the freeze-dried enzyme powder, dissolving to obtain a mixture, adding 5.8 of the mixture into another 0.2mL of new tube, adding 0.8uL of deionized water and 0.15uL of primer group, and finally adding 0.4uL of magnesium acetate solution to obtain an RPA amplification system;
(2) fully and uniformly mixing the RPA amplification system, and placing the mixture on a metal bath or a water bath kettle at 36 ℃ for reaction for 25min to obtain an RPA amplification product;
(3) after the reaction is finished, purifying the amplification product by using a PCR product purification kit and recovering the purified product to obtain a purified product;
(4) the band size was detected using capillary electrophoresis and the site type was calculated.
Example 3, a kit for detecting STR loci, the kit being prepared by the steps of:
the method comprises the following steps: preparing a primer group;
step two: preparing a reagent by the primer group in the step one and a standard reference single product to obtain a standby reagent;
step three: and preparing a kit by using the spare reagent in the step two.
Further, the primer group in the first step includes a single-stranded DNA molecule shown in SEQ ID NO.1 to a single-stranded DNA molecule shown in SEQ ID NO.8, the name of the single-stranded DNA molecule shown in SEQ ID NO.1 is vWA-F, and the sequence of the single-stranded DNA molecule shown in SEQ ID NO.1 is GCCCTAGTGGATGATAAGAATAATCAGTATGTG; the name of the single-stranded DNA molecule shown by SEQ ID NO.2 is vWA-R, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.2 is GGACAGATGATAAATACATAGGATGGATGG; the name of the single-stranded DNA molecule shown by SEQ ID NO.3 is D8S1179-F, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.3 is ATTGCAACTTATATGTATTTTTGTATTTCATG; the name of the single-stranded DNA molecule shown by SEQ ID NO.4 is D8S1179-R, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.4 is TTACCAAATTGTGTTCATGAGTATAGTTTC; the name of the single-stranded DNA molecule shown by SEQ ID NO.5 is TPOX-F, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.5 is GACTGGCACAGAACAGGCACTTAGGGAACC; the name of the single-stranded DNA molecule shown by SEQ ID NO.6 is TPOX-R, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.6 is AAACGTGAGGTTGACTCTACTGTCCTGGG; the name of the single-stranded DNA molecule shown by SEQ ID NO.7 is FGA-F, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.7 is TTGAACTCACAGATTAAACTGTAACCAA; the name of the single-stranded DNA molecule shown by SEQ ID NO.8 is FGA-R, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.8 is GCTGGATATGCTGTACTTTTTCTATGAC.
Further, the final concentration of the primer sets in the first step is 0.2 umol/L.
Further, the reagent in the second step includes a reference standard substance composed of 22 bands, the size of the band is from 60bp to 600bp, the fragment interval of the band is 20bp between 60bp and 200bp, the fragment interval of the band is 25bp between 200bp and 500bp, the fragment interval of the band is 50bp between 500bp and 600bp, that is, the band size is 60bp, 80bp, 100bp, 120bp, 140bp, 160bp, 180bp, 200bp, 225bp, 250bp, 275bp, 300bp, 325bp, 350bp, 375bp, 400bp, 425bp, 450bp, 475bp, 500bp, 550bp and 600bp, and the fluorescence intensity of the band of 100bp is twice that of other bands to simplify size distribution.
Further, the kit in the third step comprises a freeze-dried enzyme powder tube, a rehydration buffer solution, a magnesium acetate solution and a standby reagent, wherein the concentration of the magnesium acetate solution is 280 mmol/L.
Further, a detection method of the kit for detecting STR loci is as follows:
(1) taking the genome DNA of a sample to be detected as a template, adopting the kit in the third step, adding 29.5uL of rehydration buffer solution into a 0.2mL reaction tube containing freeze-dried enzyme powder, dissolving to obtain a mixture, adding 5.9uL of the mixture into another 0.2mL new tube, adding 1uL of deionized water and 0.2uL of primer group, and finally adding 0.5uL of magnesium acetate solution to obtain an RPA amplification system;
(2) fully and uniformly mixing the RPA amplification system, and placing the mixture on a metal bath or a water bath kettle at 37 ℃ for reaction for 30min to obtain an RPA amplification product;
(3) after the reaction is finished, purifying the amplification product by using a PCR product purification kit and recovering the purified product to obtain a purified product;
(4) the band size was detected using capillary electrophoresis and the site type was calculated.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
The present invention and its embodiments have been described above, and the description is not intended to be limiting, and the drawings are only one embodiment of the present invention, and the actual structure is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (6)
1. A kit for detecting STR loci is characterized in that the kit is prepared by the following steps:
the method comprises the following steps: preparing a primer group;
step two: preparing a reagent by the primer group in the step one and a standard reference single product to obtain a standby reagent;
step three: and preparing a kit by using the spare reagent in the step two.
2. The kit for detecting STR loci of claim 1, wherein: the primer group in the first step comprises a single-stranded DNA molecule shown in SEQ ID NO.1 to a single-stranded DNA molecule shown in SEQ ID NO.8, the name of the single-stranded DNA molecule shown in SEQ ID NO.1 is vWA-F, and the sequence of the single-stranded DNA molecule shown in SEQ ID NO.1 is GCCCTAGTGGATGATAAGAATAATCAGTATGTG; the name of the single-stranded DNA molecule shown in SEQ ID NO.2 is vWA-R, and the sequence of the single-stranded DNA molecule shown in SEQ ID NO.2 is GGACAGATGATAAATACATAGGATGGATGG; the name of the single-stranded DNA molecule shown in SEQ ID NO.3 is D8S1179-F, and the sequence of the single-stranded DNA molecule shown in SEQ ID NO.3 is ATTGCAACTTATATGTATTTTTGTATTTCATG; the name of the single-stranded DNA molecule shown in SEQ ID NO.4 is D8S1179-R, and the sequence of the single-stranded DNA molecule shown in SEQ ID NO.4 is TTACCAAATTGTGTTCATGAGTATAGTTTC; the name of the single-stranded DNA molecule shown in SEQ ID NO.5 is TPOX-F, and the sequence of the single-stranded DNA molecule shown in SEQ ID NO.5 is GACTGGCACAGAACAGGCACTTAGGGAACC; the name of the single-stranded DNA molecule shown by SEQ ID NO.6 is TPOX-R, and the sequence of the single-stranded DNA molecule shown by SEQ ID NO.6 is AAACGTGAGGTTGACTCTACTGTCCTGGG; the name of the single-stranded DNA molecule shown in SEQ ID NO.7 is FGA-F, and the sequence of the single-stranded DNA molecule shown in SEQ ID NO.7 is TTGAACTCACAGATTAAACTGTAACCAA; the name of the single-stranded DNA molecule shown in SEQ ID NO.8 is FGA-R, and the sequence of the single-stranded DNA molecule shown in SEQ ID NO.8 is GCTGGATATGCTGTACTTTTTCTATGAC.
3. The kit for detecting STR loci of claim 2, wherein: the final concentration of the primer group in the first step is 0.1-0.2 umol/L.
4. The kit for detecting STR loci of claim 3, wherein: the reagent in the second step comprises a reference standard substance consisting of 22 bands, the size of the bands is from 60bp to 600bp, the fragment interval of the bands is 20bp to 60-200bp, the fragment interval of the bands is 25bp to 200bp, the fragment interval of the bands is 50bp to 500bp, namely the band size is 60bp, 80bp, 100bp, 120bp, 140bp, 160bp, 180bp, 200bp, 225bp, 250bp, 275bp, 300bp, 325bp, 350bp, 375bp, 400bp, 425bp, 450bp, 475bp, 500bp, 550bp and 600bp, and the fluorescence intensity of the band fragment of 100bp is twice that of other fragments so as to simplify size distribution.
5. The kit for detecting STR loci of claim 4, wherein: the kit in the third step comprises a freeze-dried enzyme powder tube, a rehydration buffer solution, a magnesium acetate solution and a standby reagent, wherein the concentration of the magnesium acetate solution is 260-280 mmol/L.
6. The method for detecting the kit for detecting STR loci as claimed in claims 1 to 5, wherein:
(1) taking genome DNA of a sample to be detected as a template, adopting the kit in the third step, adding 28.5-29.5 uL of rehydration buffer solution into 0.2mL of reaction tube containing freeze-dried enzyme powder, dissolving to obtain a mixture, adding 5.7-5.9 uL of the mixture into another 0.2mL of new tube, adding 0.5-1 uL of deionized water and 0.1-0.2 uL of primer group, and finally adding 0.3-0.5 uL of magnesium acetate solution to obtain an RPA amplification system;
(2) fully and uniformly mixing the RPA amplification system, and placing the mixture on a metal bath or a water bath kettle at the temperature of 35-37 ℃ for reaction for 20-30 min to obtain an RPA amplification product;
(3) after the reaction is finished, purifying the amplification product by using a PCR product purification kit and recovering the purified product to obtain a purified product;
(4) the band size was detected using capillary electrophoresis and the site type was calculated.
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| AU2011256907A1 (en) * | 2005-07-25 | 2012-01-19 | Abbott Diagnostics Scarborough, Inc. | Methods for Multiplexing Recombinase Polymerase Amplification |
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| US20100311127A1 (en) * | 2002-02-21 | 2010-12-09 | TwistDix, Inc. | Recombinase polymerase amplification |
| US20160083780A1 (en) * | 2002-02-21 | 2016-03-24 | Alere San Diego, Inc. | Recombinase Polymerase Amplification |
| US20060088874A1 (en) * | 2004-10-22 | 2006-04-27 | Promega Corporation | Methods and kits for detecting mutations |
| AU2011256907A1 (en) * | 2005-07-25 | 2012-01-19 | Abbott Diagnostics Scarborough, Inc. | Methods for Multiplexing Recombinase Polymerase Amplification |
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