Nested PCR (polymerase chain reaction) primer combination, kit and detection method for detecting STR (short tandem repeat) gene loci

Abstract

The invention discloses a nested PCR (polymerase chain reaction) primer combination, a kit and a detection method for detecting STR (short tandem repeat) gene loci, and belongs to the technical field of gene detection. The STR gene loci disclosed by the invention comprise a D19S433 locus and a D2S441 locus, and the STR gene loci comprise a D19S433 locus and a The primer combination comprises a primer combination of a D19S433 site as shown in SEQ ID NO.1-4 and / or a primer combination of a D2S441 site as shown in SEQ ID NO.5-8. The primer combination of the D19S433 site and the D2S441 site comprises a primer combination of the D19S433 site and the D2S441 site as shown The nested PCR primer combination disclosed by the invention can realize the purpose of specifically amplifying a target STR gene locus, separates and measures a rare allele sequence in a molecular cloning manner, and has the advantages of wide application range, low detection cost, simplicity in operation, easiness in result interpretation, high sensitivity and specificity, stronger pertinence and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a nested PCR primer combination, a kit and a detection method for detecting STR gene loci. Background Art

[0002] STR locus rare alleles refer to the alleles of STR loci that cannot be automatically read by conventional multiplex amplification kits. How to determine such alleles is crucial for forensic practices such as paternity testing and individual identification that rely on STR typing technology.

[0003] At present, when encountering rare alleles that cannot be automatically typed, each identification agency generally activates a spare composite amplification kit, or performs manual calculations based on the size of the electrophoresis data. Sites that cannot be calculated cannot be accurately typed in the identification opinion. In the current forensic evidence practice, some rare alleles can be confirmed by manual calculation, but many rare alleles still cannot be confirmed by manual calculation. At this time, the DNA sequence of the locus can be sequenced by large-scale parallel sequencing technology to confirm the allele, but this method has high economic costs and technical requirements, which exceeds the affordability of the person being identified, and general identification agencies do not have large-scale parallel sequencing platforms and their detection technologies. Based on this, a method for specific amplification, separation and sequencing of rare alleles by low-cost biochemical means is provided, which is low-cost and easier to apply in ordinary identification agencies. Summary of the invention

[0004] The purpose of the present invention is to provide a nested PCR primer combination, a kit and a detection method for detecting STR gene loci, so as to solve the problems existing in the above-mentioned prior art. The nested PCR primer combination of the present invention can achieve the purpose of specifically amplifying the target STR gene loci, and separate and measure the rare allele sequence by molecular cloning, and has the advantages of wide application range, low detection cost, simple operation, easy interpretation of results, high sensitivity and specificity, and stronger targeting.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a nested PCR primer combination for detecting STR gene loci, wherein the STR gene loci include a D19S433 locus and a D2S441 locus;

[0007] The primer combination includes a primer combination at the D19S433 site as shown in SEQ ID NO.1-4 and / or a primer combination at the D2S441 site as shown in SEQ ID NO.5-8.

[0008] The present invention also provides the use of the nested PCR primer combination in preparing a reagent or a kit for detecting STR gene loci.

[0009] The present invention also provides a nested PCR detection kit for detecting STR gene loci, wherein the nested PCR detection kit comprises the above-mentioned nested PCR primer combination.

[0010] Furthermore, the nested PCR detection kit is used for single detection of the D19S433 site or the D2S441 site, or double detection of the D19S433 site and the D2S441 site.

[0011] The present invention also provides a detection method of the above nested PCR detection kit, comprising the following steps:

[0012] Extracting DNA from the sample to be tested;

[0013] Using the nested PCR detection kit, performing a nested PCR amplification reaction on the DNA to obtain an amplified product;

[0014] The amplified product is sequenced using molecular cloning technology, and the genotype of the STR gene locus of the sample to be tested is analyzed based on the sequencing result.

[0015] Furthermore, the nested PCR amplification reaction is a single nested PCR amplification reaction or a double nested PCR amplification reaction.

[0016] Furthermore, the reaction system of the single nested PCR amplification reaction is:

[0017] The first round of reaction system: 1 μL of upstream outer primer and downstream outer primer, 1 μL of DNA template, 10 μL of 2×taq PCR buffer, 7 μL of ddH2O; the initial concentrations of the upstream outer primer and downstream outer primer were both 8-12 μM;

[0018] The second round reaction system is: 1 μL of upstream inner primer and downstream inner primer, 0.5 μL of the first round amplification product, 10 μL of 2×taq PCR buffer, and 7.5 μL of ddH2O; the initial concentrations of the upstream inner primer and downstream inner primer are both 8-12 μM;

[0019] The reaction procedure of the single nested PCR amplification reaction is:

[0020] The first round of reaction program was: 95°C pre-denaturation for 10 min, 26 cycles, and the cycle program was: 95°C denaturation for 40 s, 54-60°C annealing for 40 s, 72°C extension for 40 s, and 72°C extension for 5 min after the cycle.

[0021] The second round of reaction program was: pre-denaturation at 95°C for 10 min, 26 cycles, and the cycle program was: denaturation at 95°C for 40 s, annealing at 54-60°C for 40 s, extension at 72°C for 40 s, and extension at 72°C for 5 min after the cycle.

[0022] Furthermore, the reaction system of the double nested PCR amplification reaction is:

[0023] The first round of reaction system: 1 μL of the upstream outer primer and the downstream outer primer of the D19S433 site, 1 μL of the upstream outer primer and the downstream outer primer of the D2S441 site, 1 μL of DNA template, 10 μL of 2×taq PCR buffer, and 5 μL of ddH2O; the initial concentrations of the upstream outer primer and the downstream outer primer were both 8-12 μM;

[0024] The second round system is: 1 μL of the upstream inner primer and the downstream inner primer of the D19S433 site, 1 μL of the upstream inner primer and the downstream inner primer of the D2S441 site, 0.5 μL of the first round amplification product, 10 μL of 2×taq PCR buffer, and 5.5 μL of ddH2O; the initial concentrations of the upstream inner primer and the downstream inner primer are both 8-12 μM;

[0025] The reaction procedure of the double nested PCR amplification reaction is:

[0026] The first round of reaction program was: 95°C pre-denaturation for 5 min, 30 cycles, and the cycle program was: 95°C denaturation for 30 s, 58-62°C annealing for 30 s, 72°C extension for 40 s, and 72°C extension for 5 min after the cycle.

[0027] The second round of reaction program was: 95°C pre-denaturation for 5 min, 30 cycles, and the cycle program was: 95°C denaturation for 30 s, 58-62°C annealing for 30 s, 72°C extension for 40 s, and 72°C extension for 5 min after the cycle.

[0028] The present invention also provides the use of the nested PCR primer combination or the nested PCR detection kit in site analysis of genetic polymorphism, individual identification and / or parentage testing.

[0029] The present invention discloses the following technical effects:

[0030] The present invention designs a nested PCR primer combination for STR loci where rare alleles are found more frequently, which can achieve the purpose of specifically amplifying the target STR gene loci, and combines molecular cloning technology to separate and purify, and then determine the rare allele sequence, which can solve the technical problem that rare alleles cannot be determined in the process of forensic evidence individual identification and paternity testing. The primer combination and detection method of the present invention have the following advantages:

[0031] (1) Wide range of applications: Currently, most laboratories do not have a next-generation sequencing detection platform. For laboratories that cannot obtain NGS equipment, nested PCR and molecular cloning technology are an effective alternative method that can be used in a variety of different laboratory environments.

[0032] (2) Low detection cost: Nested PCR and molecular cloning technologies rely on low-cost basic equipment and reagents, and relatively low operating costs, while NGS sequencing is expensive.

[0033] (3) Simple operation: Nested PCR and molecular cloning technologies are relatively mature and laboratory technicians have a higher level of mastery, while NGS sequencing requires more professional training and experience.

[0034] (4) Results are easy to interpret: Nested PCR and molecular cloning techniques can provide more direct results, which are easier to interpret and accept in forensic evidence identification. However, the large amount of data generated by NGS sequencing requires complex bioinformatics analysis, which increases the complexity and error rate of interpretation.

[0035] (5) High sensitivity and specificity: Nested PCR amplifies the target DNA through two rounds of PCR, which can significantly improve the sensitivity and specificity of detection compared to conventional PCR, and is more effective in detecting trace amounts of DNA in mixed samples or degraded samples.

[0036] (6) More targeted: The product of D19S433 locus amplified by nested PCR in heterozygous samples was directly sequenced. The sequencing results are as follows: Figure 1 As shown in the figure, it can be seen that due to the difference in alleles, when typing a certain locus in the heterozygous sample, the amplification product is a mixed product and cannot be directly sequenced by Sanger sequencing. The amplification product of this locus was separated and purified by molecular cloning technology and then sequenced. The sequencing results are shown in Figure 2 As shown, it can be seen that molecular cloning technology has more advantages in detecting the typing of the two alleles of the heterozygote. It can effectively separate and sequence the mixed amplification products produced by the heterozygote, and can accurately type one allele of the heterozygote in a targeted manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0038] Figure 1 This is the sequencing result of the nested PCR amplification product of the heterozygous sample D19S433 site;

[0039] Figure 2 This is the sequencing result of the nested PCR amplification product of the heterozygous sample D19S433 site after molecular cloning;

[0040] Figure 3 This is the STR typing result of the Huaxia Platinum Test Kit for sample 1000707;

[0041] Figure 4 This is the STR typing result of the AGCU EX25 kit for sample 1000707;

[0042] Figure 5 The comparison result of the sequencing sequence of sample 1000707 obtained by the detection method of the present invention and the standard sequence

[0043] Figure 6 This is the STR typing result of the Huaxia Platinum Test Kit for sample 102-1;

[0044] Figure 7 This is the STR typing result of the AGCU EX25 kit for sample 102-1;

[0045] Figure 8 This is the comparison result between the sequencing sequence of sample 102-1 obtained by the detection method of the present invention and the standard sequence. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] The components of the nested PCR detection kit of the present invention include: a specific primer pair, a 2×taq PCR buffer (containing a dye), and ddH2O; wherein the sequence information of the specific primer pair is shown in Table 1.

[0052] Table 1 Sequence information of specific primer pairs

[0053]

[0054]

[0055] Single nested CPR detection method:

[0056] 1. Preparation of template DNA:

[0057] DNA was extracted and purified from samples containing DNA using QIAamp DNA Investigator Kit (Qiagen, USA) according to the instructions. The obtained DNA was quantified using Qubit 3.0 Fluorometer (Thermo Fisher Scientific, USA), and the DNA concentration was adjusted to 20-40 ng / μL with ddH2O to prepare DNA templates.

[0058] 2. Preparation of specific primer working solution:

[0059] After instant centrifugation of the specific primer powder, add the corresponding volume of ddH2O to prepare a 100 μM storage solution. When using, dilute the storage solution with ddH2O to prepare a working solution with a concentration of 8-12 μM.

[0060] 3. Nested PCR reaction:

[0061] 3.1 Single-site nested PCR reaction system and reaction procedure

[0062] The first round reaction system was 20 μL: 1 μL each of outer primer-F and outer primer-R, 1 μL DNA template, 10 μL 2× taq PCR buffer, and 7 μL ddH2O.

[0063] The first round of reaction program was: 95°C pre-denaturation for 10 min, 26 cycles, and the cycle program was: 95°C denaturation for 40 s, 54-60°C annealing for 40 s (54-60°C annealing means annealing at any temperature between 54°C and 60°C), 72°C extension for 40 s, and 72°C extension for 5 min after the cycle was completed.

[0064] The second round reaction system was 20 μL: 1 μL each of inner primer-F and inner primer-R, 0.5 μL of the first round amplification product, 10 μL of 2× taq PCR buffer, and 7.5 μL of ddH2O.

[0065] The second round of reaction program was: 95°C pre-denaturation for 10 min, 26 cycles, and the cycle program was: 95°C denaturation for 40 s, 54-60°C annealing for 40 s (54-60°C annealing means annealing at any temperature between 54°C and 60°C), 72°C extension for 40 s, and 72°C extension for 5 min after the cycle was completed.

[0066] 3.2 Multi-site multiplex nested PCR reaction system and reaction procedure

[0067] The first round reaction system was 20 μL: 1 μL each of outer primer-F and outer primer-R at the D19S433 site, 1 μL each of outer primer-F and outer primer-R at the D2S441 site, 1 μL of DNA template, 10 μL of 2×taq PCR buffer, and 5 μL of ddH2O.

[0068] The first round of reaction program was: 95°C pre-denaturation for 5 min, 30 cycles, and the cycle program was: 95°C denaturation for 30 s, 58-62°C annealing for 30 s, 72°C extension for 40 s, and 72°C extension for 5 min after the cycle.

[0069] The second round reaction system was 20 μL: 1 μL each of inner primer-F and inner primer-R at the D19S433 site, 1 μL each of inner primer-F and inner primer-R at the D2S441 site, 0.5 μL of the first round amplification product, 10 μL of 2×taqPCR buffer, and 5.5 μL of ddH2O.

[0070] The second round of reaction program was: 95°C pre-denaturation for 5 min, 30 cycles, and the cycle program was: 95°C denaturation for 30 s, 58-62°C annealing for 30 s, 72°C extension for 40 s, and 72°C extension for 5 min after the cycle.

[0071] 4. Nested PCR product detection and recovery:

[0072] Prepare 1.5% agarose gel: Calculate the required agarose and TBE content according to the needs. After adding TBE to agarose, heat it in a microwave oven until it is clear, place it at room temperature to a suitable temperature, add nucleic acid dye and pour it into the gel plate. Add 10μL of the nested PCR amplification product to the spotting well, observe the results under ultraviolet light after 100V electrophoresis for 1h.

[0073] PCR product recovery: Cut the gel at the corresponding position of the amplified product and use a gel recovery kit to recover the PCR product in the gel.

[0074] 5. Sequencing of nested PCR products:

[0075] The PCR product was ligated with the T vector. The ligation system was 10 μL: 3 μL of PCR product, 1 μL of T vector, 1 μL of T4 ligase, 1 μL of ligation buffer, and 4 μL of ddH2O. The ligation reaction conditions were: 16°C, 6 h.

[0076] After the ligation product was mixed evenly with 50 μL competent cells, it was placed in a 42°C water bath for 90 seconds, and 1 mL of liquid culture medium was added, and incubated at 37°C for 10 minutes. The incubated bacterial liquid was spread on solid culture medium, and cultured in a 37°C incubator after marking. After clones grew out, 10 single clones were randomly selected and placed in liquid culture medium for 8 hours.

[0077] The bacterial liquid was collected and the recombinant plasmid was extracted by alkaline lysis method. The size and content of the obtained recombinant plasmid were detected by agarose gel (1%) electrophoresis.

[0078] Utilize the extracted recombinant plasmid, select restriction endonuclease according to the multiple cloning site of the plasmid to digest the constructed plasmid. In some embodiments of the present invention, the restriction endonucleases used are: BamHI and HindIII. The digestion system is 20 μL: 1 μL of recombinant plasmid, 0.5 μL of BamHI, 0.5 μL of HindIII, 2 μL of K buffer, 16 μL of ddH2O; the digestion conditions are: 37°C, 1 hour. After digestion, the digestion product is subjected to agarose gel electrophoresis detection (agarose gel concentration is 1%). After digestion, a small fragment of about 350-600 bp is obtained, which can be determined as a successful recombination.

[0079] The successfully recombined plasmid can be directly sequenced by Sanger sequencing, can be sequenced directly in cooperation with a sequencing company, or can be sequenced by itself using the capillary electrophoresis platform of an identification agency.

[0080] Example 1

[0081] In the process of forensic evidence identification and investigation, the sample numbered 1000707 was subjected to STR typing using the Huaxia Platinum Composite Amplification Kit, and it was found that the D19S433 locus had only one peak. According to routine analysis, the locus of this individual was homozygous, and the allele was 13.2, that is, the genotype of this locus was 13.2, 13.2. The adjacent D2S441 locus had two peaks. According to conventional understanding, the locus of this individual was heterozygous, one allele was 11.2, and the other was the OL peak, which required manual calculation. According to conventional calculation methods, the allele corresponding to the OL peak was 16.2, and the result showed that the genotype of the D2S441 locus was 11, 16.2. Figure 3 shown.

[0082] Since the value of the D2S441 locus allele calculation process was not ideal, the STR typing results of the sample were compositely verified using the AGCU EX25 composite amplification kit. The results showed that the D19S433 locus still showed only one peak. According to conventional analysis, the locus of this individual was homozygous, and the allele was 13.2, that is, the genotype of this locus was 13.2,13.2. The D2S441 locus had only one peak. According to conventional understanding, the locus of this individual was homozygous, and the allele was 11, and the genotype of this locus was 11,11. Figure 4 shown.

[0083] Since the typing results of the AGCU EX25 and Huaxia Platinum kits at the D2S441 locus were inconsistent, the STR typing spectrum of the sample was manually reviewed and tested. The results showed that the AGCU EX25 and Huaxia Platinum kits used the length / size of the amplified product as the basis for judging the results. Since the amplified fragment of the rare allele of this locus was much smaller than the detection range of the STR typing kit, the peak of the rare allele was in front of the conventional detection range of D19S433 and was outside the detection range. The D19S433 locus in the Huaxia Platinum kit was preceded by the D2S441 locus. The rare allele peak of D19S433 appeared within the detection range of D2S441 and was misunderstood as the allele peak of D2S441. There was no other locus in front of the D19S433 locus in the AGCU EX25 kit, and the rare allele fell outside the detection range, while D2S441 was not affected. By comparing and analyzing the results of the two kits ( Figure 3 and Figure 4) It can be seen that the peak always exists on the left side of the D19S433 detection range, while the D2S441 peak only exists when it is on the left side of D19S433. Therefore, it can be determined that the peak belongs to the D19S433 locus. The reason why the genetic analyzer cannot automatically calculate the allele corresponding to the peak when the two test kits are used is that the amplified fragment of the allele is extremely small, which exceeds the instrument's recognition ability and calculation range, and therefore requires manual identification and calculation. During the analysis of this type of rare allele, the genetic analyzer cannot automatically detect this type of error, which can easily lead to STR typing errors and can only be corrected manually. Through manual correction, Figure 3 and Figure 4 Comparison analysis confirmed that the D19S433 locus of the sample was actually heterozygous, with a rare allele. After confirming that the rare allele belonged to the D19S433 locus, the allele typing was recalculated: Huaxia Platinum calculation showed that the allele was 4.1, and AGCU EX25 calculation showed that the allele was 4. Since the calculation results of the two kits were inconsistent, the locus could not be determined by calculation.

[0084] The sample is tested using the nested PCR detection kit of the present invention, and the specific steps are as follows:

[0085] 1. Use QIAamp DNA Investigator Kit (Qiagen, USA) according to the instructions to extract and purify DNA from sample 102-1. After quantitative detection, adjust the DNA concentration to 30 ng / μL with ddH2O to obtain a DNA template.

[0086] 2. Use ddH2O to prepare the specific primer pair for detecting D19S433 site (SEQ ID NO.1-4) and the specific primer pair for detecting D2S441 site (SEQ ID NO.5-8) into 10 μM working solution respectively.

[0087] 3. Prepare the reaction system for nested PCR amplification:

[0088] The first round reaction system was 20 μL: 1 μL each of outer primer-F and outer primer-R at the D19S433 site, 1 μL each of outer primer-F and outer primer-R at the D2S441 site, 1 μL of DNA template, 10 μL of 2×taq PCR buffer, and 5 μL of ddH2O.

[0089] The first round of reaction program was as follows: thermal cycle parameters were: 95°C pre-denaturation for 5 min, 30 cycles, cycle program was: 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 40 s, and 72°C extension for 5 min after the cycle.

[0090] The second round system was 20 μL: 1 μL each of inner primer-F and inner primer-R at the D19S433 site, 1 μL each of inner primer-F and inner primer-R at the D2S441 site, 0.5 μL of the first round amplification product, 10 μL of 2×taqPCR buffer, and 5.5 μL of ddH2O.

[0091] The second round of reaction program was as follows: thermal cycle parameters were: 95°C pre-denaturation for 5 min, 30 cycles, cycle program was: 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 40 s, and 72°C extension for 5 min after the cycle.

[0092] 4. The amplified product was tested by 1.5% agarose gel electrophoresis. The test results showed that there were only clear bands at 502bp and 540bp, with good specificity. The gel at the corresponding position was cut off respectively, and the PCR product in the gel was recovered using a gel recovery kit.

[0093] 5. The PCR product was ligated with the T vector (purchased from TaKaRa, Takara Biotech (Dalian) Co., Ltd.). The ligation system was 10 μL: 3 μL of PCR product, 1 μL of T vector, 1 μL of T4 ligase, 1 μL of ligation buffer, and 4 μL of ddH2O. The ligation reaction conditions were: 16°C, 6 h.

[0094] After the ligation product was mixed evenly with 50 μL Top10 competent cells (purchased from Invitrogen Life Technologies, USA), it was placed in a 42°C water bath for 90 seconds, 1 mL of liquid culture medium was added, and incubated at 37°C for 10 minutes. The incubated bacterial solution was spread on TB solid culture medium, marked and cultured in a 37°C incubator. After a clone grew out, a single clone was picked and placed in TB liquid culture medium for 8 hours. After the culture was completed, the bacterial solution was collected and the recombinant plasmid was extracted by alkaline lysis method. The recombinant plasmid was tested for plasmid size and content by 1% agarose gel electrophoresis.

[0095] Select BamHI and HindIII restriction endonucleases (purchased from New England Biolabs) to digest the extracted recombinant plasmid. The digestion system is 20 μL: 1 μL recombinant plasmid, 0.5 μL BamHI, 0.5 μL HindIII, 2 μL K buffer, 16 μL ddH2O; digestion conditions are: 37°C, 1 hour. After digestion, the digestion product is detected by 1% agarose gel electrophoresis. After digestion, a small fragment of about 540 bp or 502 bp is obtained to determine that the recombination is successful.

[0096] 6. Perform Sanger sequencing on the successfully recombined plasmid.

[0097] The DNA sequence of the sample locus was determined by nested amplification and molecular cloning and sequencing. The results were compared with the standard sequence. Figure 5 As shown, the allele type can be determined as 4.0 based on this result. Therefore, the genotype of D19S433 of the sample is 4,13.2, and the genotype of D2S441 is 11,11.

[0098] Example 2

[0099] During the forensic evidence identification practice, the sample numbered 102-1 was subjected to STR typing using the Huaxia Platinum Multiplex Amplification Kit, and it was found that there was a rare allele peak at the D2S441 locus that could not be identified by the genetic analyzer because the fragment was too small. Figure 6 As shown. The typing of the locus is calculated by the fragment size, and the allele typing is 7 repeats + 0.5 bases. It is impossible to determine whether the allele typing is 7 or 7.1 (Note: If the allele is 7 repeats + 0 bases, the allele is 7.0, and if it is 7 repeats + 1 base, the allele is 7.1. Since the number of bases must be an integer, there is no 0.5 base, and 7 repeats + 0.5 bases belong to a non-existent allele). STR typing was performed using the AGCU EX25 kit. The D2S441 locus has a rare allele peak that cannot be recognized by the genetic analyzer and is marked as OL, as shown. Figure 7 By calculating the allele typing as 7 repeats + 0.66 bases, it is still not possible to determine whether the allele is 7 or 7.1 by this value.

[0100] The sample is tested using the nested PCR detection kit of the present invention, and the specific steps are as follows:

[0101] 1. Use QIAamp DNA Investigator Kit (Qiagen, USA) according to the instructions to extract and purify DNA from sample 102-1. After quantitative detection, adjust the DNA concentration to 30 ng / μL with ddH2O to obtain a DNA template.

[0102] 2. Prepare the specific primer pair (SEQ ID NO. 5-8) for detecting the D2S441 site into a 10 μM working solution using ddH2O.

[0103] 3. Prepare the reaction system for nested PCR amplification:

[0104] The first round reaction system was 20 μL: 1 μL each of outer primer-F and outer primer-R, 1 μL DNA template, 10 μL 2× taq PCR buffer, and 7 μL ddH2O;

[0105] The first round of reaction program: thermal cycle parameters are: 95℃ pre-denaturation for 10 min, 26 cycles, cycle program is: 95℃ denaturation for 40 s, 56℃ annealing for 40 s, 72℃ extension for 40 s, and 72℃ extension for 5 min after the cycle.

[0106] The second round reaction system was 20 μL: 1 μL each of inner primer-F and inner primer-R, 0.5 μL of the first round amplification product, 10 μL of 2× taq PCR buffer, and 7.5 μL of ddH2O.

[0107] The second round of reaction program was as follows: thermal cycle parameters were: 95°C pre-denaturation for 10 min, 26 cycles, cycle program was: 95°C denaturation for 40 s, 56°C annealing for 40 s, 72°C extension for 40 s, and 72°C extension for 5 min after the cycle.

[0108] 4. The amplified product was tested by 1.5% agarose gel electrophoresis. The test results showed that there were only clear bands at 521 bp and 548 bp, with good specificity. The gel corresponding to the position of the amplified product was cut off, and the PCR product in the gel was recovered using a gel recovery kit.

[0109] 5. The PCR product was ligated with the T vector (purchased from TaKaRa, Takara Biotech (Dalian) Co., Ltd.). The ligation system was 10 μL: 3 μL of PCR product, 1 μL of T vector, 1 μL of T4 ligase, 1 μL of ligation buffer, and 4 μL of ddH2O. The ligation reaction conditions were: 16°C, 6 h.

[0110] After the ligation product was mixed evenly with 50 μL Top10 competent cells (purchased from Invitrogen Life Technologies, USA), it was placed in a 42°C water bath for 90 seconds, 1 mL of liquid culture medium was added, and incubated at 37°C for 10 minutes. The incubated bacterial solution was spread on TB solid culture medium, marked and cultured in a 37°C incubator. After a clone grew out, a single clone was picked and placed in TB liquid culture medium for 8 hours. After the culture was completed, the bacterial solution was collected and the recombinant plasmid was extracted by alkaline lysis method. The recombinant plasmid was tested for plasmid size and content by 1% agarose gel electrophoresis.

[0111] Select BamHI and HindIII restriction endonucleases (purchased from New England Biolabs) to digest the extracted recombinant plasmid. The digestion system is 20 μL: 1 μL recombinant plasmid, 0.5 μL BamHI, 0.5 μL HindIII, 2 μL K buffer, 16 μL ddH2O; digestion conditions are: 37°C, 1 hour. After digestion, the digestion product is detected by 1% agarose gel electrophoresis. After digestion, a small fragment of about 548 bp or 521 bp is obtained to determine that the recombination is successful.

[0112] 6. Perform Sanger sequencing on the successfully recombined plasmid.

[0113] The DNA sequence of the locus was determined by nested amplification and molecular cloning followed by sequencing. The results were compared with the standard sequence. Figure 8 As shown, the allele typing can be determined as 7.1 based on this result. Therefore, the genotype of D2S441 of this sample is 7.1,14.

[0114] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A nested PCR primer combination for detecting STR gene loci, characterized in that: The STR gene loci include D19S433 and D2S441; The primer combination includes a primer combination at the D19S433 site as shown in SEQ ID NO.1-4 and / or a primer combination at the D2S441 site as shown in SEQ ID NO.5-8.

2. Use of the nested PCR primer combination according to claim 1 in preparing a reagent or a kit for detecting STR gene loci.

3. A nested PCR detection kit for detecting STR gene loci, characterized in that: The nested PCR detection kit comprises the nested PCR primer combination according to claim 1.

4. The nested PCR detection kit according to claim 3, characterized in that: The nested PCR detection kit is used for single detection of the D19S433 site or the D2S441 site, or double detection of the D19S433 site and the D2S441 site.

5. A detection method of the nested PCR detection kit according to claim 3 or 4, characterized in that: The steps include: Extracting DNA from the sample to be tested; Using the nested PCR detection kit according to claim 3 or 4, performing a nested PCR amplification reaction on the DNA to obtain an amplified product; The amplified product is sequenced using molecular cloning technology, and the genotype of the STR gene locus of the sample to be tested is analyzed based on the sequencing result.

6. The detection method according to claim 5, characterized in that: The nested PCR amplification reaction is a single nested PCR amplification reaction or a double nested PCR amplification reaction.

7. The detection method according to claim 6, characterized in that: The reaction system of the single nested PCR amplification reaction is: The first round reaction system is: 1 μL of upstream outer primer and downstream outer primer, 1 μL of DNA template, 10 μL of 2×taq PCR buffer, and 7 μL of ddH2O; the initial concentrations of the upstream outer primer and downstream outer primer are both 8-12 μM; The second round reaction system is: 1 μL of upstream inner primer and downstream inner primer, 0.5 μL of the first round amplification product, 10 μL of 2×taqPCR buffer, and 7.5 μL of ddH2O; the initial concentrations of the upstream inner primer and downstream inner primer are both 8-12 μM; The reaction procedure of the single nested PCR amplification reaction is: The first round of reaction program was: 95°C pre-denaturation for 10 min, 26 cycles, and the cycle program was: 95°C denaturation for 40 s, 54-60°C annealing for 40 s, 72°C extension for 40 s, and 72°C extension for 5 min after the cycle. The second round of reaction program was: pre-denaturation at 95°C for 10 min, 26 cycles, and the cycle program was: denaturation at 95°C for 40 s, annealing at 54-60°C for 40 s, extension at 72°C for 40 s, and extension at 72°C for 5 min after the cycle.

8. The detection method according to claim 6, characterized in that: The reaction system of the double nested PCR amplification reaction is: The first round reaction system is: 1 μL of the upstream outer primer and the downstream outer primer of the D19S433 site, 1 μL of the upstream outer primer and the downstream outer primer of the D2S441 site, 1 μL of DNA template, 10 μL of 2×taq PCR buffer, and 5 μL of ddH2O; the initial concentrations of the upstream outer primer and the downstream outer primer are both 8-12 μM; The second round system is: 1 μL of the upstream inner primer and the downstream inner primer of the D19S433 site, 1 μL of the upstream inner primer and the downstream inner primer of the D2S441 site, 0.5 μL of the first round amplification product, 10 μL of 2×taq PCR buffer, and 5.5 μL of ddH2O; the initial concentrations of the upstream inner primer and the downstream inner primer are both 8-12 μM; The reaction procedure of the double nested PCR amplification reaction is: The first round of reaction program was: 95°C pre-denaturation for 5 min, 30 cycles, and the cycle program was: 95°C denaturation for 30 s, 58-62°C annealing for 30 s, 72°C extension for 40 s, and 72°C extension for 5 min after the cycle. The second round of reaction program was: 95°C pre-denaturation for 5 min, 30 cycles, and the cycle program was: 95°C denaturation for 30 s, 58-62°C annealing for 30 s, 72°C extension for 40 s, and 72°C extension for 5 min after the cycle.

9. Use of the nested PCR primer combination according to claim 1 or the nested PCR detection kit according to claim 3 in site analysis of genetic polymorphism, individual identification and / or parentage testing.

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