Composite amplification system for qPCR forensic medicine human DNA detection and application thereof

Abstract

The invention discloses a multiplex amplification system for qPCR forensic medicine human DNA detection and application of the multiplex amplification system, the multiplex amplification system simultaneously amplifies four targets of HS, HL, HY and IPC through qPCR, the HS target is used for total DNA quantification, the HL target is used for degradation degree evaluation, the HY target is used for sex proportion judgment, and the IPC target is used for inhibitor detection. The kit provided by the invention detects four targets of HS, HL, HY and IPC through a qPCR technology, realizes DNA quantification and quality analysis, and is simple and convenient to operate. The kit has the advantages of high sensitivity (the lowest detection limit is 1pg / mu L), strong inhibition resistance and good specificity, can accurately quantify male and female DNA, can also evaluate the DNA degradation degree and judge a PCR inhibitor, is suitable for various biological detection materials, provides an efficient and accurate solution for forensic DNA detection, and assists in case detection.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a multiplex amplification system for forensic human DNA detection by qPCR and its application. Background Art

[0002] Modern DNA (Deoxyribonucleic acid) detection technology has become one of the most important technical means in the field of modern forensic science, and is the most effective individual identification tool in dealing with various cases, major accidents and natural disasters and other major events. Forensic DNA analysis generally includes several steps such as sample collection, DNA extraction, DNA quantification, PCR amplification and genotyping, result analysis and comparison. Among them, DNA quantification and quality assessment are the key steps in the DNA analysis process, and also the basis for subsequent PCR amplification and genotyping detection, and are the guarantee for the accuracy and reliability of the detection results. In forensic practice, various difficult and degraded samples often need to be dealt with. The DNA molecules in these biological samples are easily degraded and broken into small fragment molecules due to being exposed to harsh environments and affected by various physical, chemical and biological factors. Some samples also show an increase in PCR inhibitors, resulting in a significant reduction in the detection rate. Conventional DNA quantification methods such as spectrophotometers cannot accurately quantify complex biological samples in forensic practice, nor can they evaluate the quality of sample DNA. Therefore, a simple and efficient forensic DNA quantification kit is needed. This kit should have a large detection range, be able to accurately measure the concentrations of constant and trace DNA samples; be able to determine whether there are inhibitors in the sample that interfere with the PCR process and the degree of inhibition; whether the sample DNA is degraded and the degree of degradation; be able to distinguish male and female DNA and give the ratio of male and female DNA in a mixed sample.

[0003] Therefore, there is an urgent need for a human DNA quantification detection technology with high sensitivity, high stability and simple operation that is compatible with STR kits. Summary of the Invention

[0004] In view of the current compatibility problems of forensic kits, complex procurement processes and high prices, the present invention provides a multiplex amplification system for forensic human DNA detection by qPCR and its application. By simultaneously amplifying four targets of HS, HL, HY and IPC by qPCR, it can be used for the quantification and quality judgment of forensic human DNA samples, with better accuracy, higher sensitivity and compatibility with STR typing detection kits.

[0005] To achieve the above object, the embodiments of the present invention adopt the following technical solutions: A multiplex amplification system for qPCR forensic human DNA detection. The multiplex amplification system simultaneously amplifies four targets, namely HS, HL, HY, and IPC, by qPCR. Among them, the HS target is used for total DNA quantification, the HL target is used for degradation degree assessment, the HY target is used for sex ratio determination, and the IPC target is used for inhibitor detection.

[0006] Preferably, the HS target is a multi-copy small fragment amplicon of autosomes, and its sequence is SEQ ID NO.1; the HL target is a multi-copy large fragment amplicon of autosomes, and its sequence is SEQ ID NO.2; the HY target is a multi-copy amplicon of the Y chromosome, and its sequence is SEQ ID NO.3; the IPC target is a synthetic sequence target not found in nature, and its sequence is SEQ ID NO.4.

[0007] Preferably, the upstream and downstream primer sequences for amplifying the HS target are as shown in SEQ ID NO.5 - 6; the upstream and downstream primer sequences for amplifying the HL target are as shown in SEQ ID NO.7 - 8; the upstream and downstream primer sequences for amplifying the HY target are as shown in SEQ ID NO.9 - 10; the upstream and downstream primer sequences for amplifying the IPC target are as shown in SEQ ID NO.11 - 12.

[0008] Preferably, the probe sequence for labeling the HS target is as shown in SEQ ID NO.13; the probe sequence for labeling the HL target is as shown in SEQ ID NO.14; the probe sequence for labeling the HY target is as shown in SEQ ID NO.15; the probe sequence for labeling the IPC target is as shown in SEQ ID NO.16.

[0009] The sequences of the present invention are shown in the following table:

[0010] Preferably, the 5'-fluorescent label of the probe for labeling the HS target is VIC, and the 3'-fluorescent label is BHQ1; the 5'-fluorescent label of the probe for labeling the HL target is CY5, and the 3'-fluorescent label is BHQ2; the 5'-fluorescent label of the probe for labeling the HY target is FAM, and the 3'-fluorescent label is BHQ1; the 5'-fluorescent label of the probe for labeling the IPC target is TAMRA, and the 3'-fluorescent label is BHQ2.

[0011] Preferably, ROX dye is also added to the multiplex amplification system for qPCR instrument calibration.

[0012] A qPCR forensic human DNA quantification kit includes the above multiplex amplification system.

[0013] Preferably, the kit also includes PCR amplification Mix and DNA quantification standards.

[0014] Preferably, the method of using the kit is as follows: Perform gradient dilution on the quantitative standard. Perform multiplex fluorescence PCR detection on the standard and forensic DNA samples using the primer-probe mixture. Convert the CT value of the sample into DNA concentration according to the obtained standard curve, determine the total amount of human DNA in the sample according to the multi-copy small fragment HS of autosomes, determine the male DNA content in the sample according to the multi-copy fragment HY of the Y chromosome (the measured value of female DNA is 0, and the measured value of male DNA is not much different from HS, and there may be a large difference in mixed samples), determine the female DNA content in the sample according to the difference between HS and HY, evaluate the degradation degree of the sample according to the degradation index DI = HS / HL, and evaluate the content of inhibitors in the sample according to the difference between the CT value of the IPC target of the sample and the CT value of the IPC target of the standard combined with the amplification situation of the target.

[0015] Use of the above multiplex amplification system or the above kit in the quantification of forensic DNA samples and the evaluation of sample quality.

[0016] Advantages of the implementation of the present invention: 1. The present invention can use qPCR amplification to detect four targets, namely HS, HL, HY, and IPC, so as to quantitatively analyze and quality analyze forensic human DNA, and the operation is simple.

[0017] 2. The present invention has good inhibitor resistance and a wide template adaptation range, and is applicable to the quantification of forensic DNA in all physical evidence cases involving cells. Any biological sample containing cells, such as oral swabs, blood stains, semen stains, saliva, hair, and other human tissues, can be used for DNA quantification.

[0018] 3. The kit of the present invention has high sensitivity and can detect a minimum template amount of 1 pg / μL in actual cases; it has good specificity, and common mammalian, avian, fish, and microbial DNAs have no interference with the system.

[0019] 4. The kit of the present invention can accurately quantify the content of male and female DNA, and can still accurately quantify the male DNA content when the male-female DNA ratio reaches 1:4000.

[0020] 5. The kit of the present invention can accurately reflect the degradation degree of DNA. The HL target specifically amplifies large fragments (240 bp) of autosomes, which can determine the degradation degree of DNA and guide whether it is necessary to use a Mini STR kit.

[0021] 6. The kit of the present invention can accurately determine the situation of PCR inhibitors in a sample. According to the change of CT values of the artificially synthesized IPC target amplification standard product and the sample, it can be determined whether the sample contains PCR inhibitors, and guide whether it is necessary to purify the sample.

[0022] 7. The kit of the present invention can accurately quantify the DNA content of various biological samples such as oral swabs, blood stains, semen stains, saliva, and hair, and guide the use of subsequent STR typing kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0024] Figure 1 It is the amplification map of a male semen stain DNA sample amplified by the kit of the present invention.

[0025] Figure 2 It is the standard curve graph drawn according to the CT values of different concentration standard products of three targets HS, HL, and HY obtained by using the kit of the present invention to amplify male standard products with concentrations of 50 / 5 / 0.5 / 0.05 / 0.005 ng / μL.

[0026] Figure 3 It is the CT value graph of using the kit of the present invention to amplify samples of pigeon, mouse, fish, dog, and Escherichia coli at 10 ng / μL, cat DNA sample at 2 ng / μL, and male DNA sample at 0.1 ng / μL, characterizing the species specificity of the kit.

[0027] Figure 4 It is the efficacy graph of using the kit of the present invention to detect male-female mixed samples.

[0028] Figure 5 It is the correlation graph of the degradation index DI of the simulated degraded DNA sample detected by the kit of the present invention and the typing system of the product named Microreader 28A ID System of Suzhou Yuewei Gene Technology Co., Ltd., where 28A amplifies ultrasonicated blood DNA.

[0029] Figure 6 It is the correlation graph of the degradation index DI of the simulated degraded DNA sample detected by the kit of the present invention and the typing system of the product named Microreader 28A ID System of Suzhou Yuewei Gene Technology Co., Ltd., where 28A amplifies enzymatically digested cell DNA.

[0030] Figure 7 To detect the correlation map between the inhibitor index of an inhibitor-containing DNA sample and the genotyping system of the product named Microreader 28A ID System of Suzhou Yuewei Gene Technology Co., Ltd. using the kit of the present invention, 28A amplifies the sample containing heme.

[0031] Figure 8 To detect the correlation map between the inhibitor index of an inhibitor-containing DNA sample and the genotyping system of the product named Microreader 28A ID System of Suzhou Yuewei Gene Technology Co., Ltd. using the kit of the present invention, 28A amplifies the sample containing humic acid.

[0032] Figure 9 To detect the sample genotyping map for using the kit of the present invention to detect simulated case samples and guiding the use of the genotyping systems of the product named Microreader 28A ID System of Suzhou Yuewei Gene Technology Co., Ltd. and the product named Microreader 40Y Plus ID System of Suzhou Yuewei Gene Technology Co., Ltd. according to the measured concentration, 28A amplifies a 0.2 ng cigarette butt sample (DI = 1.78).

[0033] Figure 10 To detect the sample genotyping map for using the kit of the present invention to detect simulated case samples and guiding the use of the genotyping systems of the product named Microreader 28A ID System of Suzhou Yuewei Gene Technology Co., Ltd. and the product named Microreader 40Y Plus ID System of Suzhou Yuewei Gene Technology Co., Ltd. according to the measured concentration, 28A amplifies a 0.46 ng keyboard sample (DI = 1.16).

[0034] Figure 11 To detect the sample genotyping map for using the kit of the present invention to detect simulated case samples and guiding the use of the genotyping systems of the product named Microreader 28A ID System of Suzhou Yuewei Gene Technology Co., Ltd. and the product named Microreader 40Y Plus ID System of Suzhou Yuewei Gene Technology Co., Ltd. according to the measured concentration, 40YP amplifies a 0.2 ng cigarette butt sample (DI = 1.78). Detailed implementation mode

[0035] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0036] A qPCR forensic human DNA quantification kit is based on the fluorescence quantitative PCR platform and combines specific primers and Taqman probe technology. It uses human-specific multi-copy targets to improve detection sensitivity, including small fragment amplicons HS and large fragment amplicons HL scattered on autosomes and amplicon HY on the Y chromosome. At the same time, synthetic sequences not found in nature are used as IPC targets to monitor the qPCR process and evaluate sample inhibition. Conserved sequences in the genome are selected to design primers and probes to reduce copy number variability between different individuals and populations.

[0037] The probes of a qPCR forensic human DNA quantification kit are labeled with different fluorescent labels to distinguish the amplification signals of different targets. The 5' and 3' of the HY target probe are labeled with FAM and BHQ1, the 5' and 3' of the small fragment HS target probe on autosome are labeled with VIC and BHQ1, the 5' and 3' of the large fragment HL target probe on autosome are labeled with Cy5 and BHQ2, and the 5' and 3' of the IPC target probe are labeled with TAMRA and BHQ2.

[0038] A qPCR forensic human DNA quantification kit contains the following components, as shown in Table 1.

[0039] Table 1 Composition of the kit

[0040] The above-mentioned qPCR forensic human DNA quantification kit can simultaneously amplify 4 targets, namely HS, HL, HY and IPC. Its primer mixture contains 8 specific primers and 4 probes, and the sequences and contents are shown in Table 2.

[0041] Table 2

[0042] The above-mentioned qPCR forensic human DNA quantification kit also contains a synthetic IPC plasmid, and its sequence and content are shown in Table 3.

[0043] Table 3

[0044] The use of the described qPCR forensic human DNA quantification kit requires diluting the standard DNA into 5 concentration gradients to construct a standard curve before testing the samples. The concentrations of the standard samples are 50 / 5 / 0.5 / 0.05 / 0.005 ng / μL respectively.

[0045] The amplification system of the described qPCR forensic human DNA quantification kit is shown in Table 4.

[0046] Table 4

[0047] The amplification program of the described qPCR forensic human DNA quantification kit is shown in Table 5.

[0048] Table 5 PCR reaction program

[0049] The present invention also provides an application method of the described qPCR forensic human DNA quantification kit. By performing qPCR amplification on the DNA extracted from forensic samples using a primer and probe mixture, the CT values of each target in the sample can be measured; by comparing with the standard curve, it can be directly converted into DNA concentration. The DNA concentration measured at the HS target is the total human DNA concentration; according to the DNA measured at the HY target, it can be determined whether there is male DNA. The difference between the CT value of the IPC site and the average CT value of the IPC in the standard product is used as the inhibitor index to determine whether there is an inhibitor in the sample. The ratio of the DNA concentration measured at the HS site to the DNA concentration measured at the HL site is used as the DNA degradation index DI of the sample. Based on the detected results of the total human DNA concentration, male DNA content, DNA degradation degree, and PCR inhibition index in the sample, it can guide subsequent typing strategies. For example, if it is determined that there may be an inhibitor interfering with the PCR process in the DNA sample and the inhibitor index is greater than 2, it will affect the subsequent typing results and the sample needs to be purified. If the measured DNA content is lower than 30 pg / μL, the DNA loading amount can be increased or the number of PCR cycles can be increased to obtain good typing results. If it is determined that the DNA sample has obvious degradation and the DI is greater than 3, when using the typing kit named Microreader 28A ID System of Suzhou Yuewei Gene Technology Co., Ltd. for typing, there will be an obvious phenomenon of high at the front and low at the back. When the DI is greater than 5, when using the typing kit named Microreader 28A ID System of Suzhou Yuewei Gene Technology Co., Ltd. for typing, there will be missing points in large fragments and a Mini STR kit needs to be used for typing. If it is found that the sample is a mixed male and female sample, a Y STR kit needs to be selected for subsequent testing, which is particularly important for sexual assault cases.

[0050] Example 1

[0051] Amplify the DNA sample of a male seminal stain using a qPCR forensic human DNA quantification kit 1. The DNA sample of the seminal stain is from the Institute of Forensic Science, Ministry of Public Security 2. Reaction system: After oscillating and mixing each reaction reagent, prepare the PCR reaction mixture according to the volume ratio in Table 4 (except for the template), aliquot 18 μL into PCR reaction tubes, and finally add 2 μL of the template to each reaction tube. After centrifugation, proceed to the next step. The composition of the reaction system is shown in Table 4

[0052] 3. The PCR reaction program is shown in Table 5

[0053] Place the prepared PCR reaction plate in an ABI 7500 real-time fluorescence quantitative PCR instrument, and set the amplification target information as shown in Table 6

[0054] Table 6

[0055] Set and run the following amplification program: Step 1: Denature at 95°C for 2 minutes, Step 2: Denature at 95°C for 9 seconds, Step 3: Anneal and extend at 60°C for 30 s (set fluorescence signal collection), repeat steps 2 to 3 for 40 times

[0056] 4. Data analysis After the run is completed, use ABI 7500 Software v2.3 to analyze the amplification results and generate the standard curve for each target. The slope of the standard curve is generally between -3.1 and -3.6, the R2 value ≥ 0.99, and the target amplification efficiency is between 90% - 110%, indicating that the amplification results are valid. Then, the amplification curve, amplification CT value, quantitative results, etc. of the sample can be viewed

[0057] 5. Experimental results The seminal stain sample from the Institute of Forensic Science, Ministry of Public Security can amplify the signals of 4 targets. The baseline of the amplification curve in each color channel is flat and the curve is smooth, as shown in Figure 1 . Among them, the HS quantitative value is 5.78 ng / μL, the HL quantitative value is 5.75 ng / μL, the HY quantitative value is 5.67 ng / μL, and the CT value of the IPC target is basically the same as that of the standard product. It is determined that the sample has no DNA degradation and does not contain inhibitors, meeting the expectations

[0058] Example 2

[0059] Construct a standard curve using a qPCR forensic human DNA quantification kit 1. The quantitative standard DNA sample is purchased from the National Institute of Metrology, China

[0060] 2. Reaction system After mixing the reaction reagents, oscillate and prepare the PCR reaction mixture according to the volume ratio in Table 4 (except template), dispense 18 μL into PCR reaction tubes, and finally add 2 μL template to each reaction tube, centrifuge and proceed to the next step. The composition of the reaction system is shown in Table 4.

[0061] 3. Dilution of Standards Refer to Table 7 below to dilute the quantitative standards to concentrations of 0.005 ng / µL, 0.050 ng / µL, 0.500 ng / µL, 5.000 ng / µL, and 50.000 ng / µL.

[0062] Table 7

[0063] 4. The PCR reaction program is shown in Table 5.

[0064] Place the configured PCR reaction plate in the ABI 7500 real-time fluorescence quantitative PCR instrument and set the amplification target information as described in Table 6.

[0065] Set up the following amplification program: Step 1: denaturation at 95°C for 2 minutes, step 2: denaturation at 95°C for 9 seconds, step 3: annealing and extension at 60°C for 30 seconds (set up fluorescence signal acquisition), and repeat steps 2 to 3 40 times.

[0066] 5. Data Analysis After the run, ABI 7500 Software v2.3 was used to analyze the amplification results and generate standard curves for each target. The slope of the standard curve was generally -3.1 to -3.6, R 2 The value is ≥0.99 and the target amplification efficiency is between 90% and 110%, indicating that the amplification result is valid.

[0067] 6. Experimental Results The standard curve constructed using the quantitative standards at concentrations of 0.005 ng / µL, 0.050 ng / µL, 0.500 ng / µL, 5.000 ng / µL, and 50.000 ng / µL is shown in Figure 2 , see Table 8 for specific information.

[0068] Table 8

[0069] The slope of the linear regression equation of the standard curves of HS, HL and HY is close to -3.33, R 2 All of them were greater than 0.99, which could effectively reflect the linear relationship between CT value and concentration.

[0070] Example 3

[0071] Verify the species specificity of a qPCR forensic human DNA quantification kit 1. Use Nanodrop to preliminarily quantify the DNA of animals, livestock, and microorganisms from different sources; dilute pigeon, mouse, fish, dog, and Escherichia coli DNA to a concentration of 10 ng / μL, and dilute cat DNA to a concentration of 2 ng / μL. At the same time, use male DNA with a concentration of 0.005 ng / μL as a positive control.

[0072] 2. Reaction system After mixing each reaction reagent by oscillation, prepare a PCR reaction mixture according to the volume ratio in Table 4 (except for the template), dispense 18 μL into a PCR reaction tube, and finally add 2 μL of the template to each reaction tube. After centrifugation, proceed to the next step. The composition of the reaction system is shown in Table 4.

[0073] 3. The PCR reaction procedure is shown in Table 5.

[0074] Place the prepared PCR reaction plate in an ABI 7500 real-time fluorescence quantitative PCR instrument, and set the amplification target information as shown in Table 6.

[0075] Set the following amplification program to run: Step 1: Denature at 95°C for 2 minutes, Step 2: Denature at 95°C for 9 seconds, Step 3: Anneal and extend at 60°C for 30 s (set fluorescence signal collection), repeat steps 2 to 3 forty times.

[0076] 4. Data analysis After the run is completed, use ABI 7500 Software v2.3 to analyze the amplification results and generate a standard curve for each target. The slope of the standard curve is generally between -3.1 and -3.6, the R2 value ≥ 0.99, and the target amplification efficiency is between 90% and 110%, indicating that the amplification results are valid. Then, the amplification curve, amplification CT value, and quantification results of the samples can be viewed.

[0077] 5. Experimental results Use a qPCR forensic human DNA quantification kit to test the above DNA samples of different species. The CT values of the HS, HL, and HY targets are shown in Table 9.

[0078] Table 9: CT values of different species samples tested for each target

[0079] As Figure 3As shown, a qPCR forensic human DNA quantification kit was used to test DNA of different species. The CT values of three target genes amplified from common DNA samples such as pigeon, cat, dog, mouse, fish, and Escherichia coli were all comparable to those of the NC samples, significantly higher than those of the positive control sample P5 at 5 pg / μL, and there was no obvious cross - contamination.

[0080] Example 4

[0081] Use a qPCR forensic human DNA quantification kit to test its ability to distinguish male - female mixed samples In the R & D sample library of Yuewei Gene (samples donated by volunteers), one DNA sample extracted from the blood of male and female volunteers was selected and preliminarily quantified using Nanodrop; under the condition of controlling the concentration of male DNA samples at 20 pg / µL, they were mixed with female samples at ratios of 1:1, 1:5, 1:10, 1:20, 1:100, 1:1000, 1:1500, 1:2000, and 1:4000.

[0082] 2. Reaction system After shaking and mixing each reaction reagent, prepare a PCR reaction mixture according to the volume ratio shown in Table 4 (except for the template), aliquot 18 μL into PCR reaction tubes, and finally add 2 μL of the template to each reaction tube, followed by centrifugation and amplification. The composition of the reaction system is shown in Table 4.

[0083] 3. The PCR reaction program is shown in Table 5.

[0084] Place the prepared PCR reaction plate into an ABI 7500 real - time fluorescence quantitative PCR instrument and set the amplification target information as shown in Table 6.

[0085] Set the following amplification program to run: Step 1: Denature at 95°C for 2 minutes. Step 2: Denature at 95°C for 9 seconds. Step 3: Anneal and extend at 60°C for 30 s (set fluorescence signal collection), and repeat steps 2 and 3 for 40 times.

[0086] 4. Data analysis After the run, use ABI 7500 Software v2.3 to analyze the amplification results and generate standard curves for each target. The slope of the standard curve is generally between - 3.1 and - 3.6, and the R 2 value ≥ 0.99, and the target amplification efficiency is between 90% - 110%, indicating that the amplification results are valid. Then, the amplification curves, amplification CT values, quantitative results, etc. of the samples can be viewed.

[0087] 5. Experimental results A qPCR forensic human DNA quantification kit was used to test the above - mentioned male - female samples mixed at different ratios, and the measured concentration values of each target are shown in Table 10.

[0088] Table 10

[0089] This method can well distinguish the presence of male DNA components in male-female mixed samples. Even when the ratio of male to female DNA is 1:4000, the content of male DNA can still be accurately calibrated. The results are shown in Figure 4 .

[0090] Example 5

[0091] Apply a qPCR forensic human DNA quantification kit to test DNA samples with different degradation degrees and guide STR typing tests.

[0092] 1. Select 1 DNA sample extracted from the blood of a male volunteer and 1 DNA sample of the 9948 cell line in the R&D sample library of Yuewei Gene (the samples were donated by volunteers). Use Nanodrop for preliminary quantification. The concentration of the blood DNA sample is about 50 ng / µL, labeled as M, and the concentration of the 9948 cell line sample is about 2 ng / µL, labeled as H. The blood DNA sample is fragmented to different degrees using the ultrasonic fragmentation method, labeled as ML, MM, MH; the cell line DNA sample is fragmented to different degrees using the enzymatic digestion method (DNA endonuclease, purchased from Kangwei Century, product number CW2090S), labeled as HL, HM, HH.

[0093] 2. Reaction system After oscillating and mixing each reaction reagent, prepare a PCR reaction mixture according to the volume ratio in Table 4 (except for the template), aliquot 18 μL into PCR reaction tubes, and finally add 2 μL of the template to each reaction tube, followed by centrifugal amplification. The composition of the reaction system is shown in Table 4.

[0094] 3. The PCR reaction program is shown in Table 5.

[0095] Place the prepared PCR reaction plate in an ABI 7500 real-time fluorescence quantitative PCR instrument, and set the amplification target information as shown in Table 6.

[0096] Set the following amplification program to run: Step 1: Denature at 95°C for 2 minutes, Step 2: Denature at 95°C for 9 seconds, Step 3: Anneal and extend at 60°C for 30 s (set fluorescence signal collection), repeat steps 2 to 3 for 40 times.

[0097] 4. Data analysis After the run, use ABI 7500 Software v2.3 to analyze the amplification results and generate standard curves for each target. The slope of the standard curve is generally between -3.1 and -3.6, the R2 value is ≥ 0.99, and the target amplification efficiency is between 90% and 110%, indicating that the amplification results are valid. Then, the amplification curves, amplification CT values, quantitative results, etc. of the samples can be viewed.

[0098] 5. Experimental Results A qPCR forensic human DNA quantification kit was used to test the above simulated samples with different degradation degrees. The average IPC CT value was 27.86, indicating, as expected, no significant PCR inhibition. The measured concentration values of each target are shown in Table 11.

[0099] Table 11

[0100] This kit can accurately determine the concentration and degradation degree of degraded samples at different levels. According to the quantitative results, the blood DNA samples M, ML, MM, and MH were diluted to 1 ng / µL, and the cell line sample H was diluted to 1 ng / µL. Then, a genotyping kit with the product name Microreader 28A ID System from Suzhou Yuewei Gene Technology Co., Ltd. was used to perform genotyping tests on the above samples. The input template amount was 1 ng for all samples. The results are shown in Figures 5-6 When the degradation index DI of the sample reaches 2, there is an obvious phenomenon of high at the front and low at the back in the amplification pattern. When the degradation index DI is greater than 3, there is a risk of loss of large fragments in the genotyping pattern, and it is necessary to use a MiniSTR kit for additional testing.

[0101] Example Six

[0102] Application of a qPCR forensic human DNA quantification kit to test DNA samples containing different inhibitors and guide STR genotyping tests 1. The two most common inhibitors in forensic samples are mainly heme and humic acid. When the concentration of 9948 cell DNA (produced by Yuewei Gene) is 0.1 ng / µL, 0 - 1250 μM of heme is added to the qPCR system; when the concentration of M308 cell DNA (produced by Yuewei Gene) is 0.1 ng / µL, 0 - 800 ng / µL of humic acid is added to the qPCR system to test the tolerance of the present invention to inhibitors and the level of PCR inhibitors in the sample, and to guide the subsequent use of the genotyping kit with the product name Microreader 28A ID System from Suzhou Yuewei Gene Technology Co., Ltd.

[0103] 2. Reaction System After shaking and mixing each reaction reagent, prepare a PCR reaction mixture according to the volume ratio in Table 4 (except for the template), dispense 18 μL into PCR reaction tubes, and finally add 2 μL of the template to each reaction tube, followed by centrifugal amplification. The composition of the reaction system is shown in Table 4.

[0104] 3. The PCR reaction program is shown in Table 5.

[0105] Place the prepared PCR reaction plate in an ABI 7500 real-time fluorescence quantitative PCR instrument, and set the amplification target information as shown in Table 6.

[0106] Set the following amplification program to run: Step 1: Denature at 95 °C for 2 minutes, Step 2: Denature at 95 °C for 9 seconds, Step 3: Anneal and extend at 60 °C for 30 s (set fluorescence signal collection), repeat steps 2 to 3 for 40 times.

[0107] 4. Data analysis After the run, use ABI 7500 Software v2.3 to analyze the amplification results and generate standard curves for each target. The slope of the standard curve is generally between -3.1 and -3.6, and the R 2 value ≥ 0.99, and the target amplification efficiency is between 90% - 110%, indicating that the amplification results are valid. Then, the amplification curves, amplification CT values, quantitative results, etc. of the samples can be viewed.

[0108] 5. Experimental results A qPCR forensic human DNA quantification kit was used to test the above samples containing different concentrations of PCR inhibitors, and the measured concentration values of each target are shown in Table 12.

[0109] Table 12

[0110] This kit can accurately determine the DNA sample concentrations containing different amounts of heme and humic acid. The difference between the CT value of the IPC target and the CT of the standard product is used as the inhibitor index to indicate whether there is a PCR inhibitor. The quantitative results show that the amplification is normal and the quantification is accurate when the final concentration of heme is below 750 μM; above 1000 μM, the amplification of all targets is significantly inhibited and the quantification concentration is inaccurate. When the final concentration of humic acid is 600 ng / μL or above, the amplification of all targets is significantly inhibited and the quantification concentration is inaccurate.

[0111] Directly add 5 μL of the sample and use the typing kit with the product name Microreader28A ID System from Suzhou Yuewei Gene Technology Co., Ltd. for STR typing test, and the results are shown in Figures 7-8Large - scale data loss occurred at the final heme concentration of 1000 µM, and the spectrum was significantly higher at the front and lower at the back; large - scale data loss occurred at the final humic acid concentration of 600 ng / µL, and the spectrum was significantly higher at the front and lower at the back.

[0112] When using a qPCR forensic human DNA quantification kit, an inhibitor index greater than 2 will significantly affect qPCR quantification and subsequent genotyping, and the sample needs to be purified.

[0113] Example Seven

[0114] Use a qPCR forensic human DNA quantification kit to test simulated case samples and guide STR genotyping tests 1. The sources of forensic case samples are complex. For simulated case samples, DNA was extracted from 8 cases sourced from bloodstains, semen stains, oral swabs, cigarette butts, hair, and various swab samples. The samples were diluted 100 - fold to simulate extremely trace case samples. A qPCR forensic human DNA quantification kit was used for quantitative analysis and to guide the use of the typing kits named Microreader 28A ID System and Microreader 40Y Plus ID System produced by Suzhou Yuewei Gene Technology Co., Ltd.

[0115] 2. Reaction system After oscillating and mixing each reaction reagent, prepare the PCR reaction mixture according to the volume ratio in Table 4 (except for the template), dispense 18 μL into PCR reaction tubes, and finally add 2 μL of the template to each reaction tube, then centrifuge for amplification. The composition of the reaction system is shown in Table 4.

[0116] 3. The PCR reaction program is shown in Table 5.

[0117] Place the prepared PCR reaction plate in an ABI 7500 real - time fluorescence quantitative PCR instrument and set the amplification target information as shown in Table 6.

[0118] Set the following amplification program to run: Step 1: Denature at 95 °C for 2 minutes, Step 2: Denature at 95 °C for 9 seconds, Step 3: Anneal and extend at 60 °C for 30 s (set fluorescence signal collection), repeat steps 2 to 3 for 40 times.

[0119] 4. Data analysis After the run, use ABI 7500 Software v2.3 to analyze the amplification results and generate the standard curve for each target. The slope of the standard curve is generally between - 3.1 and - 3.6, R 2The value ≥ 0.99 and the target amplification efficiency is between 90% - 110%, indicating that the amplification result is valid. Then, the amplification curve, amplification CT value, quantitative result, etc. of the sample can be viewed.

[0120] 5. Experimental Results A qPCR forensic human DNA quantification kit was used to test the above-mentioned simulated case samples, and the concentrations of each target were measured. The detection results of STR genotyping tests using the genotyping kit with the product name Microreader 28A ID System of Suzhou Yuewei Gene Technology Co., Ltd. are shown in Table 13.

[0121] Table 13

[0122] This kit can accurately measure the concentrations and degradation conditions of simulated case samples from different sources, and guide the use of the genotyping kit with the product name Microreader 28A ID System of Suzhou Yuewei Gene Technology Co., Ltd. and the genotyping kit with the product name Microreader 40Y Plus ID System of Suzhou Yuewei Gene Technology Co., Ltd. for STR genotyping tests. The results are shown in Figures 9-11 . For the 0.2 ng cigarette butt sample amplified by 28A (DI = 1.78), it is significantly high at the front and low at the back. For the 0.2 ng cigarette butt sample amplified by 40YP (DI = 1.78), it is also significantly high at the front and low at the back. For the 0.46 ng keyboard wipe sample amplified by 28A (DI = 1.16), the obtained genotyping map has good balance. The guiding effect of the STR genotyping kit is good, which can greatly improve the sample detection rate.

[0123] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A composite amplification system for qPCR forensic human DNA detection, characterized in that: The composite amplification system simultaneously amplifies four targets, HS, HL, HY and IPC, by qPCR, wherein the HS target is used for total DNA quantification, the HL target is used for degradation evaluation, the HY target is used for sex ratio determination, and the IPC target is used for inhibitor detection.

2. The composite amplification system according to claim 1, characterized in that: The HS target is a multi-copy small-fragment amplicon of an autosome, and its sequence is SEQ ID NO.1; the HL target is a multi-copy large-fragment amplicon of an autosome, and its sequence is SEQ ID NO.2; the HY target is a multi-copy amplicon of a Y chromosome, and its sequence is SEQ ID NO.3; the IPC target is a synthetic sequence target not found in nature, and its sequence is SEQ ID NO.

4.

3. The composite amplification system according to claim 2, characterized in that: The upstream and downstream primer sequences for amplifying the HS target are shown in SEQ ID NOs.5-6; the upstream and downstream primer sequences for amplifying the HL target are shown in SEQ ID NOs.7-8; the upstream and downstream primer sequences for amplifying the HY target are shown in SEQ ID NOs.9-10; the upstream and downstream primer sequences for amplifying the IPC target are shown in SEQ ID NOs.11-12.

4. The composite amplification system according to claim 2, characterized in that: The probe sequence for marking the HS target is shown in SEQ ID NO.13; the probe sequence for marking the HL target is shown in SEQ ID NO.14; the probe sequence for marking the HY target is shown in SEQ ID NO.15; and the probe sequence for marking the IPC target is shown in SEQ ID NO.

15.

5. The composite amplification system according to claim 4, characterized in that: The HS target-labeled probe is 5' fluorescently labeled with VIC and 3' fluorescently labeled with BHQ1; the HL target-labeled probe is 5' fluorescently labeled with CY5 and 3' fluorescently labeled with BHQ2; the HY target-labeled probe is 5' fluorescently labeled with FAM and 3' fluorescently labeled with BHQ1; the IPC target-labeled probe is 5' fluorescently labeled with TAMRA and 3' fluorescently labeled with BHQ2.

6. The composite amplification system according to claim 5, characterized in that: ROX dye is also added to the composite amplification system for qPCR instrument correction.

7. A qPCR forensic human DNA quantification kit, characterized in that: Comprising the composite amplification system as described in any one of claims 1-6.

8. The kit according to claim 7, characterized in that The kit also includes PCR amplification mix and DNA quantitative standards.

9. The kit according to claim 7, characterized in that The method of using the kit is as follows: Perform serial dilutions of the quantitative standards; Multiplex fluorescence PCR detection of standards and forensic DNA samples was performed using primer-probe mixtures; The CT value of the sample was converted into DNA concentration according to the obtained standard curve, the total amount of human DNA in the sample was determined according to HS, the male DNA content in the sample was determined according to the multi-copy fragment HY of the Y chromosome, the female DNA content in the sample was determined according to the difference between HS and HY, the degree of sample degradation was evaluated according to the degradation index DI=HS / HL, and the content of inhibitors in the sample was evaluated according to the difference between the IPC target CT value of the sample and the IPC target CT value of the standard.

10. Use of the composite amplification system according to any one of claims 1 to 6, or the kit according to any one of claims 7 to 8 in forensic DNA sample quantification and sample quality assessment.

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