A multiplex amplification system for qPCR forensic human DNA detection and its application

Abstract

The present invention discloses a multiplex amplification system for forensic human DNA detection by qPCR and its application. 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 evaluation, the HY target is used for sex ratio judgment, and the IPC target is used for inhibitor detection. The kit of the present invention detects four targets, namely HS, HL, HY and IPC, by qPCR technology to achieve DNA quantification and quality analysis, and the operation is simple. It has high sensitivity (the lowest detection limit is 1 pg / μL), strong anti-inhibition ability, good specificity, can accurately quantify male and female DNA, can also evaluate the DNA degradation degree, judge PCR inhibitors, is applicable to a variety of biological samples, provides an efficient and accurate solution for forensic DNA detection, and helps solve cases.

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 handling various cases, major accidents, 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, which guarantee the accuracy and reliability of the detection results. In forensic practice, various difficult-to-degrade 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 interfering with the PCR process in the sample 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 highly sensitive, highly stable and easy-to-operate human DNA quantification detection technology 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, namely 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:

[0006] 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 evaluation, the HY target is used for sex ratio determination, and the IPC target is used for inhibitor detection.

[0007] 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.

[0008] 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.

[0009] 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.

[0010] The sequences of the present invention are shown in the following table:

[0011]

[0012] 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.

[0013] Preferably, ROX dye is also added to the multiplex amplification system for qPCR instrument calibration.

[0014] A qPCR forensic human DNA quantification kit includes the above-mentioned multiplex amplification system.

[0015] Preferably, the kit also includes PCR amplification Mix and DNA quantification standard.

[0016] Preferably, the method of using the kit is as follows:

[0017] Perform gradient dilution on the quantitative standard.

[0018] Perform multiplex fluorescence PCR detection on the standard and forensic DNA samples using the primer-probe mixture.

[0019] Convert the CT value of the sample to DNA concentration according to the obtained standard curve, determine the total amount of human DNA in the sample based on the multi-copy small fragment HS of autosomes, determine the male DNA content in the sample based on 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 in the sample and the CT value of the IPC target in the standard combined with the amplification situation of the target.

[0020] Use of the above multiplex amplification system or the above kit in the quantification of forensic DNA samples and the evaluation of sample quality.

[0021] Advantages of the implementation of the present invention:

[0022] 1. The present invention can detect four targets, HS, HL, HY, and IPC, by qPCR amplification, so as to perform quantification and quality analysis on forensic human DNA, and the operation is simple.

[0023] 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 buccal swabs, blood stains, semen stains, saliva, hair, and other human tissues, can be used for DNA quantification.

[0024] 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 the DNA of common mammals, birds, fish, and microorganisms has no interference with the system.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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

[0029] 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.

[0030] Figure 1 It is the amplification map of a male semen stain DNA sample amplified by the kit of the present invention.

[0031] Figure 2 It is to amplify male standard products with concentrations of 50 / 5 / 0.5 / 0.05 / 0.005 ng / μL using the kit of the present invention, and draw a standard curve graph based on the CT values of different concentration standard products of the three targets HS, HL, and HY obtained.

[0032] Figure 3 It is the CT value graph of amplifying 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 using the kit of the present invention, characterizing the species specificity of the kit.

[0033] Figure 4 It is the efficiency graph of detecting a male-female mixed sample using the kit of the present invention.

[0034] Figure 5 It is the correlation graph of the degradation index DI of detecting a simulated degraded DNA sample and the typing system with the product name of Microreader 28A ID System of Suzhou Yuewei Gene Technology Co., Ltd. 28A amplifies ultrasonically fragmented blood DNA.

[0035] Figure 6 It is the correlation graph of the degradation index DI of detecting a simulated degraded DNA sample and the typing system with the product name of Microreader 28A ID System of Suzhou Yuewei Gene Technology Co., Ltd. 28A amplifies enzymatically digested cell DNA.

[0036] Figure 7 To detect the correlation map between the inhibitor index of the 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.

[0037] Figure 8 To detect the correlation map between the inhibitor index of the 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.

[0038] 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 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).

[0039] 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 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).

[0040] 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 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

[0041] 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.

[0042] 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 dispersed on autosomes and amplicon HY on the Y chromosome. At the same time, a synthetic sequence not found in nature is used as the IPC target to monitor the qPCR process and evaluate sample inhibition. Primers and probes are designed based on conserved sequences in the genome to reduce copy number variability between different individuals and populations.

[0043] 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 autosomal fragment HS target probe are labeled with VIC and BHQ1, the 5' and 3' of the large autosomal target HL probe are labeled with Cy5 and BHQ2, and the 5' and 3' of the IPC target probe are labeled with TAMRA and BHQ2.

[0044] A qPCR forensic human DNA quantification kit contains the following components, as shown in Table 1.

[0045] Table 1 Composition of the kit

[0046]

[0047] 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.

[0048] Table 2

[0049]

[0050] The above-mentioned qPCR forensic human DNA quantification kit also contains an artificially synthesized IPC plasmid, and its sequence and content are shown in Table 3.

[0051] Table 3

[0052]

[0053] 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.

[0054] The amplification system of the described qPCR forensic human DNA quantification kit is shown in Table 4.

[0055] Table 4

[0056]

[0057] The amplification program of the described qPCR forensic human DNA quantification kit is shown in Table 5.

[0058] Table 5 PCR reaction program

[0059]

[0060] 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; based on 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 results of the total human DNA concentration, male DNA content, DNA degradation degree, and PCR inhibition index in the detected sample, it can guide subsequent genotyping strategies. For example, if it is determined that there may be inhibitors interfering with the PCR process in the DNA sample and the inhibitor index is greater than 2, it will affect the subsequent genotyping 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 genotyping results. If it is determined that the DNA sample has obvious degradation and the DI is greater than 3, when using the genotyping kit with the product name Microreader 28A ID System from Suzhou Yuewei Gene Technology Co., Ltd. for genotyping, there will be an obvious phenomenon of high at the beginning and low at the end. When the DI is greater than 5, when using the genotyping kit with the product name Microreader 28A ID System from Suzhou Yuewei Gene Technology Co., Ltd. for genotyping, there will be missing points in large fragments and a Mini STR kit needs to be used for genotyping. If it is found that the sample is a male-female mixed sample, a Y STR kit needs to be selected for subsequent testing, which is particularly important for sexual assault cases.

[0061] Example 1

[0062] Apply a qPCR forensic human DNA quantification kit to amplify a male semen stain DNA sample

[0063] 1. The semen stain DNA sample is from the Institute of Forensic Science, Ministry of Public Security

[0064] 2. Reaction system:

[0065] After oscillating 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 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

[0066] 3. The PCR reaction program is shown in Table 5

[0067] 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

[0068] Table 6

[0069]

[0070] Set the following amplification program to run:

[0071] 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

[0072] 4. Data analysis

[0073] 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, quantitative results, etc. of the sample can be viewed

[0074] 5. Experimental results

[0075] The semen 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. See 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

[0076] Example 2

[0077] Constructing a standard curve using a qPCR forensic human DNA quantification kit

[0078] 1. The quantitative standard DNA sample was purchased from the National Institute of Metrology, China.

[0079] 2. Reaction system

[0080] After mixing each reaction reagent by oscillation, prepare the PCR reaction mixture according to the volume ratios in Table 4 (except for the template), dispense 18 μL into the 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.

[0081] 3. Standard dilution

[0082] Dilute the quantitative standard according to Table 7 below to concentrations of 0.005 ng / µL, 0.050 ng / µL, 0.500 ng / µL, 5.000 ng / µL, and 50.000 ng / µL respectively.

[0083] Table 7

[0084]

[0085] 4. The PCR reaction program is shown in Table 5.

[0086] Place the prepared PCR reaction plate in the ABI 7500 real-time fluorescence quantitative PCR instrument and set the amplification target information as shown in Table 6.

[0087] Set the following amplification program to run:

[0088] 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.

[0089] 5. Data analysis

[0090] After the run, use ABI 7500 Software v2.3 to analyze the amplification results and generate the 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.

[0091] 6. Experimental results

[0092] The standard curves constructed using quantitative standards with concentrations of 0.005 ng / µL, 0.050 ng / µL, 0.500 ng / µL, 5.000 ng / µL, and 50.000 ng / µL are shown inFigure 2 , the specific information is shown in Table 8.

[0093] Table 8

[0094]

[0095] The slopes of the linear regression equations of the HS, HL, and HY standard curves are close to -3.33, and the R 2 values are all greater than 0.99, which can effectively reflect the linear relationship between CT value and concentration.

[0096] Example 3

[0097] Verify its species specificity by applying a qPCR forensic human DNA quantification kit

[0098] 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 standard male DNA with a concentration of 0.005 ng / μL as a positive control.

[0099] 2. Reaction system

[0100] After shaking 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 template to each reaction tube. After centrifugation, proceed to the next step. The composition of the reaction system is shown in Table 4 mentioned above.

[0101] 3. The PCR reaction procedure is shown in Table 5 mentioned above.

[0102] 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 mentioned above.

[0103] Set the following amplification program to run:

[0104] 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.

[0105] 4. Data analysis

[0106] After the run is completed, 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 ≥ 0.99, and the amplification efficiency of the target 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.

[0107] 5. Experimental Results

[0108] A qPCR forensic human DNA quantification kit was used to test the above-mentioned DNA samples of different species, and the CT values of the HS, HL, and HY targets are shown in Table 9.

[0109] Table 9: CT values of different species samples tested at each target

[0110]

[0111] As Figure 3 shown, when a qPCR forensic human DNA quantification kit was used to test DNA of different species, the CT values of the three targets with common DNA amplification 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.

[0112] Example 4

[0113] Apply a qPCR forensic human DNA quantification kit to test its discrimination ability for male-female mixed samples

[0114] One DNA sample extracted from the blood of male and female volunteers was selected from the R&D sample library of Yuewei Gene (the samples were donated by volunteers) and preliminarily quantified using Nanodrop; under the condition of controlling the concentration of the male DNA sample at 20 pg / µL, it was mixed with the female sample at ratios of 1:1, 1:5, 1:10, 1:20, 1:100, 1:1000, 1:1500, 1:2000, and 1:4000.

[0115] 2. Reaction System

[0116] After shaking 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 and perform centrifugal amplification. The composition of the reaction system is shown in Table 4.

[0117] 3. The PCR reaction procedure is shown in Table 5.

[0118] 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.

[0119] Set the following amplification program to run:

[0120] 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 to 3 for 40 times.

[0121] 4. Data Analysis

[0122] After the operation is completed, 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 curve, amplification CT value, quantitative results, etc. of the sample can be viewed.

[0123] 5. Experimental Results

[0124] A qPCR forensic human DNA quantification kit was used to test the above-mentioned male and female samples mixed in different ratios, and the concentration values of each target are shown in Table 10.

[0125] Table 10

[0126]

[0127] 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 .

[0128] Example Five

[0129] Apply a qPCR forensic human DNA quantification kit to test DNA samples with different degradation degrees and guide STR typing tests.

[0130] 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.

[0131] 2. Reaction System

[0132] After mixing each reaction reagent by oscillation, 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 and perform centrifugal amplification. The composition of the reaction system is shown in Table 4.

[0133] 3. The PCR reaction program is shown in Table 5.

[0134] Place the prepared PCR reaction plate into the ABI 7500 real-time fluorescence quantitative PCR instrument, and set the amplification target information as shown in Table 6.

[0135] Set the following amplification program to run:

[0136] 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 to 3 for 40 times.

[0137] 4. Data analysis

[0138] 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 samples can be viewed.

[0139] 5. Experimental results

[0140] 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, as expected, indicating no significant PCR inhibition. The measured concentration values of each target are shown in Table 11.

[0141] Table 11

[0142]

[0143] This kit can accurately measure the concentration and degradation degree of degraded samples of different degrees. According to the quantitative results, dilute the blood DNA samples M, ML, MM, and MH samples to 1 ng / µL, and the cell line sample H to 1 ng / µL. Then, use the genotyping kit with the product name Microreader 28A ID System from Suzhou Yuewei Gene Technology Co., Ltd. to perform genotyping tests on the above samples. The input template amount is 1 ng for all. 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 map. When the degradation index DI is greater than 3, there is a risk of loss of large fragments in the genotyping map, and it is necessary to use the MiniSTR kit for additional testing.

[0144] Example 6

[0145] Apply a qPCR forensic human DNA quantification kit to test DNA samples containing different inhibitors and guide STR genotyping tests

[0146] 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, heme with a concentration of 0 - 1250 μM is added to the qPCR system; when the concentration of M308 cell DNA (produced by Yuewei Gene) is 0.1 ng / µL, humic acid with a concentration of 0 - 800 ng / µL 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, so as to guide the subsequent use of the genotyping kit named Microreader 28A ID System produced by Suzhou Yuewei Gene Technology Co., Ltd.

[0147] 2. Reaction system

[0148] After oscillating and mixing each reaction reagent, a PCR reaction mixture is prepared according to the volume ratio shown in Table 4 (except for the template), and 18 μL is dispensed into a PCR reaction tube. Finally, 2 μL of the template is added to each reaction tube, and centrifugal amplification is performed. The composition of the reaction system is shown in Table 4.

[0149] 3. The PCR reaction procedure is shown in Table 5.

[0150] 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.

[0151] Set the following amplification program to run:

[0152] 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 to 3 for 40 times.

[0153] 4. Data analysis

[0154] After the run is completed, 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 curve, amplification CT value, quantitative results, etc. of the sample can be viewed.

[0155] 5. Experimental results

[0156] A qPCR forensic human DNA quantification kit is 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.

[0157] Table 12

[0158]

[0159] This kit can accurately measure the concentration of DNA samples 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; when it is above 1000 µM, the amplification of all targets is significantly inhibited and the quantified 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 quantified concentration is inaccurate.

[0160] Directly add 5 µL of the sample and use the genotyping kit named Microreader28A ID System produced by Suzhou Yuewei Gene Technology Co., Ltd. for STR genotyping test. The results are shown in Figures 7-8 . When the final concentration of heme is 1000 µM, large fragments are lost and the profile is significantly higher at the front and lower at the back; when the final concentration of humic acid is 600 ng / µL, large fragments are lost and the profile is significantly higher at the front and lower at the back.

[0161] When the inhibitor index measured by a qPCR forensic human DNA quantification kit is greater than 2, it will significantly affect qPCR quantification and subsequent genotyping, and the sample needs to be purified.

[0162] Example Seven

[0163] Use a qPCR forensic human DNA quantification kit to test simulated case samples and guide STR genotyping test

[0164] 1. The sources of forensic case samples are complex. For simulated case samples, DNA is extracted from 8 cases of bloodstains, semen stains, oral swabs, cigarette butts, hairs and various swab samples. The samples are diluted 100 times to simulate extremely trace case samples, and a qPCR forensic human DNA quantification kit is used for quantitative analysis and to guide the use of the genotyping kits named Microreader 28A ID System and Microreader 40Y Plus ID System produced by Suzhou Yuewei Gene Technology Co., Ltd.

[0165] 2. Reaction system

[0166] 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, and perform centrifugal amplification. The composition of the reaction system is shown in Table 4.

[0167] 3. The PCR reaction program is shown in Table 5.

[0168] Place the configured PCR reaction plate into the ABI 7500 real-time fluorescence quantitative PCR instrument, and set the amplification target information as shown in Table 6.

[0169] Set the following amplification program to run:

[0170] 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.

[0171] 4. Data analysis

[0172] After the run is completed, use ABI 7500 Software v2.3 to analyze the amplification results and generate the 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% 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.

[0173] 5. Experimental results

[0174] A qPCR forensic human DNA quantification kit was used to test the above simulated case samples, and the concentrations of each target were measured. The detection results of STR genotyping using the typing kit with the product name Microreader 28A ID System from Suzhou Yuewei Gene Technology Co., Ltd. are shown in Table 13.

[0175] Table 13

[0176]

[0177] This kit can accurately measure the concentrations and degradation conditions of simulated case samples from different sources, and guide the use of the typing kit with the product name Microreader 28A ID System from Suzhou Yuewei Gene Technology Co., Ltd. and the typing kit with the product name Microreader 40Y Plus ID System from 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 beginning and low at the end. For the 0.2 ng cigarette butt sample amplified by 40YP (DI = 1.78), it is also significantly high at the beginning and low at the end. For the 0.46 ng keyboard wipe sample amplified by 28A (DI = 1.16), a well-balanced genotyping map is obtained. The guiding effect of the STR typing kit is good, which can greatly improve the sample detection rate.

[0178] 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 shall be subject to the protection scope of the claimed rights.

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 assessment, the HY target is used for gender ratio judgment, and the IPC target is used for inhibitor detection. 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, 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; the probe sequence for marking the IPC target is shown in SEQ ID NO. As shown in NO.15, the probe marking the HS target is 5' fluorescently labeled with VIC and 3' fluorescently labeled with BHQ1; the probe marking the HL target is 5' fluorescently labeled with CY5 and 3' fluorescently labeled with BHQ2; the probe marking the HY target is 5' fluorescently labeled with FAM and 3' fluorescently labeled with BHQ1; the probe marking the IPC target is 5' fluorescently labeled with TAMRA and 3' fluorescently labeled with BHQ2.

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 1, characterized in that: ROX dye is also added to the composite amplification system for qPCR instrument correction.

4. A qPCR forensic human DNA quantification kit, characterized in that: It comprises the composite amplification system as described in any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that The kit also includes PCR amplification mix and DNA quantitative standards.

6. The kit according to claim 4, 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.

7. Use of the composite amplification system according to any one of claims 1 to 3, or the kit according to any one of claims 4 to 6 in forensic DNA sample quantification and DNA sample quality assessment.

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