Composite amplification detection system for identifying human skin based on microbial markers

Abstract

The invention relates to a composite amplification detection system for identifying human skin based on microbial markers. The multiplex amplification detection system comprises a primer combination for specifically amplifying five human skin microorganism markers, wherein the human skin microorganism markers are staphylococcus epidermidis, staphylococcus hominis, staphylococcus capitis, staphylococcus aureus and propionibacterium acnes respectively. The invention also provides a kit containing the composite amplification detection system. The multiplex amplification detection system and the kit have relatively good recognition capability on skin of parts such as forehead, cheeks, hands, chest, navel periphery and groin of a human body. Besides, the method is an effective supplement for an existing body fluid identification method, and can effectively identify various common body fluid samples in forensic cases by flexibly combining with a multiple amplification system for saliva and vaginal secretion established by a previous team, so that the accuracy and reliability of identification results are improved, and case investigation and judicial trial are assisted.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a composite amplification detection system for identifying human skin based on microbial markers. Background Art

[0002] Identifying the tissue and body fluid sources of biological evidence is particularly important for case characterization and scene reconstruction. Skin contact samples are the most common biological evidence at crime scenes. Human skin forensics can help reveal the crime process and reconstruct actions such as touching and contact.

[0003] At present, forensic laboratories mainly identify the types of human tissue and body fluids based on immunological reactions or enzymatic reactions, but only industry standards have been formed for the identification of human blood and human semen. The identification method for human skin tissue is still based on exfoliated cell smears and microscopic observations. The amount of samples required is large, and the amount of skin contact samples usually cannot meet the requirements. In addition, squamous epithelial cells can also be observed in oral, esophageal, and vaginal cells, so microscopic examination of the skin is not accurate enough. The industry has been committed to finding molecular markers in human skin tissue, such as mRNA, miRNA, and DNA methylation, but there is no suitable specific detection method for forensic practice. Summary of the invention

[0004] The purpose of the present invention is to disclose a composite amplification detection system and a kit and a detection method thereof, so as to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions: The first aspect of the present invention is to provide a composite amplification detection system.

[0006] The second aspect of the present invention is to provide a kit comprising the composite amplification detection system described in the first aspect of the present invention.

[0007] The third aspect of the present invention is to provide the application of the composite amplification detection system described in the first aspect of the present invention and the kit described in the second aspect of the present invention in human skin recognition, especially in forensic identification of evidence samples.

[0008] The composite amplification detection system of the first aspect of the present invention comprises a primer combination for specifically amplifying five human skin microbial markers, wherein the human skin microbial markers are Staphylococcus epidermidis ( Staphylococcus epidermidis ), Staphylococcus aureus ( Staphylococcus hominis ), Staphylococcus capitis ( Staphylococcus capitis ), Staphylococcus aureus ( Staphylococcus aureus ) and Propionibacterium acnes (Cutibacterium acnes ).

[0009] Staphylococcus epidermidis is a Gram-positive coccus that grows on the epidermis of organisms. It is named because it often aggregates into grape clusters. It is widely found in the skin, vagina and other parts of the human body, with a diameter of about 0.5 to 1.5 microns.

[0010] Staphylococcus aureus is a Gram-positive coccus that is widely present on human skin and mucous membranes. When the body's immunity is weakened or there is a wound, it may enter the blood and multiply in large numbers, causing infection.

[0011] Staphylococcus capitis is a common coagulase-negative staphylococcus, named because it is often isolated from the human head, face, and neck.

[0012] Staphylococcus aureus is a Gram-positive spherical bacterium without spores, flagella, and most of them without capsules. It belongs to the normal colonizing flora of human skin and mucous membranes. Due to favorable conditions or weak host immune system, it has the opportunity to cause disease through infection of damaged skin and mucous membranes.

[0013] Propionibacterium acnes is a species of the genus Propionibacterium. It is a Gram-positive rod-shaped or slightly curved bacillus that stains unevenly. It is often arranged in X, Y and V shapes, similar to pseudodiphtheria. Propionibacterium acnes is a normal flora on the surface of human skin, mainly existing in the sebum-rich pilosebaceous gland unit, but it can also be found in non-sebaceous skin areas.

[0014] The composite amplification detection system selects 5 human skin microbial markers, and the possibility of each human skin microbial marker existing in the skin tissue is an independent random event. Therefore, human skin identification can be achieved through the combination of multiple skin microbial markers.

[0015] In some embodiments of the first aspect of the present invention, the nucleotide sequences of primers for amplifying Staphylococcus epidermidis are shown in SEQ ID No: 1 and SEQ ID No: 2, the nucleotide sequences of primers for amplifying Staphylococcus hominis are shown in SEQ ID No: 3 and SEQ ID No: 4, the nucleotide sequences of primers for amplifying Staphylococcus capitis are shown in SEQ ID No: 5 and SEQ ID No: 6, the nucleotide sequences of primers for amplifying Staphylococcus aureus are shown in SEQ ID No: 7 and SEQ ID No: 8, and the nucleotide sequences of primers for amplifying Propionibacterium acnes are shown in SEQ ID No: 9 and SEQ ID No: 10. The primers shown in SEQ ID No: 1 and SEQ ID No: 2 target the acetyl-CoA acetyltransferase gene (ACAT), which plays a key role in the metabolism of Staphylococcus epidermidis and is highly conserved in different strains of Staphylococcus epidermidis. The primers shown in SEQ ID No: 3 and SEQ ID No: 4 target the YdcF gene, and the YdcF family protein is highly conserved in different strains, and is suitable for the detection and differentiation of Staphylococcus hominis. Primers shown in SEQ ID No:5 and SEQ ID No:6 target the phenylalanyl-tRNA synthetase alpha subunit gene (pheS). The pheS gene has high homogeneity in strains of the same species, but poor sequence similarity in different species. It is a rapidly evolving gene and a reliable tool for the identification of Staphylococcus capitis. Primers shown in SEQ ID No:7 and SEQ ID No:8 target the NAD(P) binding protein gene. NAD(P) binding protein plays an important role in the metabolic process of Staphylococcus aureus, regulating its activity and thus affecting the growth and survival of bacteria. It can be used as a biomarker for Staphylococcus aureus infection. Primers shown in SEQ ID No:9 and SEQ ID No:10 target the recA gene due to its prevalence and importance in Propionibacterium acnes and its application in drug resistance detection.

[0016] In some embodiments of the first aspect of the present invention, the 5' end of at least one of the primers targeting the same human skin microbial marker is labeled with a fluorescent light-emitting group.

[0017] In some embodiments of the first aspect of the present invention, the fluorescent light-emitting group is selected from FAM, HEX, SUM, LYN, PUR, TAMRA, ALEXA 568, ALEXA 594, ROX, VIC, PET, NED or TAZ.

[0018] In some embodiments of the first aspect of the present invention, the composite amplification detection system further comprises a Taq PCR premix, and the Taq PCR premix contains at least one of a PCR buffer, MgCl2, dNTPs or a DNA polymerase.

[0019] In some embodiments of the first aspect of the present invention, the amplification program is: preliminary denaturation at 95°C for 2 minutes; denaturation at 94°C for 30 seconds, annealing at 58°C for 1 minute, extension at 72°C for 50 seconds, for a total of 30 cycles; final extension at 72°C for 10 minutes.

[0020] The kit described in the second aspect of the present invention contains the above-mentioned composite amplification detection system.

[0021] In some embodiments of the second aspect of the present invention, the kit further comprises a positive control product, and the positive control product is a vector containing a nucleotide sequence as shown in SEQ ID No:11 to SEQ ID No:15.

[0022] In some embodiments of the second aspect of the present invention, the kit further comprises a negative control product; preferably, the negative control product is sterile water.

[0023] The method of using the kit is to first use QIAGEN's DNeasy PowerSoil Kit to extract the total DNA of the sample, determine the DNA concentration by measuring the absorbance values ​​at 260 nm and 280 nm using a fluorescence spectrophotometer, and determine the DNA quality by 1% agarose gel electrophoresis; DNA samples that meet the requirements are added to the composite amplification detection system provided by the kit, and after obtaining the amplified products, they are detected using capillary electrophoresis technology, and the obtained data are analyzed using GeneMapper ID-Xv1.5 analysis software.

[0024] The application described in the third aspect of the present invention refers to the application of the composite amplification detection system or the kit in human skin recognition.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The composite amplification detection system provided by the present invention has good recognition ability for the skin of the forehead, cheeks, hands, chest, around the navel, groin and other parts of the human body. When the amount of DNA input is as low as 0.0015 ng, it can still be successfully identified by the composite amplification detection system. In addition, the present invention is an effective supplement to the existing body fluid identification method. It flexibly combines the multiple amplification system for saliva and vaginal secretions previously established by this team, and can effectively identify a variety of common body fluid samples in forensic cases, thereby improving the accuracy and reliability of the identification results and providing strong evidence for judicial cases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the standard detection pattern of the composite amplification detection system described in Example 1; Figure 2 is a heat map of the sensitivity test results of the composite amplification detection system described in Example 3; Figure 3 is the stability test result of the composite amplification detection system described in Example 4, wherein A is the inhibition bar graph of calcium ions, B is the inhibition bar graph of indigo, C is the inhibition bar graph of hemoglobin, D is the inhibition bar graph of alcohol, E is the inhibition bar graph of EDTA, and F is the inhibition bar graph of humic acid; Figure 4 is a radar chart showing the applicability of the composite amplification detection system described in Example 5 to different skin parts; Figure 5 It is the detection typing diagram of different body fluid samples by the composite amplification detection system described in Example 6. DETAILED DESCRIPTION

[0027] The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the Molecular Cloning Experiment Guide (3rd edition) or in accordance with the methods and product instructions; the biological materials described in the methods, unless otherwise specified, can be obtained from commercial channels.

[0028] Example 1: Preparation of Standard DNA Typing of Skin Microbial Markers.

[0029] 1. Template DNA Preparation Staphylococcus epidermidis ( Staphylococcus epidermidis ), Staphylococcus aureus ( Staphylococcus hominis ), Staphylococcus capitis ( Staphylococcus capitis ), Staphylococcus aureus ( Staphylococcus aureus ) and Propionibacterium acnes ( Cutibacterium acnes , formerly known as Propionibacterium acnes ) was purchased from Guangdong Microbial Culture Collection Center.

[0030] 2. Fluorescence multiplex amplification Multiplex PCR amplification was performed using an Eppendorf AG 22331 Hamburg PCR amplifier (Eppendorf, Germany.). Specific composite amplification primers were designed and synthesized for the five human skin microbial markers, and the microbial DNA samples were amplified using the composite amplification detection system. The DNA mixed templates of the five human skin microbial markers were used as positive quality control products, and sterilized double distilled water was used as a blank control.

[0031] The nucleotide sequence of the Staphylococcus epidermidis positive template is shown in SEQ ID No: 11, the nucleotide sequence of the Staphylococcus hominis positive template is shown in SEQ ID No: 12, the nucleotide sequence of the Staphylococcus capitis positive template is shown in SEQ ID No: 13, the nucleotide sequence of the Staphylococcus aureus positive template is shown in SEQ ID No: 14, and the nucleotide sequence of the Propionibacterium acnes positive template is shown in SEQ ID No: 15.

[0032] The PCR amplification program was as follows: pre-denaturation at 95°C for 2 minutes; denaturation at 94°C for 30 seconds, annealing at 58°C for 1 minute, and extension at 72°C for 50 seconds, for a total of 30 cycles; and extension at 72°C for 10 minutes.

[0033] 3.Ladder construction PCR amplification was performed on the five human skin microbial markers respectively. The amplified products were analyzed by electrophoresis in a 3500xl genetic analysis system (Thermo Fisher Scientific). The amplified products of the five microbial standard DNAs that had been amplified in a single step were mixed in a certain proportion to ensure that all peaks were balanced. Finally, after purification, the target strain ladder was constructed and stored at -20°C for subsequent experiments and research.

[0034] 4. Capillary electrophoresis and analysis (1) Denaturation treatment system before electrophoresis: 1 µL PCR product, 9.5 µL deionized formamide, and 0.5 µL AGCUMaker SIZ-500 (AGCU, Wuxi, Jiangsu) were mixed thoroughly. Treatment conditions: 95°C for 3 minutes and cooled in an ice bath for 3 minutes.

[0035] (2) Through ABI ® The 3500 sequencer completes the detection of the amplified product, and the obtained data is analyzed by ABI ® GeneMapperID analysis software was completed, and 500 relative fluorescence units (RFU) were set as the threshold for DNA typing. Figure 1 These are the standard test results of five microbial markers for the skin. The corresponding serial numbers and microbial markers are shown in Table 1.

[0036]

[0037] Example 2: Sensitivity test of the composite amplification detection system for skin microbial markers.

[0038] 1. Experimental Methods Ultrapure water was used to dilute the five DNA samples of human skin microbial markers in a 10-fold gradient concentration, and PCR amplification was performed with template amounts of 0.15 ng, 0.015 ng, 0.0015 ng and 0.00015 ng, respectively. Each concentration gradient DNA sample was tested three times to evaluate the sensitivity of the composite amplification detection system.

[0039] The composite amplification detection system and reaction procedure are the same as those in Example 1.

[0040] 2. Experimental results The results are as follows Figure 2 As shown, within the set dilution gradient concentration, all tested samples can be detected by the composite amplification detection system when the DNA input amount is as low as 0.0015 ng, which can meet the detection requirements of trace samples in forensic practice.

[0041] Example 3: Specificity test of the composite amplification system of skin microbial markers.

[0042] 1. Experimental Methods Human DNA standards (9947A, 9948) and sample DNA covering 11 common animal genera (goose, chicken, cattle, dog, mouse, rabbit, duck, sheep, fish, pig) and 12 environmental bacteria (Edwardsiella mutans, Candida albicans, Streptococcus mutans, Bifidobacterium longum, Escherichia coli, Lactobacillus iners, Vibrio parahaemolyticus, Vibrio harveyi, Lactobacillus gasseri, Photorhabdus mermanii, Photorhabdus nautilus) were collected.

[0043] The composite amplification detection system constructed in Example 1 was used to amplify the above sample DNAs respectively.

[0044] 2. Experimental results The results showed that no target microorganism peaks were observed in human DNA standards, 11 common animal genera, and 12 environmental bacterial species, indicating that the composite amplification detection system has high species specificity.

[0045] Example 4: Stability test of the composite amplification detection system for skin microbial markers.

[0046] 1. Experimental Methods (1) Six common forensic inhibitors (calcium ion, indigo, hemoglobin, humic acid, alcohol and EDTA) were diluted into different concentration gradients for anti-PCR inhibition study. The concentration gradients of calcium ion, indigo and hemoglobin were 5 mmol / L, 10 mmol / L, 15 mmol / L, 30 mmol / L and 60 mmol / L; the concentration gradient of humic acid was 15.63 ng / μL, 31.25 ng / μL, 62.50 ng / μL, 125 ng / μL and 250 ng / μL; the concentration gradient of alcohol was 10%, 20%, 40%, 60% and 80%; the concentration gradient of EDTA was 2.5 mmol / L, 5 mmol / L, 10 mmol / L, 15 mmol / L and 20 mmol / L. 1uL of inhibitors at different concentrations was added to the reaction system containing 0.05 ng of mixed DNA of the five human skin microbial markers (ratio 1:1:1:1:1) for amplification, and each concentration was tested 3 times. The reaction system without inhibitor was used as a positive control, and sterilized ddH2O was used as a negative control.

[0047] (2) Composite amplification detection system: 5 μL Taq PCR premix (Sangon Biotech), 2.2 μL FK5primer mix, 0.5 μL mixed DNA template of 5 human skin microbial markers at a concentration of 0.1 ng / μL, 1 μL inhibitor (after gradient dilution), and ddH2O were added to the total volume of the system to 10 μL. Amplification was performed according to the reaction procedure provided in Example 1. After amplification, the amplified products were detected and analyzed by electrophoresis.

[0048] 2. Experimental results The results are as follows Figure 3 As shown in the figure, the composite amplification detection system has a very strong anti-inhibitory effect on indigo and heme, and can still detect a higher peak value even at a high concentration of 60 mmol / L; while calcium ions and humic acid cannot fully identify mixed DNA at a concentration as high as 60 mmol / L (60 mmol / L calcium ions can only identify Staphylococcus capitis); 80% alcohol has an inhibitory effect on the system; EDTA has a strong inhibitory effect on this system, and 15 mmol / L EDTA has a significant inhibitory effect, but a higher peak value can still be detected at a concentration of 10 ng / μL. In general, the composite amplification detection system has a certain tolerance to 6 inhibitors, including PCR inhibitors of different types and concentrations.

[0049] Example 5: Applicability test of the skin microbial marker composite amplification detection system to different skin sites.

[0050] 1. Sample collection Skin swabs were collected from 30 healthy volunteers at 10 skin sites, including forehead, left cheek, right cheek, left chest, right chest, navel, left groin, right groin, left hand and right hand, for a total of 300 samples.

[0051] The sample DNA extraction and detection steps are carried out with reference to Example 1.

[0052] 2. Experimental results Results Figure 4 Among the 300 samples, at least one human skin microbial marker was detected in all skin samples.

[0053] Example 6: Verification of the effectiveness of the detection system in distinguishing body fluids from different sources.

[0054] 1. Sample collection Samples of different body fluid types were collected from healthy volunteers, including skin swabs, saliva, stool, and semen samples.

[0055] The sample DNA extraction and detection steps are carried out with reference to Example 1.

[0056] 2. Experimental results Results Figure 5 . Including microbial marker detection profiles of skin swabs, saliva, feces and semen. The results show that the composite amplification detection system based on microbial markers for skin identification established in this study can effectively distinguish skin from other common body fluids in forensic practice and achieve efficient and rapid identification of skin samples.

[0057] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A composite amplification detection system, characterized in that: The invention comprises a primer combination for specifically amplifying five human skin microbial markers, wherein the human skin microbial markers are Staphylococcus epidermidis ( Staphylococcus epidermidis ), Staphylococcus aureus ( Staphylococcus hominis ), Staphylococcus capitis ( Staphylococcus capitis ), Staphylococcus aureus ( Staphylococcus aureus ) and Propionibacterium acnes ( Cutibacterium acnes ).

2. The composite amplification detection system according to claim 1, characterized in that: The nucleotide sequences of primers for amplifying Staphylococcus epidermidis are shown in SEQ ID No: 1 and SEQ ID No: 2, the nucleotide sequences of primers for amplifying Staphylococcus hominis are shown in SEQ ID No: 3 and SEQ ID No: 4, the nucleotide sequences of primers for amplifying Staphylococcus capitis are shown in SEQ ID No: 5 and SEQ ID No: 6, the nucleotide sequences of primers for amplifying Staphylococcus aureus are shown in SEQ ID No: 7 and SEQ ID No: 8, and the nucleotide sequences of primers for amplifying Propionibacterium acnes are shown in SEQ ID No: 9 and SEQ ID No:

10.

3. The composite amplification detection system according to claim 2, characterized in that: The 5' end of at least one primer among the primers targeting the same human skin microbial marker is labeled with a fluorescent luminescent group.

4. The composite amplification detection system according to claim 3, characterized in that: The fluorescent light-emitting group is selected from FAM, HEX, SUM, LYN, PUR, TAMRA, ALEXA 568, ALEXA 594, ROX, VIC, PET, NED or TAZ.

5. The composite amplification detection system according to any one of claims 1 to 4, characterized in that: The method also includes a Taq PCR premix, wherein the Taq PCR premix contains at least one of a PCR buffer, MgCl2, dNTPs or a DNA polymerase.

6. The composite amplification detection system according to claim 5, characterized in that: The amplification program was: 95°C for 2 min; 94°C for 30 sec, 58°C for 1 min, 72°C for 50 sec, for a total of 30 cycles; 72°C for 10 min.

7. A kit, characterized in that: Contains the composite amplification detection system as described in any one of claims 1 to 6.

8. The kit according to claim 7, characterized in that: Also included is a positive quality control product, which is a vector containing a nucleotide sequence as shown in SEQ ID No: 11 to SEQ ID No:

15.

9. The kit according to claim 7, characterized in that: A negative control product is also included; preferably, the negative control product is sterile water.

10. Use of the composite amplification detection system according to any one of claims 1 to 6 or the kit according to any one of claims 7 to 9 in human skin recognition.

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