Blood specific methylation site detection primer group, kit and blood trace identification method

Abstract

The invention provides a blood specific methylation site detection primer group, a kit and a blood trace identification method, and belongs to the technical field of molecular biological identification. The problems that in the prior art, a blood trace identification method is poor in specificity and insufficient in sensitivity, has high requirements for sample quality and quantity and cannot be compatible with subsequent DNA detection are solved. The requirements for the number and quality of body fluid detection materials can be remarkably reduced, the method can be well compatible with subsequent DNA detection, detection does not depend on a single source or a large number of samples any more, and the high stability, accuracy, sensitivity and specificity of the method enable the blood identification result to be reliable. Meanwhile, the method does not depend on a PCR instrument or complex laboratory equipment, so that professional requirements on instruments and operators are reduced. The whole detection process can be completed in a short time, the method has important significance in forensic on-site rapid identification, the case processing efficiency can be improved, and the cost is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biological identification, and in particular to a blood-specific methylation site detection primer set, a kit and a blood trace identification method. Background Art

[0002] Blood is one of the most common and valuable evidence in forensic investigations. Its presence can indicate criminal behavior, such as murder or injury, and provide important clues for reconstructing the crime scene, thus helping to narrow the scope of investigation. However, in actual investigations, non-human blood, blood contamination carriers, or low blood content often pose challenges to blood identification. At present, the traditional methods are immunochemical and optical detection methods, such as luminol reagent and benzidine reagent, but these methods have problems such as poor sensitivity and insufficient specificity, and may interfere with subsequent DNA analysis. In addition, although FOB test strips are widely used, they also have problems such as poor sensitivity, insufficient specificity, high requirements for sample quality and content, and are not compatible with subsequent DNA typing tests, which greatly limits their application in forensic practice. On the other hand, blood identification methods based on body fluid-specific genetic markers, such as traditional PCR, capillary electrophoresis (CE) platforms, and sequencing platforms, rely on laboratory conditions, cannot be tested on site, and cannot quickly obtain results. Therefore, it is urgent to develop more sensitive, specific, compatible with subsequent DNA typing tests, and suitable for rapid on-site detection of blood identification technologies.

[0003] It is very common to find blood at different crime scenes. Therefore, confirming the presence of human blood can not only indicate possible criminal behavior, but also provide key clues for the reconstruction of the crime scene, such as determining whether it is an accident, murder or injury, which helps to characterize the case and identify the suspect. Common blood identification methods used in forensic investigations include chemical testing and immunological methods, such as benzidine test, luminol test and FOB test strips. Although the benzidine test is a standard method for pre-testing blood stains, it has poor specificity and may produce toxic byproducts after contact with certain substances. In addition, blood treated with the benzidine test cannot be extracted using the conventional Chelex method for subsequent testing of DNA. FOB test strips are widely used at the grassroots level, but they have the following limitations. Such test strips are usually less sensitive when dealing with very small or tiny blood stains. For example, when the blood sample is very small, has dried up or is diluted, the test strip may not be able to effectively detect blood components. In addition, the accuracy of such test strips may be affected by environmental factors, such as temperature, humidity, storage conditions, etc., and there are certain requirements for the quality of the sample. And it may cross-react with other substances, resulting in false positive or false negative results, or may cause false negative results due to the "hook effect", affecting the characterization of subsequent cases and narrowing the scope of investigation. Studies have shown that when FOB test strips are used to detect samples treated with detergents, false negative results may also occur. More importantly, none of the above methods are compatible with subsequent DNA analysis. Therefore, there are many problems with existing chemical and immunological bloodstain identification methods, which limits their application in forensic practice. Summary of the invention

[0004] The purpose of the present invention is to provide a blood-specific methylation site detection primer set, a kit and a blood trace identification method, which do not rely on a PCR instrument or other complex detection equipment, have the advantages of high efficiency and simplicity, and are suitable for the needs of rapid blood identification at the forensic scene.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a blood-specific methylation site detection primer set, comprising the following primers:

[0007] An upstream primer having a nucleotide sequence as shown in SEQ ID NO.1;

[0008] The nucleotide sequence of the downstream primer is shown as SEQ ID NO.2.

[0009] Preferably, the 5' end of the downstream primer is labeled with biotin.

[0010] The present invention also provides a visualized detection primer probe set, comprising the above detection primer set and a fluorescent probe;

[0011] The nucleotide sequence of the fluorescent probe is shown in SEQ ID NO.3;

[0012] The 5' end of the fluorescent probe is labeled with a fluorescent group.

[0013] Preferably, the fluorescent group is selected from FAM, Cy3, Cy5, Cy5.5, ROX, Texas Red, HEX, TET, JOE, FITC, Alexa Fluor series or DAPI.

[0014] Preferably, the 3' end of the fluorescent probe is modified with a Spacer C3 group.

[0015] The present invention also provides the use of the above-mentioned blood-specific methylation site detection primer set or the above-mentioned visualization detection primer probe set in the preparation of a blood trace identification kit.

[0016] The present invention also provides a blood trace identification kit, comprising Hha I restriction endonuclease, the above-mentioned visualization detection primer probe group and a constant temperature amplification reagent.

[0017] Preferably, in the kit, the final concentration of the upstream primer is 8 to 12 μmol / L;

[0018] and / or, the final concentration of the downstream primer is 8-12 μmol / L;

[0019] And / or, the final concentration of the fluorescent probe is 8-12 μmol / L.

[0020] Preferably, the kit further contains a colloidal gold immunochromatographic test strip.

[0021] The present invention also provides a blood trace identification method, comprising the following steps:

[0022] Extracting DNA from the sample to be tested;

[0023] Digesting the DNA with Hha I restriction endonuclease to obtain a digested DNA sample;

[0024] The digested DNA sample was amplified using the above-mentioned visualized detection primer probe set, and the amplification results were interpreted using colloidal gold immunochromatographic test strips:

[0025] If both the control line and the test line are positive, the test sample contains blood;

[0026] If the control line is positive and the test line is negative, the test sample does not contain blood.

[0027] Beneficial effects of the present invention:

[0028] The rapid blood stain detection method based on isothermal amplification of blood-specific methylation sites provided by the present invention can reduce the requirements for the quantity and quality of body fluid samples, so that the detection is no longer dependent on a single source or a large number of samples, and its high stability, accuracy, sensitivity and specificity make the blood identification results reliable. At the same time, the method does not rely on PCR instruments or complex laboratory equipment, reduces the professional requirements for instruments and operators, and is compatible with subsequent DNA typing tests. The entire detection process of the present invention is fast and can complete the detection in a relatively short time.

[0029] The high body fluid specificity, sensitivity and repeatability of the method of the present invention, as well as the high detection success rate in random samples, make this technical solution of great significance in forensic on-site rapid identification, can improve case handling efficiency, reduce costs, and provide forensic workers with an efficient and accurate detection tool. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram showing the results of colloidal gold;

[0031] Figure 2 Schematic diagram of HhaI digestion of DNA;

[0032] Figure 3 Schematic diagram of RPA specific amplification of blood DNA;

[0033] Figure 4 This is a blood test chart based on LFD technology;

[0034] Figure 5 This is a diagram for interpreting the test strip results;

[0035] Figure 6 is the specificity result diagram;

[0036] Figure 7 This is a graph of three replicate specific results;

[0037] Figure 8 is the sensitivity result diagram;

[0038] Fig. 9 This is a graph of three replicate sensitivity results;

[0039] Fig.10 This is a graph of 14 random sample test results;

[0040] Fig.11 The following is the test result of 15 random samples. DETAILED DESCRIPTION

[0041] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0042] Example

[0043] Extraction of DNA from samples:

[0044] 1) Cut the body fluid sample into small pieces, add 1 ml of ultrapure water into a 2 ml microcentrifuge tube, mix well and let stand for 3-5 minutes;

[0045] 2) Centrifuge at 3000 rpm for 10-15 minutes and discard the supernatant;

[0046] 3) Add 1 ml of red blood cell lysis buffer, mix well and let stand for 15 minutes;

[0047] 4) Centrifuge at 3000 rpm for 10-15 minutes and discard the supernatant;

[0048] 5) Add 400 μL of leukocyte lysis buffer, 5 μL of 20 mg / ml PK, 8 μL of 10% SDS, and 20 μL of DTT, mix well, incubate at 56°C for 3 hours, and remove;

[0049] 6) Add 220 μL of saturated NaCl, mix and shake 100-150 times, and let stand at 4°C for 10 minutes;

[0050] 7) Centrifuge at 12,000 rpm for 10-15 minutes, transfer 600 μL of supernatant to a new microcentrifuge tube, add 600 μL of isopropanol, and mix well;

[0051] 8) Centrifuge at 12000 rpm for 10-15 minutes and discard the supernatant;

[0052] 9) Add 500 μL of 75% ethanol and shake well;

[0053] 10) Centrifuge at 12000 rpm for 10-15 minutes and discard the supernatant; dry the DNA precipitate at room temperature, add 20-30 μL of ultrapure water to dissolve the DNA, and store at 4°C for later use;

[0054] The concentration of extracted DNA was measured using a spectrophotometer.

[0055] Dilute the DNA concentration to 1 ng / μL for later use.

[0056] MSRE digestion of DNA

[0057] The specific methylation site used for blood identification in the present invention is cg04011671;

[0058] The methylation sensitive restriction enzyme (MSRE) used in the present invention is Hha I (New England Biolabs, USA), and its recognition and cleavage sequence is "5'-GCGC-3'". There are 4 Hha I enzyme recognition sites in the upstream and downstream regions of cg04011671 (upstream 220 bp and downstream 200 bp, respectively). The schematic diagram of HhaI digestion of DNA is shown in Figure 2 .

[0059] The experimental steps are as follows:

[0060] 1. Add the following reagents to a PCR tube: 1 μL DNA, 1 μL 10X Buffer Tango, 1 μL Hha I enzyme, 1 μL ddHO 2 O 7μL.

[0061] After mixing, the reaction system was digested at 37°C for 15 minutes.

[0062] 2. After digestion, inactivate the reaction system at 65°C for 20 minutes to obtain the digested DNA sample.

[0063] Isothermal PCR amplification and detection based on RPA and LFD technology. The schematic diagram of RPA specific amplification of blood DNA is shown in Figure 3 , blood testing based on LFD technology Figure 4 :

[0064] Use the Amp Future DNA Constant Temperature Rapid Amplification Kit (test strip type) to amplify the digested DNA sample, and use the Amp Future disposable nucleic acid test strip to interpret the amplification results.

[0065] Take out the required components of the kit in advance, melt them at room temperature, and shake to mix.

[0066] 1. Add 29.4μL Abuffer to each dry powder reaction tube. Buffer A must be completely melted and mixed.

[0067] 2. Add 5.8 μL melted and mixed Abuffer, 0.4 μL upstream primer, 0.4 μL downstream primer and 0.2 μL probe to each reaction tube (primer and probe concentration is 10 μM)

[0068] 3. Add 2.7 μL of digested DNA to the reaction tube.

[0069] 4. Finally, add 0.5 μL B Buffer to the reaction tube and mix thoroughly.

[0070] 5. After mixing, shake the reaction solution to the bottom of the tube, and then immediately place the reaction tube in a constant temperature device and incubate at 37℃ for 15 minutes.

[0071] 6. After the reaction is completed, add 5 μL of amplified product to 95 μL ddH 2 Dilute in 5% HO and mix well. Take 80 μL of dilution to develop the color of the test strip. Observe the quality control line and the test line within 5 minutes to read the results. See the schematic diagram of the result display. Figure 1 .

[0072] The primer sequences used for isothermal amplification are shown in Table 1:

[0073] Table 1 Primer sequence information

[0074]

[0075] The sample to be tested is interpreted according to the colloidal gold reaction results. If both the quality control line and the test line are positive, the test sample is a blood sample. If the quality control line is positive and the test line is negative, the test sample is not a blood sample. If the quality control line is negative, the test result is invalid. See the test strip result interpretation for details. Figure 5 .

[0076] The sensitivity, specificity and random samples of the above methods were tested and evaluated:

[0077] Figure 6 This is a specific result diagram, which shows the detection results of DNA amplification products of negative control, semen sample, vaginal fluid sample, saliva sample, and blood sample from left to right. Figure 7 The three-repeat specificity result graph shows the negative control, semen sample, vaginal fluid sample, saliva sample, and blood sample DNA amplification product test results from left to right. The results of these two graphs show that the detection method has a high degree of specificity, can accurately distinguish blood from other body fluids, and the results have good repeatability.

[0078] Figure 8 This is a sensitivity result chart, which shows NC and the DNA input amounts added to the detection system from left to right, which are 1 ng, 500 pg, 250 pg, 125 pg and 62.5 pg respectively. The virus can still be detected when the blood input DNA amount is as low as 125 pg.

[0079] Fig. 9 This is a graph of three repeated sensitivity results, where from left to right are NC and the DNA input amounts added to the detection system are 1 ng, 500 pg, 250 pg, 125 pg and 62.5 pg, respectively. The virus can still be detected when the blood input DNA amount is as low as 125 pg.

[0080] Random sample test results: 29 blood samples were randomly selected and DNA was extracted. After extraction, the test was performed using this test process. The test results of 14 random samples are shown in the figure below. Fig.10As shown in Figure 1, the test results of 15 random samples are shown in Figure 1. Fig.11 The results showed that correct positive test results were obtained in all 29 random blood samples (NC was a negative control sample), and the test accuracy was 100%.

[0081] It can be seen from the above embodiments that the present invention provides a rapid, sensitive and specific blood stain detection method, which can successfully detect the presence of blood in samples as low as 125pg, showing high sensitivity. At the same time, when the method detects DNA in blood and three other common body fluids (semen, saliva, vaginal fluid), only blood samples show positive results, showing high specificity; the entire detection process can be completed in a relatively short time, which is suitable for rapid on-site identification of blood; there is no need to rely on PCR instruments or other complex detection equipment, and the requirements for instruments and operators are low; compared with other detection technologies, this method is inexpensive and easy to popularize; the results are visualized by lateral flow chromatography technology (LFD), so that the test results can be judged by the naked eye, and no experimenters with professional knowledge are required. Therefore, the technical solution of the present invention provides a rapid, sensitive and specific blood stain detection method, which is particularly suitable for the needs of rapid on-site identification of blood in forensic medicine.

[0082] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A blood-specific methylation site detection primer set, characterized in that: The following primers were included: An upstream primer having a nucleotide sequence as shown in SEQ ID NO.1; The nucleotide sequence of the downstream primer is shown as SEQ ID NO.

2.

2. The blood-specific methylation site detection primer set according to claim 1, characterized in that: The 5' end of the downstream primer is labeled with biotin.

3. A visual detection primer probe set, characterized in that: Comprising the detection primer set and fluorescent probe according to claim 1; The nucleotide sequence of the fluorescent probe is shown in SEQ ID NO.3; The 5' end of the fluorescent probe is labeled with a fluorescent group.

4. The visualization detection primer probe set according to claim 3, characterized in that: The fluorescent group is selected from FAM, Cy3, Cy5, Cy5.5, ROX, Texas Red, HEX, TET, JOE, FITC, Alexa Fluor series or DAPI.

5. The visualization detection primer probe set according to claim 3, characterized in that: The 3' end of the fluorescent probe is modified with a Spacer C3 group.

6. Use of the blood-specific methylation site detection primer set according to claim 1 or 2 or the visualization detection primer probe set according to any one of claims 3 to 5 in the preparation of a blood trace identification kit.

7. A blood trace identification kit, characterized in that: The method comprises Hha I restriction endonuclease, the visualization detection primer probe set according to any one of claims 3 to 5, and a constant temperature amplification reagent.

8. The blood trace identification kit according to claim 7, characterized in that: In the kit, the final concentration of the upstream primer is 8 to 12 μmol / L; and / or, the final concentration of the downstream primer is 8-12 μmol / L; And / or, the final concentration of the fluorescent probe is 8-12 μmol / L.

9. The blood trace identification kit according to claim 7, characterized in that: The kit also contains a colloidal gold immunochromatography test strip.

10. A blood trace identification method, characterized in that: The following steps are involved: Extracting DNA from the sample to be tested; Digesting the DNA with Hha I restriction endonuclease to obtain a digested DNA sample; The digested DNA sample is amplified using the visualized detection primer probe set described in claim 3, and the amplification result is interpreted using a colloidal gold immunochromatographic test strip: If both the control line and the test line are positive, the test sample contains blood; If the control line is positive and the test line is negative, the test sample does not contain blood.

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