CN117990923A - Hemoglobin treatment fluid, efficient blood trace species identification method and application - Google Patents
Hemoglobin treatment fluid, efficient blood trace species identification method and application Download PDFInfo
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- 102000001554 Hemoglobins Human genes 0.000 title claims abstract description 75
- 108010054147 Hemoglobins Proteins 0.000 title claims abstract description 75
- 238000011282 treatment Methods 0.000 title claims abstract description 73
- 239000012530 fluid Substances 0.000 title claims abstract description 18
- 241000894007 species Species 0.000 claims abstract description 102
- 238000001514 detection method Methods 0.000 claims abstract description 96
- 239000007788 liquid Substances 0.000 claims abstract description 25
- 238000002791 soaking Methods 0.000 claims abstract description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 108090000631 Trypsin Proteins 0.000 claims abstract description 15
- 102000004142 Trypsin Human genes 0.000 claims abstract description 15
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- 239000006228 supernatant Substances 0.000 claims abstract description 8
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/795—Porphyrin- or corrin-ring-containing peptides
- G01N2333/805—Haemoglobins; Myoglobins
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Abstract
The hemoglobin treatment fluid comprises the following components in parts by weight: 50-70 parts of 0.25% trypsin digestion liquid and 50-70 parts of pure water, wherein the total amount of the hemoglobin treatment liquid is 120 parts. The high-efficiency blood mark species identification method comprises the following steps: s1: placing a blood mark sample into a centrifuge tube, and then adding hemoglobin treatment liquid into the centrifuge tube to obtain a primary sample; s2: sufficiently vibrating a centrifugal tube filled with a primary sample, soaking a blood mark sample in the centrifugal tube for 30min at 37 ℃, and vibrating again after soaking; s3: placing the centrifugal tube after the re-oscillation on a high-speed centrifugal machine, and centrifuging for 5in at 13000 r/min; s4: 100 μl of the supernatant was extracted in the centrifuge tube after centrifugation by a pipette, and the extracted supernatant was added dropwise to the end of the detection zone of the gold-labeled reagent strip for human hemoglobin detection, and the result was observed. The invention can efficiently identify blood streak species and detect old blood streak samples which cannot be detected by the industry standard.
Description
Technical Field
The invention relates to the technical field of forensic material evidence species identification, in particular to a hemoglobin treatment fluid, a high-efficiency blood mark species identification method and application.
Background
In court science DNA laboratory test specifications and related regulations, the on-site suspicious blood mark is generally required to be subjected to blood mark confirmation test, and the blood mark species identification test is carried out by a gold mark test paper test method.
However, in the current practical work, there are some problems: 1. according to the method of the industry standard GA/T765-2020 (gold-labeled reagent strip method for human hemoglobin detection) (comparison technique), FOB (human blood spot) positivity can not be detected for some old blood marks. 2. When the existing modified hemoglobin treatment fluid detects old blood marks, false positives appear or FOB (human blood spot) positives cannot be detected. 3. The trace old sample can influence the extraction of DNA after the current industry standard is used for blood trace species identification, so that the sample is insufficient, the identification work is incomplete, and the case result is influenced.
Disclosure of Invention
The invention aims to solve the technical problems, and provides a hemoglobin treatment fluid, a high-efficiency blood trace species identification method and application thereof, which can efficiently identify blood trace species, detect old blood trace samples which cannot be detected by the industry standard, and have the advantages of no influence on DNA extraction and low sample loss.
The invention is realized by the following technical scheme:
the hemoglobin treatment fluid comprises the following components in parts by weight: 50-70 parts of 0.25% trypsin digestion liquid and 50-70 parts of pure water, wherein the total amount of the hemoglobin treatment liquid is 120 parts.
A high-efficiency blood trace species identification method comprises the following steps:
S1: placing a blood mark sample into a centrifuge tube, and then adding the hemoglobin treatment liquid into the centrifuge tube to obtain a primary sample;
s2: sufficiently vibrating a centrifugal tube filled with the primary sample, soaking a blood mark sample in the centrifugal tube for 30min at 37 ℃, and vibrating again after soaking;
s3: placing the centrifugal tube after the secondary vibration on a high-speed centrifugal machine, and centrifuging for 5min at 13000 r/min;
S4: and (3) extracting 100 mu l of supernatant from the centrifugal tube after centrifugation by using a pipette, dripping the extracted supernatant onto the tail end of a detection zone of the gold-labeled reagent strip for detecting human hemoglobin, and observing to obtain a detection result.
Further, the blood trace sample is a filter paper sample or a yarn sample.
Further, the blood trace sample size: the filter paper type sample was 1mm 2 or the yarn type sample was 5mm 2, and the centrifuge tube was a 1.5ml centrifuge tube.
The application of the hemoglobin treatment fluid is that the hemoglobin treatment fluid is applied to blood trace species identification treatment.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. The invention relates to a hemoglobin treatment fluid, a high-efficiency blood trace species identification method and application, wherein the total detection rate of the blood trace species identification method for the known human blood sample in 1987, 2000-2023 is up to 94.98%, the total detection rate of the blood trace species identification for the known human blood sample in 1987, 2000-2023 is 51.55% by the current industry standard, and the blood trace species identification method greatly improves the blood trace identification detection rate of the human blood sample;
2. the hemoglobin treatment fluid, the high-efficiency blood mark species identification method and the application of the invention have good blood mark identification effect on old human blood samples, can detect the old human blood samples with the longest age of 36 years, and have false positive or can not detect FOB (human blood spot) positive when the existing improved hemoglobin treatment fluid detects the old blood marks;
3. According to the hemoglobin treatment fluid, the efficient blood trace species identification method and the application, after the blood trace species identification is carried out on the human blood sample, the subsequent DNA detection of the sample is not affected, and the DNA detection result has good balance, so that the loss of the old sample is reduced.
Drawings
The accompanying drawings, which are included to provide a further understanding of the embodiments and examples of the application and are incorporated in and constitute a part of this specification, illustrate embodiments and examples of the application and together with the description serve to explain the principles of the application. In the drawings:
fig. 1 is a physical diagram of a blood trace sample in 2008;
FIG. 2 is a diagram of a sample test of a blood trace in 2008 after overnight immersion using industry current blood trace species identification standards;
FIG. 3 is a diagram of a sample test for detecting a blood trace in 2008 using the blood trace species identification method of the present invention;
FIG. 4 is a diagram of a blood trace sample in 1987;
FIG. 5 is a diagram of a sample test of a blood trace in 1987 after soaking for 30min using an industry current blood trace species identification standard and a sample test of a blood trace in 1987 using the blood trace species identification method of the present invention;
FIG. 6 is a diagram of a sample test of blood streaks in 1987 after overnight immersion using industry current blood streak species identification criteria;
FIG. 7 is a diagram of a DNA detection sample after a blood streak sample is soaked by the blood streak species identification method of the present invention in 1987;
FIG. 8 is a diagram of a DNA detection sample after soaking for 30min using the current blood trace species identification standard of the industry and a diagram of a DNA detection sample after soaking using the blood trace species identification method of the present invention;
FIG. 9 is a comparative graphical representation of the current blood trace species identification in the 2009 blood trace sample use industry and the blood trace species identification using the present invention;
FIG. 10 (a) is a diagram of a real object detected in 1987, 2000 to 2023 using industry current blood trace species identification criteria;
FIG. 10 (b) is a diagram b of the current blood trace species identification standard test in 1987, 2000 to 2023;
FIG. 10 (c) is a diagram c of the current blood trace species identification standard test in 1987, 2000 to 2023;
FIG. 10 (d) is a diagram d of the current blood trace species identification standard test in 1987, 2000 to 2023;
FIG. 10 (e) is a diagram e of the blood trace species identification test of the present invention in 1987, 2000 to 2023;
FIG. 10 (f) is a diagram f of a blood trace species identification test object of the present invention used in 1987, 2000 to 2023;
FIG. 10 (g) is a diagram g of a blood trace species identification test object of the present invention used in 1987, 2000 to 2023;
FIG. 10 (h) is a diagram h of the blood streak species identification test of the present invention used in 1987, 2000 to 2023;
FIG. 11 is a diagram of a sample of 2009 old blood streaks identified by using 1% ammonia water as the hemoglobin treatment solution in the identification method of the present invention;
FIG. 12 is a diagram of a sample of 2009 old blood streaks identified using TES/STE buffer as the hemoglobin treatment solution for the identification method of the present invention;
FIG. 13 is a diagram of a sample of 2009 old blood streaks identified using proteinase K mixture as the hemoglobin treatment solution for the identification method of the present invention;
FIG. 14 is a diagram of a sample to be tested for identifying a blank sample using 3.5% PEG (polyethylene glycol-6000) as the hemoglobin treatment solution for the identification method of the present invention;
FIG. 15 is a diagram of a sample of 2009 old blood marks identified using 3.5% PEG (polyethylene glycol-6000) as the hemoglobin treatment solution for the identification method of the present invention;
FIG. 16 is a diagram of a sample of 2009 old blood streaks identified using 3.5% PEG (polyethylene glycol-6000) mixture as the hemoglobin treatment solution for the identification method of the present invention;
FIG. 17 is a diagram of a sample of 2009 old blood streaks identified using EDTA as the hemoglobin treatment solution of the identification method of the present invention;
FIG. 18 is a diagram of a sample of 2009 old blood streaks identified using a mixture of 1% Triton and 0.25% trypsin digestion solution as the hemoglobin treatment solution for the identification method of the present invention;
FIG. 19 is a diagram of a sample of 2009 old blood streaks identified using a 1% Triton and pure water mixture as the hemoglobin treatment solution for the identification method of the present invention;
FIG. 20 is a diagram of a sample of 2012 old blood marks identified by using TE buffer mixture as hemoglobin treatment solution in the identification method of the present invention;
FIG. 21 is a diagram of a blank sample test using the blood trace species identification method of the present invention;
FIG. 22 is a diagram of a sample detection object of an animal blood trace using the blood trace species identification method of the present invention;
FIG. 23 is a diagram showing sample detection objects such as living goods and seasonings using the blood trace species identification method of the present invention;
FIG. 24 is a diagram of the sample for detecting the ratios of the hemoglobin treatment solutions in the blood trace species identification method of the present invention;
FIG. 25 (a) is a diagram of a sample for detecting different soaking temperatures and ratios of the blood streak species identification method of the present invention and the current blood streak species identification method in the industry;
FIG. 25 (b) is a diagram b of the detected objects of the present invention for blood streak species identification and the different soaking temperatures and ratios of the current blood streak species identification method in the industry;
FIG. 25 (c) is a diagram c of the detected objects of the present invention for blood streak species identification and the different soaking temperatures and ratios of the current blood streak species identification method in the industry;
FIG. 25 (d) is a diagram d of the detected objects of the present invention for identifying blood streak species and for identifying current blood streak species in the industry at different soaking temperatures and ratios;
Detailed Description
The present invention will be further described with reference to examples and test examples. The following examples and experimental examples are merely specific embodiments of the present invention, but the design concept of the present invention is not limited thereto, and any insubstantial modification of the present invention by using the concept should be construed as falling within the scope of the present invention.
Example 1
The hemoglobin treatment fluid comprises the following components in parts by weight: 50-70 parts of 0.25% trypsin digestion liquid and 50-70 parts of pure water, wherein the total amount of the hemoglobin treatment liquid is 120 parts.
Example 2
A high-efficiency blood trace species identification method comprises the following steps:
S1: placing a blood mark sample into a centrifuge tube, and then adding the hemoglobin treatment solution described in the embodiment 1 into the centrifuge tube to obtain a primary sample;
s2: sufficiently vibrating a centrifugal tube filled with the primary sample, soaking a blood mark sample in the centrifugal tube for 30min at 37 ℃, and vibrating again after soaking;
s3: placing the centrifugal tube after the secondary vibration on a high-speed centrifugal machine, and centrifuging for 5min at 13000 r/min;
S4: and (3) extracting 100 mu l of supernatant from the centrifugal tube after centrifugation by using a pipette, dripping the extracted supernatant onto the tail end of a detection zone of the gold-labeled reagent strip for detecting human hemoglobin, and observing to obtain a detection result.
More specifically, the blood trace sample is a filter paper type sample or a yarn type sample.
More specifically, the blood trace sample size: the filter paper type sample was 1mm 2 or the yarn type sample was 5mm 2, and the centrifuge tube was a 1.5ml centrifuge tube.
Example 3
The hemoglobin treatment solution described in example 1 was applied to blood trace species identification treatment.
The specific effects of the hemoglobin treatment solutions described in examples 1 to 3 for blood trace species identification will be specifically described below by way of experimental examples. The hemoglobin treatment solution in example 1 is prepared by 50-70. Mu.l of 0.25% trypsin digestion solution and 50-70. Mu.l of pure water, wherein the total amount of the hemoglobin treatment solution is 120. Mu.l.
Experimental example 1
Hemoglobin treatment fluid proportioning analysis experiment:
1. sample: 2011 human blood trace
2. The experimental method comprises the following steps: the ratio of 0.25% trypsin digestion solution to pure water was adjusted to 120. Mu.l total hemoglobin treatment solution, 13 experimental groups were designed, 209 experiments were performed by the identification method of example 2, and the detection rate was analyzed. FIG. 24 is a diagram of examples of the ratios of hemoglobin treatments used in one round of experiments in the blood trace species identification method of the present invention.
3. Analysis of experimental results: as is clear from Table 1, the blood trace species identification and detection rate was high when the amount of hemoglobin treatment liquid added was 50 to 70. Mu.l and the amount of pure water added was 50 to 70. Mu.l.
TABLE 1 analysis table for identifying and detecting blood trace species of hemoglobin treatment solutions with different proportions
Experimental example 2
Blood trace sample soaking temperature analysis experiment:
1. Sample: human blood traces from 2000 to 2023 (sample carriers of two types: yarn and filter paper)
2. The experimental method comprises the following steps: based on the optimized formulation of experimental example 1, the blood trace sample soaking temperature was adjusted, the ratio of 0.25% trypsin digestion solution to pure water was set, the total amount of hemoglobin treatment solution was 120 μl, 24 experimental groups were designed, experiments were performed using the identification method of example 2, each experimental group was subjected to 16 rounds of experiments, and the detection rate was analyzed. The current blood mark species identification method in the industry is a control group test, the current blood mark species identification method in the industry is a method of the golden standard reagent strip method for human hemoglobin detection (comparative technique) of the industry standard GA/T765-2020, the current blood mark species identification methods in the industry mentioned in the specification are all the methods, and figures 25 (a), 25 (b), 25 (c) and 25 (d) are detection physical figures of different soaking temperatures and proportions of the current blood mark species identification method in the invention and the current blood mark species identification method in the industry in one round of test.
3. Analysis of experimental results: as shown in Table 2, the blood trace samples with different trypsin digestion solution addition amounts and sample soaking times were soaked at 37℃and the highest detection rate reached 100%.
TABLE 2 blood streak species identification and detection rate analysis table for different blood streak sample soaking temperatures
Experimental example 3
The current blood mark species identification standard in industry is compared with the blood mark species identification method of the invention:
1. 2008 human blood trace sample contrast experiment
1) Sample: a 2008 human blood trace sample is shown in fig. 1.
2) The experimental method comprises the following steps: the formula and the test method of the embodiment are adopted to implement the current blood mark species identification standard in the industry and the blood mark species identification method of the invention to detect the 2008 blood mark sample.
3) Analysis of experimental results: an object diagram of 2008 year blood mark sample detection after being soaked overnight by using the current blood mark species identification standard in industry is shown in fig. 2, and the detection results are negative; a2008 blood mark sample detection physical diagram using the blood mark species identification method of the invention is shown in figure 3, and the detection results are positive.
2. Human blood trace sample contrast experiment in 1987
1) Sample: human blood trace samples in 1987 are shown in FIG. 4.
2) The experimental method comprises the following steps: the blood mark sample in 1987 is detected by adopting the formula and the test method of the embodiment and implementing the current blood mark species identification standard in the industry and the blood mark species identification method of the invention.
3) Analysis of experimental results: the real image of the blood mark sample detection in 1987 after 30min and overnight soaking by using the current blood mark species identification standard of the industry and the real image of the blood mark sample detection in 1987 by using the blood mark species identification method of the invention are shown in fig. 5 and 6, and the result of the current blood mark species identification of the industry is negative; the results of blood streak species identification using the invention are all positive.
3. 2009 Blood mark sample comparison experiment
1) Sample: 2009 human blood trace sample.
2) The experimental method comprises the following steps: the formula and the test method of the embodiment are adopted to implement the current blood mark species identification standard in the industry and the blood mark species identification method of the invention to detect the 2009 blood mark sample.
3) Analysis of experimental results: the 2009 blood mark sample is identified by using the current blood mark species of the industry and the blood mark species identification comparison object diagram of the invention is shown in fig. 9, and the results of the current blood mark species identification of the industry are negative; the results of blood streak species identification using the invention are all positive.
In conclusion, through 3 groups of comparison experiments, the detection effect of the current blood trace species identification method in the industry on the old human blood trace sample is poor, and the blood trace species identification method can detect the old human blood trace sample.
Experimental example 4
Blood trace sample detection rate experiment:
1. Sample: human blood traces from 1987, 2000 to 2023.
2. The experimental method comprises the following steps: the blood mark samples in 1987, 2000 and 2023 are detected by adopting the formula and the detection method of the embodiment, and the current blood mark species identification standard in the industry and the detection sample quantity of the blood mark species identification method of the invention are 419 parts.
3. Analysis of experimental results: the sample detection physical figures of the blood trace samples in 1987, 2000 and 2023 after being soaked overnight by using the current blood trace species identification standard in the industry are shown in fig. 10 (a), 10 (b), 10 (c) and 10 (d), and the sample detection physical figures of the blood trace samples in 1987, 2000 and 2023 by using the blood trace species identification method of the invention are shown in fig. 10 (e), 10 (f), 10 (g) and 10 (h). The result of current blood trace species identification in the use industry is that 203 samples are not detected (54 samples are not detected after being soaked overnight by an industry standard method), 216 samples are detected to be positive, and the total detection rate is 51.55%; the result of the blood streak species identification using the present invention was 94.98%.
Experimental example 5
Analysis of the effect of blood streak species identification on subsequent DNA detection of the sample:
1. sample: the current blood trace species identification in the industry and the old human blood trace sample after the blood trace species identification are used.
2. The experimental method comprises the following steps: the specific method for carrying out DNA detection on the current blood trace species identification in the use industry and the human blood trace sample after the blood trace species identification is used comprises the following steps: the samples were directly subjected to DNA detection using a magnetic bead method workstation using autosomal STR home-made reagents (amplification system 10. Mu.l, standard cycle).
3. Analysis of experimental results: the DNA detection diagram of the old sample soaked by using the current blood trace species identification standard in the industry and the DNA detection physical diagram soaked by using the blood trace species identification method of the invention are shown in fig. 7 and 8, and the result is that: ① The blood streak species identification method of the invention does not influence the detection of DNA; ② The blood streak species identification method has the advantages of good DNA result balance, and no obvious difference between the DNA concentration and the peak value and the current industry standard result.
Experimental example 6
In the conventional method for detecting human blood trace samples, hemoglobin treatment solutions were modified, and the effects of the hemoglobin treatment solutions on old blood trace samples were tested as follows, wherein the hemoglobin treatment solutions described in the prior art comprise 1% ammonia water, TES/STE buffer, proteinase K, 3.5% polyethylene glycol (PEG), EDTA, 1% Triton or TE buffer.
1. Detection effect of 1% ammonia water treatment liquid on old blood mark sample
1) Sample: 2009 old human blood trace sample.
2) The experimental method comprises the following steps: by the detection method of the example, the hemoglobin treatment liquid was replaced with 1% ammonia water.
3) Analysis of experimental results: a detection physical diagram of the hemoglobin treatment liquid for identifying 2009 old blood streak samples by adopting 1% ammonia water as the identification method is shown in fig. 11, and the detection results are negative.
2. Detection effect of TES/STE buffer solution on old blood streak sample
1) Sample: 2009 old human blood trace sample.
2) The experimental method comprises the following steps: by the detection method of the example, the hemoglobin treatment solution was replaced with TES/STE buffer.
3) Analysis of experimental results: a detection physical diagram of a 2009 old blood mark sample identified by adopting TES/STE buffer solution as the hemoglobin treatment solution in the identification method is shown in fig. 12, and the detection results are negative.
3. Detection effect of proteinase K mixed solution on old blood streak sample
1) Sample: 2009 old human blood trace sample.
2) The experimental method comprises the following steps: using the assay method of the example, 0.25% trypsin digest was replaced with proteinase K.
3) Analysis of experimental results: a detection physical diagram of the hemoglobin treatment liquid for identifying 2009 old blood streak samples by adopting the proteinase K mixed liquid as the identification method is shown in fig. 13, and the detection results are negative.
4. The detection effect of a blank control with the proportion of 3.5 percent of PEG and pure water (the total weight of the hemoglobin treatment solution is unchanged) is adjusted
1) Sample: and no.
2) The experimental method comprises the following steps: by the detection method of the example, the hemoglobin treatment liquid was replaced with 3.5% peg, and no human blood trace sample was added.
3) Analysis of experimental results: a sample graph of the hemoglobin treatment solution identified blank sample using 3.5% PEG (polyethylene glycol-6000) as the identification method of the present invention is shown in FIG. 14, and false positive occurs in the detection result.
5. 3.5% Detection effect of PEG on old blood streak sample
1) Sample: 2009 old human blood trace sample.
2) The experimental method comprises the following steps: using the test method of the example, the hemoglobin treatment was replaced with 3.5% PEG.
3) Analysis of experimental results: a detection physical diagram of a 2009 old blood mark sample identified by using 3.5% PEG (polyethylene glycol-6000) as the hemoglobin treatment liquid in the identification method is shown in fig. 15, and the detection results are positive. Based on the blank control result analysis of the 4 th test, whether all the results are false positives cannot be judged.
6. Detection effect of 3.5% PEG mixed solution on old blood streak sample
1) Sample: 2009 old human blood trace sample.
2) The experimental method comprises the following steps: by adopting the detection method of the embodiment, 0.25% of trypsin digestion liquid is replaced by 3.5% of PEG, the proportion is adjusted, and the total weight of the hemoglobin treatment liquid is unchanged.
3) Analysis of experimental results: a detection physical diagram of the hemoglobin treatment liquid used for identifying the 2009 old blood streak sample by adopting 3.5% of PEG (polyethylene glycol-6000) mixed liquid as the identification method is shown in FIG. 16, when the 3.5% of PEG is mixed with 5-60 mu l, the detection result is negative, when the 3.5% of PEG is mixed with 70-110 mu l, the detection result is positive, and based on the blank control result analysis of the 4 th test, the result that the 3.5% of PEG is mixed with 70-110 mu l cannot be judged whether the detection result is false positive or not.
7. Detection effect of EDTA on old blood streak sample
1) Sample: 2009 old human blood trace sample.
2) The experimental method comprises the following steps: by the detection method of the example, the hemoglobin treatment liquid was replaced with EDTA.
3) Analysis of experimental results: the detection physical diagram of the 2009 old blood trace sample identified by adopting EDTA as the hemoglobin treatment liquid by the identification method is shown in fig. 17, and quality control lines disappear, so that EDTA cannot be used as a soaking liquid for human blood trace species identification.
8. Detection effect of 1% Triton and 0.25% trypsin digestion solution mixture on old blood streak sample
1) Sample: 2009 old human blood trace sample.
2) The experimental method comprises the following steps: using the test method of the example, pure water was replaced with 1% Triton.
3) Analysis of experimental results: a detection physical diagram of a 2009 old blood streak sample identified by adopting a mixed solution of 1% Triton and 0.25% trypsin digestion solution as a hemoglobin treatment solution in the identification method is shown in fig. 18, and the detection result is negative.
9. Detection effect of 1% Triton and pure water mixed solution on old blood streak sample
1) Sample: 2009 old human blood trace sample.
2) The experimental method comprises the following steps: by adopting the detection method of the embodiment, 0.25% trypsin digestion solution is replaced by 1% Triton, the proportion is adjusted, the 1% Triton weight is 5 mu l-120 mu l, the pure water weight is 0 mu l-115 mu l, and the total weight of the hemoglobin treatment solution is unchanged.
3) Analysis of experimental results: a detection physical diagram of a 2009 old blood streak sample identified by adopting a 1% Triton and pure water mixed solution as the hemoglobin treatment solution in the identification method is shown in fig. 19, and the detection result is negative.
10. Detection effect of TE buffer solution mixed solution on old blood streak sample
1) Sample: 2009 old human blood trace sample.
2) The experimental method comprises the following steps: by adopting the detection method of the embodiment, 0.25% trypsin digestion solution is replaced by TE buffer solution, the proportion is adjusted, the quantity of the TE buffer solution is 5 mu l-120 mu l, the quantity of pure water is 0 mu l-115 mu l, and the total quantity of the hemoglobin treatment solution is unchanged.
3) Analysis of experimental results: a detection physical diagram of a 2009 old blood streak sample identified by adopting a 1% Triton and pure water mixed solution as the hemoglobin treatment solution in the identification method is shown in FIG. 20, and the detection result is negative.
Experimental example 7
Specificity detection experiment:
1. Blank experiment
1) Sample: a blank control (i.e., pure hemoglobin treatment solution added, without any sample), a blank filter paper, or a yarn sample.
2) The experimental method comprises the following steps: the formula and the detection method of the embodiment are adopted to detect the blank sample.
3) Analysis of experimental results: a blank sample detection object diagram using the blood trace species identification method of the invention is shown in FIG. 21, and the detection result is negative.
2. Animal blood trace sample detection experiment
1) Sample: animal (cat, dog, pig, chicken, pigeon, fish, mouse, cow, horse, sheep, cynomolgus monkey, donkey) blood streak samples.
2) The experimental method comprises the following steps: the formula and the detection method of the embodiment are adopted to detect the animal blood trace sample.
3) Analysis of experimental results: the animal blood streak sample detection sample using the blood streak species identification method of the present invention is shown in fig. 22, and the detection results of the other animals are negative except that the detection result of the primate (cynomolgus monkey) is positive. (according to the instructions of the human blood red egg Bai Jinbiao, it is clear that the reagent strip has a positive result for primate and therefore the detection result is normal.)
3. Sample detection experiment for living goods, seasonings and the like
1) Sample: samples of daily necessities and flavoring (soy sauce, vinegar, chili oil, tomato sauce, lipstick) etc.
2) The experimental method comprises the following steps: the formula and the detection method of the embodiment are adopted to detect samples such as living goods, seasonings and the like.
3) Analysis of experimental results: a sample detection object diagram of living goods, seasonings and the like using the blood trace species identification method is shown in fig. 23, and the detection result is negative.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the scope of the invention, but to limit the invention to the particular embodiments, and any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (5)
1. The hemoglobin treatment fluid is characterized by comprising the following components in parts by weight: 50-70 parts of 0.25% trypsin digestion liquid and 50-70 parts of pure water, wherein the total amount of the hemoglobin treatment liquid is 120 parts.
2. The efficient blood trace species identification method is characterized by comprising the following steps of:
S1: placing a blood mark sample into a centrifuge tube, and then adding the hemoglobin treatment solution of claim 1 into the centrifuge tube to obtain a primary sample;
s2: sufficiently vibrating a centrifugal tube filled with the primary sample, soaking a blood mark sample in the centrifugal tube for 30min at 37 ℃, and vibrating again after soaking;
s3: placing the centrifugal tube after the secondary vibration on a high-speed centrifugal machine, and centrifuging for 5min at 13000 r/min;
S4: and (3) extracting 100 mu l of supernatant from the centrifugal tube after centrifugation by using a pipette, dripping the extracted supernatant onto the tail end of a detection zone of the gold-labeled reagent strip for detecting human hemoglobin, and observing to obtain a detection result.
3. The method of claim 2, wherein the blood trace sample is a filter paper sample or a yarn sample.
4. A method of high efficiency blood streak species identification as in claim 3 wherein the blood streak sample size is: the filter paper type sample was 1mm 2 or the yarn type sample was 5mm 2, and the centrifuge tube was a 1.5ml centrifuge tube.
5. The use of the hemoglobin treatment fluid according to claim 1, wherein the hemoglobin treatment fluid is applied to blood trace species identification treatment.
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