Diatom rapid detection kit for locking drowning place

Abstract

The invention discloses a diatom rapid detection kit for locking a drowning place, and relates to the technical field of forensic medicine detection. The diatom rapid detection kit comprises a diatom detection primer combination, and the diatom detection primer combination comprises a primer pair for detecting target genes 18SrDNA, 18S rRNA, 16S rDNA, UPA, rbcL, SSU, ITS and COI. According to the kit, a diatom multi-gene multiplex amplification rapid detection system is used, and limitation of single-target PCR is made up through multiplex amplification, so that the kit has the advantages of high specificity, high sensitivity and high accuracy. The diatom rapid detection kit for locking the drowning place, provided by the invention, can provide more scientific, objective and efficient data reference for the detection of an actual drowning place.

Description

Technical Field

[0001] The present invention relates to the technical field of forensic detection, and particularly to a rapid detection kit for diatoms for locking drowning sites. Background Art

[0002] In the field of forensic medicine, the investigation of drowning sites has always been a key point and a difficult point. As a single-celled alga widely existing in water areas, the unique biological characteristics of diatoms make them key biomarkers for inferring drowning sites. When a human body drowns, a large amount of water containing diatoms will be inhaled, and these diatoms will enter various organs of the human body through blood circulation, especially the lungs, liver, kidneys, etc. By detecting and analyzing the diatoms in these organs and comparing them with the species and distribution of diatoms in different water areas, it is possible to infer the location where drowning occurred.

[0003] However, there are many deficiencies in the current research and data management of diatoms. On the one hand, the existing diatom detection methods face many challenges in actual operation. The traditional chemical digestion method is cumbersome to operate and easily damages the diatom morphology, affecting the accuracy of detection; although the enzyme digestion method is relatively gentle, it has a high cost and a long time consumption. Advanced technologies such as microwave digestion-vacuum filtration-scanning electron microscopy method, although improving the detection efficiency and accuracy to a certain extent, also have high requirements for equipment and operators and are difficult to be widely applied in grass-roots forensic institutions. On the other hand, the lack of a unified and perfect diatom database seriously restricts the identification work of drowning sites. At present, most of the diatom data in various places are stored separately and in inconsistent formats, and an effective data sharing and comparative analysis mechanism cannot be formed. In actual identification work, forensic doctors often need to spend a lot of time and energy collecting and sorting out the diatom data of relevant water areas, which is not only inefficient, but also due to the incompleteness and uncertainty of the data, the accuracy of inferring drowning sites is greatly reduced.

[0004] In addition, the existing research mainly focuses on the classification and distribution characteristics of diatoms, and the research on the relationship between diatoms and environmental factors is not deep enough. At present, there is no systematic research on correlating these environmental factors with diatom data, so that when using diatoms to infer drowning sites, the influence of environmental factors cannot be fully considered, further reducing the accuracy of inference.

[0005] In summary, constructing a comprehensive, accurate and easy-to-apply diatom database and developing a corresponding application system have important practical significance for improving the identification efficiency and accuracy of drowning sites. Summary of the Invention

[0006] The object of the present invention is to provide a rapid detection kit for diatoms for locking drowning locations to solve the problems in the above-mentioned prior art. The kit has the advantages of high specificity, high sensitivity and high accuracy, and can provide more scientific, objective and efficient data reference for the inspection of actual drowning locations.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a primer combination for detecting diatoms for locking drowning locations, including primer pairs for detecting target genes 18SrDNA, 18S rRNA, 16S rDNA, UPA, rbcL, SSU, ITS and COI.

[0009] Further, the primer pair for detecting the target gene 18S rDNA includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2;

[0010] The primer pair for detecting the target gene 18S rRNA includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.3 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.4;

[0011] The primer pair for detecting the target gene 16S rDNA includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.6;

[0012] The primer pair for detecting the target gene UPA includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.7 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.8;

[0013] The primer pair for detecting the target gene rbcL includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.9 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.10;

[0014] The primer pair for detecting the target gene SSU includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.11 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.12;

[0015] The primer pair for detecting the target gene ITS is primer pair 1 or primer pair 2; the primer pair 1 includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.13 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.14; the primer pair 2 includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.15 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.16;

[0016] The primer pair for detecting the target gene COI includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.17 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.18.

[0017] The present invention also provides the application of the above-mentioned diatom detection primer combination in the preparation of a rapid diatom detection kit for locking the drowning location.

[0018] The present invention also provides the application of the above-mentioned diatom detection primer combination in locking the drowning location based on diatom detection.

[0019] The present invention also provides a rapid diatom detection kit for locking the drowning location, which includes the above-mentioned diatom detection primer combination.

[0020] Furthermore, the kit also includes a buffer solution.

[0021] Furthermore, the buffer solution is a 10×PCR Buffer containing Mg 2+ .

[0022] Furthermore, the kit also includes Accurate Taq DNA Polymerase and dNTP Mix.

[0023] The present invention also provides the application of the above-mentioned rapid diatom detection kit in locking the drowning location based on diatom detection.

[0024] The present invention also provides a method for locking the drowning location based on diatom detection, which includes the following steps:

[0025] Obtain the lung sample of the drowning victim and extract genomic DNA;

[0026] Using the genomic DNA as a template, perform PCR amplification with the above-mentioned diatom detection primer combination to obtain a PCR amplification product;

[0027] After sequencing the PCR amplification product, perform a comparison in the diatom database to determine the drowning location;

[0028] The diatom database is constructed using the DNA of diatom samples collected at different locations.

[0029] The present invention discloses the following technical effects:

[0030] (1) The present invention provides an identification system integrating a morphological distribution database, PCR detection, and a gene database. A diatom morphological distribution database is established by the combined method of microwave digestion, vacuum filtration, and scanning electron microscopy. Combining a multi-target multiplex amplification system based on capillary electrophoresis technology and primer design and gene database construction based on the GenBank database, a complete and multi-dimensional diatom identification platform is formed. Using genes such as diatom ITS, rbcL, UPA, SSU, 16S rDNA, 18S rRNA, 18S rDNA, and COⅠ as target genes, gene sequence retrieval is performed through the GenBank database, primers are designed and screened, a multi-target multiplex amplification system based on capillary electrophoresis technology is constructed, and performance verification and molecular saturation analysis are carried out. Through multiplex amplification, the limitations of single-target PCR are compensated, and a rapid detection system for multi-gene multiplex amplification of diatoms is constructed, which has the advantages of high specificity, high sensitivity, and high accuracy.

[0031] (2) The present invention provides a rapid detection kit for diatoms to lock the drowning location, which uses PCR technology for detection and can obtain results in about 3 - 5 hours. Compared with the 98 hours of the traditional MD-VF-AtuoSEM method for detection time, the detection time is greatly shortened.

[0032] (3) The rapid detection kit for diatoms to lock the drowning location provided by the present invention can provide more scientific, objective, and efficient data reference for the inspection of the actual drowning location. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0034] Figure 1 It is a technical comparison diagram of the diatom detection method of the present invention and the traditional method;

[0035] Figure 2 It is a schematic diagram of the research and development idea of the present invention;

[0036] Figure 3 It is a schematic diagram of the principle of the rapid detection kit for diatoms of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0038] It should be understood that the terms used in the present invention are only for describing particular embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0039] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0040] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0041] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0042] The present invention provides an identification system integrating a morphological distribution database - PCR detection - gene database. A diatom morphological distribution database is established by the combined method of microwave digestion - vacuum filtration - scanning electron microscopy, combined with a multi-target multiplex amplification system based on capillary electrophoresis technology, and primer design and gene database construction based on the GenBank database, forming a complete and multi-dimensional diatom identification platform. Using genes such as diatom ITS, rbcL, UPA, SSU, 16S rDNA, 18S rRNA, 18S rDNA, COⅠ as target genes, gene sequence retrieval is performed through the GenBank database, primers are designed and screened, a multi-target multiplex amplification system based on capillary electrophoresis technology is constructed, and performance verification and molecular saturation analysis are carried out. Through multiplex amplification, the limitations of single-target PCR are compensated, a rapid detection system for multiplex amplification of diatom multi-genes is constructed, which has the advantages of high specificity, high sensitivity, and high accuracy. Furthermore, a rapid detection kit for diatoms used to lock the drowning location is constructed. The technical comparison chart of the present invention with traditional methods is shown in Figure 1 , and the schematic diagram of the research and development idea of the present invention is shown inFigure 2 , the schematic diagram of the product principle of the present invention is shown in Figure 3 .

[0043] Example 1

[0044] A primer combination for diatom multiplex amplification, including the primers described in Table 1:

[0045] Table 1 Primer combination for diatom multiplex amplification

[0046]

[0047]

[0048] Example 2

[0049] A rapid diatom detection kit for locking drowning sites, including the following components:

[0050] The primer combination of Example 1, Accurate Taq DNAPolymerase, 10×PCR Buffer (Mg 2+ plus) and dNTP Mix.

[0051] Example 3

[0052] A rapid diatom detection method for locking drowning sites, including the following steps:

[0053] (1) Obtain the lung sample of the drowning victim and extract genomic DNA;

[0054] (2) Using the genomic DNA obtained in step (1) as a template, perform PCR amplification respectively with the primer combination of Example 1. The PCR reaction system is 25 μL: Accurate Taq DNAPolymerase 0.5 U, dNTP Mix 0.3 mmol / L, each primer 0.5 μmol / L, template 60 ng, 10×PCR Buffer (Mg 2+ plus) 2.5 μL, supplemented with ddH2O to 25 μL.

[0055] The PCR reaction program is: 94°C for 10 min; 94°C for 40 s, 50°C for 1 min, 72°C for 1 min, a total of 35 cycles, and finally 72°C for 7 min of extension. The temperature is reduced to 4°C and stored for use to obtain the PCR amplification product.

[0056] (3) After sequencing the PCR amplification product obtained in step (2), perform a comparison in the diatom database to determine the drowning site. Among them, the diatom database is constructed using the DNA of diatom samples collected at different locations.

[0057] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the spirit of the present invention's design, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer combination for detecting diatoms to lock the drowning location, characterized in that, Primer pairs for detecting target genes 18S rDNA, 18S rRNA, 16S rDNA, UPA, rbcL, SSU, ITS, and COI.

2. The diatom detection primer combination according to claim 1, wherein The primer pair for detecting the target gene 18S rDNA includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2; The primer pair for detecting the target gene 18S rRNA includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.3 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.4; The primer pair for detecting the target gene 16S rDNA includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.6; The primer pair for detecting the target gene UPA includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.7 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.8; The primer pair for detecting the target gene rbcL includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.9 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.10; The primer pair for detecting the target gene SSU includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.11 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.12; The primer pair for detecting the target gene ITS is primer pair 1 or primer pair 2; primer pair 1 includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.13 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.14; primer pair 2 includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.15 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.16; The primer pair for detecting the target gene COI includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.17 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.

18.

3. Use of the diatom detection primer combination according to claim 1 or 2 in the preparation of a rapid diatom detection kit for locking the drowning location.

4. Use of the diatom detection primer combination according to claim 1 or 2 in locking the drowning location based on diatom detection.

5. A rapid detection kit for diatoms used to lock the drowning location, characterized in that, Includes the diatom detection primer combination according to claim 1 or 2.

6. The rapid diatom detection kit according to claim 5, wherein, The kit further includes a buffer.

7. The rapid detection kit for diatoms according to claim 6, wherein The buffer is a 10×PCR Buffer containing Mg 2+ .

8. The rapid detection kit for diatoms according to claim 5, wherein The kit further includes Accurate Taq DNA Polymerase and dNTP Mix.

9. Use of the rapid diatom detection kit according to any one of claims 5-8 in locking the drowning location based on diatom detection.

10. A method for detecting and locking the drowning location based on diatom detection, characterized in that, Includes the following steps: Obtain a lung sample of the drowning victim and extract genomic DNA; Using the genomic DNA as a template, perform PCR amplification with the diatom detection primer combination according to claim 1 or 2 to obtain a PCR amplification product; After sequencing the PCR amplification product, perform a comparison in the diatom database to determine the drowning location; The diatom database is constructed using the DNA of diatom samples collected at different locations.

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