CN-122012728-A - 28 Multi-InDel label composite system based on eight-color fluorescence detection technology, detection primer and application

Abstract

The invention discloses a 28 Multi-InDel label composite system based on an eight-color fluorescence detection technology, a detection primer and application thereof, and belongs to the technical field of forensic identification. The invention applies the eight-color fluorescence technology to Multi-InDel detection for the first time, integrates 28 autosomal short segment locus genetic marker systems, and remarkably improves the detection information quantity. The system has excellent suitability for highly-degraded DNA samples, has high detection rate, shows extremely low cumulative matching probability (2.3292 multiplied by 10 ‑18 ) and extremely high cumulative exclusion probability (0.9999669), and provides a high-efficiency and reliable technical scheme for forensic degradation material identification.

Inventors

• ZHALAGABAIYILA
• LI JIENAN
• JIA HONGTAO
• WEN DAN
• QU WEIFENG
• WANG YUEPENG

Assignees

• 中南大学

Dates

Publication Date

20260512

Application Date

20260212

Claims (10)

1. 1. A genetic marker system for typing of highly degraded samples, characterized in that it consists of a Multi & # x2011 site located in the reference genome grch37.p13, inDel site as shown in the following table: 。
2. 2. a primer set for amplifying the genetic marker system according to claim 1, which comprises primers having nucleotide sequences shown in SEQ ID NOS.1 to 56, respectively.
3. 3. Use of the primer set of claim 2 in the preparation of a kit for forensic identification.
4. 4. A kit for forensic identification, comprising the primer set of claim 2.
5. 5. The kit of claim 4, further comprising an allelic typing standard mixture, a DNA standard, and a multiplex amplification reaction mixture.
6. 6. The kit of claim 5, wherein the DNA standard comprises standard DNA 2800M.
7. 7. Use of the primer set of claim 2 or the kit of any one of claims 4-6 in forensic high degradation assay identification.
8. 8. The use of claim 7, wherein the highly degraded test material is selected from at least one of a large-scale catastrophic event biological test material, old bone, a spoiled sample, formalin-fixed paraffin-embedded tissue, and non-follicular hair.
9. 9. Use of a primer set according to claim 2 or a kit according to any one of claims 4 to 6 for identification of individuals or for genetic relationship identification.
10. 10. The use according to claim 9, wherein the primer set according to claim 2 or the kit according to any one of claims 4 to 6 is used for multiplex PCR amplification of DNA of a biological test material to be tested to obtain an amplification product, the amplification product is subjected to capillary electrophoresis detection, the genotype of the biological test material to be tested at the Multi-InDel locus according to the result of capillary electrophoresis is analyzed, and the biological test material to be tested is subjected to individual identification or genetic relationship identification according to the result of the genotype analysis.

Description

28 Multi-InDel label composite system based on eight-color fluorescence detection technology, detection primer and application Technical Field The invention relates to the technical field of forensic identification, in particular to a 28 Multi-InDel label composite system based on an eight-color fluorescence detection technology, a detection primer and application. Background In the fields of forensic individual identification and paternity testing, capillary electrophoresis typing techniques for Short Tandem Repeats (STRs) are currently accepted gold standards. However, the amplified fragments of common commercial STR kits are generally larger than 150 bp, and when highly degraded DNA samples (such as old bones, putrefying tissues and the like) are analyzed, the amplified fragments of large fragment alleles are often failed due to serious fragmentation of the DNA, typing information is not complete or even completely lost, so that effective identification of the problematic materials is severely restricted. To overcome the limitations of STRs in degrading test materials, researchers have gradually turned their eyes towards Single Nucleotide Polymorphism (SNP) and InDel (InDel) markers with the advantage of short amplicons. Wherein, the multiple InDel (Multi-InDel) mark is especially suitable for analysis of highly degraded DNA due to the characteristics of low mutation rate, short amplified fragment, almost no interference of 'shadow peak', and the like. At present, multi-InDel composite amplification systems based on five-color or six-color fluorescence detection systems are developed, and the systems are compatible with the conventional forensic genetic analyzer, and the data analysis flow is similar to STR detection, so that the system is convenient for technicians to master. However, limited by the number of fluorescent channels, existing systems have a limited number of Multi-InDel sites that can be accommodated in short segments of less than 130 bp, where the effective fragments of highly degraded DNA are concentrated. Therefore, the discrimination capability of the existing system for degraded samples still has a bottleneck, and it is difficult to obtain enough genetic information within a limited fragment length to meet the high-confidence discrimination requirement. The eight-color fluorescence detection technology has shown remarkable advantages in STR composite detection, and the site accommodation capacity and information quantity of unit detection are effectively improved by adding a fluorescence channel. However, to date, this technology has not been effectively applied in the field of Multi-InDel detection, and lacks a mature eight-color fluorescent Multi-InDel detection system. If the eight-color fluorescence can be combined with the Multi-InDel marker, more genetic loci are expected to be integrated within the same amplicon length range (particularly below 130 bp), so that the typing success rate and the identification efficiency of the highly degraded detection material are greatly improved. However, the development of such systems faces multiple technical challenges, namely, firstly, multi-InDel markers which are close in coincidence position, small in length difference, good in polymorphism and suitable for multiplex amplification need to be screened from massive genome data, secondly, multichannel primer design needs to be carried out aiming at an eight-color fluorescent platform to ensure that the fragment lengths in each fluorescent channel are not overlapped, the amplification efficiency is balanced and non-specific products are avoided, thirdly, a complete detection method and a data analysis flow are required to be established, and the applicability and the reliability of the detection method in actual samples, particularly in highly degraded detection materials, need to be verified. At present, an eight-color fluorescence Multi-InDel composite detection system capable of simultaneously meeting the requirements is not disclosed. Disclosure of Invention The invention aims to provide a 28 Multi-InDel label composite system based on an eight-color fluorescence detection technology, a detection primer and application thereof, so as to solve the problems in the prior art. According to the invention, the eight-color fluorescence technology is applied to Multi-InDel detection for the first time, 28 autosomal short fragment sites are integrated, and the detection information quantity is remarkably improved. The system has excellent suitability for highly-degraded DNA samples, has high detection rate, shows extremely low cumulative matching probability (2.3292 multiplied by 10 -18) and extremely high cumulative exclusion probability (0.9999669), and provides a high-efficiency and reliable technical scheme for forensic degradation material identification. In order to achieve the above object, the present invention provides the following solutions: the invention provides a genetic marker sys