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CN-121975794-A - Method for efficiently enriching host DNA from panda fecal sample

CN121975794ACN 121975794 ACN121975794 ACN 121975794ACN-121975794-A

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a method for efficiently enriching host DNA from panda fecal samples, which comprises the following steps of enriching cells in panda fecal samples, and incubating the cells with intestinal epithelial cell specific antibodies to obtain fluorescence-labeled cells; the intestinal epithelial cell specific antibody is provided with fluorescent dye, the fluorescence marked cells are identified and sorted by a flow cytometry to obtain positive cell groups, DNA of the positive cell groups is extracted, and the extracted DNA is subjected to library construction to obtain a high-quality DNA library. The method can remarkably improve the relative content of host DNA in a sample, can obtain genome data with comprehensive coverage and good uniformity only by a lower sequencing data amount, successfully realizes high-quality and high-concentration enrichment of the host DNA, and can be successfully applied to genome high-throughput sequencing.

Inventors

  • KUANG WEIMIN
  • YU LI
  • LIU FAN
  • WANG XINLING
  • QU XIAOXUAN
  • ZHANG YUXIN

Assignees

  • 云南大学

Dates

Publication Date
20260505
Application Date
20260206

Claims (10)

  1. 1. A method for efficiently enriching host DNA from panda fecal samples, comprising the steps of: Enriching cells from panda fecal samples, and incubating the cells with intestinal epithelial cell specific antibodies to obtain fluorescently labeled cells, wherein the intestinal epithelial cell specific antibodies are provided with fluorescent dyes; Identifying and sorting the fluorescence-labeled cells by using a flow cytometer to obtain a positive cell population; Extracting DNA of positive cell population, and constructing library of extracted DNA to obtain high quality DNA library.
  2. 2. The method according to claim 1, wherein the collected panda feces is placed in RNAlater preservation solution to obtain the panda feces sample; The volume mass ratio of the RNAlater preservation solution to the panda feces is 1-2 mL/1-2 g.
  3. 3. The method according to claim 2, wherein the method for enriching cells from panda fecal sample comprises swirling 5-10 mL panda fecal sample, mixing the panda fecal sample upside down, centrifuging, collecting supernatant, mixing the supernatant with DPBS buffer to 45 mL, filtering for the first time, centrifuging the filtrate again, discarding the supernatant to obtain a precipitate, mixing the precipitate with 45 mL DPBS buffer, centrifuging for the last time, discarding the supernatant to obtain a precipitate, mixing the precipitate with 500 μL DPBS buffer, and filtering for the second time to obtain the cells.
  4. 4. The method of claim 3, wherein the rotational speed of the vortex is 200-1500 rpm and the time of the vortex is 30 s; The rotating speed of the centrifugation is 500 Xg, and the centrifugation time is 30 s; The rotational speed of the secondary centrifugation and the final centrifugation is 1500 Xg, and the time of the secondary centrifugation and the final centrifugation is 5 min; the filter screen used for the first filtering is a 70 mu M filter screen; the filter screen used for the second filtration is a 50 μm filter screen.
  5. 5. The method of claim 1, wherein the number to volume ratio of cells to intestinal epithelial cell specific antibodies is 1000 to 60000 per 1 μl.
  6. 6. The method of claim 1, wherein the incubation is at a temperature of 4 ℃ and the incubation time is 30 min.
  7. 7. The method of claim 1, wherein the intestinal epithelial cell-specific antibody comprises AE1/AE3; the fluorescent dye comprises a fluorescent dye with Alexa FluorTM 488.
  8. 8. The method of claim 1, wherein the gating strategy for sorting is to use FSC as A threshold value and A value of 15000, establish an FSC/SSC image to remove background noise and impurities, then establish an FSC-A/FSC-H image and an SSC-A/SSC-H image to perform adhesion removal twice, finally use 488 nm blue laser light source and A500-560 nm emission filter to detect, establish A FITC/SSC image to define A positive cell population, select cells with fluorescence signal intensity greater than background, obtain the positive cell population in A high-purity sorting mode, and collect at least 1000 cells per sample.
  9. 9. The method of claim 1, wherein the DNA of the extracted positive cell population is extracted using a microscale DNA extraction technique; the Micro DNA extraction technique included the extraction of DNA using QIAGEN DNA Micro Kit.
  10. 10. The method of claim 1, wherein the library construction employs a library construction strategy suitable for low input; The library construction strategy suitable for low input comprises the steps of adopting Hieff NGS cube OnePot Pro DNA Library Prep Kit V kit for library construction.

Description

Method for efficiently enriching host DNA from panda fecal sample Technical Field The invention belongs to the technical field of genetic engineering, and particularly relates to a method for efficiently enriching host DNA from panda fecal samples. Background Pandas (Ailuropoda melanoleuca) are special species in China, and population protection and management are highly dependent on accurate assessment of individual identification, population dynamics and genetic diversity. Because of the difficulty in sampling panda individuals, high interference cost and ethical limitation, genetic monitoring is mainly dependent on non-invasive samples such as feces. However, the quality of genetic information in fecal samples has been limited by a number of factors, which has long been a major bottleneck in the study of panda genetics and protective biology. First, the host-derived DNA content in panda feces is extremely low. Panda feces typically contain significant amounts of exogenous DNA derived from dietary components, intestinal microorganisms, and environmental contaminants, whereas the proportion of host DNA is often less than 5%. According to the result of the previous investigation, the success rate of STR genotyping of panda fecal samples is generally low, for example, only 473 fresh samples in 1308 of the fourth national panda investigation obtain effective STR genotyping, and only 406 of the feces samples in 971 of the cold mountain area not more than two weeks can successfully amplify the commonly used 7 STR sites. STR genotyping is highly sensitive to the number of host DNA templates, and generally reflects an insufficient number of host DNA templates entering the amplification system if it still exhibits a low typing success rate, given the relatively ideal sample preservation conditions, short amplification sites, and mature typing system. Most of samples adopted in the investigation are stool samples with fresh or short preservation time, and amplified are common short-fragment STR sites, so that the influence of factors such as serious DNA degradation or inapplicable methodology and the like is eliminated to a certain extent. Therefore, the typing success rate is maintained at a lower level for a long time, which indicates that insufficient host DNA content is a main and fundamental limiting factor for limiting individual recognition efficiency in fecal samples. Second, the chemical and biological components of panda feces are highly complex. The excrement is rich in bamboo fibers, plant secondary metabolites, bile salts and various endogenous nucleases, and the substances can accelerate the degradation of host DNA under the action of environmental factors such as illumination, temperature, humidity and the like, so that the host DNA is obvious in fragmentation characteristics, and the length of common amplified fragments is less than 200 bp. Besides degradation problems, the feces also contains a plurality of PCR inhibitors such as polysaccharide, polyphenol and the like, and the PCR inhibitors can be extracted together in the DNA extraction process, so that the downstream amplification and sequencing efficiency is obviously reduced, and the risks of amplification failure, false negative and genotyping errors are increased. In addition, the existing fecal DNA extraction methods have significant differences in applicability to different species. A large number of researches show that different DNA extraction kits have obvious differences in DNA concentration, purity, microbial community composition and amplification success rate, and the differences are closely related to the species-specific fecal components. Because pandas take bamboo plants as main food, the excrement has unique characteristics of high fiber, high inhibitor, high microorganism load and the like, the existing commercial kit cannot always remove the inhibitor and promote the DNA content of a host, so that the extracted DNA is difficult to meet the quality requirements of STR genotyping and even high-throughput sequencing. In summary, due to the problems of low host DNA content, serious degradation, abundant inhibitors, insufficient applicability of the extraction method and the like, the genetic utilization efficiency of panda fecal samples is generally low at the present stage, high-success rate individual identification is difficult to realize, and the requirements of high-resolution genetic research such as whole genome sequencing, SNP analysis and the like are also difficult to meet. Therefore, development of efficient enrichment or extraction technology capable of effectively improving host DNA level, reducing complex component interference and suitable for panda fecal characteristics is needed to break through the technical bottleneck of current non-invasive genetic monitoring. Disclosure of Invention The invention aims to provide a method for efficiently enriching host DNA from panda fecal samples. The method can remarkably im