CN113186185A - Method for efficiently enriching host DNA from mammal excrement - Google Patents
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Abstract
一种从哺乳动物粪便中高效富集宿主DNA的方法，属于分子生物学技术领域。针对目前哺乳动物粪便样本提取DNA时，宿主DNA富集效率低、对陈旧粪便提取效果差、大量细菌DNA的残留严重干扰后续反应，而且成本居高不下等问题，本发明提供了一种从粪便中高效富集宿主DNA的方法，在提取前，对粪便样本进行SDS预处理：将粪便样本与10mmol/L磷酸缓冲液PBS溶液混合后，加入SDS溶液至终浓度为0.01％～5％(质量)混匀，在室温下静置1min～30min；然后离心取上清，从上清液中提取DNA。本发明成本低廉，不受样本数量的限制，拓展了粪便样本在动物研究和保护监测中的应用。
A method for efficiently enriching host DNA from mammalian feces belongs to the technical field of molecular biology. Aiming at the problems of low host DNA enrichment efficiency, poor extraction effect on old feces, and a large amount of bacterial DNA residues seriously interfering with subsequent reactions when DNA is extracted from mammalian feces samples at present, and the high cost, the present invention provides a method for extracting DNA from feces. The method of enriching host DNA in medium and high efficiency is to perform SDS pretreatment on fecal samples before extraction: after mixing the fecal samples with 10 mmol/L phosphate buffered saline solution in PBS, add SDS solution to a final concentration of 0.01% to 5% (mass). ), and let stand at room temperature for 1 to 30 minutes; then centrifuge to take the supernatant, and extract DNA from the supernatant. The invention has low cost, is not limited by the number of samples, and expands the application of fecal samples in animal research and protection monitoring.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a method for efficiently enriching host DNA from mammal excrement.
Background
In recent years, the use of non-invasive sampling in the preservation of biology and molecular ecology has received a great deal of attention and interest, and stool is due to: (1) the intestinal epithelial cells of a research object can be obtained in a non-invasive manner, and the method has the advantages of no wound, no pain and no invasion; (2) the animal's stool is regular and rhythmic, and therefore the stool sample is abundant in quantity, providing a sufficient source of sample compared to other sample types; (3) the excrement sample can be collected under the condition that animal entities cannot be touched or even seen, particularly under the condition of aiming at fierce large carnivores, the safety of sampling personnel can be ensured to the maximum extent, and meanwhile, the interference to animals in the collection process is reduced to the minimum; (4) the excrement collection is easy to operate and the cost is low. Thus, feces are the most commonly used samples in field animal research and in the management of artificially-raised animals such as zoos. Host DNA extracted from host intestinal epithelial cells present in feces plays an important role in species identification, mating system analysis, genetic diversity evaluation, population genetic structure analysis, systematic geographic study, and the like.
The research shows that each gram of fresh excrement contains 10 percent5The individual intestinal epithelial cells, most of which remain intact, continue to sustain normal vital activities, whereas over 55% of the dry weight of the stool is made up of bacteria derived from the intestinal flora and the surrounding environment. Therefore, the total DNA extracted from feces contains little host DNA and often contains substances inhibiting molecular biological reactions, so that the analysis success rate of subsequent experiments is low, and particularly the analysis of nuclear genomes is difficult, such as the confirmation of microsatellite and SNP typing results has to be repeated for many times, thus greatly increasing the experiment cost and seriously affecting the reliability of data.
Therefore, how to improve the extraction efficiency of host DNA in feces and reduce the proportion of bacterial DNA is the key in the current feces DNA extraction technology. The existing methods for enriching host DNA in excrement mainly comprise a DNA methylation co-immunoprecipitation technology, a density gradient centrifugation method, an immunomagnetic bead sorting method, a dielectrophoresis separation method (DEP), a commercial excrement DNA extraction kit and the like, and the methods have the following characteristics and defects:
(1) the efficiency of enrichment of host DNA is still not ideal, especially for enrichment of nuclear genomes.
As for the DNA methylation co-immunoprecipitation technology, the method can enrich the methylated fragments of the host nuclear genome with high purity, but because the probability of methylation of the host mitochondrial genome is very small, the method cannot enrich the mitochondrial genetic material; as for immunomagnetic bead method and dielectrophoresis chip method, the method can enrich host DNA to reach high purity, but because the quantity of bacterial DNA is too large, the interference is serious, the capture probability of host cells is low, and finally the enrichment amount of the obtained host DNA is less; at present, the QIAamp kit which is most widely applied is provided by Qiagen company, the enrichment effect on host mitochondrial genomes on the whole can meet most experimental needs, but the enrichment effect on host nuclear DNA is still not ideal, when the microsatellite locus is detected, the same sample is repeated for at least 5-7 times, the final typing result is obtained in a 'few obeying to most' manner, and the accuracy is very low.
(2) The efficiency of host DNA extraction is worse for old stools.
The current technologies for enriching host DNA in various feces are basically suitable for fresh feces, and especially for SCSR-TTM kits, methylation coprecipitation methods, magnetic bead sorting methods, dielectrophoresis chip methods, density gradient centrifugation methods and the like, the requirements for the freshness of feces samples are more strict. At present, although the QIAamp kit has relatively wide requirements on the freshness of a sample, the QIAamp kit does not have ideal effect on enrichment of host DNA in old feces.
(3) High cost and large consumption.
The existing enrichment technology of host DNA in the excrement sample is expensive, and for a methylation co-immunoprecipitation method, a magnetic bead sorting method, a dielectrophoresis chip method and the like, one sample needs 200-500 yuan RMB, so that most of the RMB are used only when necessary. For the QIAamp fecal DNA extraction kit, each sample still requires about 50 yuan, which is also cost prohibitive for large sample analysis.
(4) The operation requirement is strict.
The methylation coprecipitation method, the magnetic bead sorting method, the dielectrophoresis chip method, the density gradient centrifugation method and the like have the disadvantages of complicated operation process, strict condition requirements and longer time consumption.
Disclosure of Invention
Aiming at the problems of strict excrement quality requirement, low host DNA enrichment efficiency, high cost and the like when extracting host DNA from an excrement sample at present, the invention provides a method for efficiently enriching host DNA from mammal excrement, wherein before extracting the excrement DNA, the excrement sample is pretreated by SDS, and the pretreatment method comprises the following steps:
1) mixing the fecal sample with 10mmol/L phosphate buffer solution PBS solution, adding SDS solution to the final concentration of 0.01-5% (by mass), mixing uniformly, and standing for 1-30 min at room temperature;
2) the supernatant was then centrifuged and used for DNA extraction.
Further limiting, when the sample is used for the excrement of the herbivorous animals in the step 1), standing for 1-15 min at room temperature after the pretreatment.
Further limiting, when the sample is used for the excrement of the carnivorous animal in the step 1), standing for 1-10 min at room temperature after the pretreatment.
Further defined, the ratio of the fecal sample to the Phosphate Buffered Saline (PBS) solution in the step 1) is 180-220 mg:300 μ L.
Further, the SDS solution is added to the solution in the step 1) to a final concentration of 1% by mass.
Further defined, the method for extracting DNA in the step 2) comprises a genomic DNA kit extraction method and an organic solvent extraction method.
Further limited, the herbivore and animal manure sample in step 1) is subjected to the pretreatment and then is kept standing for 5min at room temperature.
Further defined, the meat and animal manure sample in the step 1) is allowed to stand for 3min at room temperature after the pretreatment.
Advantageous effects
(1) The invention has good enrichment capacity for both mitochondrial DNA and nuclear DNA of hosts in excrement. Compared with the currently widely used QIAamp kitThe enrichment efficiency of host mitochondrial DNA is improved by 101～103The enrichment efficiency of the host nuclear DNA can be improved by 10 times1～104And (4) doubling.
(2) The method has relatively wide quality requirements on the stool sample, is suitable for fresh stool, also has good enrichment capacity on old stool which is preserved by conventional freezing, and can improve the enrichment efficiency of host mitochondrial DNA by 1-10 compared with a QIAamp kit3The enrichment efficiency of the host nuclear DNA is improved by 10 times1～104And (4) doubling.
(3) The invention has wide application range and has good enrichment effect on host DNA in faeces of herbivorous and carnivorous mammals.
(4) All the operations related to the invention belong to the conventional laboratory operations, the experimental steps are simple and easy to implement, and the whole process can be completed only within 30-40 min.
(5) Because the price of SDS is low, the processing cost of each fecal sample by the method is only a few money, thereby greatly reducing the cost consumption of the experiment; in actual operation, only common equipment such as a water bath kettle, a liquid transfer device, a centrifugal machine and the like is needed, and the requirements of common laboratories can be met, so that the universality is extremely strong. Due to low cost and low requirement, the method is suitable for extracting large-scale and multi-batch fecal samples, and greatly expands the application of the fecal samples in animal research.
Drawings
FIG. 1: comparing the enrichment effect (expressed as host DNA copy number/bacterial DNA copy number) of different stool samples (including different species and different years of collection) on host DNA (including nuclear DNA and mitochondrial DNA) under QIAamp kit and optimal SDS lysis conditions; the abscissa represents different fecal samples, and the ordinate represents the enrichment efficiency of host DNA; wherein:
FIG. 2: comparing the results of the amplification typing of STR sites by the host DNA obtained by partial excrement samples by the method of the invention and the blood DNA of the same individual, wherein the abscissa is the fragment length and the unit bp; the ordinate is the intensity of the detection signal. Wherein A is a parting map of a microsatellite locus FCA146 of a blood sample of an individual No. 2 northeast tiger, and B is a parting map of the same locus of the individual in a stool sample; c, a microsatellite locus 32 typing map of a blood sample of a red deer No. 6 individual, and D, a typing map of the same locus of the individual in a fecal sample; e, a microsatellite locus CPH758 parting map of a blood sample of a dog No. 4 individual, and F, a parting map of the same locus of the individual on a stool sample; g, a microsatellite locus CPH9 typing map of a blood sample of an individual blue fox No. 1, and H, a typing map of the same locus of the individual in a fecal sample.
Detailed Description
According to the method, according to the difference of the outer wall structures and the anti-damage capability of intestinal epithelial cells and bacteria in the excrement, Sodium Dodecyl Sulfate (SDS) with low price is selected as a cracking agent, and the optimal cracking condition is obtained by controlling the concentration of the SDS and the acting time of the SDS on the excrement. Under the condition, the host cells can be cracked to the maximum extent, so that the host DNA is released into the solution, the structural integrity of the bacteria is kept as much as possible, and the differential extraction of the DNA is realized. During extraction, only high-speed centrifugation is needed, supernatant is extracted, and host DNA with considerable purity and quantity can be enriched from the supernatant by using a conventional DNA extraction technology (an organic solvent extraction method, various commercialized common genome DNA extraction kits and the like), so that the residual quantity of the bacterial DNA is reduced.
The key innovation point of the invention is that the SDS pretreatment is carried out before the fecal sample is extracted, and the optimal SDS lysis conditions are respectively obtained aiming at the fecal of the carnivorous animal and the herbivorous animal by controlling the concentration and the action time of the SDS, under the conditions, the intestinal epithelial cells of the host can be fully lysed, and the integrity of the bacterial cells can be furthest maintained.
The method for efficiently enriching host DNA from mammalian feces according to the present invention is described in detail below.
Example 1. method for efficiently enriching host DNA from deer feces.
1. Cutting the excrement sample by adopting a sterile scalpel and scissors, weighing 180mg of excrement and putting the excrement into a sterilized 2ml centrifugal tube, and strictly ensuring the cleanness of tools (scissors, tweezers and the scalpel) in the process so as to avoid exogenous DNA pollution and cross contamination among samples. Add 300. mu.L of 10mmol/L PBS solution to the centrifuge tube, then add SDS to a final concentration of 1%, mix the sample and solution quickly, centrifuge instantaneously and then stand at room temperature for 5 min.
2. Then, the mixture was centrifuged at 12000 Xg for 10min, and the supernatant was aspirated for further use, and the precipitate was discarded.
The supernatant obtained by the above-mentioned treatment can be used for DNA extraction by any conventional DNA extraction techniques such as ordinary genomic DNA commercial kit method, chemical extraction method and the like. In this example, the final DNA extraction method is described by taking AxyPrep genomic DNA miniatur kit (Axygen) as an example, and the following steps are followed:
3. 150ul Buffer C-L and 20ul PK enzyme are added into the supernatant obtained after centrifugation, the mixture is mixed evenly and then centrifuged instantaneously, and the mixture is placed in a 56 ℃ water bath kettle for incubation for 10 min.
4. 350ul Buffer PD was added to each tube, shaken for 10s, and centrifuged at 12000 Xg for 10 min.
5. The DNA preparation tube was placed in a 2ml PE tube, and the centrifuged supernatant was pipetted into the preparation tube and centrifuged at 12000 Xg for 1 min.
6. The filtrate was discarded, the DNA preparation tube was returned to the original 2ml centrifuge tube, 500. mu.L BufferW1 was added, and the mixture was centrifuged at 12000 Xg for 1 min.
7. The filtrate was discarded, and the DNA preparation tube was returned to the original 2ml centrifuge tube, and 700. mu.L buffer W2 was added and centrifuged at 12000 Xg for 1 min.
8. And 7, repeating the step.
9. The filtrate was discarded, and the DNA preparation tube was returned to the original 2ml centrifuge tube and centrifuged at 12000 Xg for 1 min.
10. The DNA preparation tube was placed in another clean 1.5ml centrifuge tube and DNA was eluted by adding 100. mu.L of Eluent to the center of the membrane of the preparation tube and centrifuging at 12000 Xg for 1 min.
Example 2. example 1 was repeated, and this example describes a method for enriching DNA using the northeast tiger feces as an example.
1. Cutting the excrement sample by adopting a sterile scalpel and scissors, and weighing 220mg of excrement into a sterilized 2ml centrifugal tube, wherein the cleanliness of tools (scissors, tweezers and the scalpel) is strictly ensured in the process so as to avoid the pollution of exogenous DNA and the cross contamination among samples. Add 300. mu.L of 10mmol/L PBS solution to the centrifuge tube, then add SDS to a final concentration of 1%, mix quickly, centrifuge instantaneously, and stand at room temperature for 3 min.
2. Then, the mixture was centrifuged at 12000 Xg for 10min, the precipitate in the centrifuge tube was discarded, and the supernatant was aspirated for use.
The subsequent DNA extraction method was as described in example 1.
Example 3. example 1 was repeated, differing from example 1 (sika deer) in that the standing time in step 1 in this example was 15 min.
Example 4. example 1 was repeated, differing from example 1 (sika deer) in that SDS was added to a final concentration of 0.01% in step 1 and the standing time was 1 min.
Example 5 example 1 was repeated, differing from example 1 (sika deer) in that SDS was added to a final concentration of 5% in step 1 and the standing time was 30 min.
Example 6 example 2 was repeated, differing from example 2 (northeast tiger) in that the standing time in step 1 in this example was 10 min.
Example 7. example 2 was repeated, differing from example 2 (northeast tiger) in that SDS was added to a final concentration of 0.01% in step 1 and the standing time was 1 min.
Example 8 example 2 was repeated, differing from example 2 (northeast tiger) in that SDS was added to a final concentration of 5% in step 1 and the standing time was 30 min.
First, the same stool samples were extracted simultaneously with the QIAamp kit, and the enrichment efficiency of the QIAamp kit and the method for the host DNA (including mitochondrial DNA and nuclear DNA) in the above examples was compared (see Table 1 and Table 2), wherein the enrichment efficiency of the host mitochondrial DNA was measured by the ratio of the mitochondrial DNA to the bacterial DNA copy number, and the enrichment efficiency of the host nuclear DNA was measured by the ratio of the nuclear DNA to the bacterial DNA copy number. The result shows that the excrement of both herbivorous animals and carnivorous animals has stronger host DNA enrichment capacity when the final concentration of SDS is 0.01-5% and the action time is 1-30 min. The most economical conditions for herbivore faeces are: the final concentration of SDS is 1%, and the action time is 5 min; the most economical conditions for carnivorous animal manure are: the final concentration of SDS is 1%, and the action time is 3 min.
TABLE 1 comparison of the efficiency of enrichment of host DNA (copy number of host DNA: copy number of bacterial DNA) in herbivore faeces (represented by Cervus Nippon Temminck) with the QIAamp kit
TABLE 2 comparison of the efficiency of enrichment of host DNA (host DNA copy number: bacterial DNA copy number) in meat animal feces (represented by northeast tiger) with the QIAamp kit
And secondly, examining the effectiveness and the universality of the method.
The following species, stool samples of different storage times, were enriched for host DNA while comparing the extraction results with the QIAamp kit, respectively, with reference to the optimal processing conditions described in example 1 and example 2 above, to verify the validity and versatility of the method of the invention.
TABLE 3 stool sample information
The obtained fecal samples were stored at-20 deg.C for 2019 years. By analyzing and comparing the absolute quantitative results of copy numbers of bacterial DNA, host mitochondrial DNA and host nuclear DNA of total DNA extracted by the two methods, as shown in figure 1, the enrichment efficiency of the host DNA obtained by the method of the invention is higher than that of a QIAamp kit for each fecal sample. The copy numbers of the host mitochondrial DNA and the nuclear DNA obtained by enrichment by the method are respectively 10 of the copy number of the bacteria by taking the copy number of the bacteria as a standard-3～100Multiple sum of 10-4～10-1Double, and QIAamp kit 10-5～10-1Multiple sum of 10-7～10-4That is, the present method is 10 of the QIAamp kit for the efficiency of enrichment of mitochondrial DNA1-103Doubling, efficiency of enrichment of nuclear DNA, the method is 10 of the QIAamp kit1-104And (4) doubling. Compared with fresh excrement, the old excrement stored for more than 5 years, whether meat animals or herbivorous animals, has 10 higher nuclear DNA enrichment efficiency than that of QIAamp kit2-104And (4) doubling.
The results show that the method has universality for extracting the fecal samples of various species in mammals, has wide requirements on the quality of the fecal samples and has more advantages on the enrichment effect of old feces.
Third, microsatellite STR detection
Selecting blood samples of 6 northeast tigers, 5 dogs, 8 blue foxes and 7 red deer and corresponding stool samples, and carrying out nuclear amplification capillary electrophoresis typing of 26 STR sites on the stool DNA extracted by the method and the blood DNA of the same individual at the same time. Partial results are shown in FIG. 2. The success rate of one-time amplification typing of the excrement DNA obtained by the method is over 85 percent, wherein the success rate of 22 sites reaches 100 percent. In the literature, the success rate of one-time typing of the STR by the fecal DNA extracted by other methods is mostly below 40%.
Fourth, SNP site detection
Referring to the preparation methods of the embodiment 1 and the embodiment 2, fecal DNA samples of 7 cattle and 5 dogs are selected, 10 SNP sites of each sample are detected, blood DNA typing results of the same individual are compared, the fecal DNA extracted by the method has the SNP site typing success rate of more than 90 percent as 100 percent, and the typing success rates of the rest sites are more than 90 percent. Because of the limited quality of host DNA, there are few reports of SNP typing in the literature, and the success rate of few reports is low.
Fifth, genome re-sequencing detection
And (3) selecting excrement samples of 1 northeast tiger and 1 red deer individual and blood samples corresponding to the same individual, wherein the excrement samples are extracted with DNA by the method, and the blood samples are extracted with DNA by a conventional method. The obtained DNA was used for genome re-sequencing, and the sequencing depth of the blood DNA was 10X and that of the feces sample DNA was 30X. Comparison shows that the genome re-sequencing data obtained from the fecal DNA of two species, such as Clean Base (bp), Effective Rate (%), Error Rate (%), Q20 (%), Q30 (%), GC Content (%) and NT alignment, are almost the same as those obtained from the DNA of blood samples.
By STR, SNP and genome re-sequencing, the method verifies that the typing effect of the host DNA obtained from the feces by the method in the conventional molecular marker is close to that of blood, and the quality of the re-sequencing of the host genome reaches the level of the blood DNA.
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Citations (29)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998048837A1 (en) * | 1997-04-30 | 1998-11-05 | Enzon, Inc. | Polyalkylene oxide-modified single chain polypeptides |
| US5910407A (en) * | 1992-04-01 | 1999-06-08 | The Johns Hopkins University School Of Medicine | Method for detection of target nucleic acid by analysis of stool |
| US6406857B1 (en) * | 1997-06-16 | 2002-06-18 | Exact Sciences Corporation | Methods for stool sample preparation |
| US6410340B1 (en) * | 2000-08-23 | 2002-06-25 | Children's Research Institute | Use of an 8.4 kDa protein as an immunophilin reagent in protein binding assays for immunosuppressive drugs |
| US20040180445A1 (en) * | 2003-03-12 | 2004-09-16 | Domanico Michael J. | Methods and compositions for purification of nucleic acid from a host cell |
| JP2004298184A (en) * | 2003-03-20 | 2004-10-28 | Osaka Univ | Novel thermostable protein with phosphoglycerate kinase activity |
| CN1904066A (en) * | 2006-07-15 | 2007-01-31 | 安徽大学 | Extraction method of mammal faeces DNA |
| US20080220418A1 (en) * | 2004-09-30 | 2008-09-11 | Matthias Ballhause | Method For Providing Dna Fragments Derived From An Archived Sample |
| CN101307311A (en) * | 2008-07-14 | 2008-11-19 | 四川大学 | Process for abstracting total DNA of swine waste sample |
| DE60229805D1 (en) * | 2001-03-27 | 2008-12-24 | Fermentas Ab | nucleic acid purification |
| JP2011223885A (en) * | 2010-04-15 | 2011-11-10 | Japan Tobacco Inc | New cytidine 5'-monophosphosialic acid synthetase, gene encoding the same and method for producing the synthetase |
| CN102586234A (en) * | 2012-03-12 | 2012-07-18 | 云南师范大学 | Method for extracting high-molecular-weight genome from animal feces |
| US20120252009A1 (en) * | 2011-04-02 | 2012-10-04 | New England Biolabs, Inc. | Methods and Compositions for Enriching Either Target Polynucleotides or Non-Target Polynucleotides from a Mixture of Target and Non-Target Polynucleotides |
| CN102851277A (en) * | 2012-09-07 | 2013-01-02 | 重庆市畜牧科学院 | Simple and rapid meat duck manure sample total DNA extraction method |
| CN103146683A (en) * | 2013-02-19 | 2013-06-12 | 北京林业大学 | Method for extracting DNA from excrements of mammals and birds |
| WO2013091102A1 (en) * | 2011-12-21 | 2013-06-27 | Geneohm Sciences Canada Inc. | Enrichment & isolation of microbial cells & microbial nucleic acids from a biological sample |
| CN103911370A (en) * | 2014-04-11 | 2014-07-09 | 云南师范大学 | Method for extracting DNA from nycticebus pygmaeus feces |
| US20160257987A1 (en) * | 2013-10-30 | 2016-09-08 | Merck Patent Gmbh | Method for isolating microorganisms from a complex sample |
| US20170166955A1 (en) * | 2014-03-07 | 2017-06-15 | Dna Genotek Inc. | Composition and Method for Stabilizing Nucleic Acids in Biological Samples |
| CN108220286A (en) * | 2018-03-28 | 2018-06-29 | 上海锐翌生物科技有限公司 | Excrement host DNA methylation detecting method |
| US20180195057A1 (en) * | 2015-09-10 | 2018-07-12 | Life Technologies Corporation | Purification of nucleic acid from environmental or biological samples |
| WO2019116034A1 (en) * | 2017-12-12 | 2019-06-20 | Cell Therapy Catapult Limited | Microbial enrichment method |
| WO2019195342A1 (en) * | 2018-04-02 | 2019-10-10 | Sun Genomics, Inc. | Universal method for extracting nucleic acid molecules from a diverse population of one or more types of microbes in a sample |
| CN111154751A (en) * | 2020-01-14 | 2020-05-15 | 东北林业大学 | Method for efficiently extracting DNA (deoxyribonucleic acid) in hair shaft |
| CN111621496A (en) * | 2020-05-06 | 2020-09-04 | 南京世和基因生物技术股份有限公司 | Sputum metagenome de-hosting extraction kit |
| CN111808846A (en) * | 2020-08-07 | 2020-10-23 | 深圳谱元科技有限公司 | Method for extracting total DNA of fecal sample |
| WO2020237238A1 (en) * | 2019-05-23 | 2020-11-26 | American Molecular Laboratories, Inc. | Methods for detection of rare dna sequences in fecal samples |
| WO2021159800A1 (en) * | 2020-02-12 | 2021-08-19 | 广州微远基因科技有限公司 | Method for reducing host nucleic acids in biological sample and applications |
| CN114703173A (en) * | 2022-03-18 | 2022-07-05 | 福建省农业科学院农业质量标准与检测技术研究所 | Lambda phage DNA extraction kit and extraction method |
-
2020
- 2020-01-14 CN CN202010037073.3A patent/CN113186185B/en active Active
Patent Citations (31)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5910407A (en) * | 1992-04-01 | 1999-06-08 | The Johns Hopkins University School Of Medicine | Method for detection of target nucleic acid by analysis of stool |
| WO1998048837A1 (en) * | 1997-04-30 | 1998-11-05 | Enzon, Inc. | Polyalkylene oxide-modified single chain polypeptides |
| US6406857B1 (en) * | 1997-06-16 | 2002-06-18 | Exact Sciences Corporation | Methods for stool sample preparation |
| US6410340B1 (en) * | 2000-08-23 | 2002-06-25 | Children's Research Institute | Use of an 8.4 kDa protein as an immunophilin reagent in protein binding assays for immunosuppressive drugs |
| DE60229805D1 (en) * | 2001-03-27 | 2008-12-24 | Fermentas Ab | nucleic acid purification |
| US20040180445A1 (en) * | 2003-03-12 | 2004-09-16 | Domanico Michael J. | Methods and compositions for purification of nucleic acid from a host cell |
| CN1768265A (en) * | 2003-03-12 | 2006-05-03 | 伊彭多夫公司 | Methods and compositions for purification of nucleic acid from a host cell |
| JP2004298184A (en) * | 2003-03-20 | 2004-10-28 | Osaka Univ | Novel thermostable protein with phosphoglycerate kinase activity |
| US20080220418A1 (en) * | 2004-09-30 | 2008-09-11 | Matthias Ballhause | Method For Providing Dna Fragments Derived From An Archived Sample |
| CN1904066A (en) * | 2006-07-15 | 2007-01-31 | 安徽大学 | Extraction method of mammal faeces DNA |
| CN101307311A (en) * | 2008-07-14 | 2008-11-19 | 四川大学 | Process for abstracting total DNA of swine waste sample |
| JP2011223885A (en) * | 2010-04-15 | 2011-11-10 | Japan Tobacco Inc | New cytidine 5'-monophosphosialic acid synthetase, gene encoding the same and method for producing the synthetase |
| US20120252009A1 (en) * | 2011-04-02 | 2012-10-04 | New England Biolabs, Inc. | Methods and Compositions for Enriching Either Target Polynucleotides or Non-Target Polynucleotides from a Mixture of Target and Non-Target Polynucleotides |
| WO2013091102A1 (en) * | 2011-12-21 | 2013-06-27 | Geneohm Sciences Canada Inc. | Enrichment & isolation of microbial cells & microbial nucleic acids from a biological sample |
| US20140335522A1 (en) * | 2011-12-21 | 2014-11-13 | Geneohm Sciences Canada Inc. | Enrichment & isolation of microbial cells & microbial nucleic acids from a biological sample |
| CN102586234A (en) * | 2012-03-12 | 2012-07-18 | 云南师范大学 | Method for extracting high-molecular-weight genome from animal feces |
| CN102851277A (en) * | 2012-09-07 | 2013-01-02 | 重庆市畜牧科学院 | Simple and rapid meat duck manure sample total DNA extraction method |
| CN103146683A (en) * | 2013-02-19 | 2013-06-12 | 北京林业大学 | Method for extracting DNA from excrements of mammals and birds |
| US20160257987A1 (en) * | 2013-10-30 | 2016-09-08 | Merck Patent Gmbh | Method for isolating microorganisms from a complex sample |
| US20170166955A1 (en) * | 2014-03-07 | 2017-06-15 | Dna Genotek Inc. | Composition and Method for Stabilizing Nucleic Acids in Biological Samples |
| CN103911370A (en) * | 2014-04-11 | 2014-07-09 | 云南师范大学 | Method for extracting DNA from nycticebus pygmaeus feces |
| US20180195057A1 (en) * | 2015-09-10 | 2018-07-12 | Life Technologies Corporation | Purification of nucleic acid from environmental or biological samples |
| WO2019116034A1 (en) * | 2017-12-12 | 2019-06-20 | Cell Therapy Catapult Limited | Microbial enrichment method |
| CN108220286A (en) * | 2018-03-28 | 2018-06-29 | 上海锐翌生物科技有限公司 | Excrement host DNA methylation detecting method |
| WO2019195342A1 (en) * | 2018-04-02 | 2019-10-10 | Sun Genomics, Inc. | Universal method for extracting nucleic acid molecules from a diverse population of one or more types of microbes in a sample |
| WO2020237238A1 (en) * | 2019-05-23 | 2020-11-26 | American Molecular Laboratories, Inc. | Methods for detection of rare dna sequences in fecal samples |
| CN111154751A (en) * | 2020-01-14 | 2020-05-15 | 东北林业大学 | Method for efficiently extracting DNA (deoxyribonucleic acid) in hair shaft |
| WO2021159800A1 (en) * | 2020-02-12 | 2021-08-19 | 广州微远基因科技有限公司 | Method for reducing host nucleic acids in biological sample and applications |
| CN111621496A (en) * | 2020-05-06 | 2020-09-04 | 南京世和基因生物技术股份有限公司 | Sputum metagenome de-hosting extraction kit |
| CN111808846A (en) * | 2020-08-07 | 2020-10-23 | 深圳谱元科技有限公司 | Method for extracting total DNA of fecal sample |
| CN114703173A (en) * | 2022-03-18 | 2022-07-05 | 福建省农业科学院农业质量标准与检测技术研究所 | Lambda phage DNA extraction kit and extraction method |
Non-Patent Citations (10)
| Title |
|---|
| ANNE SALONEN等: "Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: Effective recovery of bacterial and archaeal DNA using mechanical cell lysis", 《JOURNAL OF MICROBIOLOGICAL METHODS》 * |
| MASAYUKI HORIE等: "Endogenous non-retroviral RNA virus elements in mammalian genomes", NATURE * |
| 任时好: "不同Tris-Cl及SDS质量浓度对小鼠DNA提取的影响", 《河南农业大学学报》 * |
| 伊如格勒图等: "蒙古马肠道细菌总DNA提取方法的比较", 《中国畜牧兽医》 * |
| 刘艳华;张明海;: "野生动物粪便在濒危物种遗传结构研究中的应用", 野生动物 * |
| 张保卫等: "大熊猫和小熊猫粪便DNA提取的简易方法", 《动物学报》 * |
| 田新民;张明海;: "粪便微卫星DNA在种群大小评估中的应用及若干问题的探讨", 四川动物 * |
| 贾学渊等: "鞣制皮张DNA的提取", 《东北林业大学学报》 * |
| 陈思媛等: "用于分子生物学检测的粪便DNA的提取方法优化", 《农业生物技术学报》 * |
| 骞宇等: "大鼠粪便中细菌基因组DNA提取方法的比较", 《食品工业科技》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114990201A (en) * | 2022-06-08 | 2022-09-02 | 东北林业大学 | Feasibility assessment method based on application of total DNA of excrement to different genetic analyses |
| CN114990201B (en) * | 2022-06-08 | 2024-05-03 | 东北林业大学 | Feasibility assessment method based on fecal total DNA applied to different genetic analysis |
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