CN113186185B - Method for efficiently enriching host DNA from mammal feces - Google Patents
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Abstract
A method for efficiently enriching host DNA from mammal feces belongs to the technical field of molecular biology. Aiming at the problems that when DNA is extracted from the existing mammal fecal sample, the host DNA enrichment efficiency is low, the old fecal extraction effect is poor, the residual of a large amount of bacterial DNA seriously interferes with the subsequent reaction, the cost is high, and the like, the invention provides a method for efficiently enriching the host DNA from the fecal sample, which comprises the following steps of carrying out SDS pretreatment on the fecal sample before extraction: mixing the fecal sample with 10mmol/L phosphate buffer PBS solution, adding SDS solution to a final concentration of 0.01-5% (mass), uniformly mixing, and standing for 1-30 min at room temperature; the supernatant was then centrifuged, and DNA was extracted from the supernatant. The invention has low cost, is not limited by the number of samples, and expands the application of the 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 feces.
Background
In recent years, the application of non-invasive sampling in protecting biology and molecular ecology has received a great deal of attention and importance, while faeces are due to: (1) The intestinal epithelial cells of the study object can be obtained in a non-invasive way, and the intestinal epithelial cells have the advantages of no wound, no pain and no invasiveness; (2) Animal urination is conventional and rhythmic, so that the fecal sample is in sufficient quantity to provide a sufficient sample source compared to other sample types; (3) The fecal sample can be collected without touching or even without seeing animal bodies, especially for large-scale carnivores, the safety of sampling personnel can be ensured to the greatest extent, and the interference to animals in the collection process is minimized; and (4) the excrement collection is easy to operate and has low cost. Feces are therefore the most commonly used sample in the management of artificial fed animals in field animal studies and zoos. The host DNA extracted from host intestinal epithelial cells in feces plays an important role in species identification, mating system analysis, genetic diversity evaluation, population genetic structure analysis, systematic geographic research and the like.
Studies have shown that 10 per gram of fresh feces 5 The intestinal epithelial cells, most of which remain intact, continue to maintain normal vital activity, whereas more than 55% of the dry weight of the stool is composed of bacteria derived from the intestinal flora and the surrounding environment. Therefore, the total DNA extracted from the feces has very little host DNA content and often contains substances that inhibit molecular biological reactions, resulting in very low analysis success rate of subsequent experiments, and especially difficult analysis of nuclear genomes, such as the confirmation of microsatellite and SNP typing results, which has to be repeated many times, not only greatly increases the experiment cost, but also seriously affects the reliability of data.
Therefore, how to improve the extraction efficiency of host DNA in feces and reduce the proportion of bacterial DNA is a key in the current technology of feces DNA extraction. The existing methods for enriching host DNA in feces mainly comprise a DNA methylation co-immunoprecipitation technology, a density gradient centrifugation method, an immunomagnetic bead separation method, a dielectrophoresis separation method (DEP), a commercial feces DNA extraction kit and the like, and the methods have the following characteristics and defects:
(1) The enrichment efficiency of host DNA is still less than ideal, especially for enrichment of the nuclear genome.
In terms of DNA methylation co-immunoprecipitation techniques, this approach can enrich methylated fragments of the host nuclear genome with high purity, but since the probability of methylation of the host mitochondrial genome is small, enrichment of mitochondrial genetic material cannot be achieved by this approach; in terms of an immunomagnetic bead method and a dielectrophoresis chip method, the method can achieve high purity on enrichment of host DNA, but the quantity of bacterial DNA is too large, the interference is serious, the capturing probability on host cells is low, and finally the enrichment quantity of the obtained host DNA is small; the most widely used QIAamp kit is provided by Qiagen company, the enrichment effect of the kit on the host mitochondrial genome can meet most of experimental needs, but the enrichment effect on the host nuclear DNA is still not ideal, the same sample is repeated at least 5-7 times when microsatellite locus detection is carried out, the final typing result is obtained in a 'minority-compliance majority' mode, and the accuracy is low.
(2) Host DNA extraction efficiency is worse for stale feces.
The enrichment technology of host DNA in various feces applied at present is basically more suitable for fresh feces, and especially for SCSR-TTM kit, methylation coprecipitation method, magnetic bead separation method, dielectrophoresis chip method, density gradient centrifugation method and the like, the requirement for the freshness of the feces sample is more strict. At present, although the QIAamp kit has relatively wide requirements on the freshness of samples, the enrichment effect on host DNA in old feces is not ideal.
(3) High cost and high consumption.
The existing enrichment technology of host DNA in fecal samples is relatively expensive, and for methylation co-immunoprecipitation, magnetic bead sorting, dielectrophoresis chip method and the like, one sample needs 200-500 Yuan Renzhen, so most of the techniques are only used when necessary. For QIAamp fecal DNA extraction kits, each sample still requires about 50 yuan, and analysis of a large number of samples is cost prohibitive.
(4) The operation requirements are strict.
Methylation coprecipitation method, magnetic bead separation method, dielectrophoresis chip method, density gradient centrifugation method and the like, and the operation process is complex, strict in condition requirement and long in time consumption.
Disclosure of Invention
Aiming at the problems of strict requirements on stool quality, low host DNA enrichment efficiency, high cost and the like when host DNA is extracted from a stool sample at present, the invention provides a method for efficiently enriching host DNA from mammal stool, and the stool sample is pretreated by SDS before the stool DNA is extracted, and the pretreatment method comprises the following steps:
1) Mixing the fecal sample with 10mmol/L phosphate buffer PBS solution, adding SDS solution to a final concentration of 0.01-5% (mass), uniformly mixing, and standing for 1-30 min at room temperature;
2) The supernatant was then centrifuged and used for DNA extraction.
Further defined is that when the fecal sample is for a herbivore in step 1), the pre-treatment is followed by a resting time of 1-15 min at room temperature.
Further defined is that when the faecal sample of meat animals is subjected to step 1), the pre-treatment is followed by a standing time of 1-10 min at room temperature.
Further defined, the ratio of the fecal sample to phosphate buffer PBS solution in step 1) is 180-220 mg: 300. Mu.L.
Further defined, the final concentration of the SDS added solution in step 1) is 1% by mass.
Further defined, the method of extracting DNA in step 2) includes a genomic DNA kit extraction method and an organic solvent extraction method.
Further defined, the herbivore fecal sample of step 1) is left for a period of 5 minutes at room temperature after the pretreatment.
Further defined, the meat animal fecal sample of step 1) is subjected to the pretreatment for a 3 minute period of time at room temperature.
Advantageous effects
(1) The invention has good enrichment capability for the mitochondrial DNA or the nuclear DNA of a host in feces. Compared with the QIAamp kit widely applied at present, the enrichment efficiency of the host mitochondrial DNA is improved by 10 percent 1 ～10 3 The enrichment efficiency of host nuclear DNA can be improved by 10 times 1 ～10 4 Multiple times.
(2) The invention has relatively wide quality requirement on the fecal sample, is not only suitable for fresh fecal, but also has good enrichment capability on the conventional frozen and preserved old fecal, and compared with QIAamp kit, the enrichment efficiency of host mitochondrial DNA can be improved by 1-10 3 The enrichment efficiency of host nuclear DNA is improved by 10 times 1 ～10 4 Multiple times.
(3) The invention has wide application range, and has good enrichment effect on host DNA in animal feces of herbivorous or carnivorous animals.
(4) All the operations related by the invention belong to the routine laboratory operations, the experimental steps are simple and feasible, and the whole process can be completed only by 30-40 min.
(5) Because SDS is low in price, the treatment cost of each fecal sample by the method is only a few degrees, so that the cost consumption of experiments is greatly reduced; in actual operation, common equipment such as a water bath kettle, a pipettor, a centrifuge and the like is only needed, and the common laboratory can meet the requirements, so that the universality is extremely strong. Due to low cost and low requirement, the method can be suitable for large-scale and multi-batch fecal sample extraction, and greatly expands the application of fecal samples in animal research.
Drawings
Fig. 1: the enrichment effect (expressed as host DNA copy number/bacterial DNA copy number) of different stool samples (containing different species and different harvest year) on host DNA (containing nuclear DNA and mitochondrial DNA) under QIAamp kit and optimal SDS lysis conditions was compared; the abscissa represents different stool samples and the ordinate represents the enrichment efficiency of host DNA; wherein:
Fig. 2: comparing the host DNA obtained by the method with the result of amplifying and typing STR sites by 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 of northeast tiger number 2, and B is a parting map of the same locus of the individual in a fecal sample; a microsatellite locus 32 typing map of a blood sample of an individual with a red deer number 6, and 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, F, a parting map of the same locus of the individual in a fecal sample; and G, a microsatellite locus CPH9 parting map of a blood sample of the blue fox No. 1 individual, and H, a parting map of the same locus of the individual in a fecal sample.
Detailed Description
According to the method, sodium Dodecyl Sulfate (SDS) with low price is selected as a cracking agent according to the difference of the structure and the anti-damage capability of intestinal epithelial cells and bacterial outer walls in the feces, and the optimal cracking condition is obtained by controlling the concentration of SDS and the acting time of the SDS on the feces. Under the condition, the host cells can be cracked to the greatest extent, so that the host DNA is released into the solution, meanwhile, the structural integrity of 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 conventional DNA extraction technology (an organic solvent extraction method, various commercial common genome DNA extraction kits and the like) is used for enriching the supernatant to obtain host DNA with considerable purity and quantity, so that the residual quantity of bacterial DNA is reduced.
The key innovation point of the invention is that SDS pretreatment is carried out on the fecal sample before extraction, and the concentration and the action time of SDS are controlled, so that optimal SDS cracking conditions are respectively obtained for the feces of meat and grass animals, under the conditions, the full cracking of host intestinal epithelial cells can be realized, and meanwhile, the integrity of bacterial cells can be maintained to the greatest extent.
The method of the present invention for efficiently enriching host DNA from mammalian feces is described in detail below.
Example 1. Method for efficient enrichment of host DNA from Spotted deer feces.
1. The aseptic scalpel and the scissors are adopted to cut the fecal sample, 180mg of fecal is weighed and placed in a sterilized 2ml centrifuge tube, and the cleanness of tools (scissors, forceps and the scalpel) in the process is strictly ensured so as to avoid foreign DNA pollution and cross contamination among samples. To the centrifuge tube, 300. Mu.L of 10mmol/L PBS solution was added, then SDS was added to a final concentration of 1%, the sample was rapidly and uniformly mixed with the solution, and after instantaneous centrifugation, the mixture was allowed to stand at room temperature for 5 minutes.
2. Then, the mixture was centrifuged at 12000 Xg for 10min, and the supernatant was collected for use and the pellet was discarded.
The supernatant obtained by the above-described treatment may be subjected to DNA extraction by any conventional DNA extraction technique, such as a conventional genomic DNA commercial kit method, a chemical extraction method, and the like. This example describes the final DNA extraction method using the AxyPrep genomic DNA miniprep kit (Axygen) as an example, followed by the following:
3. 150ul Buffer C-L and 20ul PK enzyme are added into the supernatant obtained after centrifugation, and after uniform mixing, the supernatant is subjected to instantaneous centrifugation and is placed in a 56 ℃ water bath for incubation for 10min.
4. 350ul Buffer PD was added to each tube, and the mixture was centrifuged at 12000 Xg for 10min with shaking for 10 s.
5. A DNA preparation tube was placed in a 2ml PE tube, and the supernatant after centrifugation was aspirated into the preparation tube, and centrifuged at 12000 Xg for 1min.
6. The filtrate was discarded, the DNA preparation tube was returned to the original 2ml centrifuge tube, 500. Mu.LBuferW 1 was added, and the mixture was centrifuged at 12000 Xg for 1min.
7. The filtrate was discarded, the DNA preparation tube was returned to the original 2ml centrifuge tube, and 700. Mu.LBuffer W2 was added thereto, and the mixture was centrifuged at 12000 Xg for 1min.
8. The 7 th step is repeated.
9. The filtrate was discarded, and the DNA preparation tube was returned to the original 2ml centrifuge tube, and centrifuged at 12000 Xg for 1min.
10. The DNA preparation tube was placed in another clean 1.5ml centrifuge tube, 100. Mu.L of Eluent was added to the center of the preparation tube membrane, and the DNA was eluted by centrifugation at 12000 Xg for 1min.
Example 2. Example 1 was repeated, this example describes a method for enriching DNA using northeast tiger feces.
1. The aseptic scalpel and scissors are adopted to cut the fecal sample, 220mg of fecal is weighed into a sterilized 2ml centrifuge tube, and the cleanliness of tools (scissors, forceps and scalpel) is strictly ensured in the process, so that the pollution of exogenous DNA and the cross pollution among samples are avoided. To the centrifuge tube, 300. Mu.L of 10mmol/L PBS solution was added, followed by SDS to a final concentration of 1%, rapidly mixed well, centrifuged instantaneously, and allowed to stand at room temperature for 3min.
2. Then, the mixture was centrifuged at 12000 Xg for 10min, the pellet in the centrifuge tube was discarded, and the supernatant was collected for use.
The subsequent DNA extraction procedure was as described in example 1.
Example 3. Example 1 was repeated, differing from example 1 (sika deer) in that the rest time in step 1 in this example was 15min.
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 in this example, and the standing time was 1min.
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 in this example, and the standing time was 30min.
Example 6. Example 2 was repeated, differing from example 2 (northeast tiger) in that the standing time in step 1 in this example was 10min.
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 in this example, and the standing time was 1min.
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 in this example, and the standing time was 30min.
1. The same stool samples were simultaneously extracted using the QIAamp kit and the enrichment efficiency of the QIAamp kit and the method for host DNA (comprising mitochondrial DNA and nuclear DNA) in the above examples (see table 1, table 2), wherein the enrichment efficiency of host mitochondrial DNA was measured as the ratio of mitochondrial DNA to bacterial DNA copy number and the enrichment efficiency of host nuclear DNA was measured as the ratio of nuclear DNA to bacterial DNA copy number. The result shows that the SDS has stronger host DNA enrichment capability when the final concentration of SDS is between 0.01 and 5 percent and the action time is between 1 and 30 minutes no matter the excrement of herbivorous animals or meat animals. The most economical conditions for herbivorous animal manure are: SDS final concentration is 1%, and the action time is 5min; the most economical conditions for the faeces of meat animals are: SDS final concentration was 1% and the duration of action was 3min.
TABLE 1 comparison of the efficiency of enrichment of host DNA (host DNA copy number: bacterial DNA copy number) with grass feces (typified by sika) by QIAamp kit
TABLE 2 comparison of the efficiency of enrichment of host DNA (host DNA copy number: bacterial DNA copy number) with the QIAamp kit for animal feces (represented by northeast tiger)
2. The validity and versatility of the method of the invention are examined.
The following species, stool samples of different preservation times were enriched for host DNA, respectively, with reference to the optimal treatment conditions described in example 1 and example 2 above, and compared with the extraction results of QIAamp kit, thereby verifying the effectiveness and versatility of the method of the present invention.
TABLE 3 information on stool samples
The stool samples were collected and stored at-20℃until 2019. By analyzing and comparing the absolute quantitative results of bacterial DNA, host mitochondrial DNA and host nuclear DNA copy numbers of total DNA extracted by the two methods, as shown in FIG. 1, the enrichment efficiency of the host DNA obtained by the method of the invention is higher than that of the QIAamp kit for each fecal sample. The bacterial DNA copy number is taken as a standard, and the copy numbers of host mitochondrial DNA and nuclear DNA obtained by enrichment in the method are respectively 10 of the bacterial copy number -3 ～10 0 Double sum 10 -4 ～10 -1 Multiple times, and QIAamp kit of 10 -5 ～10 -1 Double sum 10 -7 ～10 -4 The method is 10 times that of QIAamp kit, that is, for the enrichment efficiency of mitochondrial DNA 1 -10 3 The method is 10 times of the QIAamp kit, and the enrichment efficiency of the nuclear DNA is improved 1 -10 4 Multiple times. For old feces stored for more than 5 years, whether meat or grass, the method has 10 higher enrichment efficiency on nuclear DNA than QIAamp kit compared with fresh feces 2 -10 4 Multiple times.
The results show that the method has wide requirements on the quality of the fecal samples and has more advantages on the enrichment effect of the old feces for the universality of the fecal sample extraction of each species in mammals.
3. Microsatellite STR detection
The blood samples of 6 northeast tigers, 5 dogs, 8 blue foxes and 7 red deer and the corresponding fecal samples are selected, and the fecal DNA extracted by the method and the blood DNA of the same individual are subjected to nuclear amplification capillary electrophoresis typing of 26 STR sites. The partial results are shown in FIG. 2. The success rate of the one-time amplification and typing of the microsatellite of the fecal DNA obtained by the method is more than 85%, wherein the success rate of 22 sites reaches 100%. In the literature, the success rate of the single typing of the fecal DNA extracted by other methods on STR is mostly below 40%.
4. SNP locus detection
Referring to the preparation methods of example 1 and example 2, fecal DNA samples of 7 cattle and 5 dogs are selected, 10 SNP loci are detected for each sample, blood DNA typing results of the same individual are compared, the typing success rate of more than 90% of the fecal DNA extracted by the method is 100%, and the typing success rates of the rest loci are also more than 90%. Because of the limited host DNA quality, few SNP typing reports exist in the literature, and the success rate of few reports is low.
5. Genomic resequencing detection
The method is characterized in that 1 northeast tiger and 1 red deer individual fecal sample and a blood sample corresponding to the same individual are selected, the fecal sample is used for extracting DNA by adopting the method, and the blood sample is used for extracting DNA by adopting a conventional method. Genomic resequencing was performed with the obtained DNA, the sequencing depth of blood DNA was 10×, and the sequencing depth of stool sample DNA was 30×. Comparison shows that genomic resequencing data obtained from fecal DNA from two species, such as clear Base (bp), effective Rate (%), error Rate (%), Q20 (%), Q30 (%), GC Content (%) and NT comparison results are substantially unchanged from those of blood sample DNA.
Through STR, SNP and genome resequencing, it is verified that the typing effect of host DNA obtained from feces in the method of the invention in conventional molecular markers is close to that of blood, and the resequencing quality of host genome reaches the level of blood DNA.
Claims (7)
1. A method for enriching host DNA from mammalian feces, wherein prior to extraction of fecal DNA, a fecal sample is pretreated with PBS buffer and SDS, the pretreatment method comprising:
1) Mixing the fecal sample with 10mmol/L phosphate buffer PBS solution, adding SDS solution to a final concentration of 0.01-5% by mass, uniformly mixing, and standing for 1-30 min at room temperature; the ratio of the fecal sample to the phosphate buffer PBS solution is 180-220 mg/300 mu L;
2) Centrifuging to obtain supernatant; the centrifugation condition is 12000 Xg for 10min;
the supernatant was used for DNA extraction.
2. The method according to claim 1, wherein the standing time at room temperature for the herbivore fecal sample in step 1) is 1-15 min.
3. The method according to claim 1, wherein the time of standing at room temperature for the faecal sample of the meat animal in step 1) is 1 to 10min.
4. The method according to claim 1, wherein the final concentration of the SDS added solution in step 1) is 1% by mass.
5. The method according to claim 1, wherein the method for extracting DNA in step 2) comprises a genomic DNA kit extraction method and an organic solvent extraction method.
6. The method of claim 2, wherein the herbivorous animal manure sample in step 1) is left for a period of 5 minutes at room temperature.
7. A method according to claim 3, wherein the faecal sample of the meat animal in step 1) is left for a period of 3 minutes at room temperature.
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