阅读说明：本技术 用于高通量测序的微生物饲料添加剂的核酸提取方法 (Nucleic acid extraction method of microbial feed additive for high-throughput sequencing ) 是由 饶正华 李明 孟庆石 冯潇慧 刘娜 焦京琳 谢秀兰 于 2021-02-18 设计创作，主要内容包括：本发明属于农业技术领域,具体涉及用于高通量测序的微生物饲料添加剂的核酸提取方法。本发明用于高通量测序的微生物饲料添加剂的核酸提取方法,包括以下步骤：取样品后进行清洗；裂解样品中的微生物；去除样品中的RNA；去除样品中的蛋白质；吸附DNA,进行清洗。本发明方法提取微生物饲料添加剂得到的核酸纯度高、完整度高,可用于后续的高通量测序分析。(The invention belongs to the technical field of agriculture, and particularly relates to a nucleic acid extraction method of a microbial feed additive for high-throughput sequencing. The invention relates to a nucleic acid extraction method of a microbial feed additive for high-throughput sequencing, which comprises the following steps: cleaning the sample; lysing the microorganisms in the sample; removing RNA from the sample; removing proteins from the sample; DNA was adsorbed and washed. The nucleic acid obtained by extracting the microbial feed additive by the method has high purity and high integrity, and can be used for subsequent high-throughput sequencing analysis.)

1. A method for nucleic acid extraction of a microbial feed additive for high-throughput sequencing, comprising the steps of:

(1) taking a sample, and washing the sample by using a TE buffer solution;

(2) lysing the microorganisms in the sample;

(3) adding ribonuclease into the sample obtained in the step (2) to remove RNA in the sample;

(4) adding protease into the sample obtained in the step (3) to remove proteins in the sample;

(5) adding a magnetic bead mixed solution into the sample obtained in the step (4) to adsorb bacterial DNA, wherein the magnetic bead mixed solution comprises magnetic beads and PEG, and the volume ratio of the magnetic beads to the PEG is 1:1 to 4.

(6) And (5) washing the DNA obtained in the step (5).

2. The method for extracting nucleic acid from a microbial feed additive for high-throughput sequencing according to claim 1, wherein in the step (1), the sample is washed with 10 × TE buffer.

3. The method for extracting nucleic acid from the microbial feed additive for high-throughput sequencing according to claim 1, wherein in the step (2), normal saline is added to the sample obtained in the step (1), shaking is carried out to suspend precipitation, 40-60 mg/mL lysozyme is added to be mixed uniformly, the mixture reacts at 37 ℃ for 0.5-4.5 h, centrifugation is carried out, clear liquid is discarded, and thalli are collected.

4. The method for extracting nucleic acid from a microbial feed additive for high-throughput sequencing according to claim 3, wherein in the step (2), zirconium beads and a lysis solution are added to the collected thalli to lyse microbes in the sample, wherein the volume ratio of the sample to the lysis solution is 1: 3-7, wherein the composition of the lysis solution comprises 1-5% SDS, 0.5-10 × TE buffer solution and 0.3-3M NaCl, and the pH value of the lysis solution is 7.0-9.0.

5. The method for extracting nucleic acid from a microbial feed additive for high-throughput sequencing according to claim 4, wherein the lysate is added in the step (2), grinding and shaking are performed, the mixture is cracked at 65-75 ℃ for 5-200 min, and centrifugation is performed to obtain a supernatant.

6. The method for extracting nucleic acid from a microbial feed additive for high-throughput sequencing according to claim 1, wherein in the step (3), ribonuclease is added to the sample obtained in the step (2) at a final concentration of 1-5 mg/mL, and the sample is incubated at 36.5-37.5 ℃ for 5 minutes to 12 hours.

7. The method for extracting nucleic acid from a microbial feed additive for high-throughput sequencing according to claim 1, wherein in the step (4), proteinase K is added to the sample obtained in the step (3) at a final concentration of 1-5 mg/mL, and the sample is incubated at 65-75 ℃ for 5 minutes to 2 hours.

8. The method for extracting nucleic acid from a microbial feed additive for high-throughput sequencing according to claim 1, wherein in the step (5), the final concentration of the magnetic beads is 0.5-5 mg/mL.

9. The method for extracting nucleic acid from a microbial feed additive for high-throughput sequencing according to claim 1, wherein in the step (6), the extracted nucleic acid is washed and eluted by using 70% ethanol and water in sequence.

10. The method for extracting nucleic acid from a microbial feed additive for high-throughput sequencing according to claim 1, wherein in the step (1), the sample is taken by using a PCR tube, and the volume of the sample is taken to be 200 μ L.

Technical Field

The invention belongs to the technical field of agriculture, and particularly relates to a nucleic acid extraction method of a microbial feed additive for high-throughput sequencing.

Background

Under the background of completely forbidding the use of antibiotics, the Chinese medicinal feed additive is completely withdrawn in 2020, and the microbial feed additive is the best substitute of the antibiotics. However, microbial feed additive products generally suffer from the following safety problems: (1) the proportion of functional bacteria in the product is not high, and the product contains mixed bacteria and even pathogenic bacteria; (2) species (even genus) identification errors; (3) the functional bacteria contain drug-resistant genes; (4) the functional bacteria contain virulence genes; (5) the functional bacteria are mutated and degenerated in the production process and lose the original efficacy.

The method for evaluating the safety of the microbial feed additive in the prior art is not mature, and the traditional detection means has the defects of large labor capacity, low efficiency, poor accuracy, low flux and the like. Therefore, an easy, efficient, accurate and high-throughput evaluation method is urgently established.

High-throughput sequencing is used as a new generation sequencing technology, and can effectively evaluate the safety problem of the microbial feed additive product, thereby providing guarantee for the specification and management of the microbial feed additive market. However, since the components of the microbial feed additive are complex, including a large amount of host DNA, proteins, polysaccharides, lipids, inorganic salts, etc., the first problem to be faced in establishing a high-throughput sequencing method is the extraction of microbial nucleic acids from the feed.

The existing nucleic acid extraction method is a general method established mainly aiming at samples such as soil, animals, plants, microorganisms and the like. In the face of the complex matrix of the microbial feed additive, nucleic acid with high purity and high integrity cannot be obtained, and the subsequent library building requirement of high-throughput sequencing cannot be met. Most of nucleic acid extraction methods of microbial feed additives used in the prior art are imported kits, the kit has poor extraction effect, samples cannot be subjected to sequencing analysis, evaluation of safety of the microbial feed additives is limited, and management and industry development of the market are greatly hindered.

Disclosure of Invention

The invention aims to provide a nucleic acid extraction method of a microbial feed additive for high-throughput sequencing.

The method for extracting nucleic acid of the microbial feed additive for high-throughput sequencing comprises the following steps:

(1) taking a sample, and washing the sample by using a TE buffer solution;

(2) lysing the microorganisms in the sample;

(3) adding ribonuclease into the sample obtained in the step (2) to remove RNA in the sample;

(4) adding protease into the sample obtained in the step (3) to remove proteins in the sample;

(5) adding a magnetic bead mixed solution into the sample obtained in the step (4) to adsorb bacterial DNA, wherein the magnetic bead mixed solution comprises magnetic beads and PEG, and the volume ratio of the magnetic beads to the PEG is 1:1 to 4.

(6) And (5) washing the DNA obtained in the step (5).

According to the method for extracting nucleic acid of the microbial feed additive for high-throughput sequencing, in the step (1), the sample is washed by 10XTE buffer.

Wherein the TE buffer solution is Tris + EDTA buffer solution, the 10 × TE buffer solution is 10 times concentration solution of the TE buffer solution, and the reagent composition is Tris-HCl (100mM), EDTA (10mM) and pH8.0.

According to the nucleic acid extraction method of the microbial feed additive for high-throughput sequencing, disclosed by the embodiment of the invention, in the step (2), normal saline is added into the sample obtained in the step (1), shaking and suspending are carried out, the precipitate is added, 40-60 mg/mL lysozyme is added and mixed uniformly, the reaction is carried out for 0.5-4.5 h at 37 ℃, and then the centrifugation is carried out, so that the supernatant is obtained.

Lysozyme (also called muramidase) or N-acetylmuramidase (N-acetylmuramidase glycohydrolase) is an alkaline enzyme that hydrolyzes mucopolysaccharides in bacteria.

According to the method for extracting the nucleic acid of the microbial feed additive for high-throughput sequencing, in the step (2), zirconium beads and lysis solution are added into the collected thalli to lyse the microbes in the sample, and the volume ratio of the sample to the lysis solution is 1: 3-7, wherein the composition of the lysis solution comprises 1-5% SDS, 0.5-10 × TE buffer solution and 0.3-3M NaCl, and the pH value of the lysis solution is 7.0-9.0.

The volume of the zirconium beads is measured to be equal to the volume of the sample, and the final concentration of the zirconium beads is 0.1g/mL-0.85 g/mL.

According to the nucleic acid extraction method of the microbial feed additive for high-throughput sequencing, disclosed by the embodiment of the invention, after the lysis solution is added in the step (2), grinding and shaking are carried out, the mixture is cracked for 5-200 min at the temperature of 65-75 ℃, and then the supernatant is obtained after centrifugation.

According to the nucleic acid extraction method of the microbial feed additive for high-throughput sequencing, in the step (3), 1-5 mg/mL ribonuclease is added into the sample obtained in the step (2), and the sample is incubated at 36.5-37.5 ℃ for 5 minutes-12 hours.

According to the nucleic acid extraction method of the microbial feed additive for high-throughput sequencing, in the step (4), 1-5 mg/mL proteinase K is added into the sample obtained in the step (3), and the sample is incubated at 65-75 ℃ for 5 minutes to 2 hours.

According to the nucleic acid extraction method of the microbial feed additive for high-throughput sequencing, provided by the embodiment of the invention, in the step (5), the final concentration of the magnetic beads is 0.5-5 mg/mL.

According to the nucleic acid extraction method of the microbial feed additive for high-throughput sequencing, in the step (6), 70% ethanol and water are sequentially used for washing and elution.

According to the method for extracting the nucleic acid of the microbial feed additive for high-throughput sequencing, which is provided by the embodiment of the invention, in the step (6), 70% ethanol is added into the adsorbed DNA, and after the DNA is washed, pure water is used, the suspended magnetic beads are vibrated, and after incubation at 70 ℃, centrifugation and magnetic adsorption are carried out, so that the nucleic acid is obtained.

According to the nucleic acid extraction method of the microbial feed additive for high-throughput sequencing of the embodiment of the invention, in the step (1), a PCR tube is used for measuring a sample.

The invention has the beneficial effects that:

according to the extraction method, free DNA and impurities in the microbial feed additive sample are cleaned by using the TE buffer solution, so that the yield of a final product is increased, and the pollution of host DNA is reduced; the extraction method of the invention has larger usage amount of ribonuclease and protease, which not only can save the traditional steps of nucleic acid precipitation, nucleic acid washing and the like and save the operation time, but also can obtain purer total DNA; the extraction method of the invention respectively uses the PCR tube to measure the sample and the magnetic beads, not only has simple operation, but also has fixed weighing amount.

The nucleic acid extracted by the method has high purity and integrity; can be used for subsequent high-throughput sequencing analysis.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows the comparison of the DNA extraction effect of the method of the present invention and the DNA extraction effect of a commercially available kit;

FIG. 2 shows the types and numbers of drug-resistant genes contained in different samples;

FIG. 3 shows the types and numbers of virulence genes contained in different samples.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.

Example 1 extraction of microbial nucleic acids in feed additives

1. Sampling 200 mu L of a PCR tube, and adding the sample into a 2mL tube;

2. adding 1mL of 10xTE into the sample obtained in the step (1), shaking the grinder for 1min, and cleaning the thalli for 2 times; adding physiological saline into the thalli, shaking to suspend precipitates, adding 50mg/mL lysozyme mother liquor, mixing uniformly, reacting for 1h at 37 ℃, centrifuging, removing clear liquid, and collecting the thalli;

3. taking 200 mu L of zirconium beads from the PCR tube, adding the zirconium beads into the sample, adding 1mL of lysate into the sample, and oscillating the sample for 3min by using a grinder;

4. cracking at 70 deg.C for 15min, and reversing 6 times every 5 min;

5. 13000g at 4 ℃ for 15 min;

6. taking the supernatant into a new 1.5mL tube, adding 40 mu L of RNase A, and incubating at 37 ℃ for 15 min;

7. adding 40 μ L proteinase K, incubating at 70 deg.C for 10min, and incubating at 70 deg.C for 10 min;

8. adding 300 μ L of magnetic bead mixture (magnetic beads and 40% PEG are mixed at a volume ratio of 1: 2), reversing, mixing, and standing for 3-5 min;

9. adsorbing with magnetic frame for 2-5min, and pouring off liquid;

10. adding 700 microliters of 1mL of 70% ethanol, reversing for several times, cleaning, carrying out magnetic adsorption for 1min, pouring out liquid, and cleaning twice;

11. drying in a fume hood for 4 min;

12. adding 100 μ L of pure water, shaking the suspended magnetic beads, and incubating at 70 deg.C for 5 min;

13. after microcentrifugation, magnetic adsorption was performed for 1-3min and the liquid was transferred to a new 1.5mL tube.

In the above-mentioned method, the first step of the method,

10 × TE (pH 8.0) preparation method: 200ML 500mM Tris-HCl (pH 8.0), 100ML0.1M EDTA (pH 8.0), to 1L.

The preparation method of the lysis solution comprises the following steps: adding 1.2g SDS, 4mL 10xTE, 4mL 5M NaCl into 32mL pure water to obtain the final product.

The final concentration of the added RNase is 1-5 mg/mL, optional concentrations comprise 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL and 5mg/mL, and after the RNase is added into a sample, the sample can be incubated for 5, 10 or 15min at the temperature of 36.5, 37 or 37.5 ℃, and the incubation lasts for 12 hours at most.

The final concentration of the added proteinase K is 1-5 mg/mL, optional concentrations comprise 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL or 5mg/mL, and after the proteinase K is added into a sample, the sample can be incubated for 5, 10 or 15min at 65, 70 or 75 ℃, and the incubation time is 2 hours at most.

The volume ratio of the magnetic beads to the PEG is 1: 1-4, the volume ratio of the magnetic beads to the PEG can be 1:1, 1:2, 1:3 or 1:4, and the concentration of the PEG can be 30%, 35%, 40%, 45% or 50%.

The final concentration of the magnetic beads is 0.5-5 mg/mL, preferably 0.5mg/mL, 1mg/mL or 1.5mg/mL, and at most 5 mg/mL.

The concentration of the lysozyme is 40-60 mg/mL, and the final concentration of the lysozyme is preferably 40mg/mL, 40.5mg/mL, 41mg/mL, 41.5mg/mL, 42mg/mL, 45mg/mL, 50mg/mL, 55mg/mL or 60 mg/mL.

Example 2

2.1 comparison of the Effect of the method of the present invention with that of the existing kit

DNA in a sample of a commercially available microbial feed additive for lactic acid bacteria was extracted by the method of example 1 of the present invention, and the extraction results were compared with two foreign nucleic acid extraction kits, DNeasy PowerLyzer PowerSoil Kit (QIAGEN) and FASTDNA TM Spin Kit for soil (MPbio).

The results are shown in FIG. 1, and compared with Qiagen and MP nucleic acid extraction kits, the DNA extracted by the method of the present invention has obvious bands in the region of more than 2000bp, which indicates that the nucleic acid extracted by the method of the present invention is complete and pure, and is suitable for the construction of DNA library in high-throughput detection.

2.2 extraction of DNA for high throughput detection by the method of the invention

In order to verify the reliability of the method, the invention selects the subpackaged samples of 19 microbial feed additive products in the market, and performs bacterial component analysis, drug resistance gene analysis and virulence gene analysis on each sample respectively.

Use kitDNA Library Prep Kit V2The extracted total DNA is subjected to a process such asThe following steps.

1. Unfreezing 5 XTTBL at room temperature, and reversing the upper part and the lower part and uniformly mixing for later use. Confirm whether 5 × TS is at room temperature, and flick the tube wall to confirm whether there is precipitation. If precipitate exists, heating at 37 ℃ and vortex, shaking and mixing uniformly, and dissolving the precipitate.

2. The following reaction system was prepared in a sterile PCR tube:

3. gently flick 20 times by using a pipette and mix well.

4. The reaction tube was placed in a PCR instrument and the following reaction program was run:

5. the fragmentation product was purified using VAHTS DNA Clean Beads

Vortex, shake and mix VAHTS DNA Clean Beads evenly and suck 50 to make fragment products, vortex, shake or blow and beat 10 times by using a pipette and mix evenly, and incubate 5min at room temperature.

② the reaction tube is centrifuged for a short time and placed on a magnetic rack to separate the magnetic beads from the liquid, and the supernatant is carefully removed after the solution is clarified (about 5 min).

③ keeping the reaction tube on the magnetic frame all the time, adding 200. mu.L of freshly prepared 80% ethanol to rinse the magnetic beads, incubating at room temperature for 30sec, and carefully removing the supernatant.

And fourthly, repeating the step III and rinsing twice in total.

Keeping the reaction tube on the magnetic frame all the time, opening the cover and drying in air for about 5 min.

Sixthly, taking the reaction tube out of the magnetic frame, and adding 26 mu L of sterilized ultrapure water for elution. Vortexing or blowing and beating for 10 times by using a pipette, and fully mixing, and incubating for 5min at room temperature.

Seventhly, centrifuging the reaction tube for a short time, placing the reaction tube on a magnetic frame, separating magnetic beads from liquid, and carefully sucking 24 mu L of supernatant into a new sterilized PCR tube after the solution is clarified (about 5 min).

6. The PCR tube was placed on ice to prepare the following reaction system:

kit goods number	TD501	TD502/TD503
			ddH2O	------	4μL
Step 09-1 product	24μL	25μL
			5×TAB	10μL	10μL
PPM	5μL	------
			N5XX*	5μL	5μL
N7XX*	5μL	5μL
			TAE	1μL	1μL

7. And (3) lightly blowing and beating by using a pipettor to be fully and uniformly mixed, placing the reaction tube in a PCR instrument, and operating the following reaction program:

8. vortex and mix VAHTS DNA Clean Beads evenly and suck 30.0 uL volume to 50 uL PCR product, vortex and shake or blow with a pipette 10 times and mix well, and incubate for 5min at room temperature.

9. The reaction tube was centrifuged briefly and placed on a magnetic stand to separate the magnetic beads from the liquid, after the solution was clarified (about 5min), the supernatant was carefully transferred to a fresh sterile PCR tube and the magnetic beads discarded.

10. Vortex and mix VAHTS DNA Clean Beads evenly and suck 7.5 uL volume to supernatant, vortex and shake or use the pipettor to blow 10 times and mix well, incubate 5min at room temperature.

11. The reaction tube was briefly centrifuged and placed on a magnetic stand to separate the magnetic beads from the liquid, and the supernatant carefully removed after the solution cleared (about 5 min).

12. The reaction tube was kept on the magnetic frame all the time, and 200. mu.L of freshly prepared 80% ethanol was added to rinse the magnetic beads. Incubate at room temperature for 30sec and carefully remove the supernatant.

13. Repeat step 5 for a total of two rinses.

14. Keeping the reaction tube on the magnetic frame all the time, and opening the cover to dry the magnetic beads in air for about 5 min.

15. The reaction tube was removed from the magnetic holder, and 22. mu.L of sterilized ultrapure water was added for elution. Vortexing or blowing and beating for 10 times by using a pipette, and fully mixing, and incubating for 5min at room temperature.

16. The reaction tube was centrifuged briefly and placed on a magnetic stand to separate the magnetic beads from the liquid, and after the solution cleared (about 5min) 20. mu.L of the supernatant was carefully pipetted into a fresh sterile PCR tube and stored at-20 ℃.

The results of the analysis of the bacterial composition of the split samples of 19 microbial feed additive products by high throughput analysis are shown in table 1:

table 119 analysis results of bacterial composition of microbial feed additives

As shown in Table 1, the DNA obtained by the nucleic acid extraction method of the present invention is complete and pure, and can be used for high throughput detection, such that the content of functional bacteria can be obtained, and the content of miscellaneous bacteria in the additive is low can be detected.

2.3 analysis of drug resistance genes in microbial feed additive products

On the basis of analyzing the bacterial components of 19 samples in example 2.2, the detection of drug-resistant genes in the samples is further carried out, and the specific steps are as follows:

(1) and (3) filtering data: and filtering the data by using fastp or Soapnuke, and deleting the sequences of which the N base content is more than 10% or the Q value is less than 5 and more than 50%.

(2) And (3) sequence splicing: utilizing megahit to carry out sequence splicing on the filtered data;

(3) analysis of drug resistance genes: virulence genes in the assembled data were analyzed using the abricate program and the CARD or Resfinder database.

Virulence analysis results are shown in table 2:

TABLE 2 types and numbers of resistance genes contained in different samples

As shown in FIG. 2, all samples in example 2.2 contained 16 general classes of drug-resistant genes, respectively Aminococcarins, Aminoglicosides, Bacitracin, betalactams, Cationic antimicrobial peptides, Elfamycins, Fluoroquinolones, Fosfomycin, Lipopeptides, MLS, Multi-drug resistance, Phenicol, Rifampin, sulfoamides, Tetracyclines, Tunicamycins.

Samples 1 and 9 contained a large number of drug resistance genes, 46 and 57, respectively, indicating poor safety. While sample 10 and sample 16 did not detect any drug resistance gene, indicating better safety. After the method is used for extracting the DNA in the microbial feed additive, the DNA is analyzed by using a high-throughput sequencing technology, the risk of drug-resistant genes in different samples can be effectively evaluated, the discrimination is good, and the defects of large workload, low throughput and efficiency and the like of the traditional method are overcome.

2.4 analysis of virulence genes in microbial feed additive products

On the basis of analyzing the bacterial components of 19 samples in example 2.2, virulence genes in the samples are further detected, and the method comprises the following specific steps:

(1) and (3) filtering data: filtering the data by using fastp or Soapnuke, and deleting sequences with N base content more than 10% or bases with Q value less than 5 more than 50%;

(2) and (3) sequence splicing: utilizing megahit to carry out sequence splicing on the filtered data;

(3) analysis of virulence genes: virulence genes in the assembled data were analyzed using the abricate program and the VFDB database.

As shown in FIG. 3, different samples containing virulence genes with a large difference in total number, such as sample 18 and sample 19, all contained virulence genes above 150, indicating that the corresponding products have poor safety, while samples 10 and 11 did not detect any virulence genes, indicating that the safety is better.

Therefore, after the DNA in the microbial feed additive is extracted by the method, the DNA is analyzed by a high-throughput sequencing technology, the risk of virulence genes in different samples can be effectively evaluated, the discrimination is good, and the defects of large workload, low flux and efficiency and the like of the traditional method are overcome.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.