CN107418951B - Low-pollution and high-sensitivity low-cost general strain molecular identification method - Google Patents
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Abstract
A low-pollution and high-sensitivity low-cost general strain molecular identification method belongs to the technical field of biology. Which comprises the following steps: 1) pretreating strains; 2) preparing a column with high adsorption force; 3) bacteria lysis and nucleic acid adsorption; 4) cleaning; 5) eluting; 6) performing first round PCR amplification; 7) performing second round PCR amplification; 8) electrophoresis detection and purification; 9) and (4) sequencing and aligning by using a primer M13F to determine the genus and the species. The invention utilizes simple and efficient wall breaking and extracting technology, combines efficient nucleic acid adsorption technology and PCR twice amplification effect, can identify bacteria more accurately at the initial stage of microbial colony formation, and the primers for identification of the invention give consideration to the unity of fungi and bacterial sites, not only can identify bacteria at the seed level, but also can identify fungi to the genus or even the species level, thus facilitating the simultaneous identification of some bacteria and fungi, greatly improving the efficiency and saving the experimental cost.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a low-pollution and high-sensitivity low-cost general strain molecular identification method.
Background
The identification of strains by molecular biology means is an important means for the evolution and classification identification of the current microbial system, for example, when the separated strains are detected by utilizing a 16S rDNA/16S-23S rDNA interval sequence of bacteria and an 18S rDNA/ITS 1-5.8S-ITS2 sequence of fungi, the extraction of DNA is mostly carried out firstly, then the DNA sample is utilized for PCR amplification, and finally the classification status of the bacteria is determined by sequencing and sequence comparison of PCR products. When the existing detection means is used for extracting the DNA of a sample, the separation and culture of bacteria are usually required, the time is long, the efficiency is low, and in addition, some bacteria can only be cultured under specific conditions, the conventional culture is not easy, so that the difficulty of sample collection is caused. If a small amount of sample is extracted, the sensitivity of the identification method is often insufficient and the requirement is difficult to meet. If a PCR amplification mechanism is simply utilized to improve the detection sensitivity, PCR after directly cracking a sample is theoretically feasible, but is not easy to succeed in the actual operation process, particularly most gram-positive bacteria, actinomycetes and fungi have thick cell walls, so that target DNA is difficult to collect, and trace pollution which is easy to amplify is easy to mix in the reaction, so that the final amplification result is a large amount of false positives. In addition, the molecular identification areas of the bacteria and the fungi are different, so that the bacteria and the fungi need to be identified separately, experimental troubles are brought, particularly, some unicellular fungi are sometimes confused with the bacteria to cause repeated verification, the efficiency of the identification process is greatly reduced, and the detection cost is improved.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to design and provide a technical scheme of a low-pollution and high-sensitivity low-cost general strain molecular identification method.
The molecular identification method of the low-pollution, high-sensitivity and low-cost general strain is characterized by comprising the following steps of:
1) pretreatment of strains
Picking bacteria to be detected from a flat plate, adding the bacteria to be detected into the suspension solution A, wherein the diameter of the bacteria to be detected is more than 2mm, and then mechanically crushing and breaking the wall;
2) preparing a column with high adsorption force
Adding adsorbent suspension S into a centrifugal tube with a filter column, centrifuging to form an inclined plane in the filter column by the adsorbent, and discarding filtrate;
3) bacterial lysis and nucleic acid adsorption
Adding a lysis solution L into the bacteria solution pretreated in the step 1), shaking up and down violently, standing, centrifuging, taking the supernatant, adding the supernatant into the adsorption column prepared in the step 2), standing at room temperature, centrifuging, discarding the filtrate, and putting the collection tube back again;
4) cleaning of
Adding a cleaning solution W into the supernatant obtained in the step 3), centrifuging, cleaning, pouring off the filtrate of the collecting tube each time, and throwing away for 15s for the last time, wherein the adsorption column is not returned to the collecting tube, and the cleaning solution W is an alcohol solution with the concentration of 70%;
5) elution is carried out
Transferring the adsorption column into a new centrifuge tube, dripping 10-15ul of eluent E, tightly covering a cover, keeping the temperature at 60 ℃ for 5-10min, centrifuging, and collecting filtrate which is extracted micro nucleic acid solution, wherein the eluent E is Tris solution with sterilized PH of 8.5;
6) first round PCR amplification
Taking the trace nucleic acid solution obtained in the step 5) as a template, and carrying out PCR amplification by using primers SF1 and SR1, wherein the nucleotide sequence of the primer SF1 is shown as SEQ ID No.1, and the nucleotide sequence of the primer SR1 is shown as SEQ ID No. 2;
7) second round of PCR amplification
Taking 1/1000 of the reaction solution obtained in the step 6) as a template, and carrying out PCR amplification by using primers MSF1, SR1 and M13F, wherein the nucleotide sequence of the primer MSF1 is shown as SEQ ID No.3, and the nucleotide sequence of the primer M13F is shown as SEQ ID No. 4;
8) electrophoresis detection and purification;
9) and (4) sequencing and aligning by using a primer M13F to determine the genus and the species.
The low-pollution and high-sensitivity low-cost general strain molecular identification method is characterized in that the suspension solution A in the step 1) is as follows: 100 mM Tris-HCl, 25 mM EDTA, 2.0M NaC1, RnaseA 10ug/ml, pH 8.0.
The molecular identification method of the low-pollution, high-sensitivity and low-cost general strain is characterized in that the mechanical crushing and wall breaking treatment method in the step 1) is any one of the following methods:
a. adding sterilized 1 tungsten carbide bead of 3-5mm into the bacterial liquid, placing in boiling water bath for 5min, quickly freezing with liquid nitrogen, grinding with reciprocating type pulverizer, and recovering to normal temperature;
b. adding 20ul volume of pretreated 0.1mm-1.5mm glass beads into the bacterial liquid, placing in a boiling water bath for 5min, and after the temperature is returned to room temperature, carrying out vortex oscillation for 1 min.
The low-pollution and high-sensitivity low-cost general strain molecular identification method is characterized in that the centrifugal tube with the filter column in the step 2) is obtained by paving 1-3 layers of hydrophilic microporous filter membranes with the aperture of 1-10 mu m on the centrifugal column and compressing the hydrophilic microporous filter membranes.
The molecular identification method of the low-pollution, high-sensitivity and low-cost general strain is characterized in that the adsorbent suspension S in the step 2) is prepared by the following steps: weighing 3g of silica into a 50ml triangular flask, adding 20-30ml of double distilled water, fully shaking, standing for a moment, removing supernatant, cleaning for 3 times, adding the double distilled water to 2 times of the volume of the silica, wherein the total volume is about 20ml, and storing at normal temperature for later use after autoclaving.
The low-pollution and high-sensitivity low-cost general strain molecular identification method is characterized in that the cracking solution L in the step 3) is obtained by mixing the following components: 30g of guanidinium isothiocyanate, 40ml of sterile water, 3ml of sterilized NaAc with pH5.2, 2.5ml of sterilized 10% SDS, and 2.5ml of sterilized glycerol, wherein the total volume is 50 ml.
The molecular identification method of the low-pollution, high-sensitivity and low-cost general strain is characterized in that one side of the inclined plane faces outwards when the adsorption column is centrifuged in the step 3).
The molecular identification method of the low-pollution high-sensitivity low-cost universal strain is characterized in that the volume of the first round of PCR reaction in the step 6) is 15ul, 10 XTaq buffer1.5ul, 2.5 mM dNTP mix 1.2ul, 0.6ul of each primer of Taq 1u and 10uM, the volume of a template is 5ul, and the rest is complemented by pure water; the PCR reaction program is: 300s at 95 ℃; 15s at 94 ℃, 45s at 52 ℃ and 45s at 72 ℃ for 35-40 cycles; and the temperature is 72 ℃ for 300 s.
The molecular identification method of the low-pollution high-sensitivity low-cost universal strain is characterized in that the reaction system in the step 7) is 50-100ul, the template is 1/1000 of a first round reaction product, and the PCR is three-primer PCR, wherein the working concentration of a primer MSF1 is 0.1uM and is 1/4 of the normal PCR concentration, the working concentration of a primer SR1 is 0.4uM, and the working concentration of a primer M13F is 0.4 uM; the PCR reaction program is: circulating at 95 ℃ for 300s, 94 ℃ for 15s, 52 ℃ for 45s and 72 ℃ for 45s for 35-40 cycles; and the temperature is 72 ℃ for 300 s.
The invention utilizes simple and efficient wall breaking and extracting technology, combines efficient nucleic acid adsorption technology and PCR twice amplification effect, can identify bacteria more accurately at the initial stage of microbial colony formation, and the primers for identification of the invention give consideration to the unity of fungi and bacterial sites, not only can identify bacteria at the seed level, but also can identify fungi to the genus or even the species level, thus facilitating the simultaneous identification of some bacteria and fungi, greatly improving the efficiency and saving the experimental cost. Over the years, more than 800 strains are identified in laboratories by using the technology, and the effective rate is over 90 percent. The operation of the invention is basically closed tube operation, and the related reagents can be strictly sterilized, thus not only ensuring that the samples are not easily polluted, but also ensuring that the samples are not easily influenced to operators, therefore, the invention is particularly suitable for companies to use the technology to carry out the service in the aspect of strain identification.
Compared with the prior art, the invention has the following beneficial effects:
(1) only a small amount of colony samples are needed to extract trace DNA, so that accurate identification is realized, and the trouble of strain preparation is avoided;
(2) two simple crushing and wall breaking technologies are utilized, the operation is carried out under the conditions of tube closing and sterilization, the pollution is greatly reduced, and the operation is well protected for operators;
(3) by combining the adsorbent suspension and the adsorption column, the yield or concentration of DNA extraction is improved, so that the identification of trace bacteria samples becomes possible;
(4) the degenerate primer during amplification can accurately identify the level of bacteria to species and the level of fungi to genus or species, so that the trouble of respective amplification of fungi and bacteria is avoided;
(5) the two-time amplification method is adopted, so that the identification sensitivity is greatly improved, and the amplification product is conveniently subjected to direct sequencing by using the universal primer.
Drawings
FIG. 1 is a schematic diagram of the PCR amplification principle and the flow chart of the present invention;
FIG. 2 is an electrophoretogram in example 1.
In fig. 2: from left to right: DL2000 marker, blank, BA1, BA 2.. BA19, wherein BA4 and BA11 failed amplification.
Detailed Description
The present invention is further illustrated by the following examples.
Example 1
Extraction and purification of trace nucleic acid
1.1 Strain pretreatment
2.0ml of thickened or reinforced sterile centrifuge tubes (to prevent tube rupture when cells are disrupted) were pre-filled with 20ul of solution A, 5-10ul volumes of bacteria were picked from the plate with an inoculating needle or toothpick into a 2.0ml centrifuge tube, followed by either:
a. adding 1 tungsten carbide ball of 3-5mm, placing in boiling water bath for 5min, quickly freezing in liquid nitrogen, grinding with reciprocating type pulverizer, and recovering to normal temperature;
b. adding pretreated glass beads of 0.1-1.5 mm in volume of 20ul, placing in boiling water bath for 5min, returning to room temperature, and vortex shaking for 1 min.
1.2 preparation of high adsorption force column
Preparing a 2ml centrifuge tube with a filter column, adding 50-100ul of adsorbent suspension prepared in advance, centrifuging for 1min in a 12,000g angular rotor to enable the adsorbent to form an inclined surface in the filter column, discarding filtrate, molding the appearance of the adsorbent by a centrifugation method in the step, enabling the inclined surface to face outwards during centrifugation of the adsorbent column to ensure the optimal contact area and depth during nucleic acid adsorption, and ensuring the separation efficiency.
1.3 bacterial lysis and nucleic acid adsorption
Adding 500ul of lysate into the pretreated bacteria containing the (1.1), shaking vigorously for 10s from top to bottom, standing for more than 3min, centrifuging for 1min at 12000g, adding 400ul of supernatant into the adsorption column prepared in the (1.2), standing for more than 5min at room temperature, centrifuging for 1min at the rotating speed of 6000g-8000g, discarding the filtrate, and replacing the collection tube again. It is noted that the inclined surface faces outwards (the same applies below) during centrifugation to ensure that the filtrate passes through the adsorbent to the maximum extent, which is also an important aspect of the present invention, and uneven adsorption during centrifugation due to the general planar placement of the adsorption membrane is avoided.
1.4 cleaning
Adding 500ul of cleaning solution W, centrifuging at 12000g for 15s, cleaning for 2 times, pouring off the filtrate of the collecting tube each time, and throwing away for 15s for the last time, wherein the adsorption column is not put back into the collecting tube.
1.5 elution
Transferring the adsorption column into a new 1.5ml centrifuge tube, dripping 20-30ul of eluent E, covering the cover tightly, keeping at 60 ℃ for 5-10min, and then centrifuging at 12000g for 1min, wherein the collected filtrate is the extracted trace nucleic acid solution. The solution can be directly used for 2-4 times of PCR without concentration detection.
The composition and formula of the reagent are as follows:
suspension solution a: 100 mM Tris-HCl, 25 mM EDTA, 2.0M NaC1, RnaseA 10ug/ml (pH 8.0).
The glass bead treatment method comprises the following steps: firstly, cleaning a batch of glass beads with clear water for 1-2 times, then soaking the glass beads in 12N HCl overnight, then rinsing the glass beads, finally carrying out autoclaving and drying the glass beads for later use.
Preparation of centrifuge tube with filter column: the centrifugal column is paved with 1-3 layers of hydrophilic microporous filter membranes (the pore diameter is 1-10 um) and is compressed, the membranes do not need the function of adsorbing nucleic acid, but can be sterilized under high pressure to avoid pollution, and the method is also an important aspect for distinguishing all other products in the market.
Adsorbent suspension S: weighing 3g of silica into a 50ml triangular flask, adding 30ml of double distilled water, fully shaking, standing for a moment, removing supernatant, cleaning for 3 times in this way, adding the double distilled water to 2 times of the volume of the silica (the total volume is about 20 ml), and storing at normal temperature for later use after autoclaving. Can be subpackaged by using a 1.5ml centrifuge tube.
Lysis solution L: 1 triangular flask of 50ml is sterilized earlier, 1 bottle of sterile water, aseptic medicine spoon add in proper order on super clean bench:
30g of guanidinium isothiocyanate;
sterile water is added to about 40ml, and the mixture is fully stirred and dissolved as much as possible;
3ml of sterilized NaAc (pH 5.2);
sterilized SDS 10% SDS 2.5 ml;
2.5ml of sterilized glycerol;
the total volume is about 50ml, after fully dissolving for several hours, storing at normal temperature for standby.
Cleaning solution W: sterile water is prepared in a bottle of 1, and a 70% solution is prepared by using absolute alcohol. When in preparation, 35ml of alcohol can be added firstly, and then sterile water is added to 50ml for standby at normal temperature.
Eluent E: mainly a sterile Tris solution (pH 8.5).
The embodiment has the characteristics that the adsorbed diatomite can be naturally dispersed to a certain extent, so that the adsorption and elution are convenient, a natural inclined plane is formed in the centrifugation, and the optimal adsorption condition is convenient to form, so that even a common diatomite adsorbent can achieve a good effect, and the extraction efficiency of an extremely-trace sample is greatly improved. In addition, the whole material can be sterilized under high pressure, so that the problem that the common adsorption film cannot be sterilized under high pressure, and the pollution which exists widely is easily caused is avoided.
Example 2
And (3) PCR amplification: the nested primers are adopted for amplification, so that the generation of primer dimers is avoided. The reaction consisted of two PCRs, the principle of which is shown in FIG. 1.
1.1 preparation of PCR reaction solution
For the first PCR, the reaction volume was 15ul, 10 XTaq buffer1.5ul, 2.5 mM dNTP mix 1.2ul, Taq 1u, 10uM primers each 0.6ul, template volume was 5ul, and the remainder was made up with pure water; all PCR reactions were performed on a Genepro Thermal Cycle (bio er, Hangzhou) PCR instrument, programmed as follows: circulating at 95 ℃ for 300s, 94 ℃ for 15s, 52 ℃ for 45s and 72 ℃ for 45s for 35-40 cycles; and the temperature is 72 ℃ for 300 s.
1.2 first round PCR
The primer-upstream primer used in the first round was SF 1: CWYCYGGRAGGCAGCAG (shown as SEQ ID No. 1), and the downstream primer is SR 1: AATTCCTTTRAGTTTCARMCTTGCG (shown in SEQ ID No. 2).
2.3 second round PCR
The reaction solution was as above, and the template was 1/1000 of the above reaction solution. The reaction was a three primer PCR, one of which was a combination of M13F and the forward primer, primer MSF 1: 5'-CACGACGTTGTAAAACGACCWYCYGGRAGGCAGCAG-3' (shown in SEQ ID No. 3), with a working concentration of 0.1 uM; the downstream primer is still the primer SR 1: AATTCCTTTRAGTTTCARMCTTGCG (shown in SEQ ID No. 2), at a working concentration of 0.4 uM; the upstream primer is primer M13F 5'-CACGACGTTGTAAAACGAC-3' (shown as SEQ ID No. 4), and the working concentration is also 0.4 uM.
3. Electrophoretic detection and purification
Electrophoresis was carried out on 1.2% normal agarose in the presence of 1 XTAE buffer (pH 8.0) at an electric field strength of 10v/cm for half an hour, and a photograph was taken, which is shown in FIG. 2. From left to right in fig. 2: DL2000 marker, blank, BA1, BA 2.. BA 19. Wherein BA4 and BA11 failed in amplification.
Purification of PCR products for direct sequencing
The sequencing primer is M13F
The bands were picked, purified by the purification method described above, and sequenced using M13F sequencing primer, and the genus and species were identified by Blast alignment (Table 1). As can be seen from the results, fungi and bacteria differ in the size distribution of the amplified bands.
TABLE 1 strains inferred from BLAST alignments
Note: the numbers in parentheses in the BLAST alignment results are indicated respectively (the ratio of the sequences available for alignment measured and the similarity of the aligned sequences to the putative species in the sequence).
By the method, nearly thousands of strains are identified in an accumulated way, and the success rate reaches over 90 percent.
SEQUENCE LISTING
<110> scientific institute of forestry in Zhejiang province
<120> a low-pollution and high-sensitivity low-cost general strain molecular identification method
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<170> PatentIn version 3.3
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<213> Artificial Synthesis
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cacgacgttg taaaacgac 19
Claims (3)
1. A low-pollution and high-sensitivity low-cost general strain molecular identification method is characterized by comprising the following steps:
1) pretreatment of strains
Picking bacteria to be detected from a flat plate, adding the bacteria to be detected into the suspension solution A, wherein the diameter of the bacteria to be detected is more than 2mm, and then mechanically crushing and breaking the wall; the suspension solution A is: 100 mM Tris-HCl, 25 mM EDTA, 2.0M NaC1, RnaseA 10ug/ml, pH 8.0;
2) preparing a column with high adsorption force
Adding adsorbent suspension S into a centrifugal tube with a filter column, centrifuging to form an inclined plane in the filter column by the adsorbent, and discarding filtrate; the adsorbent suspension S is prepared by the following steps: weighing 3g of silica into a 50ml triangular flask, adding 20-30ml of double distilled water, fully shaking, standing for a moment, removing supernatant, cleaning for 3 times, adding the double distilled water to 2 times of the volume of the silica, wherein the total volume is about 20ml, and storing at normal temperature for later use after autoclaving;
3) bacterial lysis and nucleic acid adsorption
Adding a lysis solution L into the bacteria solution pretreated in the step 1), shaking up and down violently, standing, centrifuging, taking the supernatant, adding the supernatant into the adsorption column prepared in the step 2), standing at room temperature, centrifuging, discarding the filtrate when the adsorption column is centrifuged with the inclined surface facing outwards, and replacing the collection pipe; the lysate L is obtained by mixing the following components: 30g of guanidinium isothiocyanate, 40ml of sterile water, 3ml of sterilized NaAc with pH of 5.2, 2.5ml of sterilized 10% SDS and 2.5ml of sterilized glycerol, wherein the total volume is 50 ml;
4) cleaning of
Adding a cleaning solution W into the supernatant obtained in the step 3), centrifuging, cleaning, pouring off the filtrate of the collecting tube each time, and throwing away for 15s for the last time, wherein the adsorption column is not returned to the collecting tube, and the cleaning solution W is an alcohol solution with the concentration of 70%;
5) elution is carried out
Transferring the adsorption column into a new centrifuge tube, dripping 10-15ul of eluent E, tightly covering a cover, keeping the temperature at 60 ℃ for 5-10min, centrifuging, and collecting filtrate which is extracted micro nucleic acid solution, wherein the eluent E is Tris solution with sterilized PH of 8.5;
6) first round PCR amplification
Taking the trace nucleic acid solution obtained in the step 5) as a template, and carrying out PCR amplification by using primers SF1 and SR1, wherein the nucleotide sequence of the primer SF1 is shown as SEQ ID No.1, and the nucleotide sequence of the primer SR1 is shown as SEQ ID No. 2; the first PCR reaction volume was 15ul, 10 XTaq buffer1.5ul, 2.5 mM dNTP mix 1.2ul, Taq 1u, 10uM primers each 0.6ul, template volume was 5ul, the remainder was made up with pure water; the PCR reaction program is: circulating at 95 ℃ for 300s, 94 ℃ for 15s, 52 ℃ for 45s and 72 ℃ for 45s for 35-40 cycles; 300s at 72 ℃;
7) second round of PCR amplification
Taking 1/1000 of the reaction solution obtained in the step 6) as a template, and carrying out PCR amplification by using primers MSF1, SR1 and M13F, wherein the nucleotide sequence of the primer MSF1 is shown as SEQ ID No.3, and the nucleotide sequence of the primer M13F is shown as SEQ ID No. 4; the reaction system is 50-100ul, the template is 1/1000 of the first round reaction product, the PCR is three-primer PCR, wherein the working concentration of the primer MSF1 is 0.1uM and is 1/4 of the normal PCR concentration, the working concentration of the primer SR1 is 0.4uM and the working concentration of the primer M13F is 0.4 uM; the PCR reaction program is: 300s at 95 ℃; 15s at 94 ℃, 45s at 52 ℃ and 45s at 72 ℃ for 35-40 cycles; 300s at 72 ℃;
8) electrophoresis detection and purification;
9) and (4) sequencing and aligning by using a primer M13F to determine the genus and the species.
2. The molecular identification method of low-pollution and high-sensitivity low-cost general bacterial species as claimed in claim 1, wherein the mechanical pulverization wall-breaking treatment method in step 1) is any one of the following methods:
a. adding sterilized 1 tungsten carbide bead of 3-5mm into the bacterial liquid, placing in boiling water bath for 5min, quickly freezing with liquid nitrogen, grinding with reciprocating type pulverizer, and recovering to normal temperature;
b. adding 20ul volume of pretreated 0.1mm-1.5mm glass beads into the bacterial liquid, placing in a boiling water bath for 5min, and after the temperature is returned to room temperature, carrying out vortex oscillation for 1 min.
3. The method for molecular characterization of low-pollution and high-sensitivity low-cost general bacterial species according to claim 1, wherein the centrifuge tube with filter column in step 2) is obtained by compressing a centrifugal column with 1-3 layers of hydrophilic microporous filter membrane with pore size of 1-10 um.
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