CN112961930B - Primer group, kit and method for identifying environmental bacteria at genus level - Google Patents
Primer group, kit and method for identifying environmental bacteria at genus level Download PDFInfo
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Abstract
The invention provides a primer group, a kit and a method for identifying environmental microorganisms at the genus level, belonging to the technical field of environmental bacteria detection and identification, wherein the primer group comprises specific forward primers F1-F6 for amplifying staphylococcus, bacillus, pantoea, micrococcus, pseudomonas and aerococcus, a specific reverse primer R1 for amplifying the pantoea and a specific reverse primer R2 shared by staphylococcus, bacillus, micrococcus, pseudomonas and aerococcus; the nucleotide sequences of the forward primers F1-F6 are shown as SEQ ID No. 1-SEQ ID No.6, the nucleotide sequence of the reverse primer R1 is shown as SEQ ID No.7, and the nucleotide sequence of the reverse primer R2 is shown as SEQ ID No. 8. The primer group, the kit and the method provided by the invention can be used for quickly, effectively and comprehensively detecting the genus level of the environmental bacteria.
Description
Technical Field
The invention belongs to the technical field of detection and identification of environmental bacteria, and particularly relates to a primer group, a kit and a method for identifying environmental microorganisms at the genus level.
Background
Bacteria are one of the major groups of organisms, and the most abundant of all organisms. The bacteria in the environment are pathogenic, such as staphylococcus aureus, salmonella, pseudomonas aeruginosa and the like, and beneficial bacteria, such as bifidobacterium, lactobacillus and the like, and the bacteria in different species have different pathogenicity and benefits and are closely related to the survival and development of human beings. Therefore, it is of great importance to provide a method for identifying environmental bacteria at the genus level.
The 16S rRNA gene is a DNA sequence coding for rRNA in bacteria, exists in the genome of all bacteria, and has high conservation and specificity. With the advent of PCR technology and the continuous improvement of nucleic acid research technology, 16s rRNA gene detection technology has become a powerful tool for detection and identification of pathogenic bacteria, and by applying the technology, rapid, trace, accurate, simple and convenient classification and detection of pathogenic bacteria can be realized.
At present, the existing environmental bacteria identification method mainly aims at detecting specific bacteria or certain strains in the environment, cannot comprehensively identify the environmental bacteria in daily life, and has a limited identification range. Chinese patent 201410667734.5 discloses a method for rapidly detecting and identifying Listeria bacteria, which is based on PCR technology and can rapidly detect and identify six Listeria bacteria in the environment; chinese patent 201810883997.8 discloses primers and method for identifying lactobacillus in environmental sample at species level, which can accurately identify lactobacillus at species level by performing high-throughput sequencing on PCR product; the Chinese invention patent 202011021614.X discloses a method for identifying clinical difficult and complicated strains by using 16S rRNA gene sequence analysis, which utilizes PCR technology to amplify bacteria 16S rDNA, determines the strain type of bacteria to be identified by sequencing an amplified product and comparing the sequence with a gene database, establishes a new method for effectively identifying difficult and complicated strains, expands the identification spectrum of clinical strains and meets the clinical identification requirement on difficult and complicated strains.
However, the above methods have great limitations in the detection range, and cannot detect bacteria in the environment more widely and comprehensively.
Disclosure of Invention
In view of the above, the present invention aims to provide a primer set, a kit and a method for identifying environmental microorganisms at the genus level. The primer group, the kit and the method provided by the invention can be used for quickly, effectively and comprehensively detecting the genus level of the environmental bacteria.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a primer group for identifying environmental microorganisms at genus level, which comprises a specific forward primer F1 for amplifying staphylococcus, a specific forward primer F2 for amplifying bacillus, a specific forward primer F3 for amplifying pantoea, a specific forward primer F4 for amplifying micrococcus, a specific forward primer F5 for amplifying pseudomonas and a specific forward primer F6 for amplifying aerococcus, a specific reverse primer R1 for amplifying pantoea and a specific reverse primer R2 shared by staphylococcus, bacillus, micrococcus, pseudomonas and aerococcus; the nucleotide sequences of the forward primers F1-F6 are shown as SEQ ID No. 1-SEQ ID No.6, the nucleotide sequence of the reverse primer R1 is shown as SEQ ID No.7, and the nucleotide sequence of the reverse primer R2 is shown as SEQ ID No. 8.
The invention provides a kit for identifying environmental microorganisms at the genus level, which comprises the primer group.
Preferably, each primer in the primer group is independently used at a concentration of 0.2-0.4. mu.M.
Preferably, a detection reagent is also included.
Preferably, the detection reagent comprises bacterial lysate, PCR reaction solution and positive control genome DNA.
Preferably, the bacterial lysate is a Tris-HCl solution containing 0.8-1.2% of Triton X-100 by volume percentage, and the concentration of Tris-HCl in the lysate is 8-12 mM.
Preferably, the PCR reaction solution comprises 10 XTaq buffer, Taq DNA polymerase, dNTPs and sterile water.
The present invention also provides a method for identifying an environmental microorganism at the genus level, comprising the steps of:
1) collecting an environmental sample;
2) performing PCR amplification by using lysate of an environmental sample as a template and using F1 and R2, F2 and R2, F3 and R1, F4 and R2, F5 and R2, F6 and R2 primer pairs in the primer group as primers respectively to obtain an amplification product;
3) and (3) carrying out agarose gel electrophoresis on the amplification product, wherein if a target amplification band appears in the electrophoresis result of the amplification product obtained by amplifying the amplification product by using primers corresponding to staphylococcus, bacillus, pantoea, micrococcus, pseudomonas and pneumococcus, the corresponding bacterium is considered to exist in the environment, and if the target amplification band does not appear, the corresponding bacterium does not exist in the environment or the content of the bacterium in the environment does not reach the detection limit of the method.
Preferably, the collection of the environmental sample is performed with a swab.
Preferably, the PCR amplification system comprises the following components in 20 μ L:
10× Taq buffer 1×；
1-2 units of Taq DNA polymerase;
dNTPs 0.2mM；
upstream primers are 0.2-0.4 mu M;
downstream primer 0.2-0.4 mu M
2 mu L of DNA template;
supplementing sterilized water to 20 μ L;
the procedure for PCR amplification comprises the following steps: 95 ℃ for 5 min; 30s at 95 ℃, 30s at 60 ℃, 60s at 72 ℃ and 35 cycles; 72 ℃ for 5 min.
The invention has the beneficial effects that: the primer group for identifying environmental microorganisms at genus level comprises specific forward primers F1-F6 for amplifying staphylococcus, bacillus, pantoea, micrococcus, pseudomonas and aerococcus, a specific reverse primer R1 for amplifying pantoea and a specific reverse primer R2 shared by staphylococcus, bacillus, pantoea, pseudomonas and aerococcus; the primer group provided by the invention can carry out specific PCR amplification on strains of different genera, thereby identifying bacteria of staphylococcus, bacillus, pseudomonas, pantoea and aerococcus in the environment, and the primer group has the advantages of simple operation, short time consumption and wider identification range.
The primer group, the kit and the method for identifying the environmental bacteria on the genus level do not need to carry out multi-step complex operations such as culture, DNA extraction and sequencing on the bacteria in the sample, can detect the sample directly taken from the environment, are quick and time-saving, and have low cost and high sensitivity; from the beginning of sampling, the whole time flow only needs 1-2 hours, compared with other detection primers and methods, the detection time is greatly reduced, and the purpose of quickly identifying environmental bacteria is achieved.
Drawings
FIG. 1 is a technical flow chart of the present invention;
FIG. 2 is a PCR agarose gel electrophoresis of environmental bacterial colonies;
FIG. 3 is a 48-environment bacterial species analysis evolutionary tree;
FIG. 4 is a diagram of agarose gel electrophoresis for verifying the specificity of primers of different genera;
FIG. 5 is an agarose gel electrophoresis image of bacterial species identification at different locations of the environment;
FIG. 6 is an agarose gel electrophoresis image of the three bacteria sensitivity detection.
Detailed Description
The invention provides a primer group for identifying environmental microorganisms at a genus level, which comprises specific forward primers F1-F6 for amplifying staphylococcus, bacillus, pantoea, micrococcus, pseudomonas and aerococcus, a specific reverse primer R1 for amplifying pantoea and a specific reverse primer R2 shared by staphylococcus, bacillus, micrococcus, pseudomonas and aerococcus; the nucleotide sequences of the forward primers F1-F6 are shown as SEQ ID No. 1-SEQ ID No.6, the nucleotide sequence of the reverse primer R1 is shown as SEQ ID No.7, and the nucleotide sequence of the reverse primer R2 is shown as SEQ ID No. 8; the method comprises the following specific steps:
staphylococcus specific forward primer F1: 5'-ATGTGTAAGTAACTATGCACGTCT-3' (SEQ ID No. 1);
bacillus specific forward primer F25 '-CTAGTTGAATAAGCTGGCACCT-3' (SEQ ID No. 2);
pantoea-specific forward primer F35 '-CAGCGATTCGGTCGGGAACTCA-3' (SEQ ID No. 3);
micrococcus-specific forward primers F45 '-AGCGAAAGTGACGGTACCTGCAGAAG-3' (SEQ ID No. 4);
pseudomonas specific forward primer F55 '-GCAGTAAGTTAATACCTTGCTGTTT-3' (SEQ ID No. 5);
aerococcus-specific forward primers F65 '-ATTGTAGAGTAACTGCTACAGTCT-3' (SEQ ID No. 6);
pantoea-specific reverse primer R15 '-CGGYTACCTTGTTACGACTT-3' (SEQ ID No. 7);
the specific reverse primer R25 '-GGGTTGCGCTCGTTG-3' (SEQ ID No. 8) shared by staphylococci, bacilli, micrococcus, pseudomonas and pneumococcus.
In the present invention, the design of the primer set is achieved by the following steps: separating and culturing bacteria from a conventional environment sample, carrying out PCR amplification on genomic DNA of the separated and cultured bacteria to obtain an amplification product, sequencing the amplification product, comparing the amplification product with a database, constructing a phylogenetic tree for analysis, and confirming that the bacteria in the environment sample belong to 6 different genera respectively: staphylococci, bacillus, pseudomonas, aerococci, pantoea and micrococcus; and then searching the primer group obtained by designing the differential sequence according to the sequencing result, wherein the primer group can carry out specific PCR amplification on strains of different genera, so that bacteria of staphylococcus, bacillus, pseudomonas, pantoea and aerococcus in the environment can be identified, and the method is simple to operate, short in time consumption and wide in identification range.
The invention also provides a kit for identifying environmental microorganisms at the genus level, which comprises the primer group. In the present invention, each primer in the primer set is preferably used independently at a concentration of 0.2-0.4. mu.M, and more preferably at a concentration of 0.2. mu.M. In the invention, the kit preferably further comprises a detection reagent, and the detection reagent preferably comprises a bacterial lysate, a PCR reaction solution and positive control genomic DNA. In the invention, the bacterial lysate is preferably a Tris-HCl solution containing 0.8-1.2% of Triton X-100 by volume percentage, and is more preferably a Tris-HCl solution containing 1.0% of Triton X-100 by volume percentage; in the invention, the concentration of Tris-HCL in the bacterial lysate is preferably 8-12 mM, more preferably 9-11 mM, and most preferably 10 mM. In the present invention, the PCR reaction solution preferably includes 10 XTaq buffer, Taq DNA polymerase, dNTPs and sterile water. The sources of the 10 XTaq buffer, Taq DNA polymerase, dNTPs and sterile water are not particularly limited, and the conventional commercial products in the field can be adopted. In the invention, the positive control genome DNA is independently packaged genome DNA of staphylococcus, bacillus, pantoea, micrococcus, pseudomonas and staphylococcus standard strains.
The present invention also provides a method for identifying an environmental microorganism at the genus level, comprising the steps of: 1) collecting an environmental sample; 2) performing PCR amplification by using lysate of an environmental sample as a template and using the F1 and R2, F2 and R2, F3 and R1, F4 and R2, F5 and R2, F6 and R2 primer pairs as primers respectively to obtain an amplification product; 3) and (3) carrying out agarose gel electrophoresis on the amplification product, wherein if a target amplification band appears in the amplification product obtained by amplifying the amplification product by using primers corresponding to staphylococcus, bacillus, pantoea, micrococcus, pseudomonas and pneumococcus in the electrophoresis result, the corresponding bacterium is considered to exist in the environment, and if the target amplification band does not appear, the corresponding bacterium is considered to not exist in the environment.
In the present invention, an environmental sample is collected. In the present invention, said collecting of the environmental sample is preferably performed with a swab; the environmental sample is not particularly limited in the present invention, and can be any sample in a general environment, such as a table, a window, glass, a trash can, a door handle, a floor, a laboratory table, an instrument surface, and the like. In the specific implementation process of the invention, preferably, the swab is soaked in the bacterial lysate to be fully wetted, then sampling is carried out, redundant parts of the swab are cut off along a swab cut after sampling, the swab is placed in the bacterial lysate, and first shaking, standing, second shaking and centrifugation are sequentially carried out; the time of the first oscillation and the second oscillation is preferably 1min, and the time of standing is preferably 2-4 min, and more preferably 3 min; the rotation speed of the centrifugation is preferably 3000-5000 rpm, more preferably 4000rpm, the time of the centrifugation is preferably 4-6 min, more preferably 5min, after the centrifugation, the supernatant is taken as an environmental sample lysate, the environmental sample lysate is taken as a template for PCR amplification, and an additional DNA extraction process is not needed.
In the invention, lysate of an environmental sample is used as a template, and the F1 and R2, F2 and R2, F3 and R1, F4 and R2, F5 and R2, F6 and R2 primer pairs are respectively used as primers to carry out PCR amplification to obtain an amplification product. In the present invention, the PCR amplification system preferably comprises the following components in 20 μ L:
10× Taq buffer 1×；
1-2 units of Taq DNA polymerase;
dNTPs 0.2mM；
upstream primers are 0.2-0.4 mu M;
downstream primer 0.2-0.4 mu M
2 mu L of DNA template;
sterile water make up 20 μ L.
In the present invention, the procedure of PCR amplification preferably comprises the following steps: 95 ℃ for 5 min; 30s at 95 ℃, 30s at 60 ℃, 60s at 72 ℃ and 35 cycles; 72 ℃ for 5 min.
After the amplification product is obtained, the agarose gel electrophoresis is carried out on the amplification product. The specific operation of the agarose gel electrophoresis is not particularly limited, and the conventional agarose gel electrophoresis scheme in the field can be adopted. According to the invention, after the agarose gel electrophoresis, the agarose gel electrophoresis result is analyzed, if a target amplification band appears in the electrophoresis result of an amplification product obtained by amplification of primers corresponding to staphylococcus, bacillus, pantoea, micrococcus, pseudomonas and staphylococcus, the environment is considered to have corresponding bacteria, and if the target amplification band does not appear, the environment is considered to have no corresponding bacteria or the content of the bacteria in the environment does not reach the detection limit of the method. In the present invention, the presence or absence of the desired amplified band in the electrophoresis result is determined by comparing the electrophoresis result with the amplified band of the positive control.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Species identification and classification of environmental bacteria
The method comprises the following specific steps:
A. bacteria at different positions (a laboratory bench, the surface of an instrument and the like) in the environment are sampled by using swabs (the sampling solution is bacteria lysate), and the serial numbers of the sampling solution are 1-48.
B. Gradient dilution of sample to 10-6Respectively take 10-4，10-5，10-610 mu l of bacterial liquid with three concentrations is coated on an LB solid culture medium, is cultured in an incubator at 37 ℃ overnight, and after the bacterial growth, a single bacterial colony is picked up and is subjected to streaking separation culture for 3 times to obtain pure bacteria.
C. Selecting the purified single bacterial colony of the bacteria cultured by streaking, carrying out PCR amplification by using 16S rDNA universal primers 27F and 1492R, and detecting the result of the PCR amplification by agarose gel electrophoresis (the electrophoresis result is shown in figure 2, wherein 1-48 are respectively 48 collected samples, M is Marker, and the size of the Marker is 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom in sequence; the number is a sample with a weaker PCR product band or an unpaired product position, and sequencing is not carried out).
27F primer sequence: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID No. 9)
1492R primer sequence: 5'-CGGYTACCTTGTTACGACTT-3' (SEQ ID No. 10)
PCR amplification System:
10× Taq buffer 1×；
dNTPs 0.2mM；
upstream primer 0.2. mu.M;
downstream primer 0.2. mu.M
2 mu L of DNA template;
sterile water make up 20 μ L.
PCR amplification procedure: 95 ℃ for 5 min; 30s at 95 ℃, 30s at 60 ℃, 60s at 72 ℃ and 35 cycles; 72 ℃ for 5 min.
D. The PCR products were sequenced using a generation of Sanger sequencing, sequencing platform ABI 3730XL, by Competition Industrial bioengineering (Shanghai).
E. Comparing The sequencing result with The NCBI gene database sequence, determining 48 bacteria, constructing an evolutionary tree by an N-J method (The specific method is described in The following references: Saitou N. The neighbor-joining methods: A new method for constructing phylogenetic trees [ J ]. mol.biol.Evol, 1987, 4; Zhang Xinzhong, Wanxi and Ji tailed white shrimp 4 wild populations ITS1 sequence and ITS genetic relationship analysis [ C ]/2016. aquatic society academic Abstract collection of 2016.), and dividing The 48 strains into 6 different genera as shown in Table 1 and FIG. 3: staphylococcus, Bacillus, Pseudomonas, Aerococcus, Pantoea, and Micrococcus.
TABLE 1 Categories of 48 bacteria in environmental samples
Example 2
Design and detection of genus level specific primer
The method comprises the following specific steps:
A. based on the sequencing results of 48 bacteria in example 1 and finding out the difference sequence, 16S rDNA genus specific primers were designed, wherein R1 is a Pantoea specific reverse primer and R2 is a specific reverse primer common to Staphylococcus, Bacillus, Micrococcus, Pseudomonas and Bacillus.
Staphylococcus specific forward primer F1: 5'-ATGTGTAAGTAACTATGCACGTCT-3' (SEQ ID No. 1)
Bacillus specific forward primer F25 '-CTAGTTGAATAAGCTGGCACCT-3' (SEQ ID No. 2)
Pantoea-specific forward primer F35 '-CAGCGATTCGGTCGGGAACTCA-3' (SEQ ID No. 3)
Micrococcus-specific forward primer F45 '-AGCGAAAGTGACGGTACCTGCAGAAG-3' (SEQ ID No. 4)
Pseudomonas specific forward primer F55 '-GCAGTAAGTTAATACCTTGCTGTTT-3' (SEQ ID No. 5)
Aerococcus-specific forward primer F65 '-ATTGTAGAGTAACTGCTACAGTCT-3' (SEQ ID No. 6)
Pantoea-specific reverse primer R15 '-CGGYTACCTTGTTACGACTT-3' (SEQ ID No. 7)
The specific reverse primer R25 '-GGGTTGCGCTCGTTG-3' (SEQ ID No. 8) shared by staphylococci, bacilli, micrococcus, pseudomonas and pneumococcus.
B. By using the designed primers, 5 standard strains (see table 2) are selected as a reference, bacteria obtained by environmental streaking separation are used as a template for PCR amplification, and the primer pairs of the corresponding genera are found to have specificity and can be used for identifying the bacteria of different genera.
PCR reaction reagent: comprises a lysis solution A, a reaction solution B, a positive control C and a negative control D.
Lysate A comprises the following specific components:
Tris-HCL (PH 8.0) 10mM；
the reaction solution B comprises the following specific components:
10× Taq buffer 1×
taq DNA polymerase 1units
dNTPs 0.2mM
Primer pairs F1/F2/F4/F5/F6+ R2 and F3/R10.2 mu M
Sterilized water supplement
The specific components of the positive control C are as follows: separately packaged staphylococcus, bacillus, pantoea, micrococcus, pseudomonas and aerococcus genome DNA;
negative control D: and (4) a lysis solution.
And (3) PCR reaction system:
PCR reaction solution B18. mu.l
C. The results of partial agarose gel electrophoresis are shown in Table 2 and FIG. 4, from which it can be seen that primers of different genera can only amplify bacteria in the genus, indicating that the designed primer pair has high specificity and can be used for identifying bacteria of different genera.
TABLE 2 amplification identification results of bacteria and standard bacteria isolated in part of the environments
Example 3
Identification of environmental bacteria at the genus level
The method comprises the following specific steps:
A. bacteria in different positions (1, a table 2, a window 3, glass 4, a small garbage can 5, a door handle 6 and a floor) in the environment are sampled by using swabs (the sampling solution is lysate).
B. Using the collected sample as a template, PCR amplification was performed using the forward primer and the reverse primer in example 2, respectively.
C. The PCR product was subjected to agarose gel electrophoresis.
D. The results of the agarose gel electrophoresis (FIG. 5) were analyzed. As can be seen from FIG. 5, the primer pairs provided are specific and can be used to directly amplify bacterial templates sampled at different locations in the environment, and the bacterial species contained at different locations in the environment are different.
Example 4
Sensitivity detection
The method comprises the following specific steps:
A. the sensitivity of the detection method was examined by taking Pantoea 9, Bacillus 5 and Pseudomonas 47 as examples, and the overnight-cultured bacterial solutions of Pantoea 9, Bacillus 5 and Pseudomonas 47 were diluted in a gradient of 10 times. The original concentrations of Pantoea 9, Bacillus 5 and Pseudomonas 47 were 1.2X 10 respectively9 CFU/ml、1.5×109 CFU/ml、1.08×109 CFU/ml
B. And (3) cracking the bacterial liquids with different concentration gradients by using a cracking solution, and respectively using the cracked bacterial liquids as templates for PCR amplification.
C. And carrying out agarose gel electrophoresis detection on the PCR product.
D. The results of the agarose gel electrophoresis are shown in FIG. 6, and lanes 1-8 in FIG. 6 are respectively dilution 100、10-1、10-2、10-3、10-4、10-5、10-6、10-7The result of sample amplification of (1).
E. As can be seen from FIG. 5, the three bacteria were diluted to 10-7Can still be effectively amplified, and shows that the lower limit of detection of the reaction system of the invention can reach 10-7The dilution gradient has higher sensitivity.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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Claims (2)
1. A method of identifying an environmental bacterium at the genus level, comprising the steps of:
1) collecting an environmental sample;
2) performing PCR amplification by using lysate of an environmental sample as a template and F1 and R2, F2 and R2, F3 and R1, F4 and R2, F5 and R2, F6 and R2 primer pairs as primers to obtain an amplification product;
3) carrying out agarose gel electrophoresis on the amplification product, wherein if a target amplification band appears in the electrophoresis result of the amplification product obtained by amplifying the primer corresponding to staphylococcus, bacillus, pantoea, micrococcus, pseudomonas and pneumococcus, the corresponding bacterium is considered to exist in the environment, and if the target amplification band does not appear, the corresponding bacterium does not exist in the environment or the content of the bacterium in the environment does not reach the detection limit of the method;
f1 is a specific forward primer for amplifying staphylococcus, F2 is a specific forward primer for amplifying bacillus, F3 is a specific forward primer for amplifying pantoea, F4 is a specific forward primer for amplifying micrococcus, F5 is a specific forward primer for amplifying pseudomonas, F6 is a specific forward primer for amplifying aerococcus, R1 is a specific reverse primer for amplifying pantoea, and R2 is a common specific reverse primer for amplifying staphylococcus, bacillus, micrococcus, pseudomonas and aerococcus; the nucleotide sequences of F1-F6 are shown as SEQ ID No. 1-SEQ ID No.6, the nucleotide sequence of R1 is shown as SEQ ID No.7, and the nucleotide sequence of R2 is shown as SEQ ID No. 8;
the collecting of the environmental sample is performed with a swab; soaking the swab in a bacterial lysate to fully wet the swab, and then sampling; the bacterial lysate is a Tris-HCl solution containing 0.8-1.2% of Triton X-100 by volume percentage, and the concentration of Tris-HCl in the lysate is 8-12 mM;
the environmental samples include tables, windows, glass, trash cans, door handles, floors, laboratory tables, and instrument surfaces.
2. The method according to claim 1, wherein the PCR amplification system comprises the following components in 20 μ L:
10× Taq buffer 1×；
1-2 units of Taq DNA polymerase;
dNTPs 0.2mM；
upstream primers are 0.2-0.4 mu M;
downstream primer 0.2-0.4 mu M
2 mu L of DNA template;
supplementing sterilized water to 20 μ L;
the procedure for PCR amplification comprises the following steps: 95 ℃ for 5 min; 30s at 95 ℃, 30s at 60 ℃, 60s at 72 ℃ and 35 cycles; 72 ℃ for 5 min.
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| CN113444825A (en) * | 2021-08-30 | 2021-09-28 | 至善时代智能科技(北京)有限公司 | Primer group, kit and constant-temperature amplification method for species level identification of environmental bacteria |
| CN116287324B (en) * | 2022-09-20 | 2023-09-22 | 北京理工大学 | Primer and kit for detecting bacillus spatially and application of primer and kit |
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