CN112961929A - Primer group, kit and method for staphylococcus in species level identification environment - Google Patents
Primer group, kit and method for staphylococcus in species level identification environment Download PDFInfo
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Abstract
The invention provides a primer group, a kit and a method for staphylococcus in species level identification environment, belonging to the technical field of molecular biological detection, wherein the primer group comprises primers F1 and R1 for amplifying staphylococcus aureus, primers F2 and R2 for amplifying staphylococcus wowensis, primers F3 and R3 for amplifying staphylococcus epidermidis, and primers F4 and R4 for amplifying staphylococcus hominis; primers F5 and R5 for amplification of Staphylococcus capitis and primers F6 and R6 for amplification of Staphylococcus haemolyticus; the nucleotide sequences of the primers F1, R1, F2, R2, F3, R3, F4, R4, F5, R5, F6 and R6 are respectively shown as SEQ ID No. 1-SEQ ID No. 12. The primer group, the kit and the method provided by the invention are simple to operate and high in sensitivity, and can complete the detection of the environmental staphylococcus in a short time; low cost, multiple species of identified staphylococcus and convenient popularization and application.
Description
Technical Field
The invention belongs to the technical field of molecular biological detection, and particularly relates to a primer group, a kit and a method for identifying staphylococcus in an environment at a seed level.
Background
Staphylococci belongs to gram-positive bacteria, is the most common pyococcus, and is most easily infected by staphylococci in the elderly, pregnant women, newborns and the like with low body resistance, so as to cause diseases such as pemphigus, conjunctivitis, sepsis and the like. In addition, staphylococci can cause infectious diseases in poultry, such as callus, omphalitis and staphylococcal septicemia, which cause significant losses to the poultry industry.
The 16S rDNA gene is a DNA sequence corresponding to the coding rRNA of bacteria, and is present in the genome of all bacteria. The 16S rDNA has high conservation and specificity, and with the emergence of PCR technology and the continuous improvement of nucleic acid research technology, the 16S rDNA gene detection technology has become a powerful tool for detecting and identifying pathogenic bacteria, and the application of the technology can realize the rapid, trace, accurate, simple and convenient classification and detection of the pathogenic bacteria.
In bacteria, gyrase B subunit gene gyrB almost appears in a single copy form, the gene evolution rate is high, compared with 16Sr DNA, the base replacement frequency is higher, at present, no method for identifying multiple bacteria in staphylococcus simultaneously based on gyrB gene exists, and the existing method is mainly used for identifying staphylococcus aureus. Chinese invention patent 201610139846.2 discloses a gyrB primer combination for detecting staphylococcus aureus as experimental animal, the primer can be used for identifying staphylococcus aureus based on LAMP isothermal amplification, the method has small identification range, only one kind of staphylococcus can be identified, and the LAMP isothermal amplification reagent used by the method is expensive and not beneficial to popularization.
Disclosure of Invention
In view of the above, the present invention provides a primer set, a kit and a method for identifying 6 staphylococci in an environment at a species level, by designing primers based on the characteristic sequence of the gyrB gene; the primer set can identify staphylococcus aureus, staphylococcus wovensis, staphylococcus epidermidis, staphylococcus hominis, staphylococcus capitis and staphylococcus haemolyticus in the environment on the species level; the primer group, the kit and the method are simple to operate and high in sensitivity, and can finish the detection of the environmental staphylococcus in a short time; meanwhile, the method has low cost, and the identified staphylococcus has multiple varieties, thereby being convenient for popularization and application.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a primer group for staphylococcus in a species level identification environment, which comprises
Primers F1 and R1 for amplifying staphylococcus aureus, primers F2 and R2 for amplifying staphylococcus wowensis, primers F3 and R3 for amplifying staphylococcus epidermidis, and primers F4 and R4 for amplifying staphylococcus hominis; primers F5 and R5 for amplification of Staphylococcus capitis and primers F6 and R6 for amplification of Staphylococcus haemolyticus;
the nucleotide sequences of the primers F1, R1, F2, R2, F3, R3, F4, R4, F5, R5, F6 and R6 are respectively shown as SEQ ID No. 1-SEQ ID No. 12.
The invention provides a kit for identifying staphylococcus in an environment at a species 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 invention provides a method for identifying staphylococci in an environment at the species 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 the F1/R1, F2/R2, F3/R3, F4/R4, F5/R5 and F6/R6 primer pairs as primers respectively to obtain an amplification product;
3) and (3) carrying out agarose gel electrophoresis on the amplification product, wherein if an amplification target band appears in the electrophoresis result of the amplification product obtained by amplifying the amplification product by using primers corresponding to staphylococcus aureus, staphylococcus wovensis, staphylococcus epidermidis, staphylococcus hominis, staphylococcus capitis and staphylococcus haemolyticus, the environment is considered to have corresponding bacteria, and if the amplification target 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.
Preferably, the collection of the environmental sample is carried out by using a swab, and the sampling area is 20-30 cm2。
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 of staphylococcus in the species level identification environment is designed based on the characteristic sequence of the gyrB gene, can be used for specifically detecting and identifying staphylococcus aureus, staphylococcus wowensis, staphylococcus epidermidis, staphylococcus hominis, staphylococcus capitis and staphylococcus haemolyticus in the environment, and is good in specificity and high in sensitivity. The kit and the method for identifying staphylococcus in the environment at the seed level do not need to carry out multi-step complex operations such as culture, DNA extraction and sequencing on bacteria in a sample, can detect the sample directly collected from the environment, are quick and time-saving, and have low cost and high sensitivity; from the beginning of sampling, the whole process only needs 1-2 hours, and the detection time is short.
Drawings
FIG. 1 is a technical flow chart of the present invention;
FIG. 2 shows the results of the primer-specific intergeneric detection;
FIG. 3 shows the results of the primer-specific in-genus detection;
FIG. 4 shows the result of the assay of the detection sensitivity of Staphylococcus aureus;
FIG. 5 shows the results of detection of staphylococci at different environmental sites.
Detailed Description
The invention provides a primer group for staphylococcus in species level identification environment, which comprises primers F1 and R1 for amplifying staphylococcus aureus, primers F2 and R2 for amplifying staphylococcus wowensis, primers F3 and R3 for amplifying staphylococcus epidermidis, and primers F4 and R4 for amplifying staphylococcus hominis; primers F5 and R5 for amplification of Staphylococcus capitis and primers F6 and R6 for amplification of Staphylococcus haemolyticus; the nucleotide sequences of the primers F1, R1, F2, R2, F3, R3, F4, R4, F5, R5, F6 and R6 are respectively shown as SEQ ID No. 1-SEQ ID No. 12; the method comprises the following specific steps:
staphylococcus aureus
F1：5'-ACTTAAAAGAAGTTGGCAC-3'（SEQ ID No.1）
R1：5'-GTTTGACCTTCGAATTGAGGATC-3'（SEQ ID No.2）
Staphylococcus Waunderskii
F2：5'-AGACGAAGATGATATCAGAC-3'（SEQ ID No.3）
R2：5'-GTTTGACCTTCGAATTGAGGATC-3'（SEQ ID No.4）
Staphylococcus epidermidis
F3：5'-AAGATGAAAGAGAAGAGGAAG-3'（SEQ ID No.5）
R3：5'- GTTGGTTGCATATCCACTG -3'（SEQ ID No.6）
Human staphylococcus
F4：5'-TGAAGAACAACGTGAAGACACG-3'（SEQ ID No.7）
R4：5'-TACTTGAACAGTCTGCCAG-3'（SEQ ID No.8）
Staphylococcus capitis
F5：5'-GCGTGAAGAGGAAGGCCGTGAG-3'（SEQ ID No.9）
R5：5'-GTTTGTCCTTCGAATTGAGGATC -3'（SEQ ID No.10）
Hemolytic staphylococcus
F6：5'-GCGTGAAGATGTAGAGCAACGC-3'（SEQ ID No.11）
R6：5'- GTTTGTCCTTCGAATTGAGGATC-3'（SEQ ID No.12）。
The invention also provides a kit for identifying staphylococcus in an environment at a species level, which comprises the primer group. In the invention, the use concentration of each primer in the primer group is preferably 0.2-0.4 mu M independently, and more preferably 0.2 mu M independently. 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 present invention, the concentration of Tris-HCl in the 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 present invention, the positive control genomic DNA is the genomic DNA of staphylococcus aureus, staphylococcus wovensis, staphylococcus epidermidis, staphylococcus hominis, staphylococcus capitis and staphylococcus haemolyticus, which are separately packaged.
The invention provides a method for identifying staphylococci in an environment at the species 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/R1, F2/R2, F3/R3, F4/R4, F5/R5 and F6/R6 primer pairs in the primer group as primers to obtain an amplification product; 3) and (3) carrying out agarose gel electrophoresis on the amplification product, wherein if an amplification target band appears in the electrophoresis result of the amplification product obtained by amplifying the amplification product by using primers corresponding to staphylococcus aureus, staphylococcus wovensis, staphylococcus epidermidis, staphylococcus hominis, staphylococcus capitis and staphylococcus haemolyticus, the environment is considered to have corresponding bacteria, and if the amplification target band does not appear, the environment is considered to have no corresponding bacteria.
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; in the invention, the sampling area is preferably 20-30 cm2More preferably 25cm2The source of the environmental sample is not particularly limited in the present invention, and the environmental sample can be any sample in a conventional environment, such as a table, a window, glass, a garbage can, a door handle, a floor, gloves, shoes, a lab coat, a lab bench, 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, the genomic DNA of an environmental sample is taken as a template, and the F1/R1, F2/R2, F3/R3, F4/R4, F5/R5 and F6/R6 primer pairs are respectively taken 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 an amplification product obtained by amplification by using primer pairs corresponding to staphylococcus aureus, staphylococcus wowensis, staphylococcus epidermidis, staphylococcus hominis, staphylococcus capitis and staphylococcus haemolyticus has an amplification target band in the electrophoresis result, the environment is considered to have corresponding bacteria, and if the amplification target band does not appear, the environment is considered to have no corresponding bacteria.
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
Identification of species of environmental staphylococci
The method comprises the following specific steps:
A. bacteria at different positions (laboratory tables, refrigerators and the like) in the environment are sampled by using a swab (the sampling solution is lysate), and the sampling area is 25cm2。
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. Single colonies of the purified streaked bacteria were picked and PCR amplified using 16S rDNA universal primers 27F and 1492R.
27F primer sequence: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID No. 13),
1492R primer sequence: 5'-CGGYTACCTTGTTACGACTT-3' (SEQ ID No. 14);
D. the PCR product was sequenced.
E. And comparing the sequencing result with the NCBI gene database sequence to determine the species classification of 48 environmental bacteria, wherein the detailed contents are shown in Table 1, and the species classification comprises 6 staphylococcus including staphylococcus aureus, staphylococcus wovensis, staphylococcus epidermidis, staphylococcus hominis, staphylococcus capitis and staphylococcus haemolyticus.
TABLE 1 Categories of 48 bacteria in environmental samples
Example 2
Design of 6 staphylococcus specific primers
The method comprises the following specific steps:
A. and (3) downloading gyrB gene sequences of staphylococcus aureus, staphylococcus wovensis, staphylococcus epidermidis, staphylococcus hominis, staphylococcus capitis and staphylococcus haemolyticus from the Genebank gene bank.
B. Comparing gene sequences, searching for difference sequences, and designing specific primers respectively aiming at 6 staphylococci, wherein the primer sequences are as follows:
staphylococcus aureus
F1：5'-ACTTAAAAGAAGTTGGCAC-3'（SEQ ID No.1）
R1：5'-GTTTGACCTTCGAATTGAGGATC-3'（SEQ ID No.2）
Staphylococcus Waunderskii
F2：5'-AGACGAAGATGATATCAGAC-3'（SEQ ID No.3）
R2：5'-GTTTGACCTTCGAATTGAGGATC-3'（SEQ ID No.4）
Staphylococcus epidermidis
F3：5'-AAGATGAAAGAGAAGAGGAAG-3'（SEQ ID No.5）
R3：5'- GTTGGTTGCATATCCACTG -3'（SEQ ID No.6）
Human staphylococcus
F4：5'-TGAAGAACAACGTGAAGACACG-3'（SEQ ID No.7）
R4：5'-TACTTGAACAGTCTGCCAG-3'（SEQ ID No.8）
Staphylococcus capitis
F5：5'-GCGTGAAGAGGAAGGCCGTGAG-3'（SEQ ID No.9）
R5：5'-GTTTGTCCTTCGAATTGAGGATC -3'（SEQ ID No.10）
Hemolytic staphylococcus
F6：5'-GCGTGAAGATGTAGAGCAACGC-3'（SEQ ID No.11）
R6：5'- GTTTGTCCTTCGAATTGAGGATC-3'（SEQ ID No.12）。
Example 3
Detection of specificity between staphylococcus primers
A. The intergeneric specificity of staphylococcus primers is verified by taking staphylococcus aureus, staphylococcus haemolyticus, staphylococcus wovensis and human staphylococcus primers as examples. Respectively taking Pantoea, Micrococcus, Pseudomonas and Bacillus bacteria as templates to perform PCR amplification; the positive control is staphylococcus strain (P) corresponding to the primer, the negative control is lysate (N), M is Maeker, and the size of the Maeker fragment is 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom.
B. The PCR product was detected by agarose gel electrophoresis.
C. The agarose gel electrophoresis results are shown in FIG. 2, and Table 2 is a list of the numbers of strains used in agarose gel electrophoresis, and it can be seen from FIG. 2 that different staphylococcal primers can only amplify corresponding staphylococci, indicating that the designed primers have good specificity among genera.
TABLE 2 List of the numbers of strains used for the intergeneric specificity test
Example 4
Specific detection in staphylococcus primers
By using the designed primers, staphylococcus aureus standard strains ATCC8325 (P1) and NCTC6538 (P2) were used as positive controls, the negative control was lysate (N), and 6 different staphylococcus were used as templates for PCR amplification.
The PCR product was detected by agarose gel electrophoresis.
The agarose gel electrophoresis result is shown in fig. 3, and table 3 is a list of the number of the strains used in the agarose gel electrophoresis, and it can be seen from fig. 3 that different primers can only amplify staphylococcus in the species, indicating that the designed primers still have high specificity in staphylococcus, and can be used for species identification of staphylococcus aureus, staphylococcus wovensis, staphylococcus epidermidis, staphylococcus hominis, staphylococcus capitis and staphylococcus haemolyticus, respectively.
TABLE 3 List of the numbers of the strains used for the detection of the specificity within the genus
Example 5
Determination of staphylococcal detection sensitivity
The method comprises the following specific steps:
A. taking staphylococcus wowensis, staphylococcus aureus and staphylococcus capitis as examples, after three kinds of staphylococcus are cultured overnight, genome DNA is extracted, after concentration is measured, the genome DNA is diluted by 10 times of gradient, and the initial concentrations of the staphylococcus wowensis, the staphylococcus aureus and the staphylococcus capitis 107.7ng/ul, 62.5ng/ul and 114.6ng/ul respectively.
B. The genomic DNA of staphylococcus wowensis, staphylococcus aureus and staphylococcus capitis with different concentration gradients is used as a template, and three staphylococcus specific primers are respectively used for PCR amplification (a negative control is lysate).
C. And carrying out agarose gel electrophoresis detection on the PCR product.
D. The agarose gel electrophoresis detection result is shown in figure 4, wherein 1-7 in the figure are 10-fold diluted genome DNA respectively, and 8 is a negative control, and as can be seen from figure 4, the staphylococcus caprae genome DNA concentration can still be effectively amplified when being 1.146 pg/mu l, which indicates that the lower limit of the detection of the reaction system can reach 1.146 pg/mu l, and the reaction system has good sensitivity.
Example 6
Detection of staphylococci at different locations in the environment
The method comprises the following specific steps:
A. with swabs for different locations in the environment:
1. a small garbage can, 2, gloves, 3, an experimental clothes, 4, a test bed, 5, a first mixed bacterium (staphylococcus epidermidis, staphylococcus capitis, staphylococcus wowensis, staphylococcus haemolyticus and staphylococcus hominis) 6, a second mixed bacterium (staphylococcus wowensis, staphylococcus haemolyticus and staphylococcus hominis), 7, shoes, 8 and a negative control (lysate); sampling (the sampling solution is lysate) with a sampling area of 25cm2。
B. Using the collected samples as templates, PCR amplification was performed using 6 pairs of specific primers designed in example 2, respectively.
Reaction reagents: 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×
dNTPs 0.2 mM
Upstream primer 0.2. mu.M;
downstream primer 0.2. mu.M;
sterile water make up to 18. mu.l.
The specific components of the positive control C are as follows: independently subpackaging the genome DNA of staphylococcus aureus, staphylococcus wowensis, staphylococcus epidermidis, staphylococcus hominis, staphylococcus capitis and staphylococcus haemolyticus;
negative control D: and (4) a lysis solution.
And (3) PCR reaction system:
PCR reaction solution B18. mu.l
C. And carrying out agarose gel electrophoresis detection on the PCR product.
D. The agarose gel electrophoresis result is shown in fig. 5, and as can be seen from fig. 5, the detection system provided by the invention can be used for directly amplifying bacterial templates sampled at different positions in the environment, so as to identify whether one or more staphylococcus in staphylococcus aureus, staphylococcus wovensis, staphylococcus epidermidis, staphylococcus hominis, staphylococcus capitis and staphylococcus haemolyticus exists in the environment.
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 (10)
1. A primer set for identifying staphylococci in an environment at the species level, comprising
Primers F1 and R1 for amplifying staphylococcus aureus, primers F2 and R2 for amplifying staphylococcus wowensis, primers F3 and R3 for amplifying staphylococcus epidermidis, and primers F4 and R4 for amplifying staphylococcus hominis; primers F5 and R5 for amplification of Staphylococcus capitis and primers F6 and R6 for amplification of Staphylococcus haemolyticus;
the nucleotide sequences of the primers F1, R1, F2, R2, F3, R3, F4, R4, F5, R5, F6 and R6 are respectively shown as SEQ ID No. 1-SEQ ID No. 12.
2. A kit for identifying staphylococci in an environment at the species level comprising the primer set of claim 1.
3. The kit according to claim 2, wherein each primer in the primer set is used independently at a concentration of 0.2-0.4. mu.M.
4. The kit of claim 2, further comprising a detection reagent.
5. The kit of claim 4, wherein the detection reagent comprises bacterial lysate, PCR reaction solution and positive control genomic DNA.
6. The kit of claim 5, wherein the bacterial lysate is a Tris-HCl solution comprising 0.8% to 1.2% by volume of Triton X-100, and the concentration of Tris-HCl in the lysate is 8 to 12 mM.
7. The kit according to claim 5, wherein the PCR reaction solution comprises 10 XTaq buffer, Taq DNA polymerase, dNTPs and sterile water.
8. A method of identifying staphylococci in an environment at the species 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/R1, F2/R2, F3/R3, F4/R4, F5/R5 and F6/R6 primer pairs in the primer set of claim 1 as primers respectively to obtain amplification products;
3) and (3) carrying out agarose gel electrophoresis on the amplification product, wherein if an amplification target band appears in the electrophoresis result of the amplification product obtained by amplifying the amplification product by using primers corresponding to staphylococcus aureus, staphylococcus wovensis, staphylococcus epidermidis, staphylococcus hominis, staphylococcus capitis and staphylococcus haemolyticus, the environment is considered to have corresponding bacteria, and if the amplification target 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.
9. The method of claim 8, wherein the collecting of the environmental sample is performed with a swab having a sampling area of 20-30 cm2。
10. The method according to claim 8, 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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