CN113444824B - Primer for identifying 4 staphylococci in environment based on isothermal amplification technology, detection kit and application - Google Patents
Primer for identifying 4 staphylococci in environment based on isothermal amplification technology, detection kit and application Download PDFInfo
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Abstract
The invention provides a primer for identifying 4 staphylococci in an environment based on an isothermal amplification technology, a detection kit and application, belonging to the technical field of molecular biological detection. A primer group for identifying 4 staphylococcus in the environment based on an isothermal amplification technology comprises primers for amplifying staphylococcus aureus, staphylococcus wovensis, staphylococcus epidermidis and staphylococcus capitis. The invention also provides a kit for identifying staphylococcus in an environment, which comprises the primer group. When the method is used for detecting staphylococcus in the environment, multi-step complex operations such as bacterial culture, DNA extraction, sequencing and the like in a sample are not needed, the sample directly sampled from the environment can be detected, and the method is simple, convenient, rapid and low in cost; the detection time is short, meanwhile, the specific primers are designed based on the pheS characteristic sequence, the rapid detection and identification can be carried out on staphylococcus aureus, staphylococcus wovensis, staphylococcus epidermidis and staphylococcus capitis in the environment, and the detection sensitivity is high.
Description
Technical Field
The invention belongs to the technical field of molecular biological detection, and particularly relates to a primer for identifying 4 staphylococci in an environment based on an isothermal amplification technology, a detection kit and application.
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 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 phenylalanyl-tRNA synthetase alpha subunit gene (pheS) has high homogeneity in the same strain, and the gene sequence has poor similarity in different strains, so that the gene is a fast evolved gene. Research shows that the pheS gene can be used as a reliable tool for identifying environmental strains and is an effective screening method for detecting the strains.
Isothermal amplification technology is a new nucleic acid amplification technology developed in recent years, which can rapidly and efficiently amplify a nucleic acid sequence at a constant temperature. Compared with a PCR method which needs complex thermal cycle to mediate denaturation, annealing and subsequent extension, the isothermal amplification can realize the melting, renaturation and extension of double-stranded DNA by using recombinase, single-stranded binding protein and DNA polymerase under the condition of constant temperature, the DNA amplification can be completed within 5-30 minutes, the operation is simple, the reaction time is short, and the method can be used for quickly detecting bacteria.
At present, a method for simultaneously identifying multiple bacteria in environmental staphylococcus by using pheS as a target gene and using an isothermal amplification technology does not exist, and the existing method mainly utilizes a PCR technology to identify staphylococcus aureus, so that the identification variety is few, the time consumption is long, the technical requirement is high, and the popularization is not facilitated. Therefore, based on the pheS characteristic sequence, the primers and the method for identifying the 4 environmental staphylococci by using the isothermal amplification technology have very important significance.
Disclosure of Invention
In view of the above, the invention aims to provide a primer and a detection kit for identifying 4 staphylococci in an environment based on an isothermal amplification technology, and an application thereof.
The invention provides a primer group for identifying 4 staphylococcus in an environment based on an isothermal amplification technology, which comprises a primer for amplifying staphylococcus aureus, a primer for amplifying staphylococcus wowensis, a primer for amplifying staphylococcus epidermidis and a primer for amplifying staphylococcus capitis;
the primer for amplifying the staphylococcus aureus comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 1 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 2;
the primer for amplifying the staphylococcus wowense comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 3 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 4;
the primer for amplifying the staphylococcus epidermidis comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 5 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 6;
the primer for amplifying the staphylococcus capitis comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 7 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 8.
The invention provides a kit for identifying 4 staphylococci in an environment based on an isothermal amplification technology, which comprises a primer group.
Preferably, the kit comprises lysis solution A and reaction solution B;
the lysate A is a Tris-HCl solution which contains 1 percent of Triton X-100 by volume, has the concentration of 10mM and the pH value of 8.0;
the reaction solution B contains the following components in each 18 mu L system: 2-3 mu g of recA recombinase protein, 1.5-2 mu g of SSB protein, 0.5-1 mu g of Bsu DNA polymerase, 10 mu L of 2 multiplied reaction buffer solution, 0.4 mu L of 5 multiplied SYBR Green I and the balance ddH2O。
Preferably, the kit further comprises a positive control C and a negative control D;
the positive control C comprises an amplified fragment of staphylococcus aureus, an amplified fragment of staphylococcus wovensis, an amplified fragment of staphylococcus epidermidis and an amplified fragment of staphylococcus capitis;
the nucleotide sequence of the amplified fragment of the staphylococcus aureus is shown as SEQ ID NO. 9;
the nucleotide sequence of the amplified fragment of the staphylococcus Woensis is shown as SEQ ID NO. 10;
the nucleotide sequence of the amplified fragment of the staphylococcus epidermidis is shown as SEQ ID NO. 11;
the nucleotide sequence of the amplified fragment of the staphylococcus capitis is shown as SEQ ID NO 12;
the negative control D is the lysate A.
The invention provides application of the primer group or the kit in identifying 4 staphylococci in the environment;
the 4 kinds of staphylococcus are staphylococcus aureus, staphylococcus wowensis, staphylococcus epidermidis and staphylococcus capitis.
The invention provides a method for identifying 4 staphylococci in environment based on isothermal amplification technology, which comprises the following steps:
1) after an environmental sample is cracked, collecting supernatant serving as a template, and carrying out isothermal amplification and high-resolution dissolution curve analysis by using the primer group to obtain an amplification product;
2) and detecting the amplification product, comparing the detection result with each fragment in the positive control C, and judging that the environment sample contains the corresponding staphylococcus when a consistent fragment is obtained.
Preferably, the reaction procedure of the isothermal amplification is amplification at 39 ℃ for 30 min.
Preferably, the reaction system of the isothermal amplification is 20 μ L:
2-3 mu g of recA recombinase protein
SSB protein 1.5-2 mug
Bsu DNA polymerase 0.5-1. mu.g
2 × reaction buffer 10 μ L
10 μ M Forward/reaction primer 0.8 μ L/0.8 μ L
5×SYBR Green I 0.4μL
ddH2Make up to 20. mu.L of O.
Preferably, the reaction procedure for the high resolution dissolution curve analysis is as follows: at 95 ℃ for 60 s; 60s at 40 ℃; 75 ℃ for 1 s; heating to 98 deg.C at a heating rate of 0.07 deg.C/s for 1 s.
Preferably, the concentration of the single staphylococcus genomic DNA in the environmental sample is not lower than 0.6 pg/mu L.
The primer group for identifying 4 staphylococci in the environment based on the isothermal amplification technology provided by the invention takes pheS as a target gene and comprises 4 pairs of primers for amplifying staphylococcus aureus, staphylococcus wovensis, staphylococcus epidermidis and staphylococcus capitis. A specificity detection experiment shows that the 4 pairs of primers are only combined with the pheS gene of the target bacteria and are not combined with staphylococcus of other species and other microorganisms with close relativity, and the 4 pairs of primers have strong specificity. Meanwhile, sensitivity detection results show that the lowest detection limit of the staphylococcus Wolsonii can reach 33 multiplied by 10-3ng/. mu.L, i.e., 33 pg/. mu.L. The lowest detection limit of staphylococcus capitis can reach 6 multiplied by 10-4ng/. mu.L, i.e., 0.6 pg/. mu.L. The primer group has higher detection sensitivity.
The invention provides a method for identifying 4 staphylococci in an environment based on an isothermal amplification technology, which can quickly finish identification by using the isothermal amplification technology, wherein the whole identification time only needs 30-45 min from sampling to detection results, and the quick detection of a sample can be realized. Meanwhile, compared with other methods, the method can be used for detecting the sample directly collected from the environment without carrying out multi-step complex operations such as culture, DNA extraction and the like on bacteria in the sample, and the whole experimental process is simple to operate.
Drawings
FIG. 1 is a flow chart that provides a technique for identifying 4 staphylococci in an identification environment according to the invention;
FIG. 2 shows the result of agarose gel electrophoresis for detecting primer specificity;
FIG. 3 shows the result of high resolution melting curve for primer specificity detection;
FIG. 4 shows the result of detecting the sensitivity of agarose gel electrophoresis;
FIG. 5 is a high resolution melting curve showing sensitivity detection results;
FIG. 6 shows the result of agarose gel electrophoresis for detecting staphylococcus at different environmental positions;
FIG. 7 shows the result of high resolution melting curve for detecting staphylococcus at different positions in environment.
Detailed Description
The invention provides a primer group for identifying 4 staphylococcus in an environment based on an isothermal amplification technology, which comprises a primer for amplifying staphylococcus aureus, a primer for amplifying staphylococcus wowensis, a primer for amplifying staphylococcus epidermidis and a primer for amplifying staphylococcus capitis; the primer for amplifying the staphylococcus aureus comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 1 (5'-CTATTGTACGTGTTAATGGATGCTTTGAACCG-3') and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 2 (5'-GGTCAGTTAGCGGACTAATGAAATTGATGAAG-3'); the primer for amplifying the staphylococcus wowense comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 3 (5'-GTACGTGCTTGTACTGGAGACGTATGTGTTCTC-3') and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 4 (5'-CAAGAACTAGATGAAAGACAAACGATTTTAGC-3'); the primer for amplifying the staphylococcus epidermidis comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 5 (5'-AGTGGATATAAACGAAGCAAAAGATGCGCAAGCG-3') and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 6 (5'-TTGGCAATGTGACATCAATTGTTTCTTCAGAAAGC-3'); the primer for amplifying the staphylococcus capilalis comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 7 (5'-CTCAGCAAGCTGTTGGTTTAATTTCTCTTC-3') and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 8 (5'-GTCATGACTCAAAATGATACGATGGCG-3'). The source of the primer set is not particularly limited in the present invention, and the primer set may be synthesized by a gene synthesis company known in the art.
The invention provides a kit for identifying 4 staphylococci in an environment based on an isothermal amplification technology, which comprises a primer group. The kit preferably comprises lysis solution A and reaction solution B. The lysate A is preferably a 10mM Tris-HCl solution with pH 8.0 containing 1% by volume of Triton X-100. The reaction solution B preferably contains the following components in 18. mu.L of the reaction system: 2-3 mu g of recA recombinase protein, 1.5-2 mu g of SSB protein, 0.5-1 mu g of Bsu DNA polymerase, 10 mu L of 2 multiplied reaction buffer solution, 0.4 mu L of 5 multiplied SYBR Green I and the balance ddH2And O. The 2 × reaction buffer preferably comprises the following components: 200mmol/L Tris (hydroxymethyl) methylglycine (Tricine, pH 8.0), 220mmol/L vinegarMagnesium, 10mmol/L dithiothreitol, 10% PEG, 6mmol/L ATP, 200 ng/. mu.L creatine kinase, 20mmol/L dNTP.
In the present invention, the kit preferably further comprises a positive control C and a negative control D. The positive control C comprises an amplified fragment of staphylococcus aureus, an amplified fragment of staphylococcus wovensis, an amplified fragment of staphylococcus epidermidis and an amplified fragment of staphylococcus capitis. The nucleotide sequence of the amplified fragment of the staphylococcus aureus is shown as SEQ ID NO. 9; the nucleotide sequence of the amplified fragment of the staphylococcus Woensis is shown as SEQ ID NO. 10; the nucleotide sequence of the amplified fragment of the staphylococcus epidermidis is shown as SEQ ID NO. 11; the nucleotide sequence of the amplified fragment of the staphylococcus capitis is shown as SEQ ID NO. 12; the negative control D is the lysate A. The volume of each reagent in the kit is not particularly limited in the present invention, and 20, 50 or 100 detection doses well known in the art may be used.
The invention provides application of the primer group or the kit in identifying 4 staphylococci in the environment. The 4 kinds of staphylococcus are staphylococcus aureus, staphylococcus wowensis, staphylococcus epidermidis and staphylococcus capitis.
The invention provides a method for identifying 4 staphylococci in an environment based on an isothermal amplification technology, which is shown in figure 1 and specifically comprises the following steps:
1) after an environmental sample is cracked, collecting supernatant serving as a template, and carrying out isothermal amplification and high-resolution dissolution curve analysis by using the primer group to obtain an amplification product;
2) and detecting the amplification product, comparing the detection result with each fragment in the positive control C, and judging that the environment sample contains the corresponding staphylococcus when a consistent fragment is obtained.
According to the invention, after an environmental sample is cracked, the collected supernatant is used as a template, and isothermal amplification and high-resolution dissolution curve analysis are carried out by using the primer group to obtain an amplification product.
The environment is not particularly limited in the present invention, and may be any environment known in the art, such as refrigerator, glassA desktop, etc. The environment sample collection method preferably uses a swab to sample bacteria at different positions in the environment (the sampling solution is lysate A), and the sampling area is 25cm2. Inserting the swab into the sampling liquid, cutting off redundant parts, shaking and centrifuging, and taking the supernatant as a template for nucleic acid amplification. The rotation speed of the centrifugation is preferably 3500-4500 rpm, and most preferably 4000 rpm. The time for centrifugation is preferably 4-6 min, and more preferably 5 min. The environmental sample can be directly amplified without a DNA extraction step, and the identification efficiency is greatly improved. In view of the detection sensitivity of the primer set, the concentration of the single staphylococcal genomic DNA in the environmental sample is preferably not less than 0.6 pg/. mu.L.
In the present invention, the reaction procedure of the isothermal amplification is preferably an amplification at 39 ℃ for 30 min. The reaction system for isothermal amplification is preferably 20 μ L: 2-3 mu g of recA recombinase protein, 1.5-2 mu g of SSB protein, 0.5-1 mu g of Bsu DNA polymerase, 10 mu L of 2 multiplied reaction buffer solution, 0.8 mu L/0.8 mu L of 10 mu M forward primer/reaction primer, 0.4 mu L of 5 multiplied SYBR Green I, 2 mu L of template and ddH2Make up to 20. mu.L of O. The isothermal amplification is preferably performed in a PCR instrument. The reaction procedure for the high resolution dissolution curve analysis is preferably as follows: at 95 ℃ for 60 s; 60s at 40 ℃; 75 ℃ for 1 s; heating to 98 deg.C at a heating rate of 0.07 deg.C/s for 1 s.
In the present invention, the lysis curve results for 4 staphylococci are as follows: the Tm peak range of staphylococcus aureus is 83.6 +/-1 ℃, the Tm peak range of staphylococcus epidermidis is 82.6 +/-1 ℃, the Tm peak range of staphylococcus capitis is 82.5 +/-1 ℃, and the Tm peak range of staphylococcus wadskii is 83 +/-1.5 ℃.
After the amplification product is obtained, the amplification product is detected, the detection result is compared with each fragment in the positive control C, and when consistent fragments are obtained, the environment sample is judged to contain the corresponding staphylococcus.
In the present invention, the detection method preferably comprises agarose gel electrophoresis, and the determination of whether the environmental sample contains one or more of staphylococcus aureus, staphylococcus wovensis, staphylococcus epidermidis and staphylococcus capitis is made according to the position of the bands of the 4 amplified fragments in the positive control C.
The following examples are provided to illustrate the primers, detection kit and application of the present invention for identifying 4 staphylococci in the environment based on isothermal amplification technology, but they should not be construed as limiting the scope of the present invention.
Example 1
The design method of 4 staphylococcus amplification primers comprises the following specific steps:
A. the pheS gene sequences of staphylococcus aureus, staphylococcus wowensis, staphylococcus epidermidis and staphylococcus capitis were found in the NCBI database for alignment.
B. Analyzing the comparison result of the pheS gene sequences of 4 staphylococci, and respectively designing 4 pairs of specific primers of the staphylococci aiming at sequences which are conserved in species and have larger interspecies difference, wherein the primer sequences are as follows:
staphylococcus aureus:
forward primer 5'-CTATTGTACGTGTTAATGGATGCTTTGAACCG-3' (SEQ ID NO: 1);
reverse primer 5'-GGTCAGTTAGCGGACTAATGAAATTGATGAAG-3' (SEQ ID NO: 2);
staphylococcus wadwinii:
forward primer 5'-GTACGTGCTTGTACTGGAGACGTATGTGTTCTC-3' (SEQ ID NO: 3);
reverse primer 5'-CAAGAACTAGATGAAAGACAAACGATTTTAGC-3' (SEQ ID NO: 4);
staphylococcus epidermidis:
forward primer 5'-AGTGGATATAAACGAAGCAAAAGATGCGCAAGCG-3' (SEQ ID NO: 5);
reverse primer 5'-TTGGCAATGTGACATCAATTGTTTCTTCAGAAAGC-3' (SEQ ID NO: 6);
staphylococcus capitis:
forward primer 5'-CTCAGCAAGCTGTTGGTTTAATTTCTCTTC-3' (SEQ ID NO: 7);
reverse primer 5'-GTCATGACTCAAAATGATACGATGGCG-3' (SEQ ID NO: 8).
Example 2
Staphylococcus primer specificity detection
A. Using the primers for 4 pairs of staphylococci designed in example 1, the genomic DNA of the staphylococci species corresponding to the primers was used as a positive control and the negative control was lysate A, and Staphylococcus aureus, Staphylococcus wovensis, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus capitis, Staphylococcus haemolyticus, Bacillus, Pseudomonas, Micrococcus, and Pantoea (all of the above bacteria were isolated from the surrounding environment and the species was determined by sequencing the 16S rDNA and pheS gene sequences) were lysed with lysate A, and then supernatants were taken and used as templates for isothermal amplification and high-resolution melting curve analysis.
Reaction system (20 μ L):
18. mu.L of the reaction solution
The reaction liquid comprises the following specific components:
recA recombinase protein 2.5 mug
SSB protein 1.8. mu.g
Bsu DNA polymerase 0.8. mu.g
2 × reaction buffer 10 μ L
Forward/reverse primer (10. mu.M) 0.8. mu.L/0.8. mu.L
5×SYBR Green I 0.4μL
ddH2O make up to 18. mu.L.
B. The amplification product was subjected to agarose gel electrophoresis.
C. The agarose gel electrophoresis result is shown in figure 2, wherein M is Marker, the sizes of the bands are sequentially 600bp, 500bp, 400bp, 300bp, 200bp and 100bp from top to bottom, and N is negative control; FIG. 3 shows the corresponding high resolution melting curve results (the Tm peak range of Staphylococcus aureus is 83.6 + -1 deg.C, the Tm peak range of Staphylococcus epidermidis is 82.6 + -1 deg.C, the Tm peak range of Staphylococcus capitis is 82.5 + -1 deg.C, the Tm peak range of Staphylococcus Wasser is 83 + -1.5 deg.C, the abscissa is "Temperature" and the ordinate is "-dF/dT"). Table 1 shows the strain numbers used, and it can be seen from Table 1 and FIG. 2 that different staphylococcal primers can only amplify corresponding staphylococci, indicating that the designed primers have good specificity.
TABLE 1 identification number of bacteria for specificity detection
Example 3
Sensitive detection of staphylococci
The method comprises the following specific steps:
A. the detection sensitivity is taken as an example of staphylococcus wowensis and staphylococcus capitis. After two kinds of staphylococcus are cultured overnight, genome DNA is extracted, and after the concentration is determined, the genome DNA is diluted by 10 times in a gradient way.
B. And performing isothermal amplification and high-resolution melting curve analysis by using the genomic DNA of the staphylococcus wavorans and the staphylococcus capitis with different concentration gradients as templates and respectively using two staphylococcus specific primers. Wherein, Staphylococcus aureus 10-1～10-4The working concentration of each gradient genomic DNA was 33X 100 ng/. mu.L, 33X 10-1ng/μL、33×10-2ng/μL、33×10-3ng/mu L; staphylococcus capitis 10-1～10-4The working concentration of genomic DNA for each gradient was 6X 10-2ng/μL 、6×10-3ng/μL、6×10-4ng/μL、6×10-5ng/μL。
C. And carrying out agarose gel electrophoresis detection on the amplification product.
D. The agarose gel electrophoresis test result is shown in FIG. 4, wherein M is Marker, the sizes of the bands are sequentially 600bp, 500bp, 400bp, 300bp, 200bp and 100bp from top to bottom, FIG. 5 is the corresponding high resolution melting curve (the Tm peak range of the head-shaped staphylococcus is 82.5 + -1 ℃, the Tm peak range of the Wolff staphylococcus is 83 + -1.5 ℃, the abscissa is "Temperature", the ordinate is "-dF/dT"), and 10 in the graph-1～10-410-fold gradient dilution of genomic DNA, respectively, and N as a negative control. As can be seen from FIGS. 4 and 5, the lowest detection limit of Staphylococcus Wauteri can reach 33X 10-3ng/. mu.L, i.e., 33 pg/. mu.L. The lowest detection limit of staphylococcus capitis can reach 6 multiplied by 10-4ng/μL，I.e., 0.6 pg/. mu.L. The conclusion shows that the lower limit of detection of the reaction system can reach 0.6 pg/mu L, and the reaction system has good sensitivity.
Example 4
Method for identifying environmental staphylococcus
The method comprises the following specific steps of simulating environment sampling and identifying the species of staphylococcus:
A. carrying out overnight shake culture on staphylococcus aureus, staphylococcus wowensis, staphylococcus epidermidis and staphylococcus capitis, randomly selecting bacteria liquids of different bacteria and mixing the bacteria liquids in equal volumes to prepare 3 groups of mixed bacteria which are respectively numbered as 1-3, wherein the No. 1 is the mixed bacteria of the staphylococcus aureus and the staphylococcus epidermidis; no. 2 is a mixed bacterium of staphylococcus aureus, staphylococcus wovensis and staphylococcus capitis; no. 3 is a mixed strain of staphylococcus capitis, staphylococcus wovensis and staphylococcus epidermidis;
B. respectively taking 100 mu l of the 3 groups of mixed bacteria liquid and uniformly smearing the mixture liquid to 25cm2The surface of the table top, the glass and the refrigerator (the smearing area is wiped clean by 75% alcohol in advance, and staphylococcus aureus, staphylococcus wowensis, staphylococcus epidermidis or staphylococcus capitis are not detected after detection), and sampling is carried out by using a swab after drying (the sampling solution is lysate).
C. The collected samples were used as templates, and were subjected to isothermal amplification using 4 specific primers designed in example 2, the amplification method was as described in example 2, wherein the positive control was staphylococcus genomic DNA (amplified fragments of staphylococcus aureus, staphylococcus warei, staphylococcus epidermidis and staphylococcus capitis in the order of P1, P2, P3 and P4) corresponding to the 4 pairs of specific amplification primer pairs, and the negative control was lysate a (n).
D. And carrying out agarose gel electrophoresis detection on each amplification product.
E. The agarose gel electrophoresis result is shown in figure 6, wherein M is Marker, the sizes of the bands are 600bp, 500bp, 400bp, 300bp, 200bp and 100bp sequentially from top to bottom, the numbers 1, 2 and 3 are respectively No. 1 (desktop), No. 2 (glass) and No. 3 (refrigerator) mixed bacteria samples, and figure 7 is a corresponding high-resolution melting curve (the Tm peak range of staphylococcus aureus is 83.6 +/-1 ℃, the Tm peak range of staphylococcus epidermidis is 82.6 +/-1 ℃, the Tm peak range of staphylococcus capitis 82.5 +/-1 ℃, the Tm peak range of staphylococcus vollekii is 83 +/-1.5 ℃, the abscissa is "Temperature" and the ordinate is "-dF/dT").
As can be seen from FIGS. 6 and 7, the method can detect Staphylococcus aureus and Staphylococcus epidermidis of the mixed bacteria 1 on the table top; staphylococcus aureus, staphylococcus wowensis and staphylococcus capitis of the mixed bacteria 2 are detected on the glass; staphylococcus capitis, Staphylococcus wowensis and Staphylococcus epidermidis of the mixed bacteria 3 were detected on the refrigerator. The method is suitable for detecting the environmental bacteria.
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.
Sequence listing
<110> Zhi Shi Intelligent technology (Beijing) Co Ltd
To Microbiology Intelligent Technology (Xiamen) Co., Ltd.
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<213> Artificial Sequence (Artificial Sequence)
<400> 10
gtacgtgctt gtactggaga cgtatgtgtt ctcattaaag tctcttcagt aatataaaaa 60
ctatcttgca tatcacgtgc aggatgtgat ttaggtaagt ttaatgcctc aaaattataa 120
tgatcctgtt ctacttcata accatcaaca atttcataac ctaagcccaa gaataaatct 180
tctatctctt caattgtacg tgtcaatgga tgttttgagc ctatttctat ttggcgacta 240
ggtaatgtca catcaattgt ttcttctgca agttgttgat ttaacttttc attggctaaa 300
atcgtttgtc tttcatctag ttcttg 326
<210> 11
<211> 257
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
agtggatata aacgaagcaa aagatgcgca agcgttacaa gaagtaaaag tgaaatattt 60
aggtaaaaaa ggttctgtta gcggcttaat gaaaaatatg aaagatttgc ctaatgaaga 120
caaacctgcg tatggtcaaa aggtaaatga attaagacaa actattcaaa gtgaattaga 180
tgaaagacaa aagctaatca aagaagagaa attaaatcaa cagctttctg aagaaacaat 240
tgatgtcaca ttgccaa 257
<210> 12
<211> 279
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ctcagcaagc tgttggttta atttctcttc ttttaataac ttttgtcttt catctaattc 60
attctgaatt gtttgacgca attcatttac tttttgacca tatgcaggtt tctcttcatt 120
aggtaaatct ttcatatttt tcattagacc acttactgaa ccttttttac ctaaatactt 180
aacctttaca tcttgcaatg cacgttcatt atctgcttca ttaatatcaa cgagagcttg 240
ttgttttaat tccgccatcg tatcattttg agtcatgac 279
Claims (5)
1. A method for identifying 4 staphylococci in environment based on isothermal amplification technology is characterized by comprising the following steps:
1) after cracking an environmental sample, collecting supernatant serving as a template, and carrying out isothermal amplification and high-resolution dissolution curve analysis by using a primer group to obtain an amplification product;
the primer group comprises a primer for amplifying staphylococcus aureus, a primer for amplifying staphylococcus wowensis, a primer for amplifying staphylococcus epidermidis and a primer for amplifying staphylococcus capitis;
the primer for amplifying the staphylococcus aureus comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 1 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 2;
the primer for amplifying the staphylococcus wowense comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 3 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 4;
the primer for amplifying the staphylococcus epidermidis comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 5 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 6;
the primer for amplifying the staphylococcus capitis comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 7 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 8;
the reaction procedure for the high resolution dissolution curve analysis was as follows: at 95 ℃ for 60 s; 60s at 40 ℃; 75 ℃ for 1 s; heating to 98 ℃ at a heating rate of 0.07 ℃/s for 1 s;
2) detecting the amplification product, comparing the detection result with each fragment in the positive control C, and judging that the environment sample contains staphylococcus of the corresponding species when consistent fragments are obtained;
the results of the lysis curves for 4 staphylococci in positive control C are as follows: the Tm peak range of staphylococcus aureus is 83.6 +/-1 ℃, the Tm peak range of staphylococcus epidermidis is 82.6 +/-1 ℃, the Tm peak range of staphylococcus capitis is 82.5 +/-1 ℃, and the Tm peak range of staphylococcus wadskii is 83 +/-1.5 ℃;
the positive control C comprises an amplified fragment of staphylococcus aureus, an amplified fragment of staphylococcus wovensis, an amplified fragment of staphylococcus epidermidis and an amplified fragment of staphylococcus capitis;
the nucleotide sequence of the amplified fragment of the staphylococcus aureus is shown as SEQ ID NO. 9;
the nucleotide sequence of the amplified fragment of the staphylococcus Woensis is shown as SEQ ID NO. 10;
the nucleotide sequence of the amplified fragment of the staphylococcus epidermidis is shown as SEQ ID NO. 11;
the nucleotide sequence of the amplified fragment of the staphylococcus capitis is shown as SEQ ID NO. 12.
2. The method of claim 1, wherein the isothermal amplification reaction procedure is amplification at 39 ℃ for 30 min.
3. The method of claim 2, wherein the reaction system of isothermal amplification is 20 μ L:
2-3 mu g of recA recombinase protein
SSB protein 1.5-2 mug
Bsu DNA polymerase 0.5-1. mu.g
2 × reaction buffer 10 μ L
10 μ M Forward/reverse primer 0.8 μ L/0.8 μ L
5×SYBR Green I 0.4μL
Template 2. mu.L
ddH2Make up to 20. mu.L of O.
4. The method of claim 1, wherein the lysis reagent comprises lysate a;
the lysate A is a Tris-HCl solution which contains 1 percent of Triton X-100 by volume, has the concentration of 10mM and the pH value of 8.0;
the reagent for isothermal amplification comprises a reaction solution B;
the reaction solution B contains the following components in each 18 mu L system: 2-3 mu g of recA recombinase protein, 1.5-2 mu g of SSB protein, 0.5-1 mu g of Bsu DNA polymerase, 10 mu L of 2 multiplied reaction buffer solution, 0.4 mu L of 5 multiplied SYBR Green I and the balance ddH2O。
5. The method according to any one of claims 1 to 4, wherein the concentration of the genomic DNA of a single staphylococcus bacterium in the environmental sample is not less than 0.6pg/μ L.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106520923A (en) * | 2016-10-11 | 2017-03-22 | 深圳市疾病预防控制中心 | Kit and method for simultaneously detecting Staphylococcus aureus and five enterotoxins thereof |
| CN112961929A (en) * | 2021-05-18 | 2021-06-15 | 至善时代智能科技(北京)有限公司 | Primer group, kit and method for staphylococcus in species level identification environment |
| CN112980982A (en) * | 2021-05-18 | 2021-06-18 | 至善时代智能科技(北京)有限公司 | Primers and detection kit for identifying multiple staphylococci based on pheS gene and application thereof |
-
2021
- 2021-08-30 CN CN202110999815.5A patent/CN113444824B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106520923A (en) * | 2016-10-11 | 2017-03-22 | 深圳市疾病预防控制中心 | Kit and method for simultaneously detecting Staphylococcus aureus and five enterotoxins thereof |
| CN112961929A (en) * | 2021-05-18 | 2021-06-15 | 至善时代智能科技(北京)有限公司 | Primer group, kit and method for staphylococcus in species level identification environment |
| CN112980982A (en) * | 2021-05-18 | 2021-06-18 | 至善时代智能科技(北京)有限公司 | Primers and detection kit for identifying multiple staphylococci based on pheS gene and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| LAMP法检测食品中凝固酶阳性葡萄球菌;张宏伟等;《食品研究与开发》;20110505(第05期);第53-55页 * |
| 等温扩增荧光技术快速检测金黄色葡萄球菌方法的建立;王耕等;《食品界》;20160820(第08期);第86页 * |
| 重组酶等温扩增技术监测采供血环境中致病菌的应用研究;朱立苇等;《中国卫生检验杂志》;20200110(第01期);第43页摘要，第44页1.3.1部分 * |
| 高分辨率溶解曲线检测9种食源性致病菌方法的建立;胡双芳等;《现代食品科技》;20161231(第03期);第278-287页 * |
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