CN114990244A - Detection method for rapidly detecting staphylococcus aureus based on MIRA fluorescence method - Google Patents

Detection method for rapidly detecting staphylococcus aureus based on MIRA fluorescence method Download PDF

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CN114990244A
CN114990244A CN202210724003.4A CN202210724003A CN114990244A CN 114990244 A CN114990244 A CN 114990244A CN 202210724003 A CN202210724003 A CN 202210724003A CN 114990244 A CN114990244 A CN 114990244A
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staphylococcus aureus
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mira
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祁克宗
宋祥军
程越
杨侃侃
涂健
邵颖
薛挺
周育
王振宇
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Anhui Agricultural University AHAU
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Abstract

The invention relates to the technical field of genetic engineering, and discloses a detection method for rapidly detecting staphylococcus aureus based on an MIRA fluorescence method, which comprises the following steps: s1, bacterial culture: taking out staphylococcus aureus liquid stored at minus 80 ℃, performing resuscitation culture on an LB solid culture medium and a blood plate culture medium by using a four-zone scribing method, culturing for 12-16h in a constant-temperature incubator at 37 ℃, selecting a single colony in 1.5mL of LB liquid culture medium, culturing for 3-6h in a constant-temperature shaking table at 37 ℃, transferring to 20mL of liquid culture medium according to the proportion of 1: 100, and culturing until the liquid culture medium is turbid for later use; the invention detects the golden yellow grape balls by the MIRA technology, and has the advantages of short detection time, low detection temperature, low requirements on instruments and equipment, strong specificity, high sensitivity and the like, thereby providing the detection efficiency of molecular biology, reducing the detection threshold and saving the detection time.

Description

Detection method for rapidly detecting staphylococcus aureus based on MIRA fluorescence method
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a detection method for rapidly detecting staphylococcus aureus based on an MIRA fluorescence method.
Background
In recent years, food poisoning events have occurred due to contamination of fresh meat by food-borne microorganisms. At present, the food-borne pathogenic microorganism detection method is developed from traditional selective culture medium screening to nucleic acid-based PCR detection to gene probes, biosensors and the like, and the methods have the limitations of long time consumption, complex pretreatment, dependence on expensive instruments and equipment and the like, and can not quickly detect the food-borne pathogenic microorganisms in fresh food. And due to the abuse of antibiotics, the drug resistance of bacteria is increased, and the traditional detection method is easy to generate false negative results, so that the establishment of a method for quickly detecting and diagnosing pathogenic microorganisms, which is quick, simple, convenient, specific, sensitive, low in consumption and applicable, is necessary.
The research aims at establishing a rapid visual detection method of common food-borne pathogenic bacteria staphylococcus aureus by a multienzyme constant temperature rapid amplification technology (MIRA), and has important significance for timely and effectively controlling pathogenic bacteria propagation, preventing food poisoning and reducing food-borne disease events.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a detection method for rapidly detecting staphylococcus aureus based on an MIRA fluorescence method, which solves the problems that the existing detection method has the limitations of long time consumption, complex pretreatment, dependence on expensive instruments and equipment and the like, and can not rapidly detect food-borne pathogenic microorganisms in fresh food.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme: a detection method for rapidly detecting staphylococcus aureus based on an MIRA fluorescence method comprises the following steps:
s1, bacterial culture: taking out staphylococcus aureus liquid stored at minus 80 ℃, performing resuscitation culture on an LB solid culture medium and a blood plate culture medium by using a four-zone scribing method, culturing for 12-16h in a constant-temperature incubator at 37 ℃, selecting a single colony in 1.5mL of LB liquid culture medium, culturing for 3-6h in a constant-temperature shaking table at 37 ℃, transferring to 20mL of liquid culture medium according to the proportion of 1: 100, and culturing until the liquid culture medium is turbid for later use;
s2, extracting bacterial genome DNA: extracting bacterial genome by using a bacterial genome DNA extraction kit (centrifugal column type) of a certain Biochemical technology Co., Ltd;
s3, primer and probe design: searching nuc gene of staphylococcus aureus in GenBank, analyzing and comparing by Meglign software, determining respective high-specificity conserved fragment regions, and designing nuc gene-specific MIRA primers and probes with the aid of Primer Premier 5; and designing a full-length Primer under the auxiliary design of Primer Premier 5 according to the size of the nuc gene fragment of the staphylococcus aureus being 687bp, and carrying out PCR amplification;
s4, PCR product recovery and purification: according to the operation of the extraction step of the SanPrep column type DNA gel recovery kit specification, recovering the amplification product for later use;
s5, construction of standard plasmids: according to pMD TM 19-T Vector Cloning Kit instructions, construct pMD19-T-nuc recombinant plasmid;
s6, recombinant plasmid identification: taking part of culture solution for sequencing service, and performing comparison analysis on BLAST function and Meglign software of NCBI website and gene full length;
s7, plasmid DNA small-scale extraction: extracting the constructed pMD19-T-nuc recombinant plasmid for later use according to the instruction of using the SanPrep column type plasmid DNA small extraction kit;
s8 establishment of staphylococcus aureus fluorescence detection system
S81, establishing a system: establishing a fluorescence result detection system according to the instruction of the DNA constant-temperature rapid amplification kit (fluorescence type);
s82, primer screening: screening experiments are carried out on the nuc gene primer of the staphylococcus aureus by using the fluorescence result detection system established in the last step, and results are compared;
s83, temperature optimization: and (3) screening the optimal reaction temperature pair of the S.aureus system by using the fluorescence result detection system established in the first step and setting the reaction temperatures to be 38 ℃, 39 ℃, 40 ℃, 41 ℃ and 42 ℃ respectively, and using the optimal reaction temperature pair for subsequent experiments.
Preferably, the specific step of step S2 is:
s21, taking 5mL of the standby bacterial liquid, centrifuging at 9000rpm for 2min, and leaving a precipitate;
s22, adding a GA solution with the ratio of the bacterial liquid to the GA solution being 25: 1 into the precipitate, and suspending the solution by using an oscillator;
s23, adding a 10: 1 (GA solution and Proteunese K solution) solution into the tube, and uniformly blowing and beating the solution;
s24, continuously adding a protease K solution and a GB solution of which the GB solution is 1: 1, shaking and uniformly mixing, standing in a 70 ℃ water bath for 8-10min, and instantly centrifuging after the liquid in the test tube is clarified;
s25, adding absolute ethyl alcohol with the ratio of GB solution to absolute ethyl alcohol being 1: 11, and shaking and mixing uniformly;
s26, adding the mixed solution (including floccules) into an adsorption column-collecting tube CB3, centrifuging at 11000rpm for 30S, and discarding liquid in the collecting tube;
s27, adding a GD solution with the ratio of absolute ethyl alcohol to GD solution being 11: 25 into an adsorption column-collecting pipe CB3, centrifuging at 11000rpm for 30S, and discarding liquid in the collecting pipe;
s28, adding a GD solution and a PW rinsing solution in a ratio of 5: 6 into an adsorption column-collection tube CB3, centrifuging at 11000rpm for 30S, and discarding liquid in the collection tube;
s29, repeating the previous step;
s210, centrifuging an empty tube of an adsorption column-collection tube CB3 for 3min, discarding liquid in the collection tube, and standing for several minutes at indoor temperature to evaporate rinsing liquid on the adsorption column;
s211, finally, taking a clean centrifuge tube, putting an adsorption column in the clean centrifuge tube, adding 50-200 mu L of TE solution into the middle part of the adsorption column, centrifuging at 11000rpm for 3min, and collecting liquid in the test tube.
Preferably, the PCR system in step S3 is 20 μ L, wherein Mix is 10 μ L, upstream primer is 1 μ L, downstream primer is 1 μ L, DNA template is 2 μ L, and sterilized water is 6 μ L.
Preferably, the method further comprises step S9, a sensitivity test: the plasmid concentration was measured as copy number concentration (copies/. mu.L) [ 6.02X 10 ] 23 X concentration (ng/. mu.L). times.10 -9 ]/[ DNA Length X660]The copy number concentration was calculated, and the nuc gene recombinant plasmid (pMD19-T-nuc) was diluted 10-fold at 10-fold dilution concentration 0 copies/μL、10 1 copies/μL、10 2 copies/μL、10 3 copies/μL、10 4 copies/μL、10 5 copies/. mu.L, and sterile water (ddH) 2 O) is used as a negative control of the experiment, and the reaction system uses the system established in the experiment to carry out sensitivity verification and compare with the PCR detection technology.
Preferably, the method further comprises step S10, specificity test: the plasmid constructed by the extracted target gene DNA conservative fragment of the staphylococcus aureus is used as a positive sample, the genomic DNA of staphylococcus, salmonella typhimurium, avian pathogenic escherichia coli, listeria monocytogenes, listeria lnnock and enterococcus faecalis is used as a template, and ddH 2 O as a negative control, a specificity test for staphylococcus aureus was performed.
Preferably, the method further comprises step S11, repeating the test: 10 in recombinant plasmid pMD19-T-nuc 3 copies/μL、10 4 copies/. mu.L and 10 5 The copies/mu L is used as a template, 3 replicates are set at each concentration, an intragroup repeatability test is carried out, 3 independent tests are respectively carried out on plasmids at each concentration, an intergroup repeatability test is carried out, and the intragroup and intergroup repeats are countedCoefficient of variation (CV value) of sexual test to evaluate repeatability and stability of the detection method of the MIRA technology.
Preferably, the method further comprises step S12, clinical specimen testing: using 51 portions of staphylococcus aureus clinical bacterial liquid sample genome DNA as template, ddH 2 O was used as a negative control and tested using the established assay for MIRA in staphylococcus aureus in this study to evaluate the feasibility of the clinical assay for MIRA technology.
(III) advantageous effects
The invention provides a detection method for rapidly detecting staphylococcus aureus based on an MIRA fluorescence method, which has the following beneficial effects:
the invention detects golden yellow staphylococcus balls by the MIRA technology, has the advantages of short detection time, low detection temperature, low requirements on instruments and equipment, strong specificity, high sensitivity and the like, thereby providing the detection efficiency of molecular biology, reducing the detection threshold, saving the detection time and having important significance for timely and effectively controlling the spread of pathogenic bacteria, preventing food poisoning and reducing the occurrence of food-borne disease events.
Drawings
FIG. 1 is a diagram showing the result of PCR amplification of the pMD19-T-nuc plasmid gene in the present invention;
FIG. 2 is a diagram showing the results of screening and amplifying Staphylococcus aureus MIRA primers according to the present invention;
FIG. 3 is a graph showing the MIRA reaction fluorescence amplification curve of Staphylococcus aureus at 37 ℃ in the present invention;
FIG. 4 is a graph showing the MIRA reaction fluorescence amplification curve of Staphylococcus aureus at 38 ℃ in the present invention;
FIG. 5 is a graph showing the MIRA reaction fluorescence amplification curve of Staphylococcus aureus at 39 ℃ in the present invention;
FIG. 6 is a MIRA reaction fluorescence amplification plot of Staphylococcus aureus at 40 ℃ in the present invention;
FIG. 7 is a MIRA reaction fluorescence amplification plot of Staphylococcus aureus at 41 ℃ in the present invention;
FIG. 8 is a graph of the MIRA (fluorescence-type) sensitivity of Staphylococcus aureus in the present invention;
FIG. 9 is a view of the lower eye of a Staphylococcus aureus MIRA (fluorescent type) sensitivity UV lamp in the present invention;
FIG. 10 is a graph of the sensitivity of Staphylococcus aureus MIRA (fluorescent type) specificity in the present invention;
FIG. 11 is a view of the objective of the present invention under the UV lamp with specific sensitivity for Staphylococcus aureus MIRA (fluorescent type);
fig. 12 is a graph showing the results of part of s.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that: a detection method for rapidly detecting staphylococcus aureus based on an MIRA fluorescence method comprises the following steps:
s1, bacterial culture: taking out staphylococcus aureus liquid stored at minus 80 ℃, performing resuscitation culture on an LB solid culture medium and a blood plate culture medium by using a four-zone scribing method, culturing for 12-16h in a constant-temperature incubator at 37 ℃, selecting a single colony in 1.5mL of LB liquid culture medium, culturing for 3-6h in a constant-temperature shaking table at 37 ℃, transferring to 20mL of liquid culture medium according to the proportion of 1: 100, and culturing until the liquid culture medium is turbid for later use;
s2, extracting bacterial genome DNA: the bacterial genome is extracted by using a bacterial genome DNA extraction kit (centrifugal column type) of a certain biochemical technology limited company, and the method specifically comprises the following steps:
s21, taking 5mL of the standby bacterial liquid, centrifuging at 9000rpm for 2min, and reserving a precipitate;
s22, adding a GA solution with the ratio of the bacteria solution to the GA solution being 25: 1 into the precipitate, and suspending the solution by using an oscillator;
s23, adding a 10: 1 (GA solution and Proteunese K solution) solution into the tube, and uniformly blowing and beating the solution;
s24, continuously adding a protease K solution and a GB solution of which the GB solution is 1: 1, shaking and uniformly mixing, standing in a 70 ℃ water bath for 8-10min, and instantly centrifuging after the liquid in the test tube is clarified;
s25, adding absolute ethyl alcohol with the ratio of GB solution to absolute ethyl alcohol being 1: 11, and shaking and mixing uniformly;
s26, adding the mixed solution (including floccules) into an adsorption column-collecting tube CB3, centrifuging at 11000rpm for 30S, and discarding liquid in the collecting tube;
s27, adding a GD solution with the ratio of absolute ethyl alcohol to GD solution being 11: 25 into an adsorption column-collecting pipe CB3, centrifuging at 11000rpm for 30S, and discarding liquid in the collecting pipe;
s28, adding a GD solution and a PW rinsing solution in a ratio of 5: 6 into an adsorption column-collection tube CB3, centrifuging at 11000rpm for 30S, and discarding liquid in the collection tube;
s29, repeating the previous step;
s210, centrifuging an empty tube of an adsorption column-collection tube CB3 for 3min, discarding liquid in the collection tube, and standing for several minutes at indoor temperature to evaporate rinsing liquid on the adsorption column;
s211, finally, taking a clean centrifugal tube, putting an adsorption column in the clean centrifugal tube, adding 50-200 mu L of TE solution into the middle part of the adsorption column, centrifuging at 11000rpm for 3min, and collecting liquid in the test tube;
s3, primer and probe design: searching nuc gene of staphylococcus aureus in GenBank (GenBank ID: V01281.1), determining respective highly specific conserved fragment regions by analysis and comparison of Meglign software, designing nuc gene specific MIRA primers (Table 1) and probes (Table 2) with the aid of Primer Premier 5;
primer sequences of nuc of Table 1
Figure BDA0003710229540000071
TABLE 2 Probe sequences for the MIRA technique
Figure BDA0003710229540000072
And according to the fact that the size of a nuc gene fragment of staphylococcus aureus is 687bp, a full-length Primer is designed under the auxiliary design of a Primer Premier 5, PCR amplification is carried out, the PCR system is 20 mu L, wherein 10 mu L of Mix, 1 mu L of an upstream Primer, 1 mu L of a downstream Primer, 2 mu L of a DNA template and 6 mu L of sterilized water are contained, and the reaction procedure is shown in a table 3;
TABLE 3 PCR amplification procedure for fragments of interest
Figure BDA0003710229540000073
Figure BDA0003710229540000081
S4, PCR product recovery and purification: according to the operation of the extraction step of the SanPrep column type DNA gel recovery kit specification, recovering the amplification product for later use;
s5, construction of standard plasmids: according to pMD TM Constructing pMD19-T-nuc recombinant plasmid according to the instruction manual of 19-T Vector Cloning Kit;
s6, recombinant plasmid identification: taking part of culture solution for sequencing service, and performing comparison analysis on the BLAST function of NCBI website and Meglign software and the whole length of gene;
s7, plasmid DNA small extraction: extracting the constructed pMD19-T-nuc recombinant plasmid for later use according to the instruction of using the SanPrep column type plasmid DNA small extraction kit;
s8 establishment of staphylococcus aureus fluorescence detection system
S81, establishing a system: establishing a fluorescence result detection system according to the instruction of the DNA constant-temperature rapid amplification kit (fluorescence type);
s82, primer screening: screening experiments are carried out on the primers of the nuc gene of the staphylococcus aureus by using the fluorescence result detection system established in the previous step, and the results are compared, wherein the fluorescence result detection system is shown in a table 4;
TABLE 4 reaction System
Figure BDA0003710229540000082
Figure BDA0003710229540000091
S83, temperature optimization: using the fluorescence result detection system established in the first step, setting the reaction temperatures to be 38 ℃, 39 ℃, 40 ℃, 41 ℃ and 42 ℃ respectively, screening the optimal reaction temperature pair of the S.aureus system and using the optimal reaction temperature pair for subsequent experiments;
s9, sensitivity test: the plasmid concentration was measured according to the copy number concentration (copies/. mu.L) [ 6.02X 10 ] 23 X concentration (ng/. mu.L). times.10 -9 ]/[ DNA Length X660]The copy number concentration was calculated, and the nuc gene recombinant plasmid (pMD19-T-nuc) was diluted 10-fold at 10-fold dilution concentration 0 copies/μL、10 1 copies/μL、10 2 copies/μL、10 3 copies/μL、10 4 copies/μL、10 5 copies/. mu.L, and sterile water (ddH) 2 O) is used as a negative control of the experiment, and the reaction system uses the system established by the experiment to carry out sensitivity verification and compare with the PCR detection technology;
s10, specificity test: the plasmid constructed by the extracted target gene DNA conservative fragment of the staphylococcus aureus is used as a positive sample, the genomic DNA of staphylococcus, salmonella typhimurium, avian pathogenic escherichia coli, listeria monocytogenes, listeria lnnock and enterococcus faecalis is used as a template, and ddH 2 Taking O as a negative control, and carrying out a specificity experiment of staphylococcus aureus;
s11, repeatability test: 10 with recombinant plasmid pMD19-T-nuc 3 copies/μL、10 4 copies/. mu.L and 10 5 The copies/mu L is used as a template, 3 replicates are set for each concentration, an intragroup repeatability test is carried out, 3 independent tests are respectively carried out on plasmids with each concentration, an intergroup repeatability test is carried out, and an intragroup and intergroup repeatability test is countedThe tested coefficient of variation (CV value) is used for evaluating the repeatability and stability of the detection method of the MIRA technology;
s12, clinical sample detection: using 51 portions of staphylococcus aureus clinical bacterial liquid sample genome DNA as template, ddH 2 O was used as a negative control and tested using the assay for MIRA in staphylococcus aureus established in this study to evaluate the feasibility of clinical testing of the MIRA technique.
Results of the experiment
1. Construction of recombinant plasmid
1.1, constructing pMD-19T-nuc recombinant plasmid: the staphylococcus aureus nuc genome was verified by 1.5% agarose gel electrophoresis on the amplified fragment after PCR amplification, and the results are shown in fig. 1. The target gene fragment which is consistent with the expectation is cut into a target band with the assistance of a blue light gel cutter, subjected to a gel recovery experiment, connected with a pMD19-T vector and transformed into DH5 alpha.
1.2, identifying and sequencing recombinant plasmids: the recombinant plasmid is cooperated with a certain biotechnology limited company to provide sequencing service, sequencing results are compared with sequences under the assistance of a Blast website, a Genbank gene bank and Meglign software of NCBI, and results show that the corresponding rate of the sequencing results and required target gene fragments reaches 100 percent, which indicates that the target gene recombinant plasmid is qualified in construction and has no variation and can be used as positive control.
2. And (3) screening primers of the nuc gene: according to the instruction of the DNA constant temperature rapid amplification kit (fluorescent type) kit, 4 upstream primers, 2 downstream primers and a fluorescent probe are designed aiming at nuc gene by using Primer Premier 5 Primer design software, the upstream and downstream primers are randomly combined, and the nuc-MIRA-F1 and nuc-MIRA-R2 are screened to obtain the optimal effect. The amplification results are shown in FIG. 2.
3. Temperature optimization: the reaction temperatures were set at 37 deg.C, 38 deg.C, 39 deg.C, 40 deg.C and 41 deg.C respectively for screening, the MIRA reaction fluorescence amplification curves at different temperatures are shown in FIGS. 3-7, and the optimal reaction temperature is 38 deg.C.
4. Sensitivity test of staphylococcus aureus: the nuc gene recombinant plasmid of staphylococcus aureus is cultured in LB culture solution overnight, and a small amount of SanPrep column type plasmid DNA is usedAfter extracting the plasmid from the extraction kit, the extracted recombinant plasmid was diluted by 10-fold dilution, and the fluorescence results obtained by the MIRA technique are shown in FIGS. 8-9. The fluorescence result of the MIRA technology shows that the sensitivity of detecting staphylococcus aureus reaches 10 2 copies/μL。
5. Specificity test of staphylococcus aureus: a DNA plasmid miniprep kit is used for extracting a staphylococcus aureus nuc gene recombinant plasmid pMD19-T-nuc, and a specificity experiment is carried out by using an MIRA technology. Gel electrophoresis identification and fluorescence result identification of the specificity results of the MIRA technology are shown in FIGS. 10-11. The result shows that the nuc gene of the staphylococcus aureus is successfully amplified, and other pathogenic bacteria all have amplification curves without cross reaction.
6. Repeatability test of staphylococcus aureus: the research establishes that the nuc gene of staphylococcus aureus is detected based on the MIRA technology, and the number of nuc genes is 10 3 copies/μL、10 4 copies/. mu.L and 10 5 Replicate within and between groups was performed using copies/. mu.L as template. The results are shown in Table 5, in which the intra-group coefficient of variation is 0.627% to 0.950%, and the inter-group coefficient of variation is 1.446% to 2.325%. The coefficient of variation of the two is within 3 percent, which shows that the established MIRA technology of the fluorescence result has higher stability and repeatability.
TABLE 5 MiRA technical fluorescence detection of Staphylococcus aureus in-and inter-group reproducibility tests
Figure BDA0003710229540000111
7. And (3) detecting a clinical sample of staphylococcus aureus: takes 51 parts of staphylococcus aureus clinical sample genome DNA as a template, ddH 2 O is negative control, and the fluorescence amplification curve detected by the MIRA method is shown in figure 12; the experimental data were counted to obtain 51 positive results of the MIRA method, which indicated that the MIRA method for Staphylococcus aureus established in this study has good clinical applicability.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (7)

1. A detection method for rapidly detecting staphylococcus aureus based on an MIRA fluorescence method is characterized by comprising the following steps:
s1, bacterial culture: taking out staphylococcus aureus liquid stored at minus 80 ℃, performing resuscitation culture on an LB solid culture medium and a blood plate culture medium by using a four-zone scribing method, culturing for 12-16h in a constant-temperature incubator at 37 ℃, selecting a single colony in 1.5mL of LB liquid culture medium, culturing for 3-6h in a constant-temperature shaking table at 37 ℃, transferring to 20mL of liquid culture medium according to the proportion of 1: 100, and culturing until the liquid culture medium is turbid for later use;
s2, extracting bacterial genome DNA: extracting bacterial genome by using a bacterial genome DNA extraction kit (centrifugal column type) of a certain Biochemical technology Co., Ltd;
s3, primer and probe design: searching nuc gene of staphylococcus aureus in GenBank, analyzing and comparing by Meglign software, determining respective high-specificity conserved fragment regions, and designing nuc gene-specific MIRA primers and probes with the aid of Primer Premier 5; and designing a full-length Primer under the auxiliary design of Primer Premier 5 according to the size of the nuc gene fragment of the staphylococcus aureus being 687bp, and carrying out PCR amplification;
s4, PCR product recovery and purification: according to the operation of the extraction step of the SanPrep column type DNA gel recovery kit specification, recovering the amplification product for later use;
s5, construction of standard plasmids: according to pMD TM 19-T Vector Cloning Kit instructions, construct pMD19-T-nuc recombinant plasmid;
s6, recombinant plasmid identification: taking part of culture solution for sequencing service, and performing comparison analysis on the BLAST function of NCBI website and Meglign software and the whole length of gene;
s7, plasmid DNA small-scale extraction: extracting the constructed pMD19-T-nuc recombinant plasmid for later use according to the instruction of using the SanPrep column type plasmid DNA small extraction kit;
s8 establishment of Staphylococcus aureus fluorescence detection system
S81, establishing a system: establishing a fluorescence result detection system according to the instruction of the DNA constant-temperature rapid amplification kit (fluorescence type);
s82, primer screening: screening experiments are carried out on the nuc gene primer of the staphylococcus aureus by using the fluorescence result detection system established in the last step, and results are compared;
s83, temperature optimization: and (3) screening the optimal reaction temperature pair of the S.aureus system by using the fluorescence result detection system established in the first step and setting the reaction temperatures to be 38 ℃, 39 ℃, 40 ℃, 41 ℃ and 42 ℃ respectively, and using the optimal reaction temperature pair for subsequent experiments.
2. The detection method for rapidly detecting staphylococcus aureus based on the MIRA fluorescence method according to claim 1, which is characterized in that: the specific steps of step S2 are:
s21, taking 5mL of the standby bacterial liquid, centrifuging at 9000rpm for 2min, and leaving a precipitate;
s22, adding a GA solution with the ratio of the bacterial liquid to the GA solution being 25: 1 into the precipitate, and suspending the solution by using an oscillator;
s23, adding a 10: 1 (GA solution and Proteunese K solution) solution into the tube, and uniformly blowing and beating the solution;
s24, continuously adding a protease K solution and a GB solution of which the GB solution is 1: 1, shaking and uniformly mixing, standing in a 70 ℃ water bath for 8-10min, and instantly centrifuging after the liquid in the test tube is clarified;
s25, adding absolute ethyl alcohol with the ratio of GB solution to absolute ethyl alcohol being 1: 11, and shaking and mixing uniformly;
s26, adding the uniformly mixed solution (including floccules) into an adsorption column-collection tube CB3, centrifuging at 11000rpm for 30S, and discarding the liquid in the collection tube;
s27, adding a GD solution with the ratio of absolute ethyl alcohol to GD solution being 11: 25 into an adsorption column-collection tube CB3, centrifuging at 11000rpm for 30S, and discarding liquid in the collection tube;
s28, adding a GD solution and a PW rinsing solution in a ratio of 5: 6 into an adsorption column-collection pipe CB3, centrifuging at 11000rpm for 30S, and discarding liquid in the collection pipe;
s29, repeating the previous step;
s210, centrifuging an empty tube of an adsorption column-collection tube CB3 for 3min, discarding liquid in the collection tube, and standing for several minutes at indoor temperature to evaporate rinsing liquid on the adsorption column;
s211, finally, taking a clean centrifuge tube, putting an adsorption column in the clean centrifuge tube, adding 50-200 mu L of TE solution into the middle part of the adsorption column, centrifuging at 11000rpm for 3min, and collecting liquid in the test tube.
3. The detection method for rapidly detecting staphylococcus aureus based on MIRA fluorescence method according to claim 1, wherein the detection method comprises the following steps: the PCR system in the step S3 is 20 μ L, wherein Mix is 10 μ L, the upstream primer is 1 μ L, the downstream primer is 1 μ L, the DNA template is 2 μ L, and the sterilized water is 6 μ L.
4. The detection method for rapidly detecting staphylococcus aureus based on the MIRA fluorescence method according to claim 1, which is characterized in that: the method further comprises step S9, sensitivity test: the plasmid concentration was measured according to the copy number concentration (copies/. mu.L) [ 6.02X 10 ] 23 X concentration (ng/. mu.L). times.10 -9 ]/[ DNA Length X660]The copy number concentration was calculated, and the nuc gene recombinant plasmid (pMD19-T-nuc) was diluted 10-fold at 10-fold dilution concentration 0 copies/μL、10 1 copies/μL、10 2 copies/μL、10 3 copies/μL、10 4 copies/μL、10 5 copies/. mu.L, and sterile water (ddH) 2 O) is used as a negative control of the experiment, and the reaction system uses the system established in the experiment to carry out sensitivity verification and compare with the PCR detection technology.
5. The detection method for rapidly detecting staphylococcus aureus based on the MIRA fluorescence method according to claim 1, which is characterized in that: the method further comprises step S10, specificity test: the plasmid constructed by the extracted target gene DNA conservative fragment of the staphylococcus aureus is used as a positive sample, the genomic DNA of staphylococcus, salmonella typhimurium, avian pathogenic escherichia coli, listeria monocytogenes, listeria lnnock and enterococcus faecalis is used as a template, and ddH 2 O as a negative control, a specificity test for staphylococcus aureus was performed.
6. The detection method for rapidly detecting staphylococcus aureus based on the MIRA fluorescence method according to claim 1, which is characterized in that: the method further comprises step S11, a repeatability test: 10 with recombinant plasmid pMD19-T-nuc 3 copies/μL、10 4 copies/. mu.L and 10 5 The plasmid with each concentration is respectively subjected to 3 independent tests, the repeatability tests among groups are carried out, and the coefficient of variation (CV value) of the repeatability tests among groups and groups is counted so as to evaluate the repeatability and stability of the MIRA technical detection method.
7. The detection method for rapidly detecting staphylococcus aureus based on the MIRA fluorescence method according to claim 1, which is characterized in that: the method further comprises step S12, clinical specimen testing: using 51 portions of staphylococcus aureus clinical bacterial liquid sample genome DNA as template, ddH 2 O was used as a negative control and tested using the established assay for MIRA in staphylococcus aureus in this study to evaluate the feasibility of the clinical assay for MIRA technology.
CN202210724003.4A 2022-06-23 2022-06-23 Detection method for rapidly detecting staphylococcus aureus based on MIRA fluorescence method Pending CN114990244A (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116064865A (en) * 2022-09-23 2023-05-05 成都中医药大学 Kit and method for detecting staphylococcus aureus based on multienzyme isothermal nucleic acid rapid amplification technology

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116064865A (en) * 2022-09-23 2023-05-05 成都中医药大学 Kit and method for detecting staphylococcus aureus based on multienzyme isothermal nucleic acid rapid amplification technology

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