CN112501323A - Staphylococcus aureus amplification primer based on RAA-LF technology and application thereof - Google Patents
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Abstract
The invention discloses a staphylococcus aureus amplification primer based on RAA-LF technology and application thereof, wherein the amplification primer comprises an upstream primer shown as SEQ ID NO.1 and a downstream primer shown as SEQ ID NO.2, and the 5' ends of the upstream primer and the downstream primer are respectively marked by fluorescein and biotin compounds. The kit contains the amplification primer pair as described above. The invention combines the RAA technology with the lateral flow chromatography test strip, and realizes the accurate and high-efficiency detection of the staphylococcus aureus. The amplification primer disclosed by the invention is strong in specificity, high in sensitivity and good in repeatability; the detection method is simple, rapid, convenient, strong in specificity and high in sensitivity, and has great application prospects.
Description
Technical Field
The invention belongs to the technical field of molecular biology detection, and relates to a staphylococcus aureus amplification primer based on an RAA-LF technology and application thereof, in particular to an RAA-LF primer for quickly detecting staphylococcus aureus, a kit prepared from the RAA-LF primer and a detection method of the kit.
Background
Bovine Mastitis (BM) is the most common disease of cows worldwide, seriously affecting the health and welfare of infected animals. Research shows that the economic loss caused by cow mastitis reaches up to $ 350 billion every year around the world, the incidence rate of cow mastitis reaches about 50% in the countries of the United states, the United kingdom, the Japan and the like, and the incidence rate of cow mastitis in China reaches about 70%, so that the method has great influence on cow feeding management. Cow mastitis is an inflammatory reaction caused by physical, chemical or microbial factors stimulating the mammary gland of a cow. The invasion of microorganisms is a non-negligible factor, and staphylococcus aureus, which is represented by contact pathogenic bacteria, is one of the main pathogens. In modern and intensive-scale dairy farms, pathogenic bacteria are identified timely and accurately, which has a direct influence on whether mastitis can be effectively prevented and controlled, a good treatment effect is obtained, and economic loss can be reduced to the maximum extent.
The conventional methods for diagnosing the mastitis pathogenic bacteria of the dairy cattle comprise a conventional microorganism biochemical identification method, a full-automatic microorganism identification system, 16SrRNA gene sequence sequencing, PCR, a gene chip, a loop-mediated isothermal amplification technology and the like. The traditional detection method is complicated in operation steps, time-consuming and labor-consuming, and cannot meet the field implementation of detection. The gene chip technology and the loop-mediated isothermal amplification technology have the defects of high cost, poor repeatability, easy occurrence of false positive results and the like. PCR detection reagent instruments are expensive; and precise detection equipment and strict operation flow are required; the requirements of on-site rapid detection and large-scale monitoring cannot be met, and the application of the method in basic units is limited. The appearance of the nucleic acid isothermal amplification technology effectively makes up the defects of the existing molecular detection method, expands the application range of the nucleic acid detection technology, develops towards the direction of rapidness, low cost and miniaturization on the basis of ensuring high sensitivity and high specificity, and gradually becomes a research hotspot in the field of pathogenic bacteria nucleic acid detection. The RAA technology has been applied to the rapid detection of viruses, bacteria, mycoplasma, parasites, etc. CN 110564881A discloses a method for detecting vibrio parahaemolyticus by a recombinase isothermal amplification technology, RAA-LF is used for detecting pathogens for the first time, and a brand new thought is provided for detecting pathogens. However, the related research of the RAA method for detecting bovine-derived staphylococcus aureus is still in the blank stage, and the research difficulty of the RAA method for detecting staphylococcus aureus is the development of a staphylococcus aureus amplification primer based on the RAA-LF technology, and how to ensure the specificity and amplification speed of the primer can be a core problem that troubles the technicians in the field because the sample may contain not only staphylococcus aureus but also many other strains such as escherichia coli and klebsiella pneumoniae.
Therefore, it is of great practical significance to develop a RAA method capable of rapidly detecting staphylococcus aureus on site.
Disclosure of Invention
The invention aims to overcome the defects that the prior art has low detection speed and can not complete detection on site, and provides an RAA method capable of quickly detecting staphylococcus aureus on site, in particular to a staphylococcus aureus amplification primer based on an RAA-LF technology.
In order to achieve the purpose, the invention provides the following technical scheme:
a staphylococcus aureus amplification primer based on RAA-LF technology comprises an upstream primer and a downstream primer which respectively contain nucleotide sequences shown as follows:
f (upstream primer): 5'-GACAAAGGTCAAAGAACTGATAAATATGGA-3', respectively;
r (downstream primer): 5'-TTCACTTTTTCTTAAAAGTTGTTCATGTGT-3' are provided.
The staphylococcus aureus amplification primer based on the RAA-LF technology realizes amplification by aiming at a specific gene conserved region of the staphylococcus aureus, so that the specificity of the primer can be ensured, the influence of other strains on the detection of the staphylococcus aureus in the later period can be avoided, and meanwhile, the specific selection is carried out on the amplification primer so as to ensure the amplification speed (sensitivity) of the primer.
As a preferred technical scheme:
the nucleotide sequences of the upstream primer and the downstream primer of the staphylococcus aureus amplification primer based on the RAA-LF technology are as follows:
F：5’-GACAAAGGTCAAAGAACTGATAAATATGGA-3’；
R：5’-TTCACTTTTTCTTAAAAGTTGTTCATGTGT-3’。
as for the staphylococcus aureus amplification primer based on the RAA-LF technology, the 5' ends of the upstream primer and the downstream primer are respectively marked by fluorescein and biotin compounds. The scope of the present invention is not limited thereto, and the specific markers can be selected by those skilled in the art according to the actual situation, and only one possible technical solution is given here.
The staphylococcus aureus amplification primer based on the RAA-LF technology is described above, and the fluorescein is carboxyfluorescein 6-FAM; the Biotin compound is Biotin. The scope of the present invention is not limited thereto, and those skilled in the art can select appropriate fluorescein and biotin compounds according to actual needs.
The staphylococcus aureus amplification primer based on the RAA-LF technology is bovine-derived staphylococcus aureus, is not limited to the bovine-derived staphylococcus aureus and can be applied to other staphylococcus aureus.
The invention also provides a staphylococcus aureus detection kit based on the RAA-LF technology, and the kit contains the amplification primer pair.
As a preferred technical scheme:
the staphylococcus aureus detection kit based on the RAA-LF technology further comprises an RAA reaction kit and a lateral flow chromatography test strip. In addition, the staphylococcus aureus detection kit can also be matched with a staphylococcus aureus nuc gene positive control sample and a staphylococcus aureus nuc gene negative control sample, and operators can compare the staphylococcus aureus nuc gene positive control sample and the staphylococcus aureus nuc gene negative control sample conveniently.
The kit for detecting staphylococcus aureus based on the RAA-LF technology comprises a reaction tube containing freeze-dried enzyme powder, a basic buffer solution, sterile deionized water and magnesium acetate, and other articles necessary for RAA reaction are contained in the kit for RAA reaction.
The invention also provides a method for detecting RAA-LF of staphylococcus aureus, which comprises the following steps:
(1) extracting DNA of a sample to be detected;
(2) preparing the primers and the DNA of the sample to be detected obtained in the step (1) to obtain an RAA reaction system, and carrying out RAA amplification reaction on the RAA reaction system at a constant temperature of 33 ℃ for 20-30 min;
(3) dropwise adding the RAA amplification product obtained in the step (2) into a sample adding area of a lateral flow chromatography test strip, vertically inserting the tail end of the sample adding area of the lateral flow chromatography test strip into a centrifugal tube containing LF buffer solution, taking out the lateral flow chromatography test strip after 5-10 min, observing and recording the result;
if the lateral flow chromatography test strip is provided with strips at the positions of the quality control line and the detection line, the sample to be detected contains staphylococcus aureus;
if the lateral flow chromatography test strip is provided with a strip at the position of the quality control line and no strip is arranged at the position of the detection line, the sample to be detected does not contain staphylococcus aureus, or the content of staphylococcus aureus in the sample to be detected is lower than the lowest detection amount;
if the lateral flow chromatography test strip has no strip at the position of the quality control line and the detection line, the failure of the lateral flow chromatography test strip needs to be detected again.
The RAA-LF detection method for staphylococcus aureus is simple and convenient to operate integrally, low in experimental conditions (only a constant temperature environment is needed), short in required time, capable of directly confirming the test result through naked eyes, capable of achieving on-site rapid detection of staphylococcus aureus, greatly reducing hardware limitation of rapid detection of staphylococcus aureus (detection can be completed on site only through one constant temperature device), and extremely wide in application prospect.
As a preferred technical scheme:
in the above method for detecting the RAA-LF of the staphylococcus aureus, the RAA reaction system in the step (2) contains the upstream primer and the downstream primer at the concentration of 200nM and 14mM magnesium acetate.
Has the advantages that:
(1) the staphylococcus aureus amplification primer based on the RAA-LF technology has the advantages of strong specificity, high sensitivity and good repeatability;
(2) according to the RAA-LF detection method for staphylococcus aureus, double-labeled products are formed by rapid amplification in a constant temperature device, and the result can be judged through LF visual observation, so that a specific probe is not needed, the detection is more convenient, and the cost is reduced;
(3) the RAA-LF detection method for staphylococcus aureus has the characteristics of simplicity in operation, good specificity, high sensitivity, portability and the like, and is more suitable for clinical field detection;
(4) the RAA-LF detection method for staphylococcus aureus can be used for timely and accurately identifying bovine-derived staphylococcus aureus in a modern and intensive-scale dairy farm, has direct influence on whether mastitis can be effectively prevented and controlled, a good treatment effect is obtained, and economic loss is reduced to the maximum extent, and has great application prospect.
Drawings
FIG. 1 is a diagram showing the screening results of the amplification primers of the present invention (wherein 1, 2, and 3 represent the first primer pair, the second primer pair, and the third primer pair, respectively);
FIG. 2 is a graph showing the optimized result of the RAA-LF amplification reaction conditions of Staphylococcus aureus of the present invention (wherein A is the optimized result of the concentration of the primer pair, B is the optimized result of the reaction temperature, and C is the optimized result of the reaction time);
FIG. 3 is a diagram showing the result of verifying the specificity of the Staphylococcus aureus amplification primer of the present invention;
FIG. 4 is a graph showing the result of the sensitivity verification of the Staphylococcus aureus amplification primers of the present invention (wherein A is a graph showing the result of RAA-LF detection, B is a graph showing the result of conventional PCR detection, and C is a graph showing the result of RAA-LF detection of the sensitivity of a pure culture of bacteria).
Detailed Description
For a better understanding of the present invention, reference is made to the following detailed description and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Modifications and substitutions in detail and form may be made to the present invention without departing from the spirit and scope thereof, and it is intended that all such modifications and substitutions fall within the scope of the present invention.
Unless otherwise specified, the raw materials and chemical reagents in the examples are all conventional commercial products, and the technical means are conventional means used by those skilled in the art.
Example 1
Designing and screening bovine-derived staphylococcus aureus RAA-LF primers (bovine-derived staphylococcus aureus amplification primers based on RAA-LF technology):
targeting a specific nuc gene sequence of staphylococcus aureus according to the RAA Primer design principle, and designing primers through Primer-BLAST and Primer 5; downloading a plurality of specific genes nuc (LS483300.1, LR134088.1, EF529607.1, EF529597.1 and AJ938182.1) from GenBank, designing primers by comparing and analyzing homology and Conserved regions, using NCBI-BLAST to compare the homology of the primers, comparing highly Conserved regions of target sequences by means of Conserved Domain Search and evaluating the specificity of the primer sequences by means of 2% agarose gel electrophoresis amplification effect, and screening the primers. Three pairs of primers (see the table below) are obtained through common design, and after a single optimal primer is determined, fluorescein 6-FAM and Biotin are respectively labeled at the upstream and downstream 5' ends and are used for carrying out test strip detection by combining colloidal gold particles.
Through multiple repeated tests and agarose gel electrophoresis (the result is shown in figure 1), through the evaluation of stability, sensitivity and specificity, the method is preferably RAA-F2/RAA-R2, and specifically comprises the following steps:
SEQ ID No.1：
5’-GACAAAGGTCAAAGAACTGATAAATATGGA-3’；
SEQ ID No.2：
5’-TTCACTTTTTCTTAAAAGTTGTTCATGTGT-3’。
example 2
A staphylococcus aureus detection kit based on an RAA-LF technology comprises a staphylococcus aureus amplification primer based on an RAA-LF technology of SEQ ID Nos. 1 and 2, an RAA reaction kit, a lateral flow chromatography test strip, a staphylococcus aureus nuc gene positive control sample and a negative control sample, wherein the RAA reaction kit comprises a reaction tube containing freeze-dried enzyme powder, a basic buffer solution (containing dNTP), sterile deionized water and magnesium acetate.
Example 3
Reaction conditions of RAA-LF detection of bovine-derived staphylococcus aureus are optimized:
the bovine-derived staphylococcus aureus DNA template is obtained by extracting a bacterial genome DNA extraction kit (Tiangen Biochemical technology (Beijing) Co., Ltd., product number: DP302), is convenient for sample treatment in subsequent researches, and is extracted by a common thermal cracking method in bacterial pure liquid culture and simulation samples.
RAA amplification was performed using a RAA reaction kit (purchased from Kyoto Qitian Biotechnology Ltd., Jiangsu, product No. B000000A) comprising a reaction tube containing lyophilized enzyme powder, a base buffer (containing dNTP), sterile deionized water and 280mM magnesium acetate. The lateral flow chromatography test strip is purchased from Hangzhou Yosidao biotechnology limited and has the product model number of D003-03.
The RAA amplification system is specifically as follows: firstly, preparing and uniformly mixing 25 mu L of basic buffer solution, 1 mu L of each of upstream and downstream primers, 1 mu L of template DNA and 19.5 mu L of sterile deionized water, adding the mixture into a reaction unit tube, slightly and uniformly mixing, dropwise adding 2.5 mu L of magnesium acetate into the tube wall, and centrifugally and uniformly mixing, wherein the total volume of an RAA amplification reaction system is 50 mu L;
(1) optimization of upstream and downstream primer concentrations:
preparing RAA amplification systems with upstream and downstream primer concentrations of 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM and 6.25nM respectively, placing each RAA amplification system at 39 ℃ for reaction for 30min, after the reaction is finished, taking 10 microliter of nucleic acid amplification product to be dripped into a sample adding area, vertically inserting the tail end of the sample adding area of a test strip into an EP (enhanced noise amplifier) tube containing 100 microliter LF (LF) buffer solution, taking out the test strip after 5min for observation and recording the result, wherein the result is shown in figure 2A, when the upstream and downstream primer concentrations are more than or equal to 200nM, the quality control line and the detection line can both see obvious feedback, and the upstream and downstream primer concentrations are selected to be 200nM for reducing the cost;
(2) optimization of reaction temperature for RAA reaction:
each RAA amplification system (the concentration of the upstream primer and the downstream primer is 200nM) is placed at 25 ℃, 31 ℃, 33 ℃, 35 ℃, 37 ℃, 39 ℃, 41 ℃ and 43 ℃ for reaction for 30min, 10 mu L of nucleic acid amplification product is dripped to a sample adding area after the reaction is finished, the tail end of the sample adding area of a test strip is vertically inserted into an EP tube containing 100 mu L of LF buffer solution, the test strip is taken out after 5min for observation and result recording, the result is shown in figure 2B, and the feedback of a quality control line and a detection line corresponding to a group with the reaction temperature of 33 ℃ is found to be most obvious, so the reaction temperature of the selected RAA is 33 ℃;
(3) optimization of reaction time for RAA reaction:
each RAA amplification system (the concentration of the upstream primer and the downstream primer is 200nM) is placed at 33 ℃ for reaction for 5min, 10min, 15min, 20min, 25min and 30min, 10 microliter of nucleic acid amplification product is dripped into a sample adding area after the reaction is finished, the tail end of the sample adding area of a test strip is vertically inserted into an EP tube containing 100 microliter of LF buffer solution, the test strip is taken out after 5min for observation and result recording, the result is shown in figure 2C, the quality control line and the detection line corresponding to a group with the reaction time of more than or equal to 20min can both see obvious feedback, and the RAA reaction time is selected to be 20min for shortening the detection time;
the RAA amplification reaction system obtained by final optimization contains 200nM of each upstream primer and downstream primer and 14mM of magnesium acetate; the optimized RAA amplification reaction conditions are as follows: the reaction temperature was 33 ℃ and the reaction time was 20 min.
The optimized RAA-LF detection method of the staphylococcus aureus comprises the following specific steps:
1) extracting DNA of a sample to be detected;
2) firstly, preparing and uniformly mixing 25 mu L of basic buffer solution, 1 mu L of each of 10 mu M upstream primer and downstream primer, 1 mu L of DNA of a sample to be detected obtained in the step 1) and 19.5 mu L of sterile deionized water, adding the mixture into a reaction unit tube, dropwise adding 2.5 mu L of 280mM magnesium acetate into the tube wall after the mixture is lightly and uniformly mixed, centrifuging and uniformly mixing, and carrying out RAA amplification reaction for 20min at constant temperature of 33 ℃;
3) dripping 10 mu L of RAA amplification product obtained in the step 2) into a sample adding area of the lateral flow chromatography test strip, vertically inserting the tail end of the sample adding area of the lateral flow chromatography test strip into a centrifuge tube containing 100 mu L of LF buffer solution, taking out the lateral flow chromatography test strip after 5min, observing and recording the result;
if the lateral flow chromatography test strip is provided with strips at the positions of the quality control line and the detection line, the sample to be detected contains staphylococcus aureus;
if the lateral flow chromatography test strip is provided with a strip at the position of the quality control line and no strip is arranged at the position of the detection line, the sample to be detected does not contain staphylococcus aureus, or the content of staphylococcus aureus in the sample to be detected is lower than the lowest detection amount;
if the lateral flow chromatography test strip has no strip at the position of the quality control line and the detection line, the failure of the lateral flow chromatography test strip needs to be detected again.
Example 4
And (3) specificity verification of RAA-LF detection of bovine-derived staphylococcus aureus:
the RAA-LF detection method of staphylococcus aureus optimized in the embodiment 3 is used for respectively detecting 13 pathogenic bacteria (shown in the table 1) and analyzing and evaluating the specificity of the amplification primers;
TABLE 1 strains for RAA-LF detection specificity analysis
The result chart of the bovine-derived staphylococcus aureus amplification primer specificity verification is shown in fig. 3, and it can be seen from the chart that the results of other 10 pathogenic bacteria are negative except that the staphylococcus aureus nucleic acid has an amplification strip, and the result is positive, which indicates that the amplification primer of the invention has good specificity for staphylococcus aureus.
Example 5
And (3) verifying the sensitivity of RAA-LF detection of bovine-derived staphylococcus aureus:
the genomic DNA of Staphylococcus aureus CDC AB91093 was used as a template, and the concentrations thereof were diluted to 4 ng. mu.L, respectively-1，400pg·μL-1，40pg·μL-1，4pg·μL-1，400fg·μL-1，40fg·μL-1And 4 fg. mu.L-1The detection sensitivity of the RAA-LF detection method of staphylococcus aureus optimized in the embodiment 3 is adopted.
Meanwhile, the genome DNA with the same concentration is used as a template to carry out conventional PCR amplification, and the sensitivity of the method and the PCR technology is compared.
Conventional PCR was a 25 μ L system: PremixTaqTM(TaKaRa TaqTMVersion 2.0) 12.5. mu.L, upstream and downstream primers (10. mu.M) 0.5. mu.L each, template 1. mu.L, and the remainder filled with sterile deionized water. PCR reaction procedure: 94 ℃ for 5 min; 30s at 94 ℃, 30s at 56 ℃, 50s at 72 ℃ and 30 cycles; 72 ℃ for 5 min; 4 ℃ is prepared.
As shown in FIG. 4, the lowest detection limit of RAA-LF in the present invention was 4 fg. mu.L-1(FIG. 4A), the lowest detection limit of the conventional PCR was 400 fg. mu.L-1(FIG. 4B), the result shows that the method of the invention has higher sensitivity compared with the conventional PCR, and can meet the requirement of detecting the bovine-derived staphylococcus aureus with lower content.
Example 6
RAA-LF detection of bovine-derived Staphylococcus aureus pure culture:
and (3) inoculating the staphylococcus aureus to 5mL of TSB culture medium, culturing overnight at 37 ℃, counting the staphylococcus aureus by adopting a plate counting method, diluting pure bacteria liquid by using normal saline in a 10-time gradient manner, and detecting according to the RAA-LF detection method of the staphylococcus aureus optimized in the embodiment 3. The count of the pure bacterial culture solution was 1.83X 109CFU/mL, diluted to 1.83X 10 respectively6CFU/mL、1.83×105CFU/mL、1.83×104CFU/mL、1.83×103CFU/mL、1.83×102CFU/mL, 1.83 CFU/mL. The result of the pure culture detection is shown in FIG. 4C, and the lowest detection limit of the method for detecting the pure culture of bacteria is 1.83X 102CFU/mL。
Example 7
And (3) actual sample detection:
64 clinical samples are collected in a dairy farm in the peripheral area of Shanghai strictly according to a standard milk sampling procedure, stored in an ice box and taken back to the laboratory as soon as possible for relevant detection. The traditional microorganism isolation culture method, the PCR method and the visual RAA-LF method established in the research are adopted for detection, the detection results of the three methods are counted, and the sensitivity, the specificity and the total coincidence rate of each method are analyzed according to a formula.
Among the detection methods, the calculation formulas of sensitivity, specificity and coincidence rate are as follows:
sensitivity ═ true positive number)/(true positive number + false negative number;
specificity ═ (true negative number)/(true negative number + false positive number);
the total coincidence rate is (true positive number + true negative number)/(total number of test subjects).
The results are shown in the following table 2, compared with the traditional microorganism isolation culture method, the visual RAA-LF method has the sensitivity up to 100%, the specificity of 93.75%, and the total coincidence rate of the two methods is 95.31%; compared with the PCR method, the visual RAA-LF method has the sensitivity as high as 100 percent and the specificity of 97.83 percent, and the total coincidence rate of the two methods is 98.44 percent. Therefore, the RAA-LF method can be used as an effective means to detect the staphylococcus aureus cow mastitis on site.
TABLE 2 actual sample test results
The verification proves that the staphylococcus aureus amplification primer based on the RAA-LF technology has strong specificity, high sensitivity and good repeatability; the RAA-LF detection method for staphylococcus aureus can complete detection quickly, is simple, quick and convenient, has strong specificity and high sensitivity, and has wide application prospect.
Although specific embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these embodiments are merely illustrative and various changes or modifications may be made without departing from the principles and spirit of the invention.
Sequence listing
<110> Shanghai ocean university
<120> staphylococcus aureus amplification primer based on RAA-LF technology and application
<141> 2020-11-24
<150> 2020107836630
<151> 2020-08-06
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Claims (10)
1. A staphylococcus aureus amplification primer based on RAA-LF technology is characterized in that an upstream primer and a downstream primer respectively contain nucleotide sequences shown as follows:
F：5’-GACAAAGGTCAAAGAACTGATAAATATGGA-3’；
R：5’-TTCACTTTTTCTTAAAAGTTGTTCATGTGT-3’。
2. the staphylococcus aureus amplification primer based on the RAA-LF technology as claimed in claim 1, wherein the nucleotide sequences of the upstream primer and the downstream primer are as follows:
F：5’-GACAAAGGTCAAAGAACTGATAAATATGGA-3’；
R：5’-TTCACTTTTTCTTAAAAGTTGTTCATGTGT-3’。
3. the staphylococcus aureus amplification primer based on the RAA-LF technology as claimed in claim 1 or 2, wherein the 5' ends of the upstream primer and the downstream primer are labeled with fluorescein and biotin compounds, respectively.
4. The staphylococcus aureus amplification primer based on the RAA-LF technology as claimed in claim 3, wherein the fluorescein is carboxyfluorescein 6-FAM, and the Biotin compound is Biotin.
5. The staphylococcus aureus amplification primer based on the RAA-LF technology as claimed in claim 1, wherein the staphylococcus aureus is bovine-derived staphylococcus aureus.
6. A staphylococcus aureus detection kit based on the RAA-LF technique, wherein the kit comprises the amplification primer pair of claim 1 or 2.
7. The staphylococcus aureus detection kit based on the RAA-LF technology of claim 6, further comprising a RAA reaction kit and a lateral flow chromatography test strip.
8. The staphylococcus aureus detection kit based on the RAA-LF technology of claim 7, wherein the RAA reaction kit comprises a reaction tube containing lyophilized enzyme powder, a base buffer solution, sterile deionized water and magnesium acetate.
9. A RAA-LF detection method of staphylococcus aureus is characterized by comprising the following steps:
(1) extracting DNA of a sample to be detected;
(2) preparing the primers according to any one of claims 1 to 5 and the DNA of the sample to be detected obtained in the step (1) to obtain an RAA reaction system, wherein the RAA reaction system is subjected to RAA amplification reaction at a constant temperature of 33 ℃ for 20-30 min;
(3) dropwise adding the RAA amplification product obtained in the step (2) into a sample adding area of a lateral flow chromatography test strip, vertically inserting the tail end of the sample adding area of the lateral flow chromatography test strip into a centrifugal tube containing LF buffer solution, taking out the lateral flow chromatography test strip after 5-10 min, observing and recording the result;
if the lateral flow chromatography test strip is provided with strips at the positions of the quality control line and the detection line, the sample to be detected contains staphylococcus aureus;
if the lateral flow chromatography test strip is provided with a strip at the position of the quality control line and no strip is arranged at the position of the detection line, the sample to be detected does not contain staphylococcus aureus, or the content of staphylococcus aureus in the sample to be detected is lower than the lowest detection amount;
if the lateral flow chromatography test strip has no strip at the position of the quality control line and the detection line, the failure of the lateral flow chromatography test strip needs to be detected again.
10. The method according to claim 9, wherein the RAA-LF detection method for Staphylococcus aureus comprises the upstream primer and the downstream primer in the RAA reaction system in step (2) at a concentration of 200nM and 14mM magnesium acetate.
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| CN104946780A (en) * | 2015-07-14 | 2015-09-30 | 东北农业大学 | Kit capable of rapidly detecting staphylococcus aureus in meat and application |
| CN108165611A (en) * | 2017-12-26 | 2018-06-15 | 天津科技大学 | A kind of methods and applications of recombinase polymerase constant-temperature amplification combination ELISA test strip staphylococcus aureus |
| CN110195119A (en) * | 2019-06-05 | 2019-09-03 | 中国农业科学院上海兽医研究所 | A kind of kit for detecting staphylococcus aureus, primer pair, probe and method |
| CN110564881A (en) * | 2019-10-21 | 2019-12-13 | 上海海洋大学 | Method for detecting vibrio parahaemolyticus by recombinase isothermal amplification technology |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104946780A (en) * | 2015-07-14 | 2015-09-30 | 东北农业大学 | Kit capable of rapidly detecting staphylococcus aureus in meat and application |
| CN108165611A (en) * | 2017-12-26 | 2018-06-15 | 天津科技大学 | A kind of methods and applications of recombinase polymerase constant-temperature amplification combination ELISA test strip staphylococcus aureus |
| CN110195119A (en) * | 2019-06-05 | 2019-09-03 | 中国农业科学院上海兽医研究所 | A kind of kit for detecting staphylococcus aureus, primer pair, probe and method |
| CN110564881A (en) * | 2019-10-21 | 2019-12-13 | 上海海洋大学 | Method for detecting vibrio parahaemolyticus by recombinase isothermal amplification technology |
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