CN112553355A - Primer probe combination, kit and method for detecting listeria monocytogenes based on RAA technology - Google Patents
Primer probe combination, kit and method for detecting listeria monocytogenes based on RAA technology Download PDFInfo
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Abstract
The invention belongs to the technical field of microbial detection, and particularly relates to a primer probe combination, a kit and a method for detecting listeria monocytogenes based on RAA technology. The primer probe combination comprises a primer pair and a probe, wherein the primer pair comprises an upstream primer and a downstream primer, the sequence of the upstream primer is shown as SEQ ID NO.1, the sequence of the downstream primer is shown as SEQ ID NO.2, and the sequence of the probe is shown as SEQ ID NO. 5. The reagent and the method for detecting the listeria monocytogenes have the advantages of good specificity, high sensitivity, high accuracy, simple operation, low cost, capability of performing laboratory detection and field rapid detection, and good application prospect.
Description
Technical Field
The invention belongs to the technical field of microbial detection, and particularly relates to a primer probe combination, a kit and a method for detecting listeria monocytogenes based on RAA technology.
Background
Listeria monocytogenes (Listeria monocytogenes) is widely found in nature, easily contaminates meat, milk, seafood, vegetables, etc., and can survive for a long time in food products due to its easy formation of biofilm. The world health organization has reported in reports on food poisoning that 4-8% of seafood, 5-10% of milk and products and more than 30% of meat products are contaminated with listeria monocytogenes. And the bacteria can also grow and breed in foods preserved in a refrigerator at 4 ℃, so that the potential hazard of the bacteria is further increased. Eating food contaminated with listeria monocytogenes can cause myocarditis, meningitis, gastroenteritis, pneumonia, and the like, in addition to general bacterial food poisoning symptoms such as nausea, sepsis, diarrhea, and the like. In 1986, research centers for listeria were established by the WHO and the FDA to specially coordinate research works such as etiology, epidemiology and clinic of the listeria, which is listed as one of four major food pathogens in the 90 s, and clinical diseases and food pollution problems caused by listeria monocytogenes in europe, the united states and japan are the first place in bacterial food poisoning. Therefore, listeria monocytogenes is now listed as a legal test item for food hygiene in all countries of the world.
The current detection method of listeria monocytogenes mainly comprises bacterial separation and identification, immunological methods, molecular biological methods and the like. The bacteria separation and identification workload is large, the cost is low, but the detection period is long, and the sensitivity is low; the immunological method is easy to cross-contaminate, the false positive is serious, and the sensitivity is low; molecular biology methods are most widely used, mainly including PCR, real-time fluorescence PCR, LAMP, gene chip, etc., but these methods also have the following general disadvantages: the common PCR is complicated to operate and needs electrophoresis; the fluorescent PCR equipment is large and can only be used in a laboratory, and field detection cannot be carried out; LAMP isothermal amplification is fast in speed but low in specificity, and is easy to pollute; the detection sensitivity of the gene chip is often not high enough, and the cost is not very high. Therefore, a sensitive, simple, fast and low-cost method for detecting listeria monocytogenes, which can be used for both laboratory detection and field fast detection, is still lacking at present.
Disclosure of Invention
In order to solve the problems, the invention provides a primer probe combination, a kit and a method for detecting listeria monocytogenes based on an RAA technology.
The recombinase-mediated strand displacement nucleic acid amplification (RAA) and the Recombinant Polymerase Amplification (RPA) are new technologies capable of efficiently and rapidly amplifying trace nucleic acid in vitro. The RAA technique relies primarily on three enzymes: recombinases that bind single-stranded nucleic acids (oligonucleotide primers), single-stranded DNA binding proteins (SSBs), and strand-displacing DNA polymerases. The recombinase and the primer are combined to form a protein-DNA complex which can search homologous sequences in double-stranded DNA; once the primer locates the homologous sequence, a strand exchange reaction occurs to form and initiate DNA synthesis, and exponential amplification is performed on the target region on the template; the replaced DNA strand binds to SSB, preventing further replacement. In this system, a single synthesis event is initiated by two opposing primers, and the entire process proceeds very quickly, typically within ten minutes to obtain detectable levels of amplification product. Compared with the traditional PCR and isothermal amplification technology, the method has the most obvious advantages of no need of high-temperature amplification, capability of observing a detection result for about half an hour, simple operation and low equipment cost.
As a first aspect of the present invention, the present invention provides a primer probe combination for detecting listeria monocytogenes based on RAA technology, comprising a primer pair and a probe, wherein the primer pair comprises an upstream primer and a downstream primer, the sequence of the upstream primer is shown in SEQ ID No.1, the sequence of the downstream primer is shown in SEQ ID No.2, and the sequence of the probe is shown in SEQ ID No. 5; SEQ ID NO. 1: CTGTAAATAATAGCTTGAATGTAAACTTCGGCGC, respectively; SEQ ID NO. 2: TCTGCATTCACTCCAAGCGCTTGCAACTGCTC, respectively; SEQ ID No. 5: TAGTTTTAAACAAATTTACTATAACGTGAA (FAM-dt) (THF) (BHQ-dt) TAATGAACCTACAAG.
As a second aspect of the invention, the invention provides a kit for detecting Listeria monocytogenes based on RAA technology, comprising the primer probe combination.
Preferably, the kit for detecting listeria monocytogenes based on the RAA technology further comprises buffer, magnesium acetate and purified water. The buffer solution is RAA basic universal reagent, comprises components such as recombinase, single-stranded DNA binding protein, DNA polymerase, dNTP and the like, can be prepared by self, and can also be obtained by purchasing a commercial RAA amplification reagent.
As a third aspect of the present invention, the present invention provides a method for detecting listeria monocytogenes based on RAA technology, which comprises the following steps: extracting the genome DNA of a sample to be detected; performing real-time fluorescent RAA amplification by using the kit for detecting Listeria monocytogenes based on the RAA technology by using the genome DNA of a sample to be detected as a template; and analyzing whether the sample to be detected contains the listeria monocytogenes or not according to the real-time fluorescent RAA amplification curve.
Preferably, in the RAA amplification reaction system, the final concentration of the upstream primer and the downstream primer is 300-450 nmol/L, and the final concentration of the probe is 350-450 nmol/L.
Further, the final concentrations of the upstream primer, the downstream primer and the probe were 400nmol/L, respectively.
Further, the reaction system of the RAA amplification is: 25. mu.L of buffer, 2.1. mu.L of each of the forward primer and the reverse primer, 0.6. mu.L of probe, 2.5. mu.L of magnesium acetate, and 17.7. mu.L of DNA template and purified water.
Preferably, the reaction conditions for the RAA amplification are: the temperature is 40-45 deg.C, and the time is 28-33 min.
Further, the reaction conditions for RAA amplification are as follows: the temperature is 42 ℃ and the time is 30 min.
As a fourth aspect of the present invention, the present invention further provides an application of the method for detecting listeria monocytogenes based on the RAA technology in detecting listeria monocytogenes in food, which can be used for detecting listeria monocytogenes in food such as fish, meat, vegetables, milk products, etc., and has a wide application range.
The invention has the following beneficial effects:
1. the specificity is good: has no cross reaction with pseudomonas aeruginosa, proteus mirabilis, klebsiella pneumoniae, salmonella typhimurium, enterobacter aerogenes, escherichia coli, edwardsiella tarda, listeria inonotus, listeria sieboldii, listeria monocytogenes, listeria williamsii, staphylococcus aureus, enterococcus faecalis and hemolytic streptococcus.
2. The sensitivity is high: the sensitivity of the RAA test of the Listeria monocytogenes can reach 3 multiplied by 102 CFU/mL, and the detection limit in actual sample detection is lower than that of the traditional streak culture method;
3. the accuracy is high: the detection result of the actual sample is basically consistent with that of the traditional streak culture method, and the reliability is high;
4. the operation is simple: the method does not need special reagents, does not need complicated steps such as denaturation of double-stranded DNA and the like in advance, only needs a constant-temperature fluorometer, has mild conditions, is simple and quick in the whole reaction, directly judges an amplification result according to real-time fluorescence data, does not need electrophoretic detection, is not only suitable for laboratory detection, but also suitable for field detection of non-laboratory places with a large number of samples;
5. the cost is low: compared with the existing molecular biology detection method, the method has the advantages that the instrument, time and labor cost are obviously reduced, and the method has good application prospect.
Drawings
FIG. 1: RAA test results for combinations of primers F1R1, F1R 2.
FIG. 2: RAA test results for combinations of primers F2R1, F2R 2.
FIG. 3: RAA amplification result of Listeria monocytogenes at 30 ℃, 35 ℃ and 39 ℃.
FIG. 4: RAA amplification result of Listeria monocytogenes at 42 ℃ and 45 ℃.
FIG. 5: RAA amplification effect of different primer concentrations of Listeria monocytogenes.
FIG. 6: RAA amplification effect of different probe concentrations of Listeria monocytogenes.
FIG. 7: and (5) specific test results of the listeria monocytogenes.
FIG. 8: and (3) the sensitivity test result of listeria monocytogenes ATCC7644 RAA.
FIG. 9: and (4) RAA detection results of different concentrations of Listeria monocytogenes in beef after different bacteria increasing times.
FIG. 10: and (4) RAA detection results of different concentrations of Listeria monocytogenes in the salmon after different bacterium increasing times.
FIG. 11: and (4) detecting the RAA of the listeria monocytogenes with different concentrations in the cheese after different bacterium increasing time.
Detailed Description
The invention is further described below with reference to the accompanying drawings and specific embodiments.
Example 1
The embodiment provides a primer-probe combination for detecting listeria monocytogenes based on the RAA technology, which comprises a primer pair and a probe, wherein the primer pair comprises an upstream primer and a downstream primer, the sequence of the upstream primer is shown as SEQ ID No.1 in table 1, the sequence of the downstream primer is shown as SEQ ID No.2 in table 1, and the sequence of the probe is shown as SEQ ID No.5 in table 1.
The process of designing and screening the primer probe for listeria monocytogenes of this example is as follows:
(1) designing a primer probe: obtaining a listeria monocytogenes hlyA sequence from an NCBI GenBank database, and designing a fluorescent RAA primer and a probe sequence according to a RAA primer and a probe design principle. The primers and probes were synthesized by Shanghai Bioengineering Co., Ltd, and the specific sequences are shown in Table 1 below.
TABLE 1 Listeria monocytogenes RAA primer Probe sequences
Note: in the probe sequence, FAM is a luminescent group, BHQ is a quenching group, and THF is tetrahydrofuran.
(2) Primer screening: combining two upstream primers (Li-hlyA-F1 and Li-hlyA-F2) and two downstream primers (Li-hlyA-R1 and Li-hlyA-R2) with each other to form four combinations with the probe (Li-hlyA-P), testing the amplification effect of the F1R1, F1R2, F2R1 and F2R2 primers on the bound probe, and screening the optimal primers and probe combinations through the size of a fluorescence signal value and the reaction time.
Specifically, RAA amplification is carried out by using an RAA-1620 fluorescent RAA detector (Jiangsu Qitian gene Biotechnology Co., Ltd.), and the reaction conditions are as follows: the temperature is 39 ℃ and the time is 30min (the conventional RAA amplification condition); reaction ofThe system is as follows: 25 μ L of buffer (purchased from fluorescence RAA amplification kit of Qitian Gene bioscience, Jiangsu, Japan), 2.1 μ L of each of upstream and downstream primers (10 μ M), 0.6 μ L of probe (10 μ M), 2.5 μ L of magnesium acetate, 4 μ L of DNA template, and 13.7 μ L of purified water; the DNA template is 3X 10-3X 106CFU/mL six concentration gradient of Listeria monocytogenes genomic DNA, with sterile water as a negative control.
The amplification results of the four primer probe combinations are shown in FIG. 1-2, and it can be seen that the amplification curves and sensitivities of the bacterial solutions of different dilutions are most stable under the F1R1 combination, so that the F1R1 primer pair is selected as the subsequent amplification primer.
Example 2
According to the primer screening result of example 1, this example provides a kit for detecting listeria monocytogenes based on RAA technology, the kit comprising: the kit comprises an upstream primer Li-hlyA-F1, a downstream primer Li-hlyA-R1, a buffer solution, magnesium acetate and purified water, wherein the buffer solution is purchased from a fluorescent RAA amplification kit of Jiangsu Qitian gene biotechnology limited.
Example 3
The embodiment provides a method for detecting listeria monocytogenes based on an RAA technology, which comprises the following specific steps: extracting the genome DNA of a sample to be detected; the genome DNA of a sample to be detected is taken as a template, the kit of the embodiment 2 is adopted to carry out real-time fluorescence RAA amplification, and the reaction system of the RAA amplification is as follows: 25 mu L of buffer solution, 2.1 mu L of each of the upstream primer and the downstream primer, 0.6 mu L of probe, 2.5 mu L of magnesium acetate, 4 mu L of DNA template and 13.7 mu L of purified water; and analyzing whether the sample to be detected contains the listeria monocytogenes or not according to the real-time fluorescent RAA amplification curve. During operation, the buffer solution, the upstream and downstream primers, the probe and the purified water are mixed uniformly and centrifuged, then are packaged into a fluorescence basic reaction unit, the lyophilized powder is fully and uniformly redissolved by gentle hand flicking, the reaction unit is centrifuged for a short time, magnesium acetate is added to a tube cover of each reaction unit, then a DNA template is added into each reaction unit, the mixture is fully and uniformly mixed and centrifuged, and the reaction tube is placed into a fluorescence detector for reaction for 30 min.
In this example, the concentration of primer probe and the reaction conditions for RAA amplification were optimized, and the specific process was as follows:
(1) optimization of reaction temperature
The RAA reaction solution was reacted at different temperatures (30 ℃, 35 ℃, 37 ℃, 39 ℃, 42 ℃, 45 ℃) to determine the optimal amplification temperature, and the concentrations of the upstream and downstream primers and the probe were 10. mu.M (i.e., the final concentration of the primer in the reaction system was 420nmol/L, and the final concentration of the probe was 120 nmol/L). The results are shown in FIGS. 3 to 4 (in the figures, well3 is amplification and well4 is blank control well), and the optimal amplification temperature is 42 ℃ from the viewpoint of amplification curve and fluorescence intensity.
(2) Optimization of primer probe concentration
The final concentrations of the primers are respectively diluted to 100nmol/L, 200nmol/L, 400nmol/L and 800nmol/L, the final concentrations of the probes are respectively diluted to 100nmol/L, 200nmol/L, 300nmol/L and 400nmol/L for amplification, and the concentrations of the primers and the probes are screened when the optimal amplification effect is achieved. As shown in FIGS. 5 to 6, the amplification signal increased with the increase in the primer concentration at the constant probe concentration, and the amplification curve was abnormal at the primer concentration of 800nmol/L (FIG. 5); when the concentration of the primer is unchanged, the amplification signal also shows obvious enhancement along with the increase of the concentration of the probe (FIG. 6); therefore, the final concentration of the selected primer and the probe is 400 nmol/L.
In conclusion, the final concentrations of the upstream primer, the downstream primer and the probe selected in the present example were 400nmol/L respectively; the reaction conditions for RAA amplification were: the temperature is 42 ℃ and the time is 30 min.
The method for detecting listeria monocytogenes based on the RAA technique described in example 3 was evaluated for the following effects:
first, specificity detection
Listeria monocytogenes (ATCC 7644), Listeria monocytogenes isolate M2-M7, Listeria inogenes (ATCC 33090), Listeria schoensis (ATCC 35967), Listeria evanescens (ATCC 19119), Listeria formates (ATCC 700545), Listeria westermanii (ATCC 35897), Pseudomonas aeruginosa (ATCC 27853), Proteus mirabilis (ATCC 12453), Klebsiella pneumoniae (ATCC 13883), Salmonella typhimurium (ATCC 14028), Enterobacterium aerogenes (ATCC 13048), Escherichia coli (ATCC 29522), Edwardsiella tarda (ATCC 15947), Staphylococcus aureus (ATCC 6538), enterococcus faecalis (ATCC 49452), and Streptococcus hemolyticus (ATCC 21059), extracting DNA template and performing fluorescence RAA detection, and using sterile water as a negative control to verify the specificity of the method.
The listeria monocytogenes isolate M2-M7 is isolated and stored in the laboratory, all the strains are stored in 10% (w/v) glycerol broth at the temperature of-80 ℃, and the recovery and DNA extraction methods of the strains are as follows:
recovering the strain: taking 100 mu L of bacterial liquid from a glycerol cryopreservation tube, inoculating the bacterial liquid into 5mL of brain-heart infusion culture broth, and oscillating at 37 ℃ for 150 r/min overnight;
preparing a DNA template: 1mL of bacterial culture 1.5mL of EP centrifuge tube is taken, centrifuged at 12000r/min for 10min to collect bacterial liquid, and supernatant is discarded; extracting bacterial DNA according to a bacterial genome DNA extraction kit method; storing at-20 deg.C for use.
The fluorescence RAA amplification result is shown in FIG. 7, and it can be seen that only Listeria monocytogenes ATCC strain and isolate can amplify specific bands, and other strains have no amplification signal, indicating that the designed primer probe has good specificity.
Second, sensitivity detection
Vortex and uniformly mix overnight cultured listeria monocytogenes liquid, and dilute by 10 times of sterile PBS; adding 1mL of diluted bacterial liquid into a sterilized culture dish, pouring counting agar in a constant-temperature water bath at 56 ℃, shaking up gently, cooling and then placing in a biochemical incubator; the cells were incubated at 37 ℃ for 24h in an inverted fashion, and 3 replicates of each gradient were run, while PBS was used as a blank. Preparing DNA template from the bacterial liquid of each dilution according to the method in the specificity detection, detecting by the established fluorescence RAA method, observing and recording the result. Counting plates cultured overnight, selecting plates with the colony number between 30 and 300 for colony counting, and calculating the concentration of the Listeria monocytogenes culture suspension, thereby calculating the detection sensitivity of the method.
The results of the fluorescent RAA amplification are shown in FIG. 8, and it can be seen that the sensitivity of the RAA test of Listeria monocytogenes can reach 3 × 102 CFU/mL。
Third, testing the detection limit of the substrate
In order to verify the detection limit of the method for the listeria monocytogenes in the sample, beef, salmon and cheese are selected from the iced fresh meat and meat entering the Shanghai port, and are detected according to the traditional method and the RAA method respectively, wherein the traditional method is a streak culture method specified in GB 4789.30-2016 (national standard food safety food microbiology inspection) for detecting the listeria monocytogenes.
The specific operation is as follows: weighing 25 g of the sample in 225 mL LB1 broth, and pipetting the mixture into 10 tubes, 9 mL each; adding a series of bacterial suspensions with different concentration gradients into 10 test tubes respectively to prepare different initial contamination concentrations (3 × 10)6 CFU/mL~3×10-3 CFU/mL), performing pre-enrichment culture (adopting LB1 culture medium) at 30 +/-1 ℃ for 24h, and then sucking 0.1 mL sample to 10 mL LB2 for enrichment culture for 2h, 4h and 24h respectively; and extracting DNA templates from the bacteria liquid with bacteria increment of 2h, 4h and 24h, and then carrying out RAA detection, and meanwhile, carrying out detection by adopting a traditional separation and scribing method. Note: LB1 and LB2 are enrichment broth basal media from Liangchi, Inc., Beijing Luqiao technology, Inc.
The detection limit test results are as follows:
(1) comparing detection limits of RAA method and traditional method in beef
Artificially polluting Listeria monocytogenes with different concentrations in a beef sample, and showing RAA detection results and traditional streaking culture results in fig. 9 and table 2, it can be seen that after the bacteria are increased for 4h, the detection limit of RAA on Listeria monocytogenes in beef can reach 3 CFU/mL, the detection limit of RAA on Listeria monocytogenes in the traditional streaking culture method for 24h is 3 × 10 CFU/mL, and the sensitivity of the RAA method is higher than that of the traditional streaking culture method.
TABLE 2 detection results of RAA and conventional method after enrichment of Listeria monocytogenes with different concentrations in beef
(2) Comparison of detection limits of RAA method and traditional method in salmon
The artificially contaminated listeria monocytogenes with different concentrations in the salmon sample, the RAA detection result and the traditional streak culture result are shown in figure 10 and table 3, and it can be seen that the RAA is used for treating the salmon sample after 2h of bacteria proliferationThe detection limit of Listeria monocytogenes in the salmon can reach 3 CFU/mL, and after the culture of Listeria monocytogenes is carried out for 4 hours, the detection limit can reach 3 multiplied by 10-1CFU/mL; the detection limit is 3 CFU/mL after the traditional streak culture method increases the bacteria for 24h, and the sensitivity of the RAA method is obviously higher than that of the traditional streak culture method.
TABLE 3 detection results of RAA and conventional method after enrichment of Listeria monocytogenes with different concentrations in Salmon
(3) Comparison of detection limits of RAA method and traditional method in cheese
Artificially polluting listeria monocytogenes with different concentrations in the cheese sample, wherein the RAA detection result and the traditional streak culture result are shown in figure 11 and table 4, and the detection limit of the RAA to the listeria monocytogenes in the cheese can reach 3 multiplied by 10 CFU/mL after the bacteria are enriched for 4 hours; the detection limit of the traditional streak culture method for increasing the bacteria for 24h is 3 multiplied by 10 CFU/mL, and the sensitivity of the RAA method is higher than that of the traditional streak culture method.
TABLE 4 detection results of RAA and conventional method after enrichment of Listeria monocytogenes with different concentrations in cheese
Fourthly, detecting actual samples
100 parts of commercial meat, chilled meat, cheese and the like across Shanghai port and 100 parts of commercial meat, chilled meat and cheese are pretreated and enriched according to GB 4789.30-2016, genome DNA is extracted, the established RAA method is adopted for detection, and meanwhile, the traditional streak culture method recommended in GB 4789.30-2016 is adopted for detection, and the results are shown in Table 5, and it can be seen that the detection result of the RAA method is consistent with the detection result of the existing national standard GB 4789.30-2016.
TABLE 5 Listeria monocytogenes assay results in actual samples
In conclusion, the kit and the method for detecting the listeria monocytogenes have good specificity and high accuracy, and compared with the traditional streaking culture method, the kit and the method have better sensitivity and greatly shortened detection time (the kit can obtain a result only after about 30min after the enrichment, and can obtain a result only after 24h of culture after the enrichment by the traditional method), and have remarkable advantages.
This detailed description is to be construed as illustrative only and is not to be taken as limiting the invention, as any changes that may be made by a person skilled in the art after reading the present specification will be protected by the patent laws within the scope of the appended claims.
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Claims (10)
1. A primer probe combination for detecting Listeria monocytogenes based on RAA technology is characterized in that: the primer pair comprises an upstream primer and a downstream primer, wherein the sequence of the upstream primer is SEQ ID NO. 1: CTGTAAATAATAGCTTGAATGTAAACTTCGGCGC, the sequence of the downstream primer is SEQ ID NO. 2: TCTGCATTCACTCCAAGCGCTTGCAACTGCTC, the sequence of the probe is SEQ ID NO. 5: TAGTTTTAAACAAATTTACTATAACGTGAA (FAM-dt) (THF) (BHQ-dt) TAATGAACCTACAAG.
2. A kit for detecting Listeria monocytogenes based on RAA technology is characterized in that: comprising the primer probe combination of claim 1.
3. The kit for detecting listeria monocytogenes based on RAA technology of claim 2, wherein: buffer, magnesium acetate and purified water are also included.
4. A method for detecting Listeria monocytogenes based on RAA technology is characterized in that: the method comprises the following specific steps: extracting the genome DNA of a sample to be detected; performing real-time fluorescent RAA amplification by using the kit of claim 2 or 3 and using the genomic DNA of a sample to be detected as a template; and analyzing whether the sample to be detected contains the listeria monocytogenes or not according to the real-time fluorescent RAA amplification curve.
5. The method of claim 4 for detecting listeria monocytogenes based on RAA technology, wherein: in the RAA amplification reaction system, the final concentration of the upstream primer and the downstream primer is 300-450 nmol/L, and the final concentration of the probe is 350-450 nmol/L.
6. The method for detecting listeria monocytogenes based on RAA technology of claim 5, wherein: the final concentrations of the upstream primer, the downstream primer and the probe were 400nmol/L, respectively.
7. The method for detecting listeria monocytogenes based on RAA technology of claim 5, wherein: the reaction system of RAA amplification is as follows: 25. mu.L of buffer, 2.1. mu.L of each of the forward primer and the reverse primer, 0.6. mu.L of probe, 2.5. mu.L of magnesium acetate, and 17.7. mu.L of DNA template and purified water.
8. The method of claim 4 for detecting listeria monocytogenes based on RAA technology, wherein: the reaction conditions for RAA amplification are as follows: the temperature is 40-45 deg.C, and the time is 28-33 min.
9. The method for detecting listeria monocytogenes based on RAA technology of claim 8, wherein:
the reaction conditions for RAA amplification are as follows: the temperature is 42 ℃ and the time is 30 min.
10. Use of the method for detecting listeria monocytogenes based on RAA technology according to claims 4-9 for detecting listeria monocytogenes in food.
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|---|---|---|---|---|
| CN113957162A (en) * | 2021-09-01 | 2022-01-21 | 上海海关动植物与食品检验检疫技术中心 | Primer probe combination, kit and method for detecting salmonella based on fluorescence RAA technology |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105671197A (en) * | 2016-04-19 | 2016-06-15 | 宁波大学 | Detecting method for food-borne pathogenic bacteria listeria monocytogenes |
| CN108148894A (en) * | 2017-12-26 | 2018-06-12 | 中科智测(天津)科技有限公司 | A kind of methods and applications of recombinase polymerase constant-temperature amplification combination ELISA test strip Listeria monocytogenes |
-
2020
- 2020-12-29 CN CN202011585591.5A patent/CN112553355A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105671197A (en) * | 2016-04-19 | 2016-06-15 | 宁波大学 | Detecting method for food-borne pathogenic bacteria listeria monocytogenes |
| CN108148894A (en) * | 2017-12-26 | 2018-06-12 | 中科智测(天津)科技有限公司 | A kind of methods and applications of recombinase polymerase constant-temperature amplification combination ELISA test strip Listeria monocytogenes |
Non-Patent Citations (4)
| Title |
|---|
| 王金凤等: "单核细胞增生李斯特氏菌实时荧光 RPA 检测方法的建立及应用", 《现代食品科技》, vol. 34, no. 8, pages 213 - 218 * |
| 邹宇等: "单增李斯特菌在牛奶中生长曲线的拟合和预测模型的建立", 《食品工业科学》, vol. 35, no. 11, pages 69 - 73 * |
| 陈晨: "重组酶介导的扩增技术检测呼吸道合胞病毒方法的建立和初步评价", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》, no. 1, pages 060 - 709 * |
| 高继铭: "酱卤肉中代表性致病菌预测微生物学研究", 《中国优秀硕士学位论文全文数据库 工程科技I辑》, no. 2, pages 024 - 350 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113957162A (en) * | 2021-09-01 | 2022-01-21 | 上海海关动植物与食品检验检疫技术中心 | Primer probe combination, kit and method for detecting salmonella based on fluorescence RAA technology |
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