Primers, kits and methods for detecting Staphylococcus aureus

Abstract

The present invention relates to the technical field of pathogen molecular detection, and specifically to a primer, a kit and a method for detecting Staphylococcus aureus. The sequences of the primers are shown in SEQ ID NO.1 and SEQ ID NO.2. The present invention also discloses a kit comprising the primers, and a method for detecting Staphylococcus aureus using the primers. The detection limit of the method for detecting Staphylococcus aureus is 3.06 copies / μL, which is higher than similar studies, equivalent to 8.4 times that of the LAMP method, 12.5 times that of the fluorescent quantitative PCR method, and 125 times that of the conventional PCR method, and does not rely on complex variable temperature instruments, so it is suitable for grassroots front-line detection institutions and can provide a feasible technical means for the rapid detection of Staphylococcus aureus.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogen molecule detection, and in particular to a primer, a kit and a method for detecting Staphylococcus aureus. Background Art

[0002] Staphylococcus aureus (SA) is one of the common foodborne pathogens. It can parasitize on the skin, nasal cavity, throat, and stomach of humans and animals. It can also survive in harsh environments. It can withstand high temperatures of 80°C and survive in 15% NaCl solutions. Eating food contaminated with S. aureus can cause vomiting, diarrhea, and sepsis, and in severe cases, it can even endanger life. The ability to quickly, accurately and effectively detect S. aureus is an important guarantee for food quality and safety.

[0003] At present, the detection methods of Staphylococcus aureus include traditional separation and identification, biochemical detection, immunological detection, polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), real-time quantitative PCR (qPCR), gene chip, etc. Traditional separation and identification methods are cumbersome, inefficient, time-consuming, and have lower detection sensitivity than molecular biology technology, which is difficult to meet the needs of food safety testing. Biochemical testing is mainly used for the initial screening and identification of pathogenic bacteria. The target strain can grow on the selective culture medium and produce characteristic enzymes. This enzyme can produce a color reaction after combining with the color development culture medium and can be observed by the naked eye, but its accuracy is general and needs to be used in combination with other methods to further improve its accuracy; immunological testing has high sensitivity, does not require large instruments, and has low costs, but has high requirements for the reaction system environment; conventional PCR is more cumbersome and requires three processes: denaturation, annealing, and extension. The reaction time is usually more than 2 hours, and a special variable temperature PCR instrument is required. It is not suitable for grassroots front-line testing, and the conventional PCR detection sensitivity is lower than LAMP and real-time fluorescence quantitative PCR. In the process of food testing, false negatives exist; real-time fluorescence quantitative PCR combines multiple technologies, and amplification and detection can be completed in the same tube, avoiding contamination from opening the lid. Since there is a one-to-one correspondence between the generation of fluorescent signals and the amplified products, the fluorescent signals can be detected by corresponding software and the results can be obtained directly, which improves the accuracy of the test results. However, the reaction process takes 2 to 3 hours and requires a relatively expensive fluorescent quantitative PCR instrument. LAMP has lower instrument requirements during the amplification process. DNA amplification can be completed at 60℃ to 65℃, and the test results can usually be obtained within 1 hour, but the requirements for primers are particularly high, requiring 4 to 6 primers, and the amplified products cannot be used for cloning and sequencing. Aerosol contamination is easily formed during the experiment, resulting in false positives and affecting the test results. Gene chips are mainly used for high-throughput detection of pathogenic microorganisms. They have strong specificity, high sensitivity, fast detection speed, small reaction volume, and can be automatically detected, which greatly reduces labor costs. However, gene chips are expensive and are only suitable for scientific research and cannot be popularized.

[0004] At present, molecular biology has become the main means of detecting Staphylococcus aureus. Zhang Xiaoyu et al. used LAMP technology to detect Staphylococcus aureus, and the detection limit was 2.7×10 1 copies / μL, and the detection limit of conventional PCR is 2.7×10 3copies / μL, the sensitivity of LAMP technology is one hundred times that of conventional PCR. Guo Mengran et al. used fluorescence quantitative PCR to detect Staphylococcus aureus, and the minimum detection limit reached 3.5×10 1 CFU / mL. Tang Mengjun et al. found that the detection limits of real-time fluorescence quantitative PCR and LAMP were the same, both 4×10 1 copies / μL, while the detection limit of conventional PCR for Staphylococcus aureus is 4×10 2 However, the detection limit of the above-mentioned prior art needs to be further reduced. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a primer, a kit and a method for detecting Staphylococcus aureus.

[0006] In a first aspect, the present invention provides a primer for detecting Staphylococcus aureus, wherein the sequences of the primers are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0007] Enzymatic recombinase amplification (ERA) technology is an improved version of recombinase polymerase amplification (RPA) technology, which can achieve rapid amplification of target genes within 10 to 30 minutes at 37 to 42°C. It has the advantages of short amplification time, high sensitivity, strong specificity, and the integration of multiple detection modes. It is particularly suitable for grassroots and on-site instant detection. Like other isothermal amplification technologies, it can be applied to the detection of foodborne and pathogenic pathogens. The present invention designs primers for detecting Staphylococcus aureus based on ERA technology according to the conservative sequence of the thermotolerant nuclease (nuc) gene of Staphylococcus aureus.

[0008] In a second aspect, the present invention provides use of the primers in preparing a Staphylococcus aureus kit.

[0009] In a third aspect, the present invention provides use of the primers in detecting Staphylococcus aureus.

[0010] Furthermore, the primers use the Staphylococcus aureus nuc gene as a target gene.

[0011] In a fourth aspect, the present invention provides a kit for detecting Staphylococcus aureus, comprising the primers, a lytic agent, an activator and dd H2O.

[0012] In a fifth aspect, the present invention provides a method for detecting Staphylococcus aureus for non-diagnostic purposes, using enzymatic recombinant isothermal amplification technology in combination with the primers to perform isothermal amplification on the sample to be tested, and analyzing the results of the amplification products.

[0013] As a preferred embodiment of the method for detecting Staphylococcus aureus for non-diagnostic purposes of the present invention, the reaction temperature of the isothermal amplification is 37° C. and the reaction time is 20 min.

[0014] As a preferred embodiment of the method for detecting Staphylococcus aureus for non-diagnostic purposes of the present invention, the sample to be tested is dairy products, egg products, cooked meat products, and frozen foods containing milk.

[0015] As a preferred method for detecting Staphylococcus aureus for non-diagnostic purposes of the present invention, it specifically includes the following steps:

[0016] The template DNA from the sample to be tested is mixed with the ERA reaction system for enzymatic recombination isothermal amplification;

[0017] Observe the presence of amplification products to determine whether it is the target bacteria. If a single band of the expected size appears, it indicates that it is Staphylococcus aureus. If the target band does not appear, it indicates that it is not Staphylococcus aureus.

[0018] As a preferred method for detecting Staphylococcus aureus for non-diagnostic purposes of the present invention, the recombinase polymerase isothermal amplification reaction system is: solvent, 20.0 μL; activator, 2.0 μL; upstream and downstream primers, 2.5 μL each; template 4.0 μL; dd H2O, 19.0 μL.

[0019] As a preferred embodiment of the method for detecting Staphylococcus aureus for non-diagnostic purposes of the present invention, agarose gel electrophoresis is used to analyze the results of the amplification products.

[0020] The present invention has the following beneficial effects:

[0021] (1) In the present invention, the detection limit of ERA for Staphylococcus aureus is 3.06 copies / μL, which is higher than similar studies, equivalent to 8.4 times that of the LAMP method, 12.5 times that of the fluorescent quantitative PCR method, and 125 times that of the conventional PCR method.

[0022] (2) The present invention designed a total of four pairs of primers, among which primer 1S / 1-1A had the best amplification effect at 37°C for 20 min, with clear bands and no mixed bands or diffusion.

[0023] (3) Compared with conventional PCR technology for detecting Staphylococcus aureus, the method of the present invention has higher sensitivity and shorter detection time, does not rely on complex temperature-changing instruments, is suitable for grassroots frontline testing institutions, and can provide a feasible technical means for the rapid detection of Staphylococcus aureus. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the agarose gel electrophoresis diagram of the Staphylococcus aureus genome, M: DL 5000bp DNA maker; 1: Staphylococcus aureus genome.

[0025] Figure 2 Agarose gel electrophoresis diagram for PCR sensitivity detection, M: DL 100bp DNA maker; 1: 1ng / μL; 2: 100pg / μL; 3: 10pg / μL; 4: 1pg / μL; 5: 100fg / μL; 6: 10fg / μL.

[0026] Figure 3 Agarose gel electrophoresis diagram of ERA detection using four pairs of primers: M: DL 2000bp DNA Maker; 1: 1S / 1-1; 2: 1S / 1-2A; 3: 2S / 2A; 4: 3S / 3A.

[0027] Figure 4 This is the agarose gel electrophoresis diagram of ERA sensitivity detection, M: DL 2000bp DNA Maker; 1: 1ng / μL; 2: 100pg / μL; 3: 10pg / μL; 4: 1pg / μL; 5: 100fg / μL; 6: 10fg / μL7: 1fg / μL; 8: negative control.

[0028] Figure 5 Agarose gel electrophoresis diagram of ERA specific detection, M: DL 2000bp DNA maker; 1: Staphylococcus aureus; 2: Staphylococcus gallinaceus; 3: Staphylococcus kluyveri; 4: Staphylococcus epidermidis; 5: Listeria monocytogenes; 6: Bacillus subtilis; 7: Escherichia coli; 8: Bacillus cereus. DETAILED DESCRIPTION

[0029] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0030] 1 Materials and methods

[0031] 1.1 Materials

[0032] 1.1.1 Test strains

[0033] Staphylococcus aureus, Staphylococcus gallinarum, Staphylococcus kloosii, Staphylococcus epidermdis, Listeria mono cytogenes, Bacillus subtilis, Escherichia coli and Bacillus cereus were all kept by the laboratory. The specific information is shown in Table 1.

[0034] Table 1 Detailed information of test strains

[0035]

[0036] 1.1.2 Main reagents and instruments

[0037] Bacterial genome extraction kit, 6× Loading Buffer, DL 2000 Plus DNA Marker, Beijing Quanshijin Biotechnology Co., Ltd.; Green Taq Mix, Nanjing Novogene Biotechnology Co., Ltd.; ERA dissolution agent, ERA activator (catalog number: KS101), Suzhou Xinda Gene Technology Co., Ltd.; other reagents were domestic analytical grade.

[0038] W201B constant temperature water bath, Yuhang Xinbo Instrument Co., Ltd.; JY600PJ electrophoresis instrument, Beijing Junyi Oriental Electrophoresis Co., Ltd.; LAS500 gel imaging system, GE, USA; Biospec-nano nucleic acid concentration meter, SHIMADZU, Japan; 5425R low temperature centrifuge, Eppendorf, Germany; HZQ-X100 constant temperature shaking incubator, Harbin Donglian Electronic Technology Development Co., Ltd.

[0039] LB medium: 1% tryptone, 0.5% yeast extract, 1% sodium chloride, 2% agar powder, pH 7.4, 121°C, sterilize for 30 min, cool and place in a 4°C refrigerator for use.

[0040] 2 Test methods

[0041] 2.1 Activation and cultivation of bacteria

[0042] Under sterile conditions, Staphylococcus aureus was streaked onto LB plate culture medium and cultured at 37°C for 24 h. A single colony with vigorous growth was picked and inoculated into LB liquid culture medium, cultured overnight at 37°C, 120 rpm, and the bacterial cell pellet was collected for genomic DNA extraction.

[0043] 2.2 Extraction of Staphylococcus aureus genome

[0044] According to the steps in the instruction manual of the bacterial genome extraction kit, the genomic DNA of Staphylococcus aureus was extracted, and the centrifuge tube containing the eluted DNA was saved. The concentration and purity of the extracted DNA were measured using a nucleic acid concentration meter. If it met the requirements, it was stored in a -20°C refrigerator.

[0045] 2.3 PCR reaction system and reaction conditions

[0046] According to the PCR reaction system, Green Taq Mix, ddH2O, Staphylococcus aureus genome, and upstream and downstream primers were added to the PCR tube in sequence. The PCR reaction system is shown in Table 2, and the upstream and downstream primers are 1S and 1-1A in Table 3.

[0047] Table 2 PCR reaction system

[0048]

[0049] The reaction conditions were set in the PCR instrument: denaturation at 94°C for 1 min, annealing at 55°C for 30 s, extension at 72°C for 1 min, and finally extension at 72°C for 10 min, for 30 cycles.

[0050] 2.4 PCR sensitivity test

[0051] The genome of Staphylococcus aureus was diluted to 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, and 10 fg / μL, and 1S / 1-1A was used as upstream and downstream primers, and amplified according to the above reaction system and reaction conditions. After amplification, 1.5% agarose gel was prepared, and electrophoresis test was performed, and the results were judged using a gel imaging system.

[0052] 2.5 ERA primers

[0053] The nuc (EF 529605.1) gene was selected as the target gene for ERA detection of Staphylococcus aureus, and the primers were designed using PremierPrimer5. The primer design results are shown in Table 3.

[0054] Table 3 Primer sequence information

[0055]

[0056] 2.6 ERA reaction conditions and primer screening

[0057] The extracted Staphylococcus aureus genome was used as a template, and the four pairs of primers 1S / 1-1A, 1S / 1-2A, 2S / 2A and 3S / 3A were used for ERA amplification. 20.0 μL of dissolving agent, 19.0 μL of dd H2O, 2.5 μL of upstream and downstream primers, and 4.0 μL of template were dissolved in the ERA basic amplification reagent in sequence; the mixture was fully shaken and centrifuged briefly. For each sample, 2.0 μL of activator was added to the reaction tube cover, and the activator was introduced into the premix by brief centrifugation. After brief shaking and mixing, the reaction tube was quickly centrifuged and placed in a 37°C water bath for incubation for 15 minutes. After the incubation, the 6× Loading Buffer provided by the product was added according to the required amount and incubated at 56°C for 5 minutes. After the reaction, 1.5% agarose gel electrophoresis was used to identify the amplification effects of the four pairs of primers and screen the optimal primers. The ERA reaction system is shown in Table 4.

[0058] Table 4 ERA reaction system

[0059]

[0060] 2.7 ERA sensitivity test

[0061] The Staphylococcus aureus genome was diluted to 1ng / μL, 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, 10fg / μL, and 1fg / μL, and the optimal upstream and downstream primers were selected to perform ERA amplification according to the corresponding ERA reaction system and reaction conditions. After the amplification was completed, a 1.5% agarose gel was prepared and the amplification results were identified by electrophoresis.

[0062] 2.8 ERA-specific detection

[0063] The corresponding genomes of Staphylococcus aureus, Staphylococcus gallinarum, Staphylococcus krusei, Staphylococcus epidermidis, Listeria monocytogenes, Bacillus subtilis, Escherichia coli and Bacillus cereus were extracted using a DNA extraction kit. After obtaining the corresponding genomes, the genomes were used as templates, the optimal upstream and downstream primers were selected, and ERA amplification was performed according to the corresponding ERA reaction system and reaction conditions. After the amplification was completed, a 1.5% agarose gel was prepared for electrophoresis test, and the results were judged using a gel imaging system.

[0064] 2.9 Sample addition test

[0065] Three samples of pork and three samples of beef were purchased from the market. According to the "GB4789.10-2016 National Food Safety Standard - Food Microbiology Inspection - Staphylococcus aureus Inspection", it was proved that there was no Staphylococcus aureus contamination.

[0066] 100 μL of Staphylococcus aureus suspension (1-100 CFU / mL) was added to 10 g of the above sample to prepare an artificially contaminated sample, which was then mixed and homogenized with 90 mL of saline solution. After removing large particles, 1 mL of the homogenate was taken to extract DNA. The Staphylococcus aureus suspension was replaced with 100 μL of saline solution, and a blank control was prepared according to the above method. The test was carried out according to the conditions of 2.4 to verify the practicality of this detection method.

[0067] 3 Results and analysis

[0068] 3.1 Results of S. aureus genome extraction

[0069] The DNA extraction kit was used to extract the genome of Staphylococcus aureus. The genome size was in line with the expected result, the band brightness was large, and there were no mixed bands, which could meet the requirements of subsequent experiments. The identification results of the extracted genome were as follows: Figure 1 shown.

[0070] 3.2 PCR sensitivity test results

[0071] The genome concentrations in lanes 1 to 6 are 1ng / μL, 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, and 10fg / μL, respectively. There are bands in lanes 1 to 5, but no band in lane 6. Therefore, in the PCR test, the detection limit of Staphylococcus aureus using primers 1S / 1A is 100fg / μL. The PCR sensitivity test results are shown in Figure 2. Figure 2 shown.

[0072] 3.3 ERA primer screening results

[0073] The four pairs of primers in Table 3 were verified using the extracted Staphylococcus aureus genome as a template. The results of agarose gel electrophoresis were as follows: Figure 3 As shown, lanes 1 to 4 correspond to the four pairs of primers 1S / 1-1A, 1S / 1-2A, 2S / 2A, and 3S / 3A in Table 3, and the product sizes are 158bp, 156bp, 160bp, and 164bp, respectively, and the results are in line with the expected size. The amplification product of the 1S / 1-1A primer in lane 2 is brighter than the other three pairs of primers. The concentration of the amplification product of the four pairs of primers was measured using a nucleic acid concentration meter. The amplification product of the 1S / 1-1A primer had the highest concentration of 2056ng / μL, indicating that its amplification effect was better. Therefore, the 1S / 1-1A primer was selected for subsequent experiments.

[0074] Table 5 ERA amplification product concentrations of different primer pairs

[0075]

[0076] 3.4 ERA sensitivity test results

[0077] like Figure 4 As shown, the genome concentrations in lanes 1 to 7 are 1ng / μL, 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, 10fg / μL, and 1fg / μL, respectively. Lane 8 is a ddH2O negative control. Target bands appear in lanes 1 to 6, and the fragment sizes are between 250 and 100bp, which is consistent with the expected results. There is no target band in lane 7. Therefore, for primers 1S / 1A, the minimum detection limit of ERA is 10fg / μL, that is, 3.06copies / μL, without non-specific bands, and the primer specificity is good. No target bands or non-specific bands were amplified in lane 8, indicating that the amplification process was not affected by interfering factors such as aerosols, and the experimental data is reliable.

[0078] 3.5 ERA specificity test results

[0079] Depend on Figure 5 It can be seen that only Staphylococcus aureus in lane 1 amplified the target band, and the test result was positive. The other lanes were the test strains Staphylococcus gallinarum, Staphylococcus kluyveri, Staphylococcus epidermidis, Listeria monocytogenes, Bacillus subtilis, Escherichia coli and Bacillus cereus, and no target band appeared, and the test results were negative. Therefore, the use of 1S / 1-1A primers to amplify the nuc gene and detect Staphylococcus aureus has good specificity.

[0080] 3.6 Sample addition test

[0081] As shown in Table 6, in the artificially contaminated meat samples with a Staphylococcus aureus concentration of 1 to 100 CFU / mL, the detection rate of the ERA detection method was 100%. In all control samples, the results were negative, indicating that this method has good applicability.

[0082] Table 6 Sample addition test results

[0083]

[0084] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0085] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0086] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A primer for detecting Staphylococcus aureus, It is characterized in that The sequences of the primers are shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. Use of the primers according to claim 1 in preparing a kit for detecting Staphylococcus aureus.

3. Use of the primers according to claim 1 in detecting Staphylococcus aureus for non-diagnostic purposes.

4. A kit for detecting Staphylococcus aureus, It is characterized in that Comprising the primer according to claim 1.

5. A method for detecting Staphylococcus aureus for non-diagnostic purposes, It is characterized in that The enzymatic recombination isothermal amplification technology is combined with the primers described in claim 1 to perform isothermal amplification on the sample to be tested, and the results of the amplification products are analyzed.

6. The method according to claim 5, It is characterized in that The reaction temperature of the isothermal amplification is 37° C., and the reaction time is 20 min.

7. The method according to claim 5, It is characterized in that The samples to be tested are dairy products, egg products, cooked meat products, and frozen foods containing milk.

8. The method according to claim 5, It is characterized in that The amplified products were analyzed by agarose gel electrophoresis.

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