Primer and probe, kit and quantitative detection method for salmonella detection

Abstract

The present invention belongs to the field of genetic engineering and biological detection, and provides a primer and probe, a kit and a quantitative detection method for detecting Salmonella. The present invention establishes a detection method that can be widely used in the detection of Salmonella by designing a group of specific primers and probes, which has strong specificity, high sensitivity, good repeatability, rapid detection, simple operation and low cost, and can be used not only for the rapid identification of Salmonella, but also for the absolute quantitative detection of Salmonella in the sample to be tested.

Description

Technical Field

[0001] The invention relates to a primer and a probe, a kit and a quantitative detection method for detecting salmonella, and belongs to the field of genetic engineering and biological detection. Background Art

[0002] As a foodborne pathogen, Salmonella can contaminate meat, eggs, milk and other foods, posing a great threat to human and animal health. Currently, Salmonella infection has been considered a major public health problem worldwide. Therefore, the detection of Salmonella is a very important challenge in food safety and microbiological analysis.

[0003] The existing methods for detecting Salmonella mainly include selective culture medium observation method, immune-based detection method and nucleic acid-based detection method.

[0004] The culture observation method generally takes 3-4 days to complete and is prone to false positive or false negative results. The immune-based detection method is mainly the enzyme-linked immunosorbent assay (ELISA), which greatly improves the specificity of the detection, but the detection sensitivity is insufficient. The nucleic acid-based detection method is currently mainly based on the principle of polymerase chain reaction (PCR). Compared with the other two methods, this method has relatively improved specificity and sensitivity, but false negative and false positive results still occur, which invisibly causes the detection time to be too long and the detection cost to be high.

[0005] In addition, since its development, loop-mediated isothermal amplification (LAMP) has been widely used in the detection of pathogens due to its simple operation and high amplification efficiency. However, this detection method still has certain problems, such as the easy generation of by-products in the reaction system, which leads to false positive problems. In addition, if the lid needs to be opened during the reaction, the amplification product will be directly exposed to the environment, resulting in amplicon aerosol contamination problems.

[0006] Therefore, there is an urgent need to develop a Salmonella detection method with strong specificity, high sensitivity, good repeatability, rapid detection, simple operation and low cost, which can be used for the rapid identification of Salmonella and the absolute quantitative detection of Salmonella-infected tissue samples, providing a basis for high-throughput rapid detection of Salmonella infection. Summary of the invention

[0007] In the present invention, unless otherwise specified, the scientific and technical terms used in the present invention have the meanings commonly understood by those skilled in the art. In addition, the relevant terms and laboratory procedures of protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used in the present invention are the terms and conventional procedures widely used in the corresponding fields.

[0008] In view of the above defects in the prior art, the present invention provides a primer and a probe for detecting Salmonella, wherein the upstream primer is shown as SEQ ID No.8, the downstream primer is shown as SEQ ID No.9, and the probe is shown as SEQ ID No.10.

[0009] In a preferred embodiment of the present invention, the fluorescent reporter group at the 5' end of the probe is FAM, and the fluorescent quencher group labeled at the 3' end is MGB.

[0010] Another aspect of the present invention provides a kit containing the primers and probes of the present invention.

[0011] Another aspect of the present invention provides use of the primers and probes of the present invention, or the kit of the present invention, in quantitative detection of Salmonella, wherein the detection is for non-diagnostic purposes.

[0012] In another aspect, the present invention provides a method for quantitatively detecting Salmonella, comprising the following steps: (1) Preparation of standard samples; (2) Using the primers and probes of the present invention or the kit of the present invention, perform real-time fluorescence PCR amplification on the sample to be tested and the standard substance, and draw a standard curve; (3) Quantitative detection of Salmonella is performed using a standard curve and the Cq value of the sample to be tested, and the detection is used for non-diagnostic purposes.

[0013] In a preferred embodiment of the present invention, in step (1), the conserved gene fragment of Salmonella invA is inserted into a vector to obtain a recombinant plasmid containing the conserved gene fragment of Salmonella invA as a standard.

[0014] In a further preferred embodiment of the present invention, the vector is pMD19-T, and the conserved gene fragment of Salmonella is the nucleotide sequence shown in SEQ ID No.1.

[0015] In a preferred embodiment of the present invention, the reaction system in step (2) is 10 μL of Premix Ex Taq (2X), 0.4 μL of each of the upstream and downstream primers with a concentration of 10 μM, 0.1 μL of the probe with a concentration of 10 μM, 2.5 μL of the template, and 20 μL of sterile water.

[0016] In a preferred embodiment of the present invention, the reaction conditions in step (2) are 95° C. for 30 seconds, 95° C. for 5 seconds, 60° C. for 30 seconds, and 40 cycles.

[0017] In a preferred embodiment of the present invention, the standard curve equation in step (2) is prepared based on each reaction, and the formula is: Y = -alogX + b; Where Y is the Cq value of the amplified standard, and X is the starting copy number of the standard DNA.

[0018] In a preferred embodiment of the present invention, the Cq value of the test sample is substituted into the formula in step (3) to calculate the starting copy number of Salmonella DNA in the test sample, and then the content of Salmonella in the test sample can be deduced based on the mass of the test sample used for DNA extraction, thereby quantitatively detecting the Salmonella in the test sample.

[0019] The present invention achieves the following beneficial effects by adopting the above technical solution: The invention establishes a detection method which can be widely used in the detection of Salmonella, and has strong specificity, high sensitivity, good repeatability, rapid detection, simple operation and low cost, by designing a group of specific primers and probes for detecting Salmonella.

[0020] The detection method of the present invention can not only be applied to the rapid identification of Salmonella, but also can perform absolute quantitative detection of Salmonella in the sample to be tested according to the cycle number Cq value of the sample to be tested and the standard curve equation.

[0021] The detection method of the present invention only takes less than 1 hour, and even with DNA extraction, it only takes 2-3 hours, and high-throughput sample quantitative detection can be performed simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a diagram showing the PCR amplification results of the Salmonella invA gene fragment detected by agarose gel electrophoresis; M: 200 bp DNA Marker; 1-2: PCR amplification product of invA gene fragment.

[0023] Figure 2 This is the result of colony PCR amplification of positive transformants detected by agarose gel electrophoresis; M: 200 bp DNA Marker; 1-8: 8 positive transformant single clones picked randomly; √: Single clone sent for sequencing.

[0024] Figure 3 It is the standard curve diagram of Salmonella of the present invention.

[0025] Figure 4 This is a graph showing the specificity evaluation results of the method of the present invention; a: Salmonella genomic DNA sample; b: Salmonella genomic DNA sample diluted 10 times; c: Salmonella genomic DNA sample diluted 100 times; d: Escherichia coli genomic DNA sample; e: Nuclease-free water.

[0026] Figure 5 It is a sensitivity evaluation result diagram of the method described in the present invention; a: Plasmid concentration is 4.00×10 6 copies / μL; b: Plasmid concentration is 4.00×10 5 copies / μL; c: Plasmid concentration is 4.00×10 4 copies / μL; d: Plasmid concentration is 4.00×10 3 copies / μL; e: Plasmid concentration is 4.00×10 2 copies / μL; f: Plasmid concentration is 4.00×10 1 copies / μL; g: Negative control.

[0027] Fig. 6A It is the amplification curve diagram of the first experiment in the repeatability evaluation of the present invention; a: Plasmid concentration is 4.00×10 6 copies / μL; b: Plasmid concentration is 4.00×10 5 copies / μL; c: Plasmid concentration is 4.00×10 4 copies / μL; d: Plasmid concentration is 4.00×10 3 copies / μL; e: Plasmid concentration is 4.00×10 2 copies / μL; f: Plasmid concentration is 4.00×10 1 copies / μL; g: negative control; Figure 6B This is the standard curve diagram of the first experiment in the repeatability evaluation of the present invention; Figure 6C It is the amplification curve diagram of the second experiment in the repeatability evaluation of the present invention; a: Plasmid concentration is 4.00×10 6 copies / μL; b: Plasmid concentration is 4.00×10 5 copies / μL; c: Plasmid concentration is 4.00×10 4 copies / μL; d: Plasmid concentration is 4.00×10 3 copies / μL; e: Plasmid concentration is 4.00×10 2 copies / μL; f: Plasmid concentration is 4.00×10 1 copies / μL; g: negative control; Fig.6D This is the standard curve diagram of the second experiment in the repeatability evaluation of the present invention; Fig. 6E It is the amplification curve diagram of the third experiment in the repeatability evaluation of the present invention; a: Plasmid concentration is 4.00×10 6 copies / μL; b: Plasmid concentration is 4.00×10 5 copies / μL; c: Plasmid concentration is 4.00×10 4 copies / μL; d: Plasmid concentration is 4.00×10 3 copies / μL; e: Plasmid concentration is 4.00×10 2 copies / μL; f: Plasmid concentration is 4.00×10 1 copies / μL; g: negative control; Fig. 6F This is the standard curve diagram of the third experiment in the repeatability evaluation of the present invention.

[0028] Figure 7 The figure shows the detection results of Salmonella in spleen samples of Salmonella-infected mice and control mice.

[0029] Figure 8 This is a comparison diagram of the positions of the primers and probes in the invA gene in this application and patent application CN105296664A; 1: upstream primer of this application; 2: Downstream primer of this application; 3: The probe of the present application; 4: Upstream primer of primer pair 1 of patent application CN105296664A; 5: Downstream primer of primer pair 1 of patent application CN105296664A; 6: Upstream primer of primer pair 2 of patent application CN105296664A; 7: Downstream primer of primer pair 2 of patent application CN105296664A; 8: Upstream primer of primer pair 3 of patent application CN105296664A; 9: Downstream primer of primer pair 3 of patent application CN105296664A; 10: Upstream primer of primer pair 4 of patent application CN105296664A; 11: Downstream primer of primer pair 4 of patent application CN105296664A; 12: Probe of patent application CN105296664A. DETAILED DESCRIPTION

[0030] The present application is further described in detail below through the accompanying drawings and embodiments. Through these descriptions, the characteristics and advantages of the present application will become clearer and more specific.

[0031] Example 1: Preparation of invA gene standard To establish a real-time fluorescence quantitative PCR method, the required external standards must first be prepared. The standards should contain highly conserved and specific sequences to ensure high specificity of the reaction.

[0032] The invA gene of Salmonella is highly conservative. Therefore, the present invention uses the invA gene of Salmonella as the target sequence.

[0033] In this example, the invA gene of Salmonella was amplified by PCR technology, and connected to the plasmid vector pMD19-T by gene recombination technology to construct the recombinant plasmid pMD19-T-invA, and the corresponding PCR identification and sequencing identification were performed. Finally, it was quantified as a standard for the method to be established, laying the foundation for the next step of the method and evaluation.

[0034] 1. Preparation of template DNA Template DNA was prepared using Takara's bacterial genome extraction kit (Takara 9763 kit).

[0035] The genomic DNA of Salmonella (standard strain) was extracted and used as a template for PCR amplification of the invA gene. The specific method is as follows: 1. Take 1 mL of bacterial suspension, centrifuge at 12000 rpm for 2 minutes, and discard the supernatant; 2. Add 180 μL of Buffer GL, 20 μL of Proteinase K (20 mg / mL), and 10 μL of RNase A (10 mg / mL), and shake thoroughly to mix. The above three reagents are all components of the Takara 9765 kit. Incubate in a 56°C water bath for 10 minutes. At this time, the solution should be transparent and clear. 3. Add 200 μL of Buffer GB and 200 μL of 100% ethanol, and mix thoroughly by pipetting; 4. Install the Spin Column on the Collection Tube (both are components of the Takara 9765 kit), transfer the solution to the Spin Column, centrifuge at 12,000 rpm for 2 minutes, and discard the solution; 5. Add 500 μL buffer WA to the adsorption column, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid; 6. Add 700 μL buffer WB to the adsorption column, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid; 7. Add 700 μL buffer WB to the adsorption column, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid; 8. Place the Spin Column on the Collection Tube and centrifuge at 12,000 rpm for 2 minutes. Discard the collection tube, transfer the adsorption column to a new 1.5 mL centrifuge tube, add 80 μL double distilled ultrapure water for elution, let stand at room temperature for 1 minute, and centrifuge at 12,000 rpm for 2 minutes.

[0036] 9. Use Nanodrop to detect the extraction concentration, dilute the concentration to 50-100 ng / μL, and store at -20℃.

[0037] The concentration of Salmonella genomic DNA extracted by Nanodrop is shown in Table 1.

[0038] Table 1

[0039] 2. PCR amplification of invA gene fragment 1. Primer design and synthesis The present invention performs bioinformatics comparison and analysis on the complete sequence of Salmonella invA gene in the NCBI database, selects a conserved fragment sequence (SEQ ID No.1) suitable for designing primers and probes as the target, and uses Primerexpress3 software, PrimerPremier5 software and Oligo7 software to design a set of real-time fluorescence quantitative PCR primers and probes and a set of peripheral primers of related sequences.

[0040] The amplification sequence selected by the present invention is shown as SEQ ID No.1, and the sequences of the designed upstream primer invA-19F and downstream primer invA-883R are shown as SEQ ID No.2 and SEQ ID No.3.

[0041] The amplified fragment of the present invention is a conservative fragment sequence, and the size is 865 bp.

[0042] 2. PCR reaction system and reaction conditions Using DNA as a template, the upstream primer invA-19F and the downstream primer invA-883R as amplification primers, PCR amplification was performed using the following reaction system and reaction conditions.

[0043] The PCR reaction system is shown in Table 2.

[0044] Table 2

[0045] The primers used were invA-19F / invA-883R, the Taq enzyme used was Takara ExTaq enzyme, and the PCR amplification instrument was Bio-Rad T100 Thermal Cycler.

[0046] The amplification procedure / reaction conditions are as follows: Pre-denaturation at 95°C for 5 minutes; 40 cycles of 95°C for 30 seconds, 57°C for 30 seconds, and 72°C for 1 minute; 72°C for 10 minutes.

[0047] After the amplification, 3.5 μL of the amplified product was taken for 1% agarose gel electrophoresis to detect the size of the PCR product, and then the DNA gel recovery kit produced by Takara was used to purify and recover the remaining PCR amplified product.

[0048] The results of agarose gel electrophoresis detection of PCR products are shown in the attached Figure 1 shown.

[0049] (III) Construction and transformation of recombinant plasmid pMD19-T-invA 1. Ligation reaction The PCR amplification product obtained by the above purification was ligated with pMD19-T (Takara) and prepared using the ligation system shown in Table 3.

[0050] Table 3

[0051] After preparation, incubate at 25°C for 1 hour to perform the ligation reaction.

[0052] 2. Transformation of pMD19-T-invA plasmid and PCR identification (1) Take out the frozen DH10B competent cells from the -80°C ultra-low temperature freezer and place them on an ice box to allow them to thaw naturally; (2) Take 5 μL of the ligation product and add it to 50 μL of DH10B competent cells, shake gently and incubate on ice for 25 minutes; (3) Heat shock at 42°C for 45 seconds, and immediately cool on ice for 2 minutes; (4) Add 700 μL of LB liquid medium without antibiotics to the 1.5 mL EP tube, mix well, and resuscitate at 32°C and 200 rpm for 30 min. (5) Centrifuge the Eppendorf tube at 5000 rpm for 1 minute, remove 600 μL of the supernatant, and evenly spread the remaining 100 μL of the bacteria on the LB plate containing Amp. Place it face up for 30 minutes. After the bacterial solution is completely absorbed by the culture medium, invert the culture dish and culture it in a 32°C incubator overnight. (6) Observe the next day and randomly pick 8 white monoclonal colonies from the plate with a pipette and place them on a new LB plate. Then transfer them into eight tubes with 10 μL ddH2O added and pipette several times to obtain the template for colony PCR. Use colony PCR primers pMD19T-JLPCR1-F / 1-R (product size 976 bp), pMD19T-JLPCR1-F / 2-R (product size 602 bp), and pMD19T-JLPCR3-F / 1-R (product size 467 bp) for amplification. The primer sequences are shown in SEQ ID No. 4 to SEQ ID No. 7, respectively.

[0053] The PCR reaction system is shown in Table 4.

[0054] Table 4

[0055] The PCR amplification program / reaction conditions are as follows: Pre-denaturation at 95°C for 5 minutes; 40 cycles of 95°C for 30 seconds, 57°C for 30 seconds, and 72°C for 1 minute; 72°C for 10 minutes.

[0056] The PCR products were detected by 1% agarose gel electrophoresis, and the positive transformants were identified by detecting the size of the PCR products.

[0057] The results of agarose gel electrophoresis detection of PCR products are shown in the attached Figure 2 shown.

[0058] (7) Select clones 1, 4, 5, and 7 with positive PCR results, inoculate them into 5 mL of LB liquid culture medium, and culture them overnight at 32°C and 200 rpm for 16 hours. Use the plasmid preparation kit produced by Takara to extract the positive recombinant plasmid, and use Nanodrop to measure the concentration. At the same time, take a portion of the purified plasmid and send it to Beijing Liuhe BGI Gene Technology Co., Ltd. for sequencing. The results show that the gene sequence of the inserted fragment of clone No. 4 is consistent with the target sequence. The plasmid extracted from this clone will be used as a plasmid standard for subsequent experiments.

[0059] The concentration of the extracted plasmid detected by Nanodrop is shown in Table 5.

[0060] Table 5

[0061] 4. Acquisition and Quantification of Standards Taking the conservative fragment sequence of the invA gene of Salmonella selected above as the target, a set of real-time fluorescence quantitative PCR primers and probes were designed using Primerexpress3 software, PrimerPremier5 software and Oligo7 software.

[0062] As the core of the present invention, a set of nucleotide sequences of specific primers and probes for real-time fluorescence quantitative PCR detection of Salmonella are shown as SEQ ID No.8-SEQ ID No.10.

[0063] The fluorescent reporter group at the 5' end of the probe is FAM, and the 3' end is labeled with the fluorescent quencher group MGB. Other fluorescent reporter groups such as TET, JOE, HEX, VIC and fluorescent quenchers such as DABCYL, BHQ, etc. can also be labeled.

[0064] The plasmid standard (plasmid extracted from clone No. 4) was diluted to a concentration of 50-100 ng / μL, and the concentration (copy number) of plasmid pMD19-T-invA was calculated as follows.

[0065] 1. When performing real-time fluorescence quantitative PCR, the unit is "copy number", so the unit needs to be converted into copies / μL. The following formula is used to calculate the plasmid copy number:

[0066] 2. Perform gradient dilution of plasmid standards according to Table 6 (taking 80.3 ng / μL as an example): Table 6

[0067] 3. Agilent AriaMx G8830A real-time fluorescence quantitative PCR instrument was used for fluorescence quantitative PCR, and Premix Ex Taq TM (Probe qPCR) (Bao-Nichi Biotech Co., Ltd., Japan).

[0068] The reaction system is: Premix Ex Taq (2X) 10μL, 0.4μL of 10μM upstream and downstream primers, 0.1μL of 10μM probe, 2.5μL of template, and make up the system to 20μL with sterile water.

[0069] The real-time fluorescence quantitative PCR program was set up as follows: Quantitative PCR was selected for Experiment Types, FAM was selected for Fluorescence Channel, and the copy number concentration of each diluted standard was entered in Standard Quantities.

[0070] The reaction conditions were: 95°C for 30 seconds (1 cycle), 95°C for 5 seconds, 60°C for 30 seconds (40 cycles). After the reaction was completed, the machine automatically generated a standard curve for the standard product.

[0071] The standard curve is as attached Figure 3 shown.

[0072] Example 2: Performance evaluation of TaqMan real-time fluorescence quantitative PCR method In this example, TaqMan real-time fluorescence quantitative PCR was performed using specific primers and probes shown in SEQ ID No. 8 to SEQ ID No. 10, respectively, so as to evaluate the specificity, sensitivity and repeatability of the method.

[0073] The fluorescent reporter group at the 5' end of the probe is FAM, and the 3' end is labeled with the fluorescent quencher group MGB.

[0074] Agilent AriaMx G8830A real-time fluorescence quantitative PCR instrument was used for fluorescence quantitative PCR, and Premix Ex Taq TM (Probe qPCR) (Bao-Nichi Biotech Co., Ltd., Japan).

[0075] The reaction system is: Premix Ex Taq (2X) 10μL, 0.4μL of 10μM upstream and downstream primers, 0.1μL of 10μM probe, 2.5μL of template, and make up the system to 20μL with sterile water.

[0076] The real-time fluorescence quantitative PCR program settings are as follows: In the specificity evaluation, select Quantitative PCR in Experiment Types and select FAM in the fluorescence channel. In the sensitivity and repeatability evaluation, select Quantitative PCR in Experiment Types and select FAM in the fluorescence channel, and enter the copy number concentration of each diluted standard in Standard Quantities.

[0077] The reaction conditions were: 95°C for 30 seconds (1 cycle), 95°C for 5 seconds, 60°C for 30 seconds (40 cycles).

[0078] 1. Specificity evaluation The DNA of strains preserved in the laboratory, including Escherichia coli (78.6 ng / μL) and Salmonella (59.3 ng / μL), was used as the template for detection, and nuclease-free water was used as the template as the negative control.

[0079] The results showed that all Salmonella and diluted samples were positive, while E. coli had no amplification signal, which was consistent with the negative control. This shows that the primers and probes in the real-time fluorescence quantitative PCR system are highly specific. Figure 4 shown.

[0080] 2. Sensitivity evaluation The standard plasmid was serially diluted with sterile ultrapure water in a 10-fold ratio to a concentration of 4 × 10 6 , 4×10 5 , 4×10 4 , 4×10 3 , 4×10 2 , 4×10 1 Plasmid DNA was used as a template for detection, and blank matrix was used as a template as a negative control.

[0081] The results showed that 4×10 1 The standard sample with the highest concentration has a good amplification curve, while the negative control shows no amplification signal. This shows that the real-time fluorescence quantitative PCR system can use 40 copies / μl as the lowest detection line. Figure 5 shown.

[0082] 3. Repeatability evaluation The standard plasmid was serially diluted with sterile ultrapure water in a 10-fold ratio to a concentration of 4 × 10 6 to 4×10 1 Plasmid DNA was used as a template to detect the coefficient of variation of real-time fluorescence quantitative PCR, and blank matrix was used as a template as a negative control.

[0083] For intra-assay reproducibility, all samples were tested in triplicate. For inter-assay reproducibility, the same experiment was repeated three times.

[0084] The results showed that the intra-batch coefficient of variation of the reaction system was 0.06-1.69%, and the inter-batch coefficient of variation was 0.16-0.49%, as shown in Table 7.

[0085] Table 7

[0086] Wherein, the amplification curve and the standard curve are shown in FIG6 .

[0087] The above results indicate that the real-time fluorescence quantitative PCR system has good repeatability and high stability.

[0088] Example 3: Application of TaqMan real-time fluorescence quantitative PCR method in rapid quantification of Salmonella 1. Design and synthesis of specific primers and probes In this example, the nucleotide sequences of specific primers and probes used for real-time fluorescence quantitative PCR detection of Salmonella are shown in SEQ ID No.8 to SEQ ID No.10.

[0089] The fluorescent reporter group at the 5' end of the probe is FAM, and the 3' end is labeled with the fluorescent quencher group MGB. Other fluorescent reporter groups such as TET, JOE, HEX, VIC and fluorescent quenchers such as DABCYL, BHQ, etc. can also be labeled.

[0090] 2. Extraction of genomic DNA from strains and infected tissue samples Female healthy BALB / c mice aged 6-8 weeks were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. Shanghai Branch. After a 3-day adaptation period, the mice were infected with VNP20009 Salmonella (Shanghai Collection of Microorganisms, D25149) by intravenous injection. One hour later, spleen samples from 10 Salmonella-infected mice and uninfected control mice were collected, a total of 11 samples, and genomic DNA was extracted.

[0091] 1. Place the tissue in a 1.5 mL Eppendorf tube, add 180 μL of Buffer GL, 20 μL of Proteinase K (20 mg / mL), 10 μL of RNase A, and 2 3 mm beads to each tube, and shake to mix.

[0092] 2. Place the centrifuge tube in a fully automatic sample rapid grinding instrument with a frequency of 60 Hz and a time of 90 s.

[0093] 3. Take out the ground sample, tap the tube wall lightly, and separate it instantly.

[0094] 4. Place the centrifuge tube in a 56°C water bath for 1-3 hours.

[0095] 5. Add 200 μL Buffer GB and 200 μL 100% ethanol to the centrifuge tube and mix by shaking.

[0096] 6. Install the Spin Column on the Collection Tube, transfer the solution to the Spin Column, centrifuge at 12,000 rpm for 2 minutes, and discard the filtrate.

[0097] 7. Add 500 μL buffer WA to the adsorption column, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid.

[0098] 8. Add 700 μL buffer WB to the adsorption column, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid.

[0099] 9. Add 700 μL buffer WB to the adsorption column, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid.

[0100] 10. Place the Spin Column on the Collection Tube, centrifuge at 12000rpm for 2 minutes, discard the collection tube, and transfer the adsorption column to a new 1.5mL centrifuge tube.

[0101] 11. Add 30-100 μL of water for elution and let stand at room temperature for 5 minutes. Centrifuge at 12000 rpm for 2 minutes to elute DNA, measure concentration and purity with Nanodrop, and store at -20℃.

[0102] 3. TaqMan real-time fluorescence quantitative PCR for rapid identification of Salmonella Agilent AriaMx G8830A real-time fluorescence quantitative PCR instrument was used for fluorescence quantitative PCR, and Premix Ex Taq TM (Probe qPCR) (Bao-Nichi Biotech Co., Ltd., Japan).

[0103] The reaction system is: Premix Ex Taq (2X) 10μL, 0.4μL of 10μM upstream and downstream primers, 0.1μL of 10μM probe, 2.5μL of template, and make up the system to 20μL with sterile water.

[0104] The real-time fluorescence quantitative PCR program was set up as follows: Quantitative PCR was selected for Experiment Types, FAM was selected for Fluorescence Channel, and the copy number concentration of each diluted standard was entered in Standard Quantities.

[0105] The reaction conditions were: 95°C for 30 seconds (1 cycle), 95°C for 5 seconds, 60°C for 30 seconds (40 cycles).

[0106] Fluorescence is collected, and samples to be tested are considered negative if the Cq value is greater than the lowest point of the standard curve or if there is no amplification. Substitute the Cq value of the test sample into the standard curve equation to calculate the copy number concentration of Salmonella in the DNA sample.

[0107] The results showed that all spleen samples of mice in the Salmonella infection group were detected, while none were detected in the spleen of the control group. Figure 7 shown.

[0108] Table 8

[0109] Example 4: Comparison of the real-time fluorescence quantitative PCR method of the present invention with existing methods In order to further demonstrate the advantages of the real-time fluorescence quantitative PCR method of the present invention, this example comprehensively compares the real-time fluorescence quantitative PCR system and method of the present invention with the systems and methods disclosed in patent applications CN105296664A and CN105087799A, respectively. The contents disclosed in the above two patent applications are fully introduced into this application.

[0110] 1. Advantages in primer and probe design 1. Primers and probes of the present application The primers and probes of the present application are designed for the invA gene of Salmonella, and the specific sequences are shown in SEQ ID No. 8 to SEQ ID No. 10.

[0111] The upstream primers were located at positions 197-219 of the invA gene, with a length of 23 bp and a GC content of 43%; the downstream primers were located at positions 238-261 of the invA gene, with a length of 24 bp and a GC content of 46%; the probes were located at positions 221-233 of the invA gene, with a length of 13 bp. The amplified product size was 65 bp.

[0112] The analysis shows that (1) the length of the upstream and downstream primers is moderate, and the GC content is within the ideal range of 40%-50%, which helps to improve the binding stability and amplification efficiency of the primers. (2) When the primer specificity is good, a relatively short probe is designed to increase the reaction speed and reduce the cost. (3) The amplification product is shorter, which can complete the amplification process faster and improve the reaction efficiency. This is especially advantageous when the template concentration is low or rapid amplification is required. In addition, when detecting the fluorescent signal, due to the shorter product length, the binding efficiency of the probe and the product is higher, which can more accurately reflect the amount of the amplification product, thereby improving the accuracy of quantification.

[0113] 2. Primers and probes of patent application CN105296664A Patent application CN105296664A designed four sets of primers and probes for the invA gene of Salmonella. The specific sequences of primer pairs 1 to 4 and the probes are shown in the table in paragraph

[0263] of its specification.

[0114] Among them, the probe is located at the 715-736th position of the invA gene, and the length is 22bp. The upstream primer in primer pair 1 is located at the 668-687th position of the invA gene, the length is 20bp, and the GC content is 60%; the downstream primer is located at the 740-758th position of the invA gene, the length is 19bp, and the GC content is 53%. The size of the amplified product is 91bp. The upstream primer in primer pair 2 is located at the 547-567th position of the invA gene, the length is 21bp, and the GC content is 57%; the downstream primer is located at the 870-890th position of the invA gene, the length is 21bp, and the GC content is 52%. The size of the amplified product is 344bp. The upstream primer in primer pair 3 is located at positions 597-618 of the invA gene, with a length of 22 bp and a GC content of 36%; the downstream primer is located at positions 774-794 of the invA gene, with a length of 21 bp and a GC content of 43%. The size of the amplified product is 198 bp. The upstream primer in primer pair 4 is located at positions 597-618 of the invA gene, with a length of 22 bp and a GC content of 36%; the downstream primer is located at positions 776-799 of the invA gene, with a length of 24 bp and a GC content of 38%. The size of the amplified product is 203 bp.

[0115] The analysis showed that (1) in primer pair 1, the overall length of the upstream and downstream primers was relatively short, and the binding stability of the shorter primers to the template was relatively low. They may be easily dissociated from the template during the PCR reaction, affecting the amplification efficiency. (2) In primer pair 2, the amplification product size was 344 bp. Longer amplification products require more time and enzyme activity during PCR amplification, and are prone to incomplete amplification or non-specific amplification. (3) In primer pairs 3 and 4, the GC content is low, resulting in weaker binding between the primers and the template, requiring a lower annealing temperature, and increasing the risk of non-specific amplification.

[0116] The specific positions of the primers and probes in the invA gene in this application and the patent application are shown in the attached Figure 8 shown.

[0117] 3. Primers and probes of patent application CN105087799A Patent application CN105087799A designs upstream primers, downstream primers and probes for the STM4493 gene of Salmonella typhimurium. For specific sequences, please refer to paragraph

[0036] of its specification.

[0118] The upstream primers were located at positions 534-557 of the STM4493 gene, with a length of 24 bp and a GC content of 38%; the downstream primers were located at positions 624-642 of the STM4493 gene, with a length of 19 bp and a GC content of 58%; the probes were located at positions 559-582 of the STM4493 gene, with a length of 24 bp. The amplified product size was 108 bp.

[0119] After analysis, it can be seen that (1) the primers and probes in the patent application are not designed for the same gene as those in the present application. (2) In the primers and probes, the GC content of the upstream and downstream primers is quite different, which is not conducive to the specific binding of the primers to the template.

[0120] In summary, the primers and probes designed in this patent application show great advantages in terms of length, GC content, amplification product size, etc., and are suitable for efficient and specific PCR amplification and subsequent analysis.

[0121] 2. Advantages in the amount of primers and probes used 1. The amount of primers and probes used in this application In the 20 μL real-time fluorescence quantitative PCR system of the present application, 0.4 μL of 10 μM upstream and downstream primers and 0.1 μL of 10 μM probe are used. Therefore, the amount of primers and probes used is small.

[0122] 2. The amount of primers and probes in patent application CN105296664A In the 20 μL real-time fluorescence quantitative PCR system of patent application CN105296664A, as shown in paragraph

[0255] of the specification, 1 μL of each of the upstream and downstream primers of 10 μM and 1.5 μL of the probe of 4 μM are used. Therefore, the amount of primers and probes used is large, which is 2.5 times and 6 times that of the present application.

[0123] 3. The amount of primers and probes in patent application CN105087799A In the 20 μL real-time fluorescence quantitative PCR system of patent application CN105087799A, as shown in paragraphs

[0042] and

[0043] of the specification, 1.6 μL of 5 μM upstream and downstream primers and 1.6 μL of 5 μM probe are used. Therefore, the amount of primers and probes used is large, which is 2 times and 8 times that of the present application.

[0124] 3. Advantages in real-time fluorescence quantitative PCR reaction procedures 1. Fluorescence quantitative PCR reaction procedure of this application The fluorescence quantitative PCR reaction program used in this application is 95°C for 30 seconds (1 cycle), 95°C for 5 seconds, 60°C for 30 seconds (40 cycles). Therefore, the theoretical reaction time of this application is about 24 minutes.

[0125] 2. Fluorescence quantitative PCR reaction procedure of patent application CN105296664A As shown in paragraphs

[0256] to

[0258] of the specification, the fluorescent quantitative PCR reaction program adopted by patent application CN105296664A is 95°C for 10 minutes, 95°C for 15 seconds, 60°C for 1 minute, and 40 cycles. Therefore, the theoretical reaction time of the application is 60 minutes, which is 2.5 times that of the present application.

[0126] 3. Fluorescence quantitative PCR reaction procedure of patent application CN105087799A As shown in paragraph

[0042] of the specification, the fluorescent quantitative PCR reaction procedure adopted by patent application CN105087799A is 94°C pre-denaturation for 2 minutes, 94°C for 15 seconds, 60°C for 40 seconds, and 40 cycles. Therefore, the theoretical reaction time of the application is 39 minutes, which is 1.6 times that of the present application.

[0127] Since the primers and probes of the present application are not designed for the same gene as those of patent application CN105087799A, they are no longer suitable for direct comparison in terms of specificity, sensitivity and repeatability of real-time fluorescence quantitative PCR reactions. Therefore, the following only compares the specificity, sensitivity and repeatability of the present application and patent application CN105296664A in real-time fluorescence quantitative PCR reactions.

[0128] 4. Advantages in real-time fluorescence quantitative PCR reaction specificity 1. Reaction specificity of this application This application uses Escherichia coli and Salmonella as test templates, and nuclease-free water as a template as a negative control. All Salmonella and diluted samples tested positive, while Escherichia coli had no amplification signal, and the results were consistent with the negative control.

[0129] Therefore, the present application sets nuclease-free water as a template as a negative control, thereby verifying that the primers and probes in the real-time fluorescence quantitative PCR system are highly specific.

[0130] 2. Reaction specificity of patent application CN105296664A As shown in paragraph

[0259] of the specification, the plasmid standard molecule pXL10 prepared in Example 1 was diluted to different concentrations of plasmid standard molecules (7.30×10 6 copies / μL, 7.30×10 5 copies / μL, 7.30×10 4 copies / μL, 7.30×10 3 copies / μL, 7.30×10 2 copies / μL, 7.30×10 1 copies / μL, 7.30×10 0 copies / μL), and used plasmid standard molecules of different concentrations as templates, and performed real-time fluorescence PCR amplification according to the method in the literature.

[0131] At the same time, as shown in paragraph

[0264] of the specification, for the invA gene sequence of Salmonella, primer pair 1 has the best detection effect, strong specificity, no non-specific bands appear after amplification, and the highest detection sensitivity, with a minimum detection of 7.30 copies / μL of the target sequence; primer pairs 2 and 3 have lower sensitivities, and can detect 7.30×10 2 copies / μL of target sequence; primer pair 4 can detect 7.30×10 1 copies / μL of the target sequence, but the specificity was poor and non-specific bands appeared.

[0132] From this, we can see that (1) the application did not set up a negative control, and the specificity of primer pair 4 was poor, with non-specific bands appearing. (2) All four primer pairs used the same probe, and the upstream primers of primer pair 3 and primer pair 4 were exactly the same, and the downstream primers were also basically the same. Therefore, the specificity of amplification should be consistent in theory, but the result showed that primer pair 3 was specific, while primer pair 4 was less specific, which was contrary to the theory, so the specificity test results of this application were questionable.

[0133] (V) Advantages in sensitivity of real-time fluorescence quantitative PCR reaction 1. The reaction sensitivity of this application In this application, the standard plasmid was serially diluted with sterile ultrapure water at a 10-fold ratio to a concentration of 4×10 6 , 4×10 5 , 4×10 4 , 4×10 3 , 4×10 2 , 4×10 1 Plasmid DNA was used as template for detection, and blank matrix was used as template as negative control. The results showed that 4×10 1 The standard with the concentration of copies / μL had a good amplification curve, while the negative control showed no amplification signal.

[0134] Therefore, the present application sets a blank matrix as a template as a negative control, and the real-time fluorescence quantitative PCR system can use 40 copies / μL as the minimum detection line.

[0135] 2. Reaction sensitivity of patent application CN105296664A As shown in paragraph

[0259] of the specification, the plasmid standard molecule pXL10 prepared in Example 1 was diluted to different concentrations of plasmid standard molecules (7.30×10 6 copies / μL, 7.30×10 5 copies / μL, 7.30×10 4 copies / μL, 7.30×10 3 copies / μL, 7.30×10 2 copies / μL, 7.30×10 1 copies / μL, 7.30×10 0 copies / μL), and used plasmid standard molecules of different concentrations as templates, and performed real-time fluorescence PCR amplification according to the method in the literature.

[0136] At the same time, as shown in paragraph

[0264] of the specification, for the invA gene sequence of Salmonella, primer pair 1 has the best detection effect, strong specificity, no non-specific bands appear after amplification, and the highest detection sensitivity, with a minimum detection of 7.30 copies / μL of the target sequence; primer pairs 2 and 3 have lower sensitivities, and can detect 7.30×10 2 copies / μL of target sequence; primer pair 4 can detect 7.30×10 1 copies / μL of the target sequence, but the specificity was poor and non-specific bands appeared.

[0137] From this, it can be seen that the application did not set up a negative control or a no-template control, and its lower limit of detection cannot be determined. Therefore, the detection value of the lower concentration standard is not credible, that is, its sensitivity of 7.30 copies / μL is questionable.

[0138] (VI) Advantages in the repeatability of real-time fluorescence quantitative PCR reactions 1. Reaction repeatability of this application In this application, the standard plasmid was serially diluted with sterile ultrapure water at a 10-fold ratio to a concentration of 4×10 6 to 4×10 1 Plasmid DNA was used as a template to detect the coefficient of variation of real-time fluorescence quantitative PCR, and blank matrix was used as a template as a negative control. For intra-batch repeatability, all samples were tested three times. The inter-batch repeatability was determined by repeating this experiment three times. The results showed that the intra-batch coefficient of variation of the reaction system was 0.06-1.69%, and the inter-batch coefficient of variation was 0.16-0.49%.

[0139] Therefore, the present application sets a blank matrix as a template as a negative control, detects the intra-batch coefficient of variation and the inter-batch coefficient of variation, and the real-time fluorescence quantitative PCR system has good repeatability and high stability.

[0140] 2. Reaction repeatability of patent application CN105296664A As shown in paragraph

[0179] of the specification, the statistical results of the uniformity of pXL10 are shown in Table 2. At a confidence level of 95%, the F value is less than F 0.05 (14, 30), demonstrating that the plasmid standard molecule pXL10 is homogeneous.

[0141] At the same time, as shown in paragraph

[0211] of the specification, it can be seen from statistical results that the plasmid standard molecule pXL10 is stable when stored at -20°C for 12 months.

[0142] It can be seen that in this application, the above uniformity and stability experiments were conducted on the plasmid standard molecule pXL10. Therefore, this application did not conduct a repeatability experiment on the real-time fluorescence quantitative PCR reaction and lacked repeatability experimental data.

[0143] 7. Advantages of real-time fluorescence quantitative PCR in practical application in sample detection 1. Practical application of this application in testing samples The present application infected mice by intravenous injection, and collected spleen samples from 10 Salmonella-infected mice and uninfected control mice 1 hour later, totaling 11 samples, extracted genomic DNA, and performed real-time fluorescence quantitative PCR.

[0144] Therefore, the present application established a mouse model infected with Salmonella by intravenous injection, set up an uninfected negative control, and performed real-time fluorescence quantitative PCR on spleen samples of all mice. All spleen samples of mice in the Salmonella infection group were detected, while spleen samples of the control group were not detected. A standard equation was established to calculate the Cq mean and copy number.

[0145] 2. Practical application of patent application CN105296664A in testing samples The real-time fluorescence quantitative PCR reaction in this patent application has not been actually applied in the detection of samples, and there is a lack of experimental data on the real-time fluorescence quantitative PCR reaction in actual sample detection. It is still uncertain whether it can be successfully applied in actual detection.

[0146] 3. Practical application of patent application CN105087799A in testing samples As shown in paragraphs

[0048] to

[0051] of the specification, using the cDNA of the sample to be tested and the DNA of the standard as templates, using the specific primers of Salmonella typhimurium, the fluorescent quantitative PCR amplification of the gene fragment of Salmonella typhimurium is carried out simultaneously in the same system. Rapid quantitative detection of Salmonella typhimurium is performed based on the standard curve and the Ct value of the sample to be tested.

[0147] It can be seen that the application only describes the use of real-time fluorescence quantitative PCR reaction to perform quantitative detection in the sample to be tested, but does not provide any experimental data related to the detection, let alone the standard equation, Cq mean value, copy number and other experimental data. Therefore, it is uncertain whether it can be successfully applied in actual detection.

[0148] In summary, through the above comprehensive comparative analysis, it can be seen that the primer and probe design of the present application is more ingenious and reasonable; the amount of primers and probes used in the amplification system is small, saving detection costs; the real-time fluorescence quantitative PCR reaction procedure takes a short time, saving time costs; the specificity, sensitivity and repeatability of the reaction are good; and it has been verified by quantitative detection of actual samples. On this basis, it is guaranteed that the real-time fluorescence quantitative PCR detection method of the present application can perform quantitative detection on the detection sample on the basis of saving detection costs and time costs, that is, the present application provides a Salmonella detection method with strong specificity, high sensitivity, good repeatability, fast detection, simple operation and low cost, which can be used for the rapid identification of Salmonella and the absolute quantitative detection in Salmonella infected tissue samples, providing a basis for high-throughput rapid detection of Salmonella infection. These are all patent applications CN105296664A and patent application CN105087799A cannot achieve.

[0149] The detailed information of all sequences of the present invention is shown in Table 9.

[0150] Table 9

[0151] The present application has been described above in conjunction with preferred embodiments, but these embodiments are only exemplary and serve only as an illustration. On this basis, various replacements and improvements may be made to the present application, all of which fall within the scope of protection of the present application.

Claims

1. A primer and a probe for detecting Salmonella, wherein the upstream primer is shown as SEQ ID No.8, the downstream primer is shown as SEQ ID No.9, and the probe is shown as SEQ ID No.

10.

2. The primer and probe according to claim 1, wherein the fluorescent reporter group at the 5' end of the probe is FAM, and the fluorescent quencher group labeled at the 3' end is MGB.

3. A kit comprising the primers and probes according to claim 1 or 2.

4. Use of the primers and probes according to claim 1 or 2 in the quantitative detection of Salmonella, wherein the detection is for non-diagnostic purposes.

5. Use of the kit according to claim 3 in the quantitative detection of Salmonella, wherein the detection is for non-diagnostic purposes.

6. A method for quantitative detection of Salmonella, comprising the following steps: (1) Preparation of standard samples; (2) Using the primers and probes described in claim 1 or 2, or the kit described in claim 3, perform real-time fluorescence PCR amplification on the standard and the sample to be tested, and draw a standard curve; (3) Quantitative detection of Salmonella is performed using a standard curve and the Cq value of the sample to be tested, and the detection is used for non-diagnostic purposes.

7. The method according to claim 6, wherein in step (1), the conserved gene fragment of Salmonella invA is inserted into a vector to obtain a recombinant plasmid containing the conserved gene fragment of Salmonella invA as a standard.

8. The method according to claim 7, wherein the vector is pMD19-T, and the conserved gene fragment of Salmonella is the nucleotide sequence shown in SEQ ID No.

1.

9. The method according to claim 6 or 7, wherein the reaction system in step (2) is 10 μL of Premix Ex Taq (2X), 0.4 μL of each of the upstream and downstream primers with a concentration of 10 μM, 0.1 μL of the probe with a concentration of 10 μM, 2.5 μL of the template, and 20 μL of sterile water.

10. The method according to claim 6 or 7, wherein the reaction conditions in step (2) are 95°C for 30 seconds, 95°C for 5 seconds, 60°C for 30 seconds, and 40 cycles.

11. The method according to claim 6 or 7, wherein the standard curve equation in step (2) is prepared based on each reaction, and the formula is: Y = -alogX + b; in, Y is the Cq value of the amplified standard, and X is the starting copy number of the standard DNA.

12. The method according to claim 6 or 7, wherein in step (3), the Cq value of the test sample is substituted into the formula to calculate the starting copy number of Salmonella DNA in the test sample, and then the content of Salmonella in the test sample can be deduced based on the mass of the test sample used for DNA extraction, thereby quantitatively detecting the Salmonella in the test sample.

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