CN115873966A - Novel staphylococcus aureus enterotoxin gene quadruple real-time fluorescence PCR (polymerase chain reaction) detection kit and method - Google Patents

Abstract

The invention discloses a quadruple real-time fluorescence PCR detection kit and a method for novel enterotoxin genes of staphylococcus aureus. Respectively designing primers and probes according to SEL, SED, SEM and SEQ gene sequences, analyzing the sequence specificity of an amplification product by blast to ensure that no homologous or sequence consistency biological gene information exists, fully considering whether specific sequences of four target genes can be amplified in the same system, carrying out elaborate optimization and repeated tests to obtain the detection primers and the detection probes of the invention, carrying out quadruple real-time fluorescence PCR detection on a sample by using the detection primers and the detection probes according to the method of the invention, and completing the specificity detection of staphylococcus aureus enterotoxin SED, SEL, SEM and SEQ genes of the sample to be detected in one amplification reaction.

Description

Quadruple real-time fluorescence PCR detection kit and method for novel enterotoxin gene of Staphylococcus aureus

Technical Field

The invention belongs to the field of biotechnology and relates to a kit and a method capable of simultaneously detecting traditional and novel enterotoxin genes of Staphylococcus aureus.

Background Art

Staphylococcus aureus can easily contaminate dairy products, meat, starch and other foods, and is one of the important pathogens that cause bacterial food poisoning. Studies have shown that Staphylococcal enterotoxins (SEs) are the main virulence factors that cause Staphylococcus aureus poisoning. They are a class of low-molecular-weight proteins with similar structures and similar toxicity. Only high nanogram to low microgram levels of enterotoxins are needed to specifically attack small intestinal cells and enter the digestive system to cause intestinal diseases such as diarrhea and vomiting. Enterotoxins generally have strong thermal stability and are still active after being treated at 100°C for 30 minutes. Therefore, even if the bacteria have been killed during processing, the enterotoxins they produce can still cause food poisoning. So far, more than 20 types of enterotoxins and enterotoxin-like substances have been discovered, and this number continues to expand. Among them, superantigens that can cause vomiting in primate models after oral administration are called Staphylococcal enterotoxins (SEs), including five classic enterotoxins SEA~SEE and new enterotoxins SEG-SEI, SEK~SET, SEY; while enterotoxins that lack emetic activity or have not been tested are called Staphylococcal enterotoxin-like proteins (SEls), including SElJ, SElU, SElV, SElW, SElX, and SElZ. Many enterotoxin encoding genes are linked, such as enterotoxins sed, selj, and ser, which can exist simultaneously on plasmid pIB485; sek and seq can exist in tandem on phages and Staphylococcus aureus virulence islands; enterotoxins sec and sel are also located on virulence islands; the most common egc cluster can express 5 enterotoxin genes, seg, sei, sem, sen, and seo, in a linked manner.

At present, the commonly used methods for detecting enterotoxins are mainly molecular biology methods and immunology methods. Molecular biology methods are mainly experimental methods based on PCR technology, which have the advantages of sensitivity, rapidity, simplicity, etc. In particular, real-time fluorescence PCR technology is often used to detect various toxin genes in food. There are no nucleic acid detection kits for new enterotoxins on the market, and the nucleic acids of classic enterotoxins SEA~SEE are only single-plex fluorescence PCR detection methods. Some scholars have successfully detected 9 enterotoxin genes from sea to selj using RT-PCR reactions, but one tube for multiple tests has not yet been achieved, and the detection of gene levels does not mean the expression of protein levels. Enzyme-linked immunosorbent assay (ELISA) is the most commonly used enterotoxin protein detection method based on antigen-antibody specific recognition, but complex components in the food matrix such as phosphatase, catalase, and IgG non-specifically bound by Staphylococcus a protein will lead to reduced sensitivity and specificity of ELISA kits and the occurrence of false positive reactions. Enterotoxins are a class of proteins with extremely similar structures, and cross-reactions are very likely to occur during ELISA detection. In addition, the commercial kits currently available can only detect classic enterotoxins SEA-SEE, and lack protein detection technology for new enterotoxins. In recent years, there have been reports that new enterotoxins can also be involved in food poisoning incidents, and the carrying rate of new enterotoxins is very high, so there is an urgent need to establish a detection method for new enterotoxins.

Summary of the invention

The first purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide a multiplex fluorescence PCR detection kit, which can simultaneously realize the typing of four Staphylococcus aureus enterotoxins in one PCR reaction, and through the phenomenon of tandem expression of enterotoxin genes, it is possible to infer the detection of 12 genes including sec-sel, sed-selj-ser, seg-sei-sem-sen-seo, and sek-seq.

To achieve the above purpose, the present invention downloads the base sequences of Staphylococcus aureus enterotoxin genes sel, sed, sem, and seq from GenBank, and then performs homology comparison analysis to find the conserved regions of the genes. Specific primers and target probes are designed in the conserved nucleic acid sequence regions, respectively. The primer sequences are shown in the following table:

Table 1 Primer sequences

The second object of the present invention is a non-diagnostic method for detecting a novel enterotoxin of Staphylococcus aureus using quadruple real-time fluorescence PCR, comprising the following steps:

a) Pre-treat the sample to be tested;

b) extracting bacterial DNA from the sample to be tested, preferably using a Promega genome extraction kit (Lot: A1120);

c) using 2 μl of the bacterial DNA extracted in step (b) as a template, and using the optimized Taqman fluorescent PCR reaction system and reaction conditions to perform real-time fluorescent quantitative Real-time PCR detection;

d) After the reaction is completed, read and record the PCR amplification cycle number (Ct) value of each test sample based on the fluorescent signal recorded by the fluorescent reporter group labeled with the probe. According to the established interpretation criteria: a Ct value of 0 or greater than 40 indicates a negative result, a Ct value less than 38 indicates a positive result; if the Ct value is between 38-40, the sample needs to be retested. The Ct value of each sample is used to determine whether the sample contains enterotoxin genes.

The reaction system is as follows:

Table 2 PCR reaction system

The reaction program was as follows: pre-denaturation at 95°C for 2 min, 40 cycles of denaturation at 95°C for 20 s, annealing at 58°C for 45 s, and fluorescence detection during the annealing stage.

The dNTP Mixture is a mixed solution of 2.5 mM dATP, 2.5 mM dCTP, 2.5 mM dGTP and 2.5 mM dTTP.

Step b) The applicable scope of samples includes food samples, feces, vomitus and other specimens. For feces and vomitus specimens, depending on the amount, suspend and boil with 100-200μl of saline, centrifuge at 10000rpm for 2 minutes, and take 2μl of supernatant for PCR reaction. After the food sample is enriched in the enrichment solution of the bacteria to be tested, take 1ml of the enrichment solution and centrifuge at 10000rpm for 5 minutes, discard the supernatant, extract DNA with Qiagen DNA extraction kit, and take 2μl for PCR reaction.

Primers and probes are designed according to the gene sequences of sel, sed, sem and seq, and the sequence specificity of the amplified products is analyzed by blast to ensure that there is no homologous or sequence consistency biological gene information, and whether the specific sequences of the four target genes can be amplified in the same system is fully considered, and the detection primers and detection probes of the present invention are carefully optimized and repeatedly tested to obtain the detection primers and detection probes of the present invention, and the detection primers and detection probes are used to perform quadruple real-time fluorescence PCR detection on the sample according to the method of the present invention, and the specific detection of the SED, SEL, SEM and SEQ genes of the Staphylococcus aureus enterotoxins of the sample to be tested is completed in one amplification reaction, and the detection of 12 genes including sec-sel, sed-selj-ser, seg-sei-sem-sen-seo, and sek-seq in the sample to be tested is inferred through the phenomenon of tandem expression of the enterotoxin genes. The detection primers and probes of the present invention are used to perform quadruple real-time fluorescence PCR detection on the sample according to the method of the present invention, and the detection is fast, and the process from sample preparation to test result issuance can be completed in 8-10 hours, without interference from false positives and cross contamination, etc., and the results are reliable, with high sensitivity and strong specificity.

Staphylococcus aureus is very easy to contaminate milk powder, pork and pastry foods, and produces enterotoxins after co-culture, thereby causing gastroenteritis symptoms such as nausea, vomiting, and diarrhea. The present invention can provide a favorable tool for epidemiological investigation of Staphylococcus aureus food poisoning incidents, and is suitable for use by food and water inspection, disease control centers, and quality supervision departments. The present invention not only meets the needs of rapid diagnosis and food testing of Staphylococcus aureus food poisoning incidents, but also has important significance for the monitoring of Staphylococcus aureus in the production process of the food industry and the quality control of the final product.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A-D shows the PCR amplification results of strains ATCC 13565, 19-51, 19-65 and 18-95; 1: sed; 2: sel; 3: sem; 4: seq;

Figure 1E shows the PCR amplification results of strain 20-38; 1: sed; 2: sel; 3: sem; 4: seq;

Figure 1F shows the PCR amplification results of strain 20-122; 1: sed; 2: sel; 3: sem; 4: seq;

Figure 1G shows the PCR amplification results of strain 18-79; 1: sed; 2: sel; 3: sem; 4: seq;

Figure 1H shows the PCR amplification results of strain 19-63; 1: sed; 2: sel; 3: sem; 4: seq;

Figure 1I shows the PCR amplification results of strains 18-95 and 19-63; 1: sed; 2: sel; 3: sem; 4: seq;

Figure 1J shows the PCR amplification results of Staphylococcus epidermidis ATCC12228, Escherichia coli ATCC25922, Staphylococcus aureus ATCC29213, ATCC14458, ATCC27664 (see), 18-66, 19-50, 20-46; 1: sed; 2: sel; 3: sem; 4: seq;

Figure 2A is the PCR amplification result of sed of strain 19-63; 1: 10 ng/ml; 2: 1 ng/ml; 3: 0.1 ng/ml; 4: 0.01 ng/ml;

Figure 2B is the PCR amplification result of sel of strain 19-63; 1: 10 ng/ml; 2: 1 ng/ml; 3: 0.1 ng/ml; 4: 0.01 ng/ml;

Figure 2C is the PCR amplification result of sem of strain 19-63; 1: 10 ng/ml; 2: 1 ng/ml; 3: 0.1 ng/ml; 4: 0.01 ng/ml;

Figure 2D shows the PCR amplification results of the seq of strain 18-95; 1: 10 ng/ml; 2: 1 ng/ml; 3: 0.1 ng/ml; 4: 0.01 ng/ml;

FIG3A shows the PCR amplification results of pork contaminated with 21-68 producing novel enterotoxins D, J, and R;

FIG3B shows the PCR amplification results of pork contaminated with 19-51 producing novel enterotoxins C and L;

FIG3C shows the PCR amplification results of pork contaminated with 19-65 producing novel enterotoxins G, I, M, N, O, and U;

Figure 3D shows the PCR amplification results of pork contaminated with 18-95 producing novel enterotoxins K and Q.

DETAILED DESCRIPTION

The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

Example 1

The 12 tested bacterial strains, namely Staphylococcus epidermidis ATCC12228, Escherichia coli ATCC25922, Staphylococcus aureus ATCC29213, ATCC14458 (seb), ATCC13565 (sea, sed), and ATCC27664 (see), were purchased from the National Institute for Food and Drug Control and the China Center for General Microbiological Culture Collection and confirmed by VITEK2;

Whole genome sequencing verified Staphylococcus aureus strains 19-63 (SEC, SED, SELJ, SEL, SEG, SEE, SEM, SEN, SEO, SER, SELU), 19-50 (SEH), 19-52 (SEC, SEEK, SEE, SEL, SEQ, TSST), 18-66 (SEP), 19-65 (SEG, SEE, SEM, SEN, SEO, SELU), 20-46 (SEA), 19-51 (SEC, SEL), 18-95 (SEK, SEQ), 20-38 (SED, SELJ, SEG, SEE, SEM, SEN, SEO, SER, SELU), 20-122 (SEC, SEL, SEG, SEE, SEM, SEN, SEO, SER, SELU), and 18-79 (SEC, SEL, SEEK, SEQ).

The Staphylococcus aureus genome was extracted by referring to the experimental steps of the Promega genome extraction kit (Lot: A1120) and making appropriate adjustments. The specific method is as follows: centrifuge 1 ml of culture medium and discard the supernatant; add 200 μl TE and 2 μl lysostaphin (Sigma, 1 mg/ml), suspend the precipitate, and place it in a 37°C metal bath for 2 to 4 hours; add 200 μl Nuclei Lysis Solution, 80°C, 5 minutes, and cool to room temperature; add 4 μl RNase Solution, place it in a 37°C metal bath for 15 to 60 minutes; add 200 μl Protein Precipitation Solution, shake vigorously, mix thoroughly, and incubate on ice for 5 minutes; 14000 rpm, 3 minutes, take the supernatant, add 600 μl isopropanol, mix, and stand at room temperature for 1 hour; 14000 rpm, 3 minutes, discard the supernatant, add 600 μl 70% ethanol, rinse the precipitate; 14000rpm, 1min, discard the supernatant, dry naturally at room temperature; add 50μl DNA Rehydration Solution, 65℃, 30min, dissolve the precipitate, and store at -20℃ for later use. The sample addition system and PCR amplification program are shown in Table 2. The fluorescence intensity was detected during the annealing stage using a QDX real-time PCR amplifier (ABI).

Table 2 PCR reaction system

The reaction program was as follows: pre-denaturation at 95°C for 2 min, 40 cycles of denaturation at 95°C for 20 s, annealing at 58°C for 45 s, and fluorescence detection during the annealing stage.

The experimental results showed that there were no peaks in all channels of Staphylococcus epidermidis ATCC12228, Escherichia coli ATCC25922, Staphylococcus aureus ATCC29213, ATCC14458, ATCC27664 (see), 18-66, 19-50, and 20-46, and all were negative (see Figure 1J).

The strains ATCC 13565, 19-51, 19-65, and 18-95 only amplified the amplification curves of sed(VIC), sel(FAM), sem(Cy5), and seq(ROX), respectively (Figure 1A-D); the strain 20-38 amplified the amplification curves of sed(VIC) and sem(Cy5) (Figure 1E); the strain 20-122 amplified the amplification curves of sel(FAM) and sem(Cy5) (Figure 1F); 8-79 amplified the amplification curves of seq(ROX) and sel(FAM) (Figure 1G); strain 19-63 amplified the amplification curves of sed(VIC), sem(Cy5), and sel(FAM) (Figure 1H); the mixture of strains 18-95 and 19-63 amplified the amplification curves of sed(VIC), sem(Cy5), sel(FAM), and seq(ROX) (Figure 1I), which were consistent with the sequencing results. After random combinations of single, double, triple, and quadruple, and detection using the above-mentioned multiplex fluorescence PCR detection method, each strain showed a highly specific fluorescence signal curve, and the detection sensitivity was high.

Example 2

Test strain: 19-63

DNA was extracted from the broth culture medium of strains 19-63 and 18-95 according to the above method, and the DNA concentration was measured by Nanodrop2000, and diluted to 100 ng/ml with deionized water, and then 10 times gradient dilution was performed to 0.01 ng/ml. 2 μl of culture medium was taken from each dilution, and fluorescent PCR amplification was performed according to the quadruple fluorescent PCR reaction system in the above method. The standard mode was selected on the real-time fluorescent PCR instrument, and the instrument automatically drew a standard curve.

As shown in Figure 2, there is a good linear relationship between the initial template concentration and the Ct value. The minimum detection concentrations of sel, sed and seq are 0.01 ng/ml, and the minimum detection concentration of sem is 0.1 ng/ml. This shows that the designed primers and probes are highly specific and the amplification system optimization effect is also good.

Example 3

Test sample: pork sold in a market

The purchased commercial pork was tested. Before artificial contamination with Staphylococcus aureus, the product was sterilized and tested according to national standards to confirm that it did not contain Staphylococcus aureus. Strains 21-68 (sed), 19-65 (seg, sei, sem, sen, seo, selu), 19-51 (sec, sel), and 18-95 (sek, seq) were artificially contaminated into pork, that is, 25g of sample was added to 225mL of buffered peptone water and homogenized, and then the homogenate was artificially contaminated and cultured at 37℃ for 12h. Take 10ml of the homogenate, centrifuge at 800rpm for 5min to remove the pork and leave the bacterial solution in the slurry. Pipette the supernatant into a centrifuge tube, centrifuge at 10000rpm for 10min, and discard the supernatant. Refer to the experimental steps of the Promega genome extraction kit (Lot: A1120) and make appropriate adjustments to extract the total DNA of bacteria in the raw material. The steps are the same as Example 1. The sample loading system and PCR amplification procedure are shown in Table 2. The fluorescence intensity was detected during the annealing stage using a QDX real-time PCR amplifier (ABI).

Table 2 PCR reaction system

The reaction procedure was as follows: pre-denaturation at 95°C for 2 min, 40 cycles of denaturation at 95°C for 20 s, annealing at 58°C for 45 s, and fluorescence detection during the annealing stage.

The results are shown in Figure 3. The negative control of commercial pork samples had no specific bands; the amplification curve of sed(VIC) was amplified from the pork contaminated with 21-68 of the novel enterotoxins SED, SEJ, and SER (Figure 3A); the amplification curve of sel(FAM) was amplified from the pork contaminated with 19-51 of the novel enterotoxins SEC and SEL (Figure 3B); the amplification curve of sem(Cy5) was amplified from the pork contaminated with 19-65 of the novel enterotoxins SEG, SEI, SEM, SEN, SEO, and SEU (Figure 3C); the amplification curve of seq(ROX) was amplified from the pork contaminated with 18-95 of the novel enterotoxins SEK and SEQ (Figure 3D). There was no cross-reaction in each fluorescent channel, and the specificity was strong.

The multiplex fluorescence PCR detection kit only takes 1-2 days (including sample pre-treatment) to detect food samples, and can detect multiple enterotoxin genes at the same time. Therefore, the multiplex fluorescence PCR method saves time and effort, has high accuracy, can meet the rapid diagnosis of Staphylococcus aureus enterotoxin, and the consistency rate with the results of traditional detection methods is 100%.

Claims (8)

1. A quadruple real-time fluorescent PCR detection kit for novel enterotoxins of Staphylococcus aureus, characterized by comprising enterotoxin SED, SEL, SEM and SEQ gene detection primers and probes; the enterotoxin SED, SEL, SEM and SEQ gene detection primers and probes are composed of the following:

2. The kit according to claim 1 is characterized in that it also includes TaKaRa heat-stable DNA polymerase, 10×ExTaqBuffer (Mg2+plus), and dNTP Mixture. 3. A non-diagnostic method for detecting a novel enterotoxin of Staphylococcus aureus by quadruple real-time fluorescence PCR, characterized in that it comprises the following steps: Step S1: pre-treating the sample to be tested; Step S2: extracting bacterial DNA from the sample to be tested; Step S3: Using 2 μl of bacterial DNA extracted in step S2 as a template, and using the optimized Taqman fluorescent PCR reaction system and reaction conditions, perform real-time fluorescent quantitative Real-time PCR detection; Step S4: After the reaction is completed, the PCR amplification cycle number Ct value of each test sample is read and recorded according to the fluorescent signal recorded by the fluorescent reporter group labeled with the probe; if the Ct value is 0 or greater than 40, it indicates a negative result, and a Ct value less than 38 indicates a positive result; a Ct value between 38-40 indicates that the sample needs to be retested. The Ct value of each sample is used to determine whether the sample contains enterotoxin genes. 4. The method according to claim 3, characterized in that step S2 uses Promega genome extraction kit Lot: A1120. 5. The method according to claim 3, characterized in that the real-time fluorescence quantitative Real-time PCR reaction system in step S3 is as follows:

The reaction procedure was as follows: pre-denaturation at 95°C for 2 min, 40 cycles of denaturation at 95°C for 20 s, annealing at 58°C for 45 s, and fluorescence detection during the annealing stage; The enterotoxin SED, SEL, SEM and SEQ gene detection primers and probes are composed of the following:

6. The method according to claim 3, characterized in that the sample to be tested in step S1 includes a food sample, feces or vomitus. 7. The method according to claim 6, characterized in that if the sample to be tested is feces or vomitus, it is suspended and boiled with 100-200 μl of physiological saline, centrifuged at 10000 rpm for 2 minutes, and 2 μl of supernatant is taken for PCR reaction. 8. The method according to claim 6, characterized in that if the sample to be tested is a food sample, after enriching the bacteria to be tested in the enrichment solution, 1 ml of the enrichment solution is taken and centrifuged at 10000 rpm for 5 minutes to take the precipitate.

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