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

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

Technical Field

The invention belongs to the technical field of biology, and relates to a kit and a method capable of simultaneously detecting traditional and novel enterotoxin genes of staphylococcus aureus.

Background

Staphylococcus aureus is very easy to pollute food such as dairy products, meat, starch and the like, and is one of important pathogenic bacteria causing bacterial food poisoning. Studies have shown that Staphylococcal Enterotoxins (SEs) are the major virulence factors responsible for S.aureus intoxication. They are low molecular weight proteins with similar structure and toxicity, and can specifically attack small intestinal cells only by enterotoxin with high nanogram to low microgram order, and enter a digestive system to cause intestinal diseases such as diarrhea, vomiting and the like. The enterotoxin generally has strong thermal stability and still has activity after being treated for 30min at 100 ℃, so even if the thalli are killed in the processing process, the produced enterotoxin can still cause food poisoning. To date, over twenty enterotoxins and enterotoxoids have been discovered, and this number is continuing to be expanded. The superantigen which can cause the vomiting of the primate model after being taken orally is called staphylococcus aureus enterotoxin (SEs), and comprises five classical enterotoxins SEA-SEE and novel enterotoxin SEG-SEI, SEK-SET and SEY; while enterotoxins that lack emetic activity or have not been tested are called staphylococcus aureus enterotoxins (SEls), including SElJ, SElU, SElV, SElW, SElX, SElZ. Many enterotoxin-encoding genes are linked, e.g., enterotoxin sed, selj, ser can be present on plasmid pIB485 at the same time; sek and seq can exist in tandem in bacteriophage and staphylococcus aureus virulence islands; also located on the virulence island are enterotoxins sec and sel; the most common egc cluster can express seg, sei, sem, sen and seo5 enterotoxin genes in a linked manner.

The enterotoxin detection methods commonly used at present mainly comprise molecular biological methods and immunological methods. The molecular biological method is mainly an experimental method established based on the PCR technology, has the advantages of sensitivity, rapidness, simplicity and the like, and particularly, the real-time fluorescence PCR technology is often used for detecting various toxin genes in food. No nucleic acid detection kit aiming at novel enterotoxins exists in the market, and the nucleic acid of the classical enterotoxins SEA-SEE is only a single fluorescence PCR detection method. Although some researchers successfully detect sea-selj 9 enterotoxin genes by using RT-PCR reaction, one-tube multi-detection has not been realized, and the detection of gene level does not mean the expression of protein level. Enzyme-linked immunosorbent assay (ELISA) is the most commonly used method for detecting enterotoxin protein based on antigen-antibody specific recognition, but complex components in food matrix such as phosphatase, catalase, igG non-specifically bound by staphylococcal protein A all cause the reduction of sensitivity and specificity of ELISA kit and the occurrence of false positive reaction. Enterotoxin is a protein with extremely similar structure, and cross reaction is easy to occur in the ELISA detection process. In addition, the existing commercial kit can only detect the classical enterotoxins SEA-SEE, and a novel enterotoxin protein detection technology is lacked. In recent years, it is reported that the novel enterotoxin can also participate in food poisoning, and the carrying rate of the novel enterotoxin is very high, so that a method for detecting the novel enterotoxin is urgently needed to be established.

Disclosure of Invention

The first objective of the present invention is to overcome the above-mentioned deficiencies of the prior art, and to provide a multiplex fluorescence PCR detection kit, which can simultaneously realize the typing of 4 staphylococcal enterotoxins in one PCR reaction, and is based on the phenomenon of serial expression of enterotoxin genes, which is presumed to include the detection of 12 genes, sec-sel, sed-selj-ser, seg-sei-sen-seo, and sek-seq.

In order to achieve the purpose, the invention downloads the base sequences of staphylococcus aureus enterotoxin genes sel, sed, sem and seq from GenBank, and then carries out homologous alignment analysis to search the conserved region of the genes. Designing specific primers and target probes in the region of conserved nucleic acid sequence. The primer sequences are shown in the following table:

TABLE 1 primer sequences

The invention also provides a quadruple real-time fluorescence PCR detection method for the novel staphylococcal enterotoxin without diagnosis, which comprises the following steps:

a) Pretreating a sample to be detected;

b) Extracting thallus DNA in a sample to be detected, preferably adopting a Promega genome extraction kit (Lot: A1120);

c) Taking 2 mu l of thallus DNA extracted in the step (b) as a template, and carrying out Real-time fluorescent quantitative Real-time PCR detection by using an optimized Taqman fluorescent PCR reaction system and reaction conditions;

d) After the reaction is finished, reading and recording the PCR amplification cycle number (Ct) value of each detection sample according to the fluorescent signal recorded by the fluorescent reporter group marked by the probe. According to the established interpretation standards: the Ct value is 0 or more than 40, which indicates negative, and the Ct value is less than 38, which indicates positive; ct values ranged from 38-40, and samples needed to be retested. And (4) judging whether the samples contain enterotoxin genes or not according to the Ct value of each sample.

The reaction system is as follows:

TABLE 2PCR reaction System

Reaction procedure: pre-denaturation at 95 ℃ for 2 min, denaturation at 95 ℃ for 20 sec in 40 cycles, annealing at 58 ℃ for 45 sec, and fluorescence detection during the annealing phase.

The dNTP mix was a mixed solution of 2.5mM dATP, 2.5mM dCTP, 2.5mM dGTP and 2.5mM dTTP.

The sample in the step b) has the application range of food samples, feces, vomit and other samples. For stool and vomit samples, according to the amount, 100-200. Mu.l of physiological saline is used for suspension and boiling, centrifugation is carried out at 10000rpm for 2 minutes, and 2. Mu.l of supernatant is taken for PCR reaction. After the enrichment of the food sample in the enrichment liquid of the bacteria to be detected, 1ml of the enrichment liquid is taken to centrifuge at 10000rpm for 5 minutes, the supernatant is discarded, DNA is extracted by a Qiagen DNA extraction kit, and 2 mu l of the enrichment liquid is taken to carry out PCR reaction.

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 there is no homologous or sequence consistency biological gene information, and 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 detection probes of the invention, carrying out quadruple real-time fluorescence PCR detection on a sample by using the detection primers and detection probes according to the method of the invention, completing the specificity detection of staphylococcus aureus enterotoxin SED, SEL, SEM and SEQ genes of the sample to be detected in one amplification reaction, and presuming the detection conditions of 12 genes including sec-SEL, SED-selj-ser, seg-sei-SEM-seo and sek-SEQ in the sample to be detected through the phenomenon of enterotoxin gene tandem expression. The detection primers and the probes are used for carrying out quadruple real-time fluorescent PCR detection on the sample according to the method of the invention, the detection is rapid, the process from sample preparation to detection result presentation can be completed within 8-10 hours, the interference of false positive, cross contamination and the like is avoided, the result is reliable, the sensitivity is high, and the specificity is strong.

Staphylococcus aureus easily pollutes milk powder, pork and cake foods, and enterotoxin is generated after co-culture, so that symptoms of enterogastritis such as nausea, vomiting, diarrhea and the like are caused. The invention can provide a beneficial tool for epidemiological investigation of staphylococcus aureus food poisoning events, and is suitable for food and water inspection, disease control centers and quality supervision departments. The invention not only meets the requirements of rapid diagnosis and food detection of staphylococcus aureus food poisoning events, but also has important significance for monitoring staphylococcus aureus in the production process of the food industry and controlling the quality of final products.

Drawings

FIGS. 1A-D are the results of PCR amplification of strains ATCC13565, 19-51, 19-65 and 18-95; 1; 2, sel; 3; 4;

FIG. 1E shows the results of PCR amplification of strains 20-38; 1; 2, sel; 3; 4;

FIG. 1F shows the results of PCR amplification of strains 20-122; 1; 2, sel; 3; 4;

FIG. 1G shows the results of PCR amplification of strains 18-79; 1; 2, sel; 3; 4;

FIG. 1H shows the results of PCR amplification of strains 19-63; 1; 2, sel; 3; 4;

FIG. 1I shows the results of PCR amplification of strains 18-95 and 19-63; 1; 2, sel; 3; 4;

FIG. 1J shows the results of PCR amplification of Staphylococcus epidermidis ATCC12228, escherichia coli ATCC25922, staphylococcus aureus ATCC29213, ATCC14458, ATCC27664 (see), 18-66, 19-50, 20-46; 1; 2, sel; 3; 4;

FIG. 2A shows the results of PCR amplification of sed of strains 19-63; 1; 2, 1ng/ml;3, 0.1ng/ml;4, 0.01ng/ml;

FIG. 2B is the result of PCR amplification of sel from strain 19-63; 1; 2, 1ng/ml;3, 0.1ng/ml;4, 0.01ng/ml;

FIG. 2C is the PCR amplification result of sem of strains 19-63; 1; 2; 3, 0.1ng/ml;4, 0.01ng/ml;

FIG. 2D is the result of PCR amplification of seq for strain 18-95; 1; 2; 3, 0.1ng/ml;4, 0.01ng/ml;

FIG. 3A shows the PCR amplification results of 21-68 contaminated pork with enterotoxins D, J, and R;

FIG. 3B shows the PCR amplification results of 19-51 contaminated pork producing novel enterotoxins C and L;

FIG. 3C shows the PCR amplification results of 19-65 contaminated pork with novel enterotoxins G, I, M, N, O, U;

FIG. 3D shows the PCR amplification results of 18-95 contaminated pork with neoenterotoxin K and Q.

Detailed Description

The invention is further described with reference to the following detailed description and accompanying drawings.

Example 1

The test strains 12, namely staphylococcus epidermidis ATCC12228, escherichia coli ATCC25922, staphylococcus aureus ATCC29213, ATCC14458 (seb), ATCC13565 (sea, sed) and ATCC27664 (see), are purchased from China food and drug institute and China general microbiological culture Collection center and are confirmed by VITEK 2;

whole genome sequencing verified S.aureus strains 19-63 (sec, sed, selj, sel, seg, sei, sem, sen, seo, ser, selu), 19-50 (seh), 19-52 (sec, sek, seh, sel, seq, tsst), 18-66 (sep), 19-65 (seg, sei, sem, sen, seo, selu), 20-46 (sea), 19-51 (sec, sel), 18-95 (sek, seq), 20-38 (sed, selj, seg, sei, sem, sen, seo, ser, selu), 20-122 (sec, sel, seg, sei, sem, sen, seo, ser, selu), 18-79 (sec, sel, sek, seq).

The method refers to the experimental steps of a Promega genome extraction kit (Lot: A1120) and properly adjusts and extracts the staphylococcus aureus genome, and comprises the following steps: centrifuging 1ml of culture solution, and removing supernatant; adding 200 mu l of TE and 2 mu l of lysostaphin (Sigma, 1 mg/ml), carrying out suspension precipitation, and placing in a metal bath at 37 ℃ for 2-4 h; adding 200 μ l of nucleic lysine Solution, at 80 deg.C for 5min, and cooling to room temperature; adding 4 mu l of RNase Solution, and placing the mixture in a metal bath at 37 ℃ for 15-60 min; adding 200 μ l Protein Precipitation Solution, shaking vigorously, mixing well, and incubating on ice for 5min;14000rpm,3min, taking supernatant, adding 600 mul of isopropanol, mixing evenly, and standing for 1h at room temperature; 14000rpm,3min, abandoning the supernatant, adding 600 μ l of 70% ethanol, rinsing and precipitating; 14000rpm for 1min, discarding the supernatant, and naturally drying at room temperature; adding 50 μ l DNA hydration Solution, dissolving the precipitate at 65 deg.C for 30min, and storing at-20 deg.C for use. The sample application system and PCR amplification procedure are shown in Table 2, and fluorescence intensity was measured at the annealing stage using a QDX real-time PCR amplification apparatus (ABI Co.).

TABLE 2PCR reaction System

Reaction procedures are as follows: pre-denaturation at 95 ℃ for 2 min, denaturation at 95 ℃ for 20 sec in 40 cycles, annealing at 58 ℃ for 45 sec, and fluorescence detection during the annealing phase.

The experimental results showed that all channels of Staphylococcus epidermidis ATCC12228, escherichia coli ATCC25922, staphylococcus aureus ATCC29213, ATCC14458, ATCC27664 (see), 18-66, 19-50 and 20-46 did not peak and were all negative (see FIG. 1J).

The strains ATCC13565, 19-51, 19-65 and 18-95 amplified only amplification curves for sed (VIC), sel (FAM), sem (Cy 5) and seq (ROX), respectively (FIGS. 1A-D); the strains 20-38 amplified the amplification curves for sed (VIC) and sem (Cy 5) (FIG. 1E); strains 20-122 amplified sel (FAM) and sem (Cy 5) amplification curves (FIG. 1F); 18-79 amplification curves for seq (ROX) and sel (FAM) (FIG. 1G); the strains 19-63 amplified amplification curves for sed (VIC), sem (Cy 5), sel (FAM) (FIG. 1H); the mixture of strains 18-95 and 19-63 amplified amplification curves for sed (VIC), sem (Cy 5), sel (FAM), seq (ROX) (FIG. 1I), consistent with the sequencing results. Through the random combination of singleness, double, triple, quadruple and the like and the detection by the multiplex fluorescence PCR detection method, each strain has a fluorescence signal curve with strong specificity, and the detection sensitivity is higher.

Example 2

The test strains are: 19-63

The broth culture of strains 19-63 and 18-95 was used for DNA extraction as described above, DNA concentration determination was performed using Nanodrop2000, dilution to 100ng/ml with deionized water, 10-fold gradient dilution to 0.01ng/ml, 2. Mu.l of culture broth from each dilution, and fluorescent PCR amplification was performed according to the quadruple fluorescent PCR reaction system described above. And (3) selecting a standard mode on the real-time fluorescent PCR instrument, and automatically drawing a standard curve by the instrument.

The result is shown in FIG. 2, a good linear relationship also appears between the initial template concentration and the Ct value, the minimum detection concentration of sel, sed and seq reaches 0.01ng/ml, and the minimum detection concentration of sem reaches 0.1ng/ml. The designed primer and probe are shown to have higher specificity, and the optimization effect of the amplification system is also better.

Example 3

Receiving a sample book: pork sold in certain market

The method comprises the steps of detecting some purchased pork sold in the market, sterilizing the product before artificially polluting staphylococcus aureus, and detecting according to national standards to confirm that the product does 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) are respectively and artificially polluted into pork, namely 25g of sample is added into 225mL of buffer peptone water to be homogenized and mixed, then the homogenized liquid is artificially polluted, and the homogenized liquid is shake-cultured for 12h at 37 ℃. 10ml of homogenate was centrifuged at 800rpm for 5min to remove pork and leave the broth in the serum. And (4) sucking the supernatant into a centrifuge tube, centrifuging at 10000rpm for 10min, and discarding the supernatant. The procedure of the Promega genome extraction kit (Lot: A1120) was followed in the same manner as in example 1 with appropriate adjustments to extract total bacterial DNA from the starting material. The sample application system and PCR amplification procedure are shown in Table 2, and fluorescence intensity was measured at the annealing stage using a QDX real-time PCR amplification apparatus (ABI Co.).

TABLE 2PCR reaction System

Reaction procedure: pre-denaturation at 95 ℃ for 2 min, denaturation at 95 ℃ for 20 sec in 40 cycles, annealing at 58 ℃ for 45 sec, and fluorescence detection during the annealing phase.

The results are shown in fig. 3, with no specific band for the commercial negative control of pork samples; amplification curves of SED (VIC) were amplified from 21-68 contaminated pork producing the novel enterotoxin SED, SEJ, SER (FIG. 3A); amplification curves for SEL (FAM) were amplified from 19-51 contaminated pork producing new enterotoxin SEC and SEL (fig. 3B); amplification curves of SEM (Cy 5) from 19-65 contaminated pork producing novel enterotoxin SEG, SEI, SEM, SEN, SEO, SEU (FIG. 3C); amplification curves for SEQ (ROX) were amplified from the novel enterotoxin-producing SEK and the 18-95 contaminated pork of SEQ (FIG. 3D). Each fluorescence channel has no cross reaction and strong specificity.

The multiplex fluorescence PCR detection kit only needs 1-2 days (including the pretreatment of samples) for detecting food samples, and can simultaneously detect a plurality of enterotoxin genes. Therefore, the multiple fluorescence PCR method is time-saving and labor-saving, has high accuracy, can meet the requirement of rapid diagnosis of staphylococcus aureus enterotoxin, and has a coincidence rate with the result of the traditional detection method of 100 percent.

Claims (8)

1. A novel staphylococcus aureus enterotoxin quadruple real-time fluorescent PCR detection kit is characterized by comprising enterotoxin SED, SEL, SEM, SEQ gene detection primers and probes; the enterotoxin SED, SEL, SEM and SEQ gene detection primers and probes consist of the following components:

2. the kit according to claim 1, further comprising TaKaRa thermostable DNA polymerase, 10 XExTaqBuffer (Mg 2+ plus), dNTPMixture.

3. A non-diagnostic purpose staphylococcus aureus novel enterotoxin quadruple real-time fluorescence PCR detection method is characterized by comprising the following steps:

step S1: pretreating a sample to be detected;

step S2: extracting thallus DNA in a sample to be detected;

and step S3: taking 2 mu l of thallus DNA extracted in the step S2 as a template, and carrying out Real-time fluorescent quantitative Real-time PCR detection by using an optimized Taqman fluorescent PCR reaction system and reaction conditions;

and step S4: after the reaction is finished, reading and recording the Ct value of the PCR amplification cycle times of each detection sample according to the fluorescent signal recorded by the fluorescent reporter group marked by the probe; if the Ct value is 0 or more than 40, the result is negative, and if the Ct value is less than 38, the result is positive; ct values between 38 and 40 indicate that the sample needs to be retested. And (4) judging whether the samples contain enterotoxin genes or not according to the Ct value of each sample.

4. The method according to claim 3, wherein the Promega genome extraction kit Lot: A1120 is used in the step S2.

5. The method of claim 3, wherein the Real-time quantitative Real-time fluorescence PCR reaction system in step S3 is as follows:

reaction procedure: pre-denaturation at 95 ℃ for 2 min, denaturation at 95 ℃ for 20 sec in 40 cycles, annealing at 58 ℃ for 45 sec, and fluorescence detection at the annealing stage;

the enterotoxin SED, SEL, SEM and SEQ gene detection primers and probes consist of the following components:

6. the method of claim 3, wherein the sample to be tested in step S1 comprises a food sample, stool or vomit.

7. The method according to claim 6, wherein the PCR reaction is carried out by suspending and boiling the sample to be tested in 100-200. Mu.l of physiological saline, centrifuging at 10000rpm for 2 minutes, and collecting 2. Mu.l of the supernatant.

8. The method according to claim 6, wherein if the sample to be tested is a food sample, the enrichment is performed on the enriched liquid of the bacteria to be tested, 1ml of the enriched liquid is centrifuged at 10000rpm for 5 minutes, and the precipitate is taken.

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