CN114480731B - Combined kit for phage bioamplification-real-time fluorescent quantitative PCR (polymerase chain reaction) for rapidly detecting staphylococcus aureus and method thereof - Google Patents
Combined kit for phage bioamplification-real-time fluorescent quantitative PCR (polymerase chain reaction) for rapidly detecting staphylococcus aureus and method thereof Download PDFInfo
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Abstract
The invention discloses a phage bioamplification-real-time fluorescence quantitative PCR combined kit for rapidly detecting staphylococcus aureus and a method thereof, wherein the combined kit comprises a phosphate buffer solution containing staphylococcus aureus phage LSA2311 and a primer pair tsp for real-time fluorescence quantitative PCR detection; the phage bioamplification and qPCR method can achieve better detection effect when used for detecting staphylococcus aureus in food matrix, and the detection limit is 10 1 CFU/mL. Compared with the traditional culture method, the detection time is greatly shortened, and the detection of the sample is completed within 2-3 hours. The invention can provide experimental basis and theoretical basis for the establishment of a rapid detection method of staphylococcus aureus based on phage biological amplification.
Description
Technical Field
The invention relates to the field of food safety detection, in particular to a phage bioamplification-real-time fluorescent quantitative PCR combined kit for rapidly detecting staphylococcus aureus and a method thereof.
Background
Staphylococcus aureus (Staphylococcus aureus, s.aureus) is a gram positive bacterium, is coccoid, tends to be clustered, and is described as "staphyloid". It is widely distributed in nature, such as air, soil, water and other environments, and is also present in the normal human flora of the skin and mucous membranes (usually the nasal area) of most healthy individuals. Staphylococcus aureus is widely distributed in air, water, humans, cows and other livestock and poultry animals and their excretions, which makes foods more easily contaminated. Food-borne diseases due to staphylococcus aureus contamination are reported to be global health problems. Staphylococcus aureus is one of the most common bacterial infections present in humans, and can cause a variety of infections and diseases in humans, including abdominal pain, diarrhea, nausea, vomiting, severe cases also resulting in sepsis, infectious endocarditis, and even shock. In recent decades, advances have been made in detection, prevention and control of staphylococcus aureus, and effective methods of detecting staphylococcus aureus are essential for food safety and human health.
Phages are small viruses that lack the self-metabolic mechanism, whose structure includes the protein capsid and genetic material within the capsid, but they can be propagated using host bacterial cells. Phage are very widely distributed in nature, the most abundant and ubiquitous organisms on earth, estimated to be 10 on earth 31 Individual phage particles, in some ecosystems, can even exceed the number of bacteria by about ten times. Phages are viruses which can infect and replicate and proliferate in bacteria, and which can specifically adsorb host bacteria, thusPhage-based pathogen detection methods have been developed to take advantage of this specific relationship between phage and bacteria. Molecular biological methods that do not require colony culture have evolved to a great extent in microbiological detection. Molecular biological detection methods have been developed primarily around polymerase chain reaction (Polymerase Chain Reaction, PCR), the most widely used nucleic acid-based technique in point of care diagnostics, primarily for detecting, identifying and quantifying pathogens or beneficial populations, such as fermenting microorganisms or probiotics. But molecular biology methods have the following disadvantages:
requiring a trained laboratory operator, requiring the use of radioactive materials, or having a high potential for cross-contamination. Foremost are false positive results due to the expansion of non-viable or "dead" cells.
Phage bioamplification (Phage Amplification Assay, PAA) is a method for indirectly detecting a target bacterium by detecting progeny phage that lyses and releases a lytic phage after it specifically adsorbs and infects host cells. The key point of phage bioamplification is the efficacy of the virucide selected for inactivation of phage particles, as any residual free phage will produce false positive results by infecting the added helper cells. In 1998 Stewart et al used phage bioamplification for the first time for rapid detection and identification of specific bacteria, 40CFU/mL of P.aeruginosa and 600CFU/mL of Salmonella respectively were detected in 4 hours using a Pomegranate Rind Extract (PRE) in combination with ferrous sulfate as a virucide. However, the traditional phage bioamplification method indirectly detects the existence of pathogenic bacteria by observing plaque formed by progeny phage on an agar plate, but the time required for the plaque formation process is generally 3-4 hours, and the time is longer.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a phage bioamplification-real-time fluorescence quantitative PCR combined kit for rapidly detecting staphylococcus aureus and a method thereof, wherein the combined kit utilizes staphylococcus aureus phage LSA2311 to jointly detect staphylococcus aureus, and the detection limit is 10 1 CFU/mL has the characteristics of high speed, high sensitivity, low cost, strong specificity and capability of distinguishing dead bacteria from living bacteria.
In order to achieve the aim, the invention designs a phage bioamplification-real-time fluorescence quantitative PCR combined kit for rapidly detecting staphylococcus aureus, which comprises phosphate (phosphate buffered saline, PBS) buffer solution containing staphylococcus aureus phage LSA2311,
among them, staphylococcus aureus phage LSA2311 was sent to the chinese collection of typical cultures for preservation in 9 months 29 of 2020, and named after classification: staphylococcus aureus phage (Staphylococcus aureus bacteriophage) LSA2311 with a deposit number of university of martial arts, hubei province: cctccc NO: m2020562; the staphylococcus aureus phage is disclosed in Chinese patent application with application number of 202011210849.3 and invention name of staphylococcus aureus phage LSA2311 and application thereof;
and primer pair tsp for real-time fluorescent quantitative PCR detection:
forward primer tsp-F:5'-TGCGTGCTATTCTAGGTGGA-3';
reverse primer tsp-R:5'-ACCACTTACCAAACCTCCGA-3'.
Further, the combined kit further comprises a negative control, a positive control, a Ferrous Ammonium Sulfate (FAS) solution and a trisodium citrate solution; wherein,
the negative reference substance is PBS buffer solution, and the molar concentration of the PBS buffer solution is 0.05mol/L;
the positive reference is staphylococcus aureus, and the staphylococcus aureus ATCC25923;
ferrous Ammonium Sulphate (FAS) solution concentration was 25mM;
the trisodium citrate solution was 10mM.
The invention provides a method for detecting staphylococcus aureus by using the combined kit, which comprises the following steps:
1) Phage bioamplification detection
a. Mixing the staphylococcus aureus phage LSA2311 suspension with a sample to be detected, and culturing at 37 ℃ to obtain a culture;
b. mixing the culture with Ferrous Ammonium Sulfate (FAS) solution, adding LB liquid medium, supplementing to 1mL, and culturing at 37deg.C; then adding trisodium citrate solution (neutralizing the activity of the virucide);
c. centrifuging the mixed solution to obtain a supernatant, namely the progeny phage;
2) Real-time fluorescent quantitative PCR detection of progeny phage:
a. the progeny phage were DNA extracted and stored at-20 ℃.
b. Taking DNA of a progeny phage as a template, and carrying out real-time fluorescence quantitative PCR (polymerase chain reaction) by using a primer pair tsp to obtain a PCR product, wherein the primer pair tsp is as follows:
forward primer tsp-F:5'-TGCGTGCTATTCTAGGTGGA-3';
reverse primer tsp-R:5'-ACCACTTACCAAACCTCCGA-3'.
And c, analyzing the Ct value of the PCR product and an established standard curve to obtain the content of staphylococcus aureus in the sample to be detected.
Further, in step 1) step a, the suspension of the staphylococcus aureus phage LSA2311 is mixed with the sample to be detected according to the volume of 1:1, and the concentration of the suspension of the staphylococcus aureus phage LSA2311 is 10 7 PFU/mL, concentration of sample to be detected is 10 8 CFU/mL。
Still further, in step 1) step b, the culture is mixed with a Ferrous Ammonium Sulfate (FAS) solution at a volume of 1:1, the concentration of the Ferrous Ammonium Sulfate (FAS) solution is 25mM, and the culture time is 5min;
the addition amount of the trisodium citrate solution was 100. Mu.L/L, and the concentration of the trisodium citrate solution was 10mM.
Still further, the PCR reaction amplification system is as follows:
| 2×qPCR Mix | 10μL |
| forward primer tsp-F | 0.4μL |
| Reverse primer tsp-R | 0.4μL |
| Template (DNA of progeny phage) | 1μL |
| ddH 2 O to | 20μL |
The invention has the beneficial effects that:
the invention establishes a simple, low-cost and high-sensitivity phage bioamplification-real-time fluorescence quantitative PCR combined kit for rapidly detecting staphylococcus aureus, and combines a phage bioamplification method (Phage Amplification Assay, PAA for short) for detecting staphylococcus aureus with qPCR for detecting progeny phage, and establishes PAA-qPCR for detecting staphylococcus aureus so as to further shorten detection time. The phage bioamplification and qPCR method are combined to detect staphylococcus aureus in food matrixes, so that a better detection effect can be achieved, and compared with a traditional culture method, the detection time is greatly shortened, and the detection of samples can be completed within 2-3 hours; the invention can provide experimental basis and theoretical basis for the establishment of a rapid detection method of staphylococcus aureus based on phage biological amplification.
In conclusion, the invention utilizes staphylococcus aureus phage LSA2311 to establish a phage bioamplification method (PAA), and optimizes experimental conditions to shorten detection time. On the other hand, in combination with the real-time fluorescent quantitative polymerase chain reaction (qPCR) method for detecting progeny phage generated by phage bioamplification during phage infection of host bacteriaAn increase in DNA, the detection limit of this method is 10 1 CFU/mL solves the defect that dead bacteria and living bacteria cannot be distinguished when pathogenic bacteria are detected by the traditional PCR method.
Drawings
FIG. 1 is a graph showing the biological characteristics of Staphylococcus aureus phage LSA2311,
in the figure, A is a proliferation amount diagram of phage LSA2311 co-cultured with staphylococcus aureus with different concentrations,
panel B is a plot of the optimal multiplicity of infection (MOI) for phage LSA 2311;
FIG. 2 is a conditional optimization diagram of phage bioamplification (PAA),
in the figure, FIG. 2A is a graph showing the killing effect of a biocide on phage LSA2311,
FIG. 2B is a graph showing the effect of Ferrous Ammonium Sulfate (FAS) on Staphylococcus aureus,
FIG. 2C is a graph of the neutralization effect of different types of neutralizing agents on a biocide;
FIG. 3 is a standard graph of phage bioamplification for detection of Staphylococcus aureus,
FIG. 4 is a diagram showing the specificity of the primer tsp,
in the figure, FIG. 4A is a diagram showing the specificity of primer tsp in the Polymerase Chain Reaction (PCR),
FIG. 4B is a diagram showing the specificity of primer tsp in real-time fluorescent quantitative PCR (qPCR);
the labels in fig. 4B are in the order: s.aureus 25923 (alive) a S.aureus 25923 (dead), S.aureus 6538, S.aureus 196, S.aureus 142, SE 13311 b 、E.coli BL21 c 、L.monocytogenes 19115 d 、 b + c + d 、 a + b + c + d 、LSA2311(NC)；
FIG. 5 is a standard graph of real-time fluorescent quantitative PCR (qPCR) detection of Staphylococcus aureus;
FIG. 6 is a diagram showing the detection of Staphylococcus aureus in food by phage bioamplification combined with real-time fluorescent quantitative PCR (PAA-qPCR),
in the figure, FIG. 6A is a graph of PAA-qPCR detection of Staphylococcus aureus in labeled milk,
FIG. 6B is a graph of PAA-qPCR detection of Staphylococcus aureus in the labeled lettuce.
Detailed Description
The present invention is described in further detail below in conjunction with specific embodiments for understanding by those skilled in the art.
Example 1 phage activation
1. Activation of host bacteria
Taking out staphylococcus aureus ATCC25923 preserved in a glycerol pipe from a refrigerator at the temperature of minus 80 ℃ to be streaked and inoculated on a Baird Parker solid culture medium, culturing for 12 hours at the temperature of 37 ℃, picking up single bacterial colonies to be inoculated in 3mL of LB liquid culture medium, culturing for 8 hours at the temperature of 37 ℃ in a 160r/min shaking table, taking 100 mu L of cultured bacterial liquid, and measuring the concentration of the bacterial liquid by adopting a dilution coating method;
bacterial concentration (CFU/mL) =number of single colonies x dilution x 10;
2. activation of phage
Taking out the phage LSA2311 stored in the glycerol pipe from the refrigerator at the temperature of minus 80 ℃ and mixing with the logarithmic phase host bacteria liquid for culturing for 12-18 hours, centrifuging the cultured mixed liquid at the temperature of 4 ℃ for 10min at 8000r/min, filtering the supernatant with a microporous filter membrane of 0.22 mu m to remove the host bacteria, obtaining phage liquid, and placing in the refrigerator at the temperature of 4 ℃ for standby.
Example 2 establishment of phage biological amplification Process
1. Influence of host bacteria concentration
The staphylococcus aureus ATCC25923 bacterial liquid was serially diluted 10-fold in gradient (10 5 -10 9 CFU/mL), 100. Mu.L each was mixed with phage LSA2311 in the same volume (10) 7 PFU/mL), the volume was made up to 1mL with LB medium, placed in a shaking table at 37℃and cultured with 160r/min shaking for 24 hours, centrifuged and sterilized by filtration, and phage titer was determined by a double-layer plate method. The optimum host bacteria concentration is selected by progeny phage titers.
As can be seen from fig. 1A: 10 8 Phage titer after amplification of the host bacteria at CFU/mL concentration is obviously higher than 10 9 And 10 7 CFU/mL concentration group, thus determining the concentration of host bacteria in the culture system to be 10 8 CFU/mL。
2. Selection of phage titers
Phage titers in the amplification system were determined by optimal multiplicity of infection. The multiplicity of infection (Multiplicity of Infection, MOI) refers to the ratio of the number of phages to the number of host bacteria at the time of initial infection. Phage suspensions were mixed with 500. Mu.L of host bacterial solutions at a given MOI value (0.001, 0.01, 0.1, 1, 10, 100, 1000), incubated at 37℃for 3.5h, centrifuged at 8000r/min for 10min, and phage titers of supernatants at different MOI values were determined by double-layer plate method. The test was repeated 3 times with 2 replicates each.
As can be seen from fig. 1B: phage LSA2311 reached a maximum phage titer at MOI=0.1, indicating that more progeny phage could be propagated when the phage titer was infected with the host bacterial load at a ratio of 0.1, thus selecting a titer of 1X 10 7 PFU/mL phage.
3. Influence of different virucide classes
mu.L of phage LSA2311 suspension (10 7 PFU/mL) was mixed with 100. Mu.L of FAS (25 mM) and 100. Mu.L of MAS (25 mM) solution, and after allowing to act at room temperature for 10min, the mixture was centrifuged at 4℃and 8500r/min for 15min, 100. Mu.L of the supernatant was aspirated, and phage titer was measured by the double-layer plate method. A negative control was also set up with 200. Mu.L LB medium instead of the virus killing agent.
As shown in fig. 2A: the difference of the plaque numbers formed on the control plate and the plate added with MAS bactericide is less than 20%, no obvious difference exists, and the plaque number formed on the plate added with FAS bactericide is obviously reduced, which shows that the FAS has better effect of killing staphylococcus aureus phage LSA2311 and can completely kill the phage which is free outside host bacteria, thus FAS is selected as the bactericide of the experiment.
4. Effect of virucide on host bacteria
100. Mu.L of FAS (25 mM) was separated from 900. Mu.L of FAS (10 mM) 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 、10 0 CFU/mL) staphylococcus aureus ATCC25923 was mixed and incubated at room temperature for 15min, dilutedPerforming colony counting on the mixed bacterial liquid by a coating plate method, wherein 100 mu L of LB culture medium of a control group replaces FAS;
as can be seen from fig. 2B: the counts of staphylococcus aureus ATCC25923 were very similar to the counts of the non-killed plates with the addition of the killing agent FAS, the difference being statistically significant (T4.682, p < 0.05); thus, it is believed that the addition of the biocide FAS has no effect on the growth of the host bacterium staphylococcus aureus ATCC 25923.
5. Selection of neutralizing agent
100. Mu.L of phage LSA2311 suspension (final concentration 10) 7 PFU/mL) and 100. Mu.L of Staphylococcus aureus ATCC25923 bacterial liquid (final concentration 10) 8 CFU/mL), culturing at 37deg.C for 15min, taking out, mixing with 100 μL FAS (final concentration of 200 mM) FAS, incubating at room temperature for 10min, and adding 100 μL 2% Tween-80, 10mM CaCl2, and 10mM trisodium citrate solution to neutralize virus killing agent activity. The mixture was centrifuged at 8500r/min for 15min at 4℃to aspirate 100. Mu.L of supernatant, and phage titer was determined by double-layer plate method.
As can be seen from fig. 2C: the neutralizing agent is selected from 2% Tween-80 and 10mM CaCl 2 In solution, the number of phages formed on agar plates was reduced to 10mM trisodium citrate (C 8 H 5 Na 3 O 7 ) As compared with the solution, the solution was drastically reduced, 2% Tween-80 and 10mM CaCl 2 The neutralization effect of the solution on the FAS is not obvious, so that the phage killing effect of the FAS can be sustained, and the released progeny phage can be killed, thereby influencing the subsequent test result. And trisodium citrate (sodium citrate) oxidizes ferrous ions to ferric ions, thereby terminating the killing effect of FAS on phage. Thus, a 10mM trisodium citrate solution was selected as the neutralizing agent.
6. Selection of time and concentration of virucide
100. Mu.L of phage LSA2311 suspension (final concentration 10) 7 PFU/mL) and 100. Mu.L of Staphylococcus aureus ATCC25923 bacterial liquid (final concentration 10) 8 CFU/mL), culturing at 37deg.C for 15min, taking out, mixing with 100 μl of FAS with different final concentrations (200 mM, 100mM, 50mM, 25mM, 10mM, 5mM, 2.5mM, 0 mM), and adding LB mediumThe solution was added to 1mL, mixed and incubated at room temperature, and 100. Mu.L of the mixture was taken out of the corresponding test tube after 0, 5, 10, 15, 20, 25 and 30min, and 100. Mu.L of trisodium citrate solution (final concentration: 10 mM) was added to neutralize the activity of the virus killing agent. The mixture was centrifuged at 8500r/min for 15min at 4℃to aspirate 100. Mu.L of supernatant, and phage titer was determined by double-layer plate method.
As can be seen from table 1: FAS with the concentration of 2.5mM and 5mM can not thoroughly kill the free phage outside the host bacteria, has no obvious effect after 30 minutes of action, and has the quantity slightly lower than that of positive control without FAS; FAS at a concentration of 10mM was effective for more than 25min and small amounts of plaques were still visible; 25. FAS at concentrations of 50, 100 and 200mM works for 5min with the same effect, and free phage LSA2311 can be completely inactivated.
Thus, FAS was selected at a concentration of 25mM for 5min to kill free phage.
TABLE 1 concentration of the biocidal FAS and time to kill phage
EXAMPLE 3 evaluation of phage bioamplification method in Staphylococcus aureus detection
1. Specificity of phage bioamplification
Phage bioamplification was performed on 40 strains of bacteria (see Table 2). Meanwhile, staphylococcus aureus ATCC25923 inactivated by heat treatment at 100 ℃ for 10min is set as an experimental group, unheated living bacteria are set as a control group, and the capability of detecting staphylococcus aureus by a phage bioamplification method is observed.
Detecting 29 staphylococcus aureus in 40 bacteria by using a phage bioamplification method to form plaque; the 11 non-staphylococcus aureus bacteria had no plaque formation; the heat-inactivated staphylococcus aureus ATCC25923 was free of plaque formation, while the non-inactivated staphylococcus aureus ATCC25923 was plaque-formed. It is known that phage bioamplification can only detect live staphylococcus aureus, but has no detection effect on dead bacteria and other species bacteria, and the specificity of the method is good.
TABLE 2 specificity of detection of Staphylococcus aureus based on phage LSA2311 bioamplification
a ATCC,American Type Culture Collection；LS,Laboratory storage.
b Y,positive result；N,negative result.
2. Standard curve and minimum detection limit of phage biological amplification method
100. Mu.L of phage LSA2311 suspension (final concentration 10) 7 PFU/mL) and 100. Mu.L of different final concentration (10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 、10 0 CFU/mL) of staphylococcus aureus ATCC25923 was mixed uniformly, incubated at 37 ℃ for 15min, taken out, mixed with 100 μl of FAS (final concentration of 25 mM) FAS, incubated at room temperature for 5min, taken out of the corresponding experimental tube, and 100 μl of the mixed solution was added to 100 μl of trisodium citrate solution (final concentration of 10 mM) to neutralize the virus killing agent activity. The mixture was centrifuged at 8500r/min for 15min at 4℃to aspirate 100. Mu.L of supernatant, and phage titer was determined by double-layer plate method. And drawing a standard curve by taking the logarithmic value of the theoretical staphylococcus aureus concentration as an abscissa and the logarithmic value of the phage titer as an ordinate.
The results are shown in FIG. 3: standard curve R 2 A PAA of 0.9967 is shown to be a good linear relationship. In the standard curve of the PAA method for detecting staphylococcus aureus ATCC25923, the PAA method can detect 10 1 CFU/mL Staphylococcus aureus ATCC 25923.
3. Stability of phage bioamplification methods
Staphylococcus aureus ATCC25923, 142 was tested 2 times per day using phage method for 5 days in succession, and the inter-batch coefficient of variation was calculated; the staphylococcus aureus ATCC25923, 142 was repeatedly measured 5 times within 1d, and the intra-batch variation coefficient was calculated. As can be seen from table 5, the maximum CV (%) value of the inter-group repeated experiments was 0.34, and the maximum CV (%) value of the intra-group repeated experiments was 0.09. The measurement results in the repeated experiments in the groups and between the groups are demonstrated to have small data dispersion degree, and the established phage bioamplification method has good repeatability and high stability (Table 3).
TABLE 3 stability of phage bioamplification (PAA) to detect Staphylococcus aureus
Example 4
The phage bioamplification-real-time fluorescence quantitative PCR combined kit for rapidly detecting staphylococcus aureus comprises phosphate (phosphate buffered saline, PBS) buffer solution containing staphylococcus aureus phage LSA2311, a primer pair tsp for real-time fluorescence quantitative PCR detection, a negative control, a positive control, a Ferrous Ammonium Sulfate (FAS) solution and a trisodium citrate solution; wherein, primer pair tsp for real-time fluorescence quantitative PCR detection:
forward primer tsp-F:5'-TGCGTGCTATTCTAGGTGGA-3';
reverse primer tsp-R:5'-ACCACTTACCAAACCTCCGA-3'.
The negative reference substance is PBS buffer solution, and the molar concentration of the PBS buffer solution is 0.05mol/L;
the positive reference is staphylococcus aureus, and the staphylococcus aureus ATCC25923;
ferrous Ammonium Sulphate (FAS) solution concentration was 25mM;
the trisodium citrate solution was 10mM.
The method for detecting staphylococcus aureus by using the combined kit comprises the following steps of:
1) Phage bioamplification detection
a. Mixing the staphylococcus aureus phage LSA2311 suspension with a sample to be detected according to the volume of 1:1, and culturing to obtain a culture; wherein the concentration of the staphylococcus aureus phage LSA2311 suspension is 10 7 PFU/mL, concentration of sample to be detected is 10 8 CFU/mL；
b. Mixing the culture with a solution of Ferrous Ammonium Sulfate (FAS) with the concentration of 25mM according to the volume of 1:1, adding LB liquid culture medium and supplementing to 1mL, and culturing for 5min; then adding 10mM trisodium citrate solution (neutralizing the activity of the virucide) at a concentration of 100. Mu.L/L;
c. centrifuging the mixed solution to obtain a supernatant, namely the progeny phage;
2) Real-time fluorescent quantitative PCR detection of progeny phage:
a. the progeny phage were DNA extracted and stored at-20 ℃.
b. Taking DNA of a progeny phage as a template, and carrying out real-time fluorescence quantitative PCR (polymerase chain reaction) by using a primer pair tsp to obtain a PCR product, wherein the primer pair tsp is as follows:
forward primer tsp-F:5'-TGCGTGCTATTCTAGGTGGA-3';
reverse primer tsp-R:5'-ACCACTTACCAAACCTCCGA-3'.
c, analyzing the Ct value of the PCR product and an established standard curve to obtain the content of staphylococcus aureus in the sample to be detected; wherein, the reaction amplification system of PCR is as follows:
| 2×qPCR Mix | 10μL |
| forward primer tsp-F | 0.4μL |
| Reverse primer tsp-R | 0.4μL |
| Template (DNA of progeny phage) | 1μL |
| ddH 2 O to | 20μL |
Example 5 verification of the detection method of the above-described Combined kit
1. Primer design and amplification system
qPCR primers were designed based on the sequence alignment of the Staphylococcus aureus phage LSA2311 genomic sequence with the Staphylococcus aureus genomic sequence data downloaded from NCBI using Primer3 Input (https:// bioinfo. Ut. Ee/Primer3 /), and tail sheath gene (tail shealth protein, TSP) fragments of Staphylococcus aureus phage LSA2311 were amplified. The primer pairs tsp were synthesized by the biological engineering (Shanghai) Co., ltd as follows:
forward primer tsp-F:5'-TGCGTGCTATTCTAGGTGGA-3';
reverse primer tsp-R:5'-ACCACTTACCAAACCTCCGA-3';
the product length is 200bp;
the PAA-qPCR detection kit adopts a reaction amplification system of 20 mu L,
the PCR reaction amplification system is as follows:
| 2×qPCR Mix | 10μL |
| forward primer tsp-F | 0.4μL |
| Reverse primer tsp-R | 0.4μL |
| Template (DNA of progeny phage) | 1μL |
| ddH 2 O to | 20μL |
According to qPCR/>Real-time fluorescent quantitative PCR (Real-time PCR) analysis is performed by the Green Master Mix product Specification; experiments were set up in three replicates, and the reproducibility of the experiments was assessed by running samples independently at different times.
2. Extraction of phage DNA
DNA of phage LSA2311 was extracted by thermal lysis: heating the sample in water bath at 100deg.C for 10min, taking out, centrifuging at 4deg.C and 4000g for 10min, collecting supernatant, and storing at-20deg.C;
3. primer specificity assay
Respectively with the bacterial liquid concentration of 10 8 CFU/mL of staphylococcus aureus, salmonella, escherichia coli, listeria and mixed bacterial liquid are used as specific samples for phage biological amplification, 1 mu L of supernatant obtained by thermal cleavage extraction is taken for qPCR detection, and a culture medium added with phage only is used as Negative Control (NC), and delta Ct is calculated.
After 4 staphylococcus aureus, staphylococcus aureus phage LSA2311 and phage DNA thereof, salmonella phage T155 and 3 common bacteria (salmonella 13311, escherichia coli BL21 and listeria 19115) were biologically amplified, 1 μl of the supernatant extracted by thermal lysis was taken for PCR, and ultrapure water was set as a negative control group (NC). Agarose gel results of the PCR products (as shown in FIG. 4A) showed that the designed primer pair was specific for LSA2311 (LSA 2311 with a target band of 200bp, slightly lower than 250bp in FIG. 4B). Because the primers were designed based on the sequence of LSA2311 and by bioinformatic analysis the host sequences were avoided without PCR target bands against any other strains and phages, fragments of staphylococcus aureus phage LSA2311 could be specifically amplified without amplifying other species of bacteria.
There are two points to be met when the sample contains staphylococcus aureus. First, the Ct value at a time point should be less than that at 0min of incubation (i.e., the negative control group to which phage LSA2311 alone was added), indicating that phage at that time point had been amplified and that the sample may contain Staphylococcus aureus. Second, considering that acceptable Ct value deviations for qPCR detection are typically below 0.5,0.5 is the threshold for Ct change. If the Ct change value (ΔCt) between the initial time point and the other time points of the sample is greater than 0.5, the sample contains Staphylococcus aureus. Otherwise, there is no staphylococcus aureus in the sample.
In the experiment of detecting the specificity of staphylococcus aureus by PAA-qPCR, as shown in FIG. 4B, the live staphylococcus aureus ATCC25923, 6538, 196 and 142 are obviously amplified, while specific samples such as staphylococcus aureus dead bacteria, salmonella, escherichia coli, listeria and the like are not amplified, and the mixed bacterial liquid containing the live staphylococcus aureus also has the amplification phenomenon (Table 4).
TABLE 4 real-time fluorescent quantitative PCR (qPCR) detection specificity
Note that: a : live staphylococcus aureus ATCC25923; b : salmonella typhimurium 13311; c : coli BL 21; d : listeria monocytogenes 19115.
4. Standard curve
Ten-fold gradient dilution of Staphylococcus aureus ATCC25923 bacterial liquid (10 8 -10 0 CFU/mL), extracting DNA by thermal decomposition after biological amplification, taking 1 mu L of each as a template for qPCR detection, and repeating each concentration gradient for 3 timesAnd taking a culture medium with only phage as a Negative Control (NC), and adopting the established PAA-qPCR method to detect the lowest detection limit of the established PAA-qPCR method for detecting staphylococcus aureus. And drawing a standard curve by taking the logarithmic value of the staphylococcus aureus concentration as an abscissa and the Ct value as an ordinate. The minimum limit of detection of staphylococcus aureus 25923 by PAA-qPCR is shown in FIG. 5, 10 was detected 1 The Ct value of CFU/mL was 26.31, so the minimum detection limit of the method was 10 1 CFU/mL. As can be seen from FIG. 5, the standard curve R 2 The amplification efficiency was 99% at 0.998, indicating that PAA-qPCR was a good linear relationship and high amplification efficiency.
PAA-qPCR kit stability
To evaluate the reproducibility and stability of PAA-qPCR assay for staphylococcus aureus ATCC25923, samples were subjected to an Intra-group repeat experiment (n=3) and coefficient of variation values (Coefficient of Variation, CV) were calculated, evaluating the reproducibility and stability of the method (table 5);
TABLE 5 real-time fluorescent quantitative PCR (qPCR) detection stability
Example 6 application of PAA-qPCR kit to detection of staphylococcus aureus in labeled food
1. Application of staphylococcus aureus detection in milk sample
(1) Sample aseptic treatment: 10g of skimmed milk powder is weighed and dissolved in 100mL of distilled water, and the mixture is sterilized at the temperature of 115 ℃ for 15min for standby.
(2) The PAA-qPCR kit detects staphylococcus aureus in milk: contains different concentrations of Staphylococcus aureus ATCC25923 (10) 8 -10 0 CFU/mL) of milk prepared according to sample: staphylococcus aureus phage LSA2311 suspension = 1:1 (volume ratio)The phage bioamplification mixture was prepared and phage bioamplified in example 4 to obtain a progeny phage suspension.
(3) After extracting progeny phage DNA by thermal decomposition, 1 μL of each of the phage DNA is taken as a template for qPCR detection, each concentration gradient is repeated 3 times, and a culture medium with phage added only is used as a Negative Control (NC), and the established PAA-qPCR method is adopted to detect the lowest detection limit of the established PAA-qPCR method for detecting staphylococcus aureus. And drawing a standard curve by taking the logarithmic value of the theoretical staphylococcus aureus concentration as an abscissa and the Ct value as an ordinate.
The minimum limit of detection of staphylococcus aureus 25923 in milk by PAA-qPCR is shown in FIG. 6A, 10 1 The Ct value of CFU/mL was 28.25, so the minimum detection limit of the method was 10 1 CFU/mL. As can be seen from FIG. 6A, the standard curve R 2 The amplification efficiency is 105% for 0.9680, which shows that the PAA-qPCR detection of staphylococcus aureus 25923 in milk has good linear relation and high amplification efficiency.
(4) Specificity of PAA-qPCR kit for detection in milk: the sterile milk samples were inoculated with 100. Mu.L of Staphylococcus aureus 25923 (live), 6538, 196, 142 (final bacterial load 10) 2 CFU/mL), staphylococcus aureus 25923 (dead), salmonella 13311, escherichia coli BL21, listeria 19115 (final bacterial load 10) 8 CFU/mL) and the mixed bacterial liquid as specific samples, performing phage bioamplification, taking 1 μl of supernatant obtained by thermal cleavage extraction, performing qPCR detection, and calculating Δct with a medium to which only phage is added as Negative Control (NC).
The traditional culture method is used as a standard: the bacterial liquid is inoculated on a Baird Parker plate after being enriched for 18-24 hours at 37 ℃, the plate is placed upside down on the 37 ℃ for culturing for 24-48 hours, and whether characteristic colonies appear on the Baird Parker plate is observed.
In the specific experiment for detecting staphylococcus aureus by PAA-qPCR, the results are shown in Table 6, the live staphylococcus aureus ATCC25923, 6538, 196 and 142 are obviously amplified, the specific samples such as staphylococcus aureus dead bacteria and other species bacteria are not amplified, and the mixed bacterial liquid containing the live staphylococcus aureus also has amplification phenomenon. The qualitative test result of staphylococcus aureus is the same as that of national standard GB 4789.10-2016.
TABLE 6 specificity of PAA-qPCR kit in milk samples
Note that: "+" indicates that colonies on the Baird-Parker plates are black circular protrusions; "-" indicates no-feature colonies on Baird-Parker plates. a : live staphylococcus aureus ATCC25923; b : salmonella typhimurium 13311; c : coli BL 21; d : listeria monocytogenes 19115.
2. Application of staphylococcus aureus detection in raw vegetable sample
(1) Sample aseptic treatment: washing fresh lettuce purchased in fresh vegetable market with distilled water for 2min, and taking fresh and tender parts of lettuce to specific size (area 1 cm) 2 ) And placing the lettuce sample in a sterile culture dish, and irradiating the front and back sides of the lettuce sample for half an hour by ultraviolet rays to ensure sterility for later use.
(2) The PAA-qPCR kit detects staphylococcus aureus in lettuce: as in embodiment 5.1.
The minimum limit of detection of staphylococcus aureus 25923 in lettuce by PAA-qPCR is shown in FIG. 6B, 10 1 The Ct value of CFU/mL was 26.31, so the minimum detection limit of the method was 10 1 CFU/mL. As can be seen from FIG. 6B, the standard curve R 2 The amplification efficiency is 110% when the ratio is 0.9980, which shows that the PAA-qPCR detection of staphylococcus aureus 25923 in lettuce has good linear relation and high amplification efficiency.
(3) Specificity of PAA-qPCR kit for detection in lettuce: as in example 6.1.
In the experiment of detecting the specificity of staphylococcus aureus in lettuce by PAA-qPCR, the results are shown in Table 7, the live staphylococcus aureus ATCC25923, 6538, 196 and 142 are obviously amplified, and the specific samples such as staphylococcus aureus dead bacteria, salmonella, escherichia coli, listeria and the like are not amplified, so that the mixed bacterial liquid containing the live staphylococcus aureus also has the amplification phenomenon. The qualitative test result of staphylococcus aureus is the same as that of national standard GB 4789.10-2016.
TABLE 7 specificity of PAA-qPCR kit in lettuce samples
Note that: "+" indicates that colonies on the Baird-Parker plates are black circular protrusions; "-" indicates no-feature colonies on Baird-Parker plates. a : live staphylococcus aureus ATCC25923; b : salmonella typhimurium 13311; c : coli BL 21; d : listeria monocytogenes 19115.
Other parts not described in detail are prior art. Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (1)
1. A phage bioamplification-real-time fluorescent quantitative PCR combined kit for rapidly detecting staphylococcus aureus is characterized in that: the combined kit comprises a phosphate buffer solution containing staphylococcus aureus phage LSA2311, a negative control, a positive control, a ferrous ammonium sulfate solution, a trisodium citrate solution and a primer pair tsp for real-time fluorescence quantitative PCR detection:
forward primer tsp-F:5'-TGCGTGCTATTCTAGGTGGA-3';
reverse primer tsp-R:5'-ACCACTTACCAAACCTCCGA-3'; wherein the negative control is PBS buffer solution, and the molar concentration of the PBS buffer solution is 0.05mol/L;
the positive reference is staphylococcus aureus, and the staphylococcus aureus ATCC25923;
the concentration of the ferrous ammonium sulfate solution is 25mM;
trisodium citrate solution at 10mM; staphylococcus aureus phage (Staphylococcus aureus bacteriophage) LSA2311 with a preservation number of CCTCC NO: m2020562 phosphate buffer containing Staphylococcus aureus phage LSA2311 at a concentration of 10 7 PFU/mL。
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