Novel method for detecting salmonella, application and detection kit

Abstract

The invention belongs to the technical field of food-borne pathogenic bacterium detection, and discloses a detection method for targeted analysis and detection of salmonella based on a characteristic peptide, and the method detects salmonella pollution in a dairy product by analyzing the following characteristic peptides: I1: (LLADDLVPSR; i2: (IGTISTTGEMSPLDAR), and I3: ( I3: (IELALFLDSYIPEPER), and a method for preparing the same. And I4: (AVAAVEELK). The method for identifying the type of the strain by taking the characteristic peptide as an index is a relatively safe food detection method, the characteristic peptide of a specific microorganism is used for screening, and the characteristic peptide is detected through a targeted mass spectrometry analysis technology, so that the detection of pathogenic bacteria is converted into the detection of the characteristic peptide. The method has the advantages of rapidness, high throughput, high reliability, no dependence on bacterial colony purity and the like. By obtaining a characteristic peptide combination with good specificity, the technology can realize detection of various specific microorganisms.

Description

Technical Field

[0001] The invention belongs to the technical field of foodborne pathogenic bacteria detection, in particular to a new method for detecting Salmonella, an application and a detection kit. Background Art

[0002] Foodborne pathogens can cause a variety of foodborne diseases, such as infection, diarrhea and poisoning, by contaminating food, and they continue to threaten human health today. Therefore, efficient and rigorous inspection and quarantine measures are extremely important for controlling the hazards of foodborne pathogens.

[0003] Salmonella is a Gram-negative anaerobic bacterium that belongs to the intestinal flora. Its colony appearance is grayish white or milky white, surrounded by brown or black precipitation rings, with a flat or slightly convex surface and neat edges. The colony morphology of Salmonella is mainly affected by factors such as the type of strain and the growth environment, so the colony morphology of different strains in the laboratory may be different. Salmonella can adapt to high temperatures (below 80°C), has a wide pH range (pH>3), and can usually grow in an environment containing 10% or more salt. This characteristic is largely adapted to the intestinal environment of animals, including poultry, livestock, wild animals, insects, water bodies, etc. Through contaminated food and water, people can be exposed to this pathogen, causing infection and foodborne diseases. Studies have shown that the pathogenicity of Salmonella is caused by the interaction of a large number of virulence factors, mainly including: virulence islands, virulence plasmids, enterotoxins and endotoxins. Salmonella also carries virulence plasmids to enhance its adhesion to the intestine and improve its survival ability. In addition, enterotoxins also help to enhance the invasive ability of bacteria and induce salmonella enteritis in animals. The endotoxin lipopolysaccharide (LPS) is a component of the Salmonella cell wall. When the bacteria die and dissolve, it will be released and can cause salmonella sepsis. If the immune system cannot eliminate the bacteria, Salmonella will multiply in the spleen, causing lesions and obvious swelling of the liver and spleen. Salmonella infection often leads to anemia and sepsis, which in turn causes gastrointestinal bleeding, massive inflammatory infiltration and intestinal wall ulceration, and ultimately leads to the death of the animal. Due to the large number of Salmonella virulence factors, subsequent prevention and detection have caused great difficulties.

[0004] Existing detection technologies for Salmonella include traditional biochemical identification methods, as well as modern enzyme-linked immunosorbent assay, PCR detection method, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Among them, traditional biochemical identification methods can only detect single microorganisms, cannot detect multiple microorganisms at the same time, and have certain subjectivity and misjudgment rates. Enzyme-linked immunosorbent assay requires a high target concentration in the sample, cannot distinguish between living and non-living organisms, and is difficult to simultaneously detect the synergistic presence of multiple pathogens in a complex system. PCR has excellent specificity and detection speed, but this target-based method is limited to specific primers, and amplification inhibitors in the tested product will also interfere with the detection results. The detection method based on MALDI-TOF can only be used for colonies that have been separated and purified, so it cannot be directly used for the detection of mixed bacteria. Although each has its advantages, there are certain shortcomings. Therefore, it is of great significance to develop a method for rapid and accurate detection of Salmonella. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a new method, application and detection kit for detecting Salmonella.

[0006] The technical solution adopted by the present invention to solve its technical problem is:

[0007] A detection method for detecting Salmonella based on targeted analysis of combined characteristic peptides, wherein the method detects Salmonella in a sample by analyzing the following characteristic peptides:

[0008] Amino acid sequence of characteristic peptide 1: IGTISTTGEMSPLDAR;

[0009] Amino acid sequence of characteristic peptide 2: IIELAGFLDSYIPEPER;

[0010] Amino acid sequence of characteristic peptide 3: AVAAAVEELK.

[0011] Furthermore, if all of the characteristic peptides are detected, it can be determined that the sample is positive for Salmonella.

[0012] Furthermore, a liquid chromatography-mass spectrometry system was used to detect the combined characteristic peptides of Salmonella.

[0013] Furthermore, LC-ESI / QQQ MRM mode was used for detection.

[0014] The above-mentioned detection method for detecting Salmonella by targeted analysis based on combined characteristic peptides is applied in the detection of Salmonella.

[0015] A Salmonella detection kit based on a combination of characteristic peptides, the kit comprising the above-mentioned combined characteristic peptides of Salmonella:

[0016] Furthermore, the kit also includes Salmonella enrichment solution, physiological saline, bacterial denaturation solution, iodoacetamide solution, trypsin, formic acid, and characteristic peptide standards; the bacterial denaturation solution is a 50mM ammonium bicarbonate solution with a pH of 8, containing 10mM dithiothreitol and 1.2M urea; and the iodoacetamide solution is sterile water containing 0.5M iodoacetamide.

[0017] Furthermore, the detection method of the kit comprises the following steps:

[0018] (1) Bacterial cell collection and sample pretreatment: Collect the cells by centrifugation, wash the precipitate twice with physiological saline, add 0.5 ml of bacterial denaturation solution to the bacterial precipitate, mix well and then ultrasonically disrupt for 10 min; then, add 10 μL of iodoacetamide solution and mix quickly, and react in the dark for 10 min; then add 20 μL of trypsin and digest in a 50°C water bath for 15 min; add 3.5 μL of formic acid to terminate the trypsin digestion and use directly for analysis or store at -20°C.

[0019] (2) LC-ESI / QQQ analysis: LC-ESI-Q-TOF-MS / MS mass spectrometer was used to identify and analyze the non-targeted proteome of the samples, confirm the retention time and characteristic fragments of the standard peptides, and then use Triple Quad TM The 5500UPLC-MS / MS liquid chromatography-mass spectrometry system was used to perform targeted analysis of candidate characteristic peptides of MRM.

[0020] (3) Salmonella contamination identification method: If all three characteristic peptide combinations are detected, the sample can be determined to be positive for Salmonella.

[0021] The advantages and positive effects achieved by the present invention are:

[0022] The present invention uses characteristic peptides as indicators to identify the type of bacterial species, which is a relatively safe food detection method. It uses characteristic peptides of specific microorganisms for screening, and detects their characteristic peptides through targeted mass spectrometry analysis technology, thereby converting the detection of pathogenic bacteria into the detection of their characteristic peptides. This method has the advantages of being rapid, high-throughput, highly reliable, and independent of colony purity. By obtaining a combination of characteristic peptides with good specificity, this technology can achieve the detection of a variety of specific microorganisms.

[0023] Compared with the method in the national standard for Salmonella inspection (GB4789.40-2024), the method of the present invention does not rely on human judgment and selection of suspicious colonies, does not rely on cumbersome biochemical inspection steps, and the detection process is efficient and reliable, and is easy to achieve standardized and automated operations.

[0024] The application of this technology can be extended to the application detection of other bacterial species, improving the efficiency and quality of bacterial species detection in food, and bringing new technical methods to food safety detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figure is a screening diagram of the characteristic peptides of Salmonella in the present invention; wherein (a) the peptide screening is derived from the whole proteomic analysis of 21 strains of Salmonella; (b) the proteomes of Escherichia coli and Salmonella are virtually digested by computer, and the candidate characteristic peptides of Salmonella are eliminated due to their poor specificity; (c) the PCA analysis diagram of the candidate characteristic peptides; (d) the PCA loading diagram, which reflects the contribution of the peptides to the separation of Salmonella from other strains;

[0026] Figure 2 The figure is a comparison and optimization diagram of candidate characteristic peptides of Salmonella in the present invention; wherein, (a) is a diagram of the coverage rate of Salmonella species, and (b) is the coverage rate of each characteristic peptide to Salmonella.

[0027] Figure 3 Comparison of the response intensity of candidate characteristic peptides of Salmonella in 29 Salmonella samples. DETAILED DESCRIPTION

[0028] The embodiments of the present invention are described in detail below. It should be noted that the embodiments are descriptive rather than restrictive and cannot be used to limit the protection scope of the present invention.

[0029] The raw materials used in the present invention, unless otherwise specified, are conventional commercially available products; the methods used in the present invention, unless otherwise specified, are conventional methods in the art.

[0030] The specific preparation and testing are as follows:

[0031] Example 1: Mining of characteristic peptides

[0032] (1) Non-targeted proteomics analysis: LC-ESI-Q-TOF-MS / MS mass spectrometer was used to identify and analyze the non-targeted proteome of the strain, and Mascot software was used to analyze and process the data collected by the instrument. Liquid chromatography conditions: flow rate 0.2mL / mL; injection volume 10μL; elution steps: 0-5 minutes, B phase maintained at 5%; 5-25 minutes, B phase increased from 5% to 40%; 25-35 minutes, B phase decreased from 40% to 5%, 35-45 minutes, B phase maintained at 5%. Mass spectrometry analysis conditions: use heated electrospray ion source (HESI); adopt positive ion mode; adopt full scan mode, so that the particle range is 3200m / z; resolution is 70,000; Ctrap maximum capacity is 100000; Ctrap maximum injection time is 100ms; sheath gas speed is 40arb; auxiliary gas flow is 3arb; spray voltage is 3.5kV; capillary temperature is 300℃. Mascot software identifies proteins and limits its related conditions: select SwissProt for protein database, trypsin for protease type, maximum missing cleavage site 1, carbamidomethyl (C) for fixed modification, oxidation (M) for variable modification, molecular weight error: 1.2Da; charge type is 2,3. First, 24 Salmonella standard strains were screened using non-targeted proteome analysis methods, and 94 candidate characteristic peptides ( Figure 1 (a)), these candidate signature peptides covered at least 95% of the tested strains, and the MS1 area of ​​the signature peptides was greater than 200.

[0033] (2) Specificity analysis: The local BLAST program was used to specifically analyze the protein libraries of Salmonella, diarrheagenic Escherichia coli and amoeba. Skyline software was used for computer virtual digestion, and the background proteome was customized to the Salmonella library. Trypsin was virtually digested under the condition of prohibiting missing cleavage. The length of the polypeptide chain was limited to 6 to 25 amino acids. The charge type of the parent ion was selected as 2,3; the type of the daughter ion was selected as b and y. With the help of computer virtual digestion technology, further screening was carried out to exclude the theoretical hydrolysis peptides of Salmonella-related strains. Computer virtual digestion technology uses the known cleavage sites of the enzyme to cut the protein sequence to predict the sequence of the enzymatic peptide. Due to the large size of the database protein sequence, the use of computer virtual digestion technology can greatly improve the experimental efficiency. Finally, 48 candidate specific peptides were screened out ( Figure 1 (b)).

[0034] (3) PCA principal component evaluation: PCA analysis of 48 candidate characteristic peptides revealed that these characteristic peptides could clearly distinguish Salmonella from its related strains ( Figure 1(c)). Finally, by analyzing the loading graph, eight characteristic peptides with the strongest influence on cluster analysis were screened out ( Figure 1 (d)).

[0035] Example 2: Specificity verification and optimization of candidate characteristic peptides

[0036] (1) Standard strains of Salmonella and Escherichia coli were cultured in LB solid medium (1% peptone, 0.5% yeast extract, 1% NaCl, 2% agar) at 37°C for 24 h.

[0037] (2) Pretreatment and digestion of Salmonella and diarrheagenic Escherichia coli: Scrape the colonies on the solid culture medium and resuspend them in 2 mL of normal saline. Centrifuge at 8,000 r / min for 15 minutes to allow the bacteria to aggregate at the bottom of the tube, discard the supernatant, resuspend the precipitate in bacterial washing solution, centrifuge and wash, and finally resuspend the bacteria in 0.5 mL of bacterial washing solution. Add 0.5 mL of bacterial denaturation solution to the above test tube, shake to mix, and then sonicate for 10 minutes. Add 10 μL of iodoacetamide and react in the dark for 10 minutes for alkylation. Add 20 μL of trypsin and digest in a 50°C water bath for 15 minutes. Stop the trypsin digestion by adding 3.5 μL of formic acid, and the sample can be directly analyzed or stored at -20°C.

[0038] (3) Targeted analysis of LC-DSI / QQQ in MRM mode. Chromatographic conditions: The chromatographic column used was an XBridge C18 column (2.1 mm × 150 mm, 5 μm) (Waters, USA). The flow rate was 0.3 mL / min; the injection volume was 2 μL; the elution program was: 0-3 min, phase B was maintained at 2%; 3-13 min, phase B increased from 5% to 40%. 13-16 min, phase B decreased from 40% to 5%, and 16-20 min, phase B was maintained at 5%. Mass spectrometry conditions: positive ion mode and MRM mode were used; the Turbo V ion source nebulization temperature was set to 550°C; the ion spray voltage was set to 5500V; the curtain gas flow was set to 172.2kPa; the collision gas flow was set to 68.3kPa; the GS1 (ion source Gas1) nebulization gas flow was set to 413.7kPa; the GS2 (ion source Gas2) auxiliary gas flow was set to 379.2kPa; the MRM mode setting parameters for the Salmonella characteristic peptide are shown in Table 1.

[0039] Table 1. MRM mode setting parameters for Salmonella

[0040]

[0041] (4) Qualitative judgment method: LC-ESI / QQQ data are analyzed by Skyline software. In order to confirm the characteristic peptide in the sample by comparing with the characteristic peptide standard data, the following conditions must be met: a) The deviation of the retention time of the target ion should be less than 0.1min. B) The signal-to-noise ratio is greater than 3; C) The deviation value of the peak area ratio (Pr) of the most abundant product ion is within ±0.05. The calculation formula of the Pr deviation value is as follows:

[0042]

[0043] Pr(sample)-Pr(standard)|≤0.05 Formula (2-2)

[0044] Where P i -The daughter ions with the highest abundance in response to the characteristic peptide, P1, P2, P3 - the three daughter ions of the characteristic peptide.

[0045] The coverage of characteristic peptides I1, I2, and I3 against Salmonella is as high as 100%. The evaluation results are as follows: Figure 2 (b)).

[0046] Since it is impossible to compare all Salmonella and non-Salmonella samples, it is necessary to check and compare candidate characteristic peptides through the database. This can provide theoretical guidance and experimental basis for the evaluation and screening of characteristic peptides. The UniProt protein database (https: / / www.uniprot.org) contains a large amount of extensive protein information. So far, it has included 563,972 manually tested and annotated protein sequences and 209,157,139 unfinished protein sequences. The UniProt protein database BLAST analysis of 8 candidate characteristic peptides of Salmonella was used to examine their specificity. The results are as follows ( Figure 2 (b)). Among them, four candidate characteristic peptides (I 1, I 2, I 3, I 8) have good specificity for Salmonella, with a coverage rate of up to 75% ( Figure 2 (a)), indicating that these candidate characteristic peptides have good specificity. BLAST analysis using only manual testing and annotated sequences found that the three characteristic peptides (I 1, I 2, I 3) had a coverage rate of 100% for Salmonella, and I 8 had a coverage rate of 78%, further verifying their specificity. In actual biological samples, the applicability of peptides also needs to consider their detection efficacy. Figure 3 The response intensity of each characteristic peptide of 33 strains of Salmonella was displayed, among which I2, I3, and I8 had stronger responses and could be used as ideal characteristic peptides, while the response intensity of I1 was weaker and was not suitable as a characteristic peptide. Therefore, I2, I3, and I8 were finally selected as the combined characteristic peptides.

[0047] Example 3: Application of characteristic peptides in detecting salmonella contamination in milk powder samples

[0048] The target bacteria were added to infant formula (Yili, China) to a bacterial concentration of 100 CFU / mL. 0.1 mL of milk powder contaminated with bacterial solution was added to 0.9 mL of LB medium and cultured at 37°C for 16 h. The subsequent steps were consistent with the treatment method in Example 2. The evaluation results are shown in Table 2.

[0049] Table 3-5 Detection of trace salmonella contamination in milk powder samples

[0050]

[0051]

[0052] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for detecting Salmonella based on targeted analysis of combined characteristic peptides, wherein the method detects Salmonella in a sample by analyzing the following characteristic peptides: Amino acid sequence of characteristic peptide 1: IGTISTTGEMSPLDAR; Amino acid sequence of characteristic peptide 2: IIELAGFLDSYIPEPER; Amino acid sequence of characteristic peptide 3: AVAAAVEELK.

2. The method for detecting Salmonella based on targeted analysis of combined characteristic peptides according to claim 1, characterized in that: If all of I2, I3, and I4 in the characteristic peptides are detected, it can be determined that the sample is positive for Salmonella.

3. The method for detecting Salmonella based on targeted analysis of combined characteristic peptides according to claim 1, characterized in that: The combined characteristic peptides of Salmonella were detected by liquid chromatography-mass spectrometry system.

4. The method for detecting Salmonella based on targeted analysis of combined characteristic peptides according to claim 3, characterized in that: Detection was performed using LC-ESI / QQQ MRM mode.

5. Application of the method for detecting Salmonella by targeted analysis based on combined characteristic peptides as claimed in any one of claims 1 to 4 in detecting Salmonella.

6. A Salmonella detection kit based on combined characteristic peptides, the kit comprising the three characteristic peptides according to claim 1.

7. The Salmonella detection kit based on combined characteristic peptides according to claim 6, characterized in that: The kit also includes Salmonella enrichment solution, physiological saline, bacterial denaturation solution, iodoacetamide solution, trypsin, formic acid, and characteristic peptide standards; the bacterial denaturation solution is a 50 mM ammonium bicarbonate solution with a pH of 8 and containing 10 mM dithiothreitol and 1.2 M urea; and the iodoacetamide solution is sterile water containing 0.5 M iodoacetamide.

8. The Salmonella detection kit based on combined characteristic peptides according to claim 6 or 7, characterized in that: The detection method of the kit comprises the following steps: (1) Bacterial cell collection and sample pretreatment: Collect the cells by centrifugation, wash the precipitate twice with physiological saline, add 0.5 ml of bacterial denaturation solution to the bacterial precipitate, mix well, and then ultrasonically disrupt for 10 min; Subsequently, 10 μL of iodoacetamide solution was added and mixed quickly, and the mixture was allowed to react in the dark for 10 min. Subsequently, 20 μL of trypsin was added and the mixture was digested in a 50°C water bath for 15 min. 3.5 μL of formic acid was added to terminate the trypsin digestion, and the mixture was directly used for analysis or stored at -20°C. (2) LC-ESI / QQQ analysis: Use LC-ESI-Q-TOF-MS / MS mass spectrometer to identify and analyze the non-targeted proteome of the sample, confirm the retention time and characteristic fragments of the standard peptides, and then use liquid chromatography-mass spectrometry system to perform targeted analysis of candidate characteristic peptides; (3) Salmonella contamination identification method: If the characteristic peptide combinations I2, I3, and I4 are all detected, the sample can be determined to be positive for Salmonella.

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