CN116064874A - Primer probe group, kit and method for rapidly detecting food-borne salmonella - Google Patents

Abstract

The invention provides a primer probe group, a kit and a method for rapidly detecting food-borne salmonella. The method is designed and synthesized by aiming at determining specific species identification genes stn and invA of salmonella, and uses standard strains and sample isolates to carry out repeated verification in a large batch, so that the method has the characteristics of rapidness, accuracy, sensitivity, convenience in operation, universality, independence of a database, proper cost, high throughput and the like, and can realize rapid identification and high-throughput screening of the salmonella, particularly the food-borne salmonella.

Description

Primer probe set, kit and method for rapid detection of foodborne Salmonella

Technical Field

The invention belongs to the technical field of gene molecular detection, and in particular relates to a primer probe set, a kit and a method for rapid detection of foodborne Salmonella.

Background Art

Salmonella is an important foodborne pathogen that is widely distributed in nature and can colonize in the intestines of a variety of animal hosts, including pigs, cattle, poultry and wild animals, as well as companion animals such as dogs, cats, birds and reptiles. It is most common in the intestines of poultry and livestock. In addition to humans and animals, Salmonella can also be detected in plants, water bodies, soil, etc. The common presence of Salmonella in nature and its transmission through the fecal-oral route make salmonellosis the most important foodborne bacterial zoonosis. As one of the most common foodborne pathogens, Salmonella mainly causes human infection through food, and the contaminated food categories are diverse, mainly meat and eggs and their products. It is estimated that more than 90 million cases of diarrhea-related diseases are caused by Salmonella each year worldwide, of which 85% are related to food. Salmonellosis has long become an important global public health issue.

Taxonomically, Salmonella belongs to the phylum Proteobacteria, class Gammaproteobacteria, order Enterobacteriales, family Enterobacteriaceae. The genus includes two species, Salmonella enterica and Salmonella bongore. The former contains six subspecies (enteric subspecies I; Salam subspecies II; Arizona subspecies IIIa; biphasic Arizona subspecies IIIb; Hoton subspecies IV; Indica subspecies VI). The original subspecies V has now been upgraded to Bongore species. At the same time, each species or subspecies of Salmonella contains multiple serotypes. There are differences in pathogenicity, drug resistance and host range between different serotypes of Salmonella. So far, more than 2,600 serotypes have been discovered, of which more than 1,500 can cause salmonellosis. Because Salmonella has an extremely complex classification system and a large number of serotypes, this places high demands on the inspection of Salmonella in various sample sources in daily testing and scientific research.

In 2009, my country promulgated the Food Safety Law, established and implemented the food safety risk monitoring system, and established a national food safety risk monitoring network to conduct continuous monitoring of various pathogenic microorganisms in various food samples across the country. Among them, Salmonella testing is one of the most important monitoring items that must be inspected for many types of food samples every year. Relying on this food safety risk monitoring network, the number of Salmonella isolates collected and reported from all over the country has reached up to 2,500 strains per year so far. Whether it is the identification of suspicious colonies of Salmonella in samples isolated from each monitoring point, or the review and preservation of reported isolates at the national level, new requirements have been put forward for high-throughput, rapid and accurate identification methods of Salmonella.

At present, the final confirmation method of the Salmonella identification step in the domestic and foreign food inspection standards (such as ISO, FDA, USDA, AOAC, GB, etc.) is mainly based on the traditional biochemical identification method. However, this process has many steps, expensive reagents, and high requirements for the experience of the inspectors. In particular, when all suspicious colonies picked in the sample separation need to be biochemically identified to obtain accurate results, it is impossible to achieve rapid and accurate screening and identification, which brings a large workload to the development of food safety risk monitoring and increases the difficulty of related research on Salmonella contamination, prevalence, and typing in various food matrices in China. The development of accurate and reliable rapid detection and identification methods for foodborne Salmonella is of great significance to the development of risk monitoring, early warning and assessment in my country's food, and even to a greater extent, the rapid diagnosis of Salmonella infection in animals and humans in agriculture and clinical practice.

At present, the identification and rapid detection technologies for foodborne Salmonella generally include the following categories:

(1) Fully automatic or semi-automatic biochemical identification method: For suspected Salmonella colonies obtained during or after sample separation, inoculate them on a non-selective culture medium for a certain period of time, pick an appropriate amount of colonies to prepare a bacterial suspension with a certain turbidity, and use imported or domestic biochemical reagent strips for inoculation reaction, or use fully automatic biochemical identification instruments combined with biochemical identification cards for identification, such as the pathogenic bacteria series biochemical identification reagent strips produced by France's BioMérieux and China's Luqiao, the Vitek2 Compact automatic biochemical identification instrument supporting identification cards produced by France's BioMérieux, or the Biolog series bacterial detectors and supporting identification cards produced by the United States' BIOLOG (see "National Food Safety Standard Microbiological Testing for Salmonella Testing GB 4789.4-2016" or the reagent manufacturer's operating requirements). This method has certain requirements for reagent quality, strain culture status, identification instrument throughput, strain database size, etc. The automatic identification instrument is relatively expensive; identification reagent strips or identification cards are special matching reagents. If the number of strains to be identified is large, the reagent price is not cheap; in addition, if the strain is a rare bacteria or the instrument database is incomplete, the final result may not be detected; in addition, when there are many strains, it is easy to be limited by the instrument throughput, resulting in a long batch identification time. Although this method is regarded as the gold standard method for microbial identification in terms of identification results by multiple standards, this method is not suitable for rapid screening of a large number of strains or preliminary screening and identification experiments.

(2) Molecular biological methods: ① Molecular biological detection technology based on in vitro specific amplification of characteristic gene nucleic acid sequences in pathogenic bacteria, with polymerase chain reaction (PCR) being the most widely used. By designing PCR primers and amplification systems for specific target genes of Salmonella, and using different numbers of Salmonella strains for verification, it is used for rapid detection and identification of Salmonella. Compared with selective culture, monoclonal selection and other methods, the amplification of specific target fragments by PCR reaction reduces the probability of false positives, and can obtain lower detection limits in terms of sensitivity than traditional methods. The test operation and convenience are simpler, the instrument penetration rate is high, and it takes less time and is suitable for high-throughput screening. The limitations of this method are: it may be affected by PCR inhibitors and produce false negatives, the quality of primer design will directly affect the accuracy (specificity and sensitivity) of the method, it is impossible to distinguish whether the foodborne microorganisms in the sample are alive, and the detection sensitivity is greatly affected by the design and is sometimes not high enough. ②Real-time fluorescence quantitative PCR technology is based on ordinary PCR, designing primers and fluorescent labeled probes, adding fluorescent groups to the reaction system, and using the accumulation of fluorescent signals to monitor the entire nucleic acid amplification process in real time. Finally, the concentration of unknown templates is quantitatively analyzed according to the standard curve, which can achieve qualitative and quantitative simultaneous analysis, overcoming the shortcomings of conventional PCR. Since the amplification signal can be observed in real time during the amplification process, gel electrophoresis of the amplified product is not required, which can shorten the nucleic acid detection cycle, reduce costs and improve efficiency. If the primers, probes, amplification system and conditions are well designed, the method can have the characteristics of high throughput, high sensitivity, rapid specificity, etc. In addition to the limitations of this method, since the method is more sensitive, it puts forward higher requirements on the design of primers and probes and the number of control strains and sample strains used for method verification, otherwise it is more likely to have false positives or false negatives; in addition, compared with the ordinary PCR method, the detection cost is significantly increased in the instrument for amplifying nucleic acids (fluorescent quantitative PCR instrument) and the synthesis of probe reagents. If a commercial fluorescent quantitative PCR detection kit is used, the cost may be very high.

(3) Protein spectrum method: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) technology is a new type of soft ionization organic mass spectrometry developed in recent years. By introducing matrix molecules, the molecules to be tested do not produce fragments, solving the problem of desorption and ionization of non-volatile and thermally unstable biomacromolecules. In the field of microbial identification, by measuring the unique protein composition of bacteria themselves, mass spectrometry technology is used to arrange the measured proteins and peptides according to molecular weight to form a unique proteome fingerprint, and rapid detection and identification of microorganisms is achieved through characteristic pattern peaks. The specific operation is as follows: for the suspected Salmonella strains that have been isolated, they are inoculated on a non-selective culture medium for a certain period of time. According to the strain classification prediction and growth situation, the direct smear method or protein extraction method is selected to treat the strain, and then an appropriate amount of colonies or treated samples are picked to a specific target plate. The sample on the target plate is then treated with a matrix. After the solvent evaporates, a co-crystal of the sample and the matrix is formed. An imported or domestic instrument is used to emit a laser, and the matrix absorbs energy from the laser to desorb the sample. Charge transfer occurs between the matrix and the sample to ionize the sample molecules. After passing through a time-of-flight detector, the sample molecules are detected according to the different flight times reaching the detector, that is, the mass-to-charge ratio (M/Z) of the ion is measured, which is proportional to the flight time of the ion to detect the molecular weight of the ion. Through analysis and comparison with dedicated software, a specific fingerprint spectrum is determined, and the strain is identified as a species through a supporting database and identification software system, such as the MALDI-TOF MS system produced by manufacturers such as France's BioMérieux, Germany's Bruker, and China's Antu Biological Company. This technology is not based on the physiological and biochemical indicators and genes of microorganisms, but on the comparison of proteomic expression spectra of various microorganisms. Since each microorganism has its own unique protein composition that is different from other species, it has a unique protein fingerprint, which is determined by the genetic characteristics of the species and is less affected by external environmental conditions. Therefore, compared with other commonly used microbial testing technologies, proteomic fingerprints are more accurate and direct, and have the characteristics of sensitivity, accuracy and speed, suitable for high-throughput rapid detection. This method does not require high reagents. If there are ready-made instruments, the price of reagents will also be relatively cheap, but the experimental results will be affected by the strain culture status, sample processing, operator proficiency, etc.; secondly, this method has a high dependence and requirements on the size of the strain database in the software. For microorganisms that do not obtain standard spectra in the database, qualified identification results cannot be obtained or the corresponding classification or typing level cannot be identified; in addition, this method cannot obtain a good fingerprint for some strains, which may result in the inability to obtain a good identification result.

(4) Whole genome sequencing method: Extract genomic DNA from the strain to be tested, use the second or third generation whole genome sequencing (WGS) technology, perform bioinformatics analysis and comparison on the sequencing data, and search in relevant databases to identify the strain. In recent years, with the improvement of genome sequencers and the rapid development of sequencing technology, this method has developed rapidly. The accuracy of sequencing and the improvement of analysis and processing have made this technology more and more widely used, but there are also some problems: the price of sequencing is still not cheap enough for the whole people, especially the price of third generation sequencing is still relatively high; the sequencing library construction step requires the use of kits to extract high-quality genomes in the early stage; the analysis of sequencing results requires certain bioinformatics analysis capabilities and mastery of relevant genome processing software; in addition, the time to obtain sequencing results is still not fast enough, ranging from one or two weeks to one month. The above problems limit the application of whole genome sequencing in the field of rapid identification of microorganisms or high-throughput screening.

Therefore, it is necessary to develop a new rapid detection method for Salmonella to overcome the problems and defects of the existing Salmonella detection methods and realize the rapid identification and high-throughput screening of Salmonella, especially foodborne Salmonella.

Summary of the invention

In view of the above existing problems, the present invention provides a primer probe set, a kit and a method for rapid detection of foodborne Salmonella, the main purpose of which is to overcome the various problems existing in the existing rapid detection methods of foodborne Salmonella, including time-consuming detection, expensive reagents, database size requirements, high experience and operation requirements, inability to perform high-throughput rapid screening, insufficient specificity and accuracy, etc. We designed and optimized the specific species identification genes that determine Salmonella, and used standard strains and sample isolates for repeated large-scale verification, and established a high-throughput, rapid screening foodborne Salmonella fluorescent quantitative PCR method for rapid and accurate detection and identification of foodborne Salmonella.

In a first aspect, the present invention provides a primer-probe set for rapid detection of foodborne Salmonella, comprising an upstream primer F1, a downstream primer R1 and a probe P1 for specific detection of the Salmonella gene stn, and an upstream primer F2, a downstream primer R2 and a probe P2 for specific detection of the Salmonella gene invA:

The upstream primer F1 is: 5'-CGC CAT GCT GTT CGA TGA TA-3';

The downstream primer R1 is: 5'-GGA TCA GYT GGA GGC GAT TT-3';

The probe P1 is: 5'-FAM-TGT CCC GTC AGC TTT GGT CGT AAA-3'-BHQ1;

The upstream primer F2 is: 5'-AGC GTA CTG GAA AGG GAA AG-3';

The downstream primer R2 is: 5'-CAC CGA AAT ACC GCC AAT AAA G-3';

The probe P2 is: 5'-ROX-TTA CGG TTC CTT TGA CGG TGC GAT-3'-BHQ2.

In a second aspect, the present invention provides a fluorescent quantitative detection kit for rapid detection of foodborne Salmonella, which contains the primer probe set as described above.

In one embodiment, the PCR reaction system for fluorescence quantitative detection is 25 μL, including 12.5 μL of reaction premix (Beijing Tiangen, SuperReal PreMix Probe, FP206), 0.8 μL of upstream and downstream primers, 0.5 μL of probe, 3.3 μL of ddH ₂ O, and 5.0 μL of template DNA. The reaction system can be expanded or reduced as needed.

In a third aspect, the present invention provides a method for detecting foodborne Salmonella in vitro, comprising the following steps:

1) Extracting genomic DNA from the sample to be tested;

2) using the sample DNA extracted in step 1) and the kit described in claim 2 or 3 to form a PCR reaction system for fluorescence quantitative detection, and detecting whether stn and invA are contained in the sample to be tested;

3) Fluorescence quantitative PCR amplification.

In one embodiment, the conditions of the fluorescent quantitative PCR are: pre-denaturation at 95°C for 15 min; denaturation at 95°C for 15 s, annealing at 60°C for 20 s, extension at 72°C for 30 s, 35 cycles, and fluorescence collection at 72°C.

In one embodiment, if the Ct values of stn and invA detection in the sample to be tested are both less than or equal to 30, it is determined that the sample to be tested contains Salmonella.

In a fourth aspect, the present invention provides use of the primer probe set described above in the preparation of a product for detecting or diagnosing Salmonella.

The beneficial effects of the present invention are:

Compared with several existing foodborne Salmonella rapid detection methods, this application has been improved and perfected in the following aspects:

1. Compared with some existing molecular biological methods, this method selects Salmonella-specific genes as target genes and redesigns and optimizes primers and probes, which has higher accuracy in method specificity and reduces false positives and false negatives in existing methods;

2. Compared with some existing methods, this method uses more standard strains of Salmonella of different serotypes, standard strains of non-Salmonella of different species, and isolates from a large number of food samples for method establishment and verification. The verification strains have a good time and geographical span. After verification and improvement, the method has better accuracy and representativeness for the detection of foodborne Salmonella in my country.

3. Compared with the throughput, detection time, operation process, reagent price and result comparability of some existing methods, this method has higher detection throughput (based on the fluorescence quantitative PCR instrument, two specific genes of 96 samples can be detected simultaneously), shorter detection time (DNA extraction time is 0.5 hours, detection time is 1.5 hours, and the results can be observed after amplification without further analysis and waiting, which is much shorter than traditional biochemical methods of 2 to 3 days, protein spectrometry 1 to 2 days, WGS 1 to 3 weeks, etc.), better operation simplicity (the instruments, reagents, consumables and processes required for ordinary fluorescence quantitative PCR detection are sufficient, and no more detection or analysis technology is involved, and the experience requirements or special experimental skills or analysis requirements for operators are relatively low), lower reagent prices (compared with traditional biochemical methods, WGS and other instrument equipment or detection reagents, etc.), and other indicators are more advantageous.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the fluorescence amplification curves of two genes in the positive sample and the negative control.

DETAILED DESCRIPTION

The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example 1: Primer and probe synthesis

Specific primers and probes were designed and synthesized for the Salmonella-specific species detection genes stn and invA, which were subsequently used to perform real-time fluorescence quantitative PCR detection on whether the test strains contained the target genes, and the upstream primer F1, downstream primer R1 and probe P1 for the specific detection of stn, as well as the upstream primer F2, downstream primer R2 and probe P2 for the specific detection of invA were obtained.

Upstream primer F1: 5′-CGC CAT GCT GTT CGA TGA TA-3′;

Downstream primer R1: 5′-GGA TCA GYT GGA GGC GAT TT-3′;

Probe P1: 5′-FAM-TGT CCC GTC AGC TTT GGT CGT AAA-3′-BHQ1;

Upstream primer F2: 5′-AGC GTA CTG GAA AGG GAA AG-3′;

Downstream primer R2: 5′-CAC CGAAAT ACC GCC AAT AAA G-3′;

Probe P2: 5′-ROX-TTA CGG TTC CTT TGA CGG TGC GAT-3′-BHQ2.

Example 2: Salmonella DNA extraction method

(1) 90 standard strains of Salmonella (including the main serotypes in my country), 120 standard strains of non-Salmonella of different species, and more than 5,900 foodborne Salmonella isolates obtained from different food sample sources by the my country Food Safety Risk Monitoring Network from 2018 to 2021;

(2) The strain to be tested was streaked onto a brain heart infusion agar (BHA) plate and cultured in a 37°C incubator overnight;

(3) Use a sterile cotton swab to scrape an appropriate amount of bacterial moss from the BHA plate into a 1.5 ml EP tube containing 1 ml of sterile water, grind the colonies into sterile water to make a bacterial suspension with a turbidity of about 1 McFarland turbidity;

(4) Place the EP tube in a boiling water bath or a 100°C metal bath for 10 min, then quickly place it in an ice water bath or a refrigerator freezer for 5 min. Centrifuge it at 12,000 rpm for 10 min and take 200 μl of the supernatant, which is the DNA to be tested.

Example 3: Fluorescence quantitative PCR detection method

(1) Preparation of reaction system: For each sample, prepare 25 μL of reaction system, including 12.5 μL of reaction premix (Beijing Tiangen, SuperReal PreMix Probe, FP206), 0.8 μL of upstream and downstream primers, 0.5 μL of probe, 3.3 μL of ddH ₂ O, and 5.0 μL of template DNA. Alternatively, the premix except the DNA template can be uniformly prepared according to the number of samples and dispensed into a 96-well PCR plate, and then the DNA to be detected can be added respectively;

(2) Amplification: Use a plate shaker to remove the liquid from the tube wall of a 96-well PCR plate to the bottom of the tube. Set the amplification conditions on a fluorescent quantitative PCR instrument as follows: pre-denaturation at 95°C for 15 min; denaturation at 95°C for 15 s, annealing at 60°C for 20 s, extension at 72°C for 30 s, 35 cycles, and collect fluorescence at 72°C;

(3) Result interpretation: The reaction result file is exported from the instrument, and the Ct value of the two target genes in each well is obtained using the associated software (as shown in Figure 1), and the result is interpreted according to the following criteria: positive (Ct≤30), negative (Ct＞30). Based on the two reaction results, samples with positive reactions are selected and judged as Salmonella.

90 standard strains of Salmonella (including the main serotypes in my country) (Table 1), 120 standard strains of non-Salmonella of different species (Table 2), and more than 5,900 foodborne Salmonella isolates isolated from different food sample sources (involving Salmonella isolates isolated from various types of food in 31 provinces, municipalities and autonomous regions in my country in the past three years) were tested respectively. The results showed that the detection accuracy of the Salmonella detection method provided by the present invention can be as high as 100%, without false positives or false negatives, and each qPCR machine can detect 96 samples at the same time. The results can be observed after the PCR is completed, thereby realizing rapid high-throughput detection.

Table 1 Salmonella standard strains used in the present invention and detection conditions

Note: WHO EQAS: External quality assurance system assessment of the World Health Organization Global Foodborne Infection Network (GFN); EU EQA: External quality assurance system assessment; ATCC: American Type Culture Collection; DSM: German Microbiological Culture Collection; CMCC: China Medical Bacteria Culture Collection Management Center; CICC: China Industrial Microbiology Culture Collection Management Center; IQCC: Food Safety Microbiology Culture Collection Management Center of China National Institute for Food and Drug Control; NIFDC-QC: Quality Control Assessment of China Food and Drug Inspection Institutes; Monitoring Network: National Food Safety Risk Monitoring Network.

Table 2 Non-Salmonella standard strains used in the present invention and detection results

Note: WHO EQAS: External quality assurance system assessment of the World Health Organization Global Foodborne Infection Network (GFN); EU EQA: External quality assurance system assessment; ATCC: American Type Culture Collection; DSM: German Microbiological Culture Collection; CMCC: China Medical Bacteria Culture Collection Management Center; CICC: China Industrial Microbiology Culture Collection Management Center; IQCC: Food Safety Microbiology Culture Collection Management Center of China National Institute for Food and Drug Control; NIFDC-QC: Quality Control Assessment of China Food and Drug Inspection Institutes; Monitoring Network: National Food Safety Risk Monitoring Network.

The above results show that the present invention selects Salmonella-specific genes stn and invA as target genes for the first time and redesigns and optimizes primers and probes, which has higher accuracy, higher detection throughput, shorter detection time, better operation simplicity, and lower reagent price in the detection method, and has significant advantages over traditional Salmonella detection methods.

Although the present invention has been described with reference to the preferred embodiments, various modifications may be made thereto and components thereof may be replaced with equivalents without departing from the scope of the present invention. The above contents are further detailed descriptions of the present invention in conjunction with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or replacements may be made without departing from the concept of the present invention, which should all be deemed to fall within the scope of protection of the present invention.

Claims (7)

1. A primer-probe set for rapid detection of foodborne Salmonella, characterized in that it comprises an upstream primer F1, a downstream primer R1 and a probe P1 for specific detection of the Salmonella gene stn, and an upstream primer F2, a downstream primer R2 and a probe P2 for specific detection of the Salmonella gene invA: The upstream primer F1 is: 5'-CGC CAT GCT GTT CGA TGA TA-3'; The downstream primer R1 is: 5'-GGA TCA GYT GGAGGC GAT TT-3'; The probe P1 is: 5'-FAM-TGT CCC GTC AGC TTT GGT CGT AAA-3'-BHQ1; The upstream primer F2 is: 5'-AGC GTA CTG GAA AGG GAA AG-3'; The downstream primer R2 is: 5'-CAC CGAAAT ACC GCC AAT AAA G-3'; The probe P2 is: 5'-ROX-TTA CGG TTC CTT TGA CGG TGC GAT-3'-BHQ2'. 2. A fluorescent quantitative detection kit for rapid detection of foodborne Salmonella, characterized in that it contains the primer probe set according to claim 1. 3. The fluorescence quantitative detection kit according to claim 2, characterized in that the PCR reaction system for fluorescence quantitative detection is 25 μL, including 12.5 μL of reaction premix, 0.8 μL of upstream and downstream primers, 0.5 μL of probe, _{3.3 μL} of ddH2O, and 5.0 μL of template DNA. The reaction system can be expanded or reduced as needed. 4. A method for detecting foodborne Salmonella in vitro, comprising the following steps: 1) Extracting genomic DNA from the sample to be tested; 2) using the sample DNA extracted in step 1) and the kit described in claim 2 or 3 to form a fluorescent quantitative PCR detection system, and detecting whether the sample to be tested contains stn and invA genes respectively; 3) Fluorescence quantitative PCR amplification. 5. The method according to claim 4 is characterized in that the conditions for the fluorescence quantitative PCR amplification are: pre-denaturation at 95°C for 15 min; denaturation at 95°C for 15 s, annealing at 60°C for 20 s, extension at 72°C for 30 s, 35 cycles, and fluorescence collection at 72°C. 6. The method according to claim 4, characterized in that if the Ct values of stn and invA detection in the sample to be tested are both less than or equal to 30, it is determined that the sample to be tested contains Salmonella. 7. Use of the primer probe set according to claim 1 in preparing a product for detecting or diagnosing Salmonella.

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