Primer-probe composition for LAMP-based detection of Staphylococcus aureus and enterotoxins and its application

Abstract

This invention discloses a primer-probe composition for detecting Staphylococcus aureus and enterotoxins based on LAMP technology and its application, belonging to the field of molecular diagnostic technology. The primer-probe composition provided by this invention can specifically recognize Staphylococcus aureus and simultaneously identify the enterotoxin genes entA, entB, entC, entD, and entE of Staphylococcus aureus. Based on this, a highly sensitive and specific detection kit can be formed, which can simultaneously perform multiple LAMP reactions in a single tube. It has the advantages of low cost, convenient operation, short detection time, and simple result interpretation, meeting the needs of grassroots testing departments for speed and convenience, and facilitating real-time on-site testing within the production line for enterprises.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostic technology, specifically relating to a primer-probe composition for detecting Staphylococcus aureus and enterotoxins based on LAMP technology and its application. Background Technology

[0002] Staphylococcus aureus is a common foodborne pathogen widely distributed in the natural environment. It is the most common pathogen causing purulent infections in humans, leading to localized purulent infections, pneumonia, pseudomembranous colitis, pericarditis, and even systemic infections such as sepsis and septicemia. Furthermore, under suitable conditions, Staphylococcus aureus can produce enterotoxins, causing food poisoning and posing a significant public health burden. Therefore, the prevention and detection of Staphylococcus aureus and its enterotoxins are crucial.

[0003] Currently, the main methods for detecting and typing Staphylococcus aureus and its enterotoxins include immunological methods based on viral antigen-antibody reactions, such as virus isolation and nucleic acid detection techniques using viral antigens and genomic DNA. With the development of nucleic acid molecular detection technology, PCR technology can amplify target sequences millions of times within hours, allowing direct sample detection. However, PCR amplification requires specialized equipment and is not suitable for widespread application in real-time detection by grassroots testing departments. Furthermore, those skilled in the art know that Staphylococcus aureus is an opportunistic pathogen. In public health events, if no enterotoxin gene is detected, even the isolation of Staphylococcus aureus fluid cannot definitively determine that the event was caused by Staphylococcus aureus. Therefore, the identification of Staphylococcus aureus enterotoxin genes is extremely important in determining the pathogenicity of Staphylococcus aureus events. To ensure food safety, it is necessary to develop a rapid, simple, and accurate method for detecting Staphylococcus aureus and its enterotoxins in food.

[0004] Loop-mediated isothermal amplification (LAMP) is a novel isothermal nucleic acid amplification method that has been developed in recent years. Compared with the well-known PCR technology, it has the following advantages: (1) It is fast, and the entire process can be completed in 15-20 minutes. The amplification fold is comparable to that of PCR amplification in 90 minutes, which is particularly advantageous in time-sensitive scenarios such as emergency, epidemic prevention and control, and outdoor testing; (2) It has lower requirements for template purity than the PCR system and is compatible with fast and convenient lysis methods; (3) It has better specificity and lower commercialization costs; (4) The reagents can be lyophilized and are easy to store. These characteristics make loop-mediated isothermal amplification technology very suitable for the needs of grassroots testing departments for speed and convenience.

[0005] The existing technology CN111088377 A discloses a rapid isothermal detection method for Staphylococcus aureus. However, this method only involves the detection of Staphylococcus aureus and does not identify the various enterotoxin genes of Staphylococcus aureus. Moreover, the detection results are presented by immunochromatography or visual colorimetry, which are subject to many human interference factors, resulting in poor method stability. Secondly, since the detection methods of loop-mediated isothermal amplification technology mainly include electrophoresis detection, turbidity detection, or colorimetric detection, each reaction system must exist separately, making it impossible to perform multiple nucleic acid amplification reactions simultaneously in one tube, which greatly reduces the detection efficiency of grassroots testing departments.

[0006] Based on this, the present invention provides a method for detecting Staphylococcus aureus and simultaneously identifying the genotype of Staphylococcus aureus enterotoxin based on LAMP technology. The detection method introduces the RNase HII enzyme digestion probe system into the reaction system, so that the LAMP reaction is like fluorescent PCR, realizing multiple nucleic acid amplification reaction in one tube at the same time, which meets the needs of grassroots testing departments for speed and convenience, and makes it convenient for enterprises to carry out real-time on-site testing within the production line. Summary of the Invention

[0007] The purpose of this invention is to provide a set of primer and probe combinations based on LAMP technology for the specific identification of Staphylococcus aureus and the simultaneous identification of the enterotoxin entA, entB, entC, entD and entE genes of Staphylococcus aureus. Based on this, a highly sensitive and specific detection kit is formed, which can perform multiple LAMP reactions simultaneously in one tube. It has the advantages of low cost, convenient operation, short detection time and simple result interpretation.

[0008] The success of LAMP detection methods, and whether their specificity and sensitivity meet requirements, hinges on primer design. In LAMP primer design, key factors that need to be considered and balanced include primer spacing, Tm values ​​of primer regions, terminal stability of primer regions, GC content, and secondary structures. Those skilled in the art know that although primer design software exists, software-designed primers may not necessarily amplify the target gene efficiently and specifically. In particular, when multiple LAMP amplification systems are run in the same tube, avoiding mutual interference between amplification systems and specifically amplifying the target gene sequence are the core technical issues of greatest concern to technicians.

[0009] Typically, the target gene sequence to be amplified is selected by technicians based on the latest microbial genome data to ensure both universality (the target sequence is common to Staphylococcus aureus) and specificity (the target sequence is specific to Staphylococcus aureus, avoiding misidentification of non-Staphylococcus aureus as Staphylococcus aureus). Therefore, the screening and confirmation of the target gene sequence is a crucial step in achieving LAMP detection. After determining the target gene sequence, the design and optimization of primers and probes to achieve multiplex LAMP reactions in the same system present another technical challenge. This invention overcomes the above-mentioned technical challenges and successfully obtains a set of LAMP primer-probe compositions capable of detecting Staphylococcus aureus and simultaneously identifying enterotoxin genes. These primer-probe compositions not only enable triplex LAMP reactions in the same reaction tube but also exhibit high sensitivity and specificity, meeting the needs for rapid, convenient, real-time, and in-situ detection.

[0010] This invention includes the following technical solutions:

[0011] In a first aspect, the present invention provides a set of LAMP primer-probe compositions, characterized in that the primer-probe compositions consist of primers and probes as shown in i)-vi) :

[0012] i) LAMP primers and probes for detecting Staphylococcus aureus, wherein the nucleotide sequences of the primers are shown in SEQ ID NO:1-SEQ ID NO:4 and the nucleotide sequences of the probes are shown in SEQ ID NO:5;

[0013] ii) LAMP primers and probes for detecting Staphylococcus aureus enterotoxin entA, wherein the nucleotide sequences of the primers are shown in SEQ ID NO:6-SEQ ID NO:9 and the nucleotide sequences of the probes are shown in SEQ ID NO:10;

[0014] iii) LAMP primers and probes for detecting Staphylococcus aureus enterotoxin entB, wherein the nucleotide sequences of the primers are shown in SEQ ID NO:11-SEQ ID NO:14 and the nucleotide sequences of the probes are shown in SEQ ID NO:15;

[0015] iv) LAMP primers and probes for detecting Staphylococcus aureus enterotoxin entC, wherein the nucleotide sequences of the primers are shown in SEQ ID NO:16-SEQ ID NO:19 and the nucleotide sequences of the probes are shown in SEQ ID NO:20;

[0016] v) LAMP primers and probes for detecting Staphylococcus aureus enterotoxin entD, wherein the nucleotide sequences of the primers are shown in SEQ ID NO:21-SEQ ID NO:24 and the nucleotide sequences of the probes are shown in SEQ ID NO:25;

[0017] vi) LAMP primers and probes for detecting Staphylococcus aureus enterotoxin entE, wherein the nucleotide sequences of the primers are shown in SEQ ID NO:26-SEQ ID NO:29 and the nucleotide sequences of the probes are shown in SEQ ID NO:30.

[0018] Furthermore, the probe is labeled with a fluorescent reporter group at its 5' end and a fluorescent quencher group at its 3' end.

[0019] The fluorescent reporter group is selected from FAM, ROX, Cy5, TET, JOE, Cy3, Cy5.5, HEX or FITC.

[0020] The fluorescence quenching group is selected from BHQ-1, BHQ-2, BHQ-3, TAMRA, or MGB-NFQ.

[0021] In this invention, probes in the same reaction system have different fluorescent reporter groups labeled at their 5' ends, achieving non-interference of fluorescence signals within the same reaction system. "Non-interference of signals" means that the fluorescent groups used by the probes are different and do not affect each other's detection, i.e., different channels can be used for detection. For example, this invention can achieve a triple LAMP reaction in the same system, where three probes can be labeled with FAM, ROX, and Cy5 respectively. These groups have significantly different excitation and emission wavelength ranges, and the signals from different channels will not interfere with each other. In a specific embodiment of this invention, the primers and probes shown in i)-iii) are in the same reaction system, and the 5' ends of the probes described in SEQ ID NO:5, SEQ ID NO:10, and SEQ ID NO:15 are labeled with FAM, ROX, and Cy5 respectively; the primers and probes shown in iv)-vi) are in the same reaction system, and the 5' ends of the probes described in SEQ ID NO:20, SEQ ID NO:25, and SEQ ID NO:30 are labeled with FAM, ROX, and Cy5 respectively.

[0022] In a second aspect, the present invention provides the use of the LAMP primer-probe composition described in the first aspect of the present invention in the preparation of products for detecting and / or assisting in the detection of Staphylococcus aureus and enterotoxins.

[0023] The Staphylococcus aureus enterotoxin is a combination of enterotoxin entA, enterotoxin entB, enterotoxin entC, enterotoxin entD, and enterotoxin entE.

[0024] The product forms used for detecting Staphylococcus aureus and enterotoxins include, but are not limited to, reagents, kits, test strips, membrane strips, chips, or detection platforms.

[0025] Thirdly, the present invention provides a kit, characterized in that the kit comprises the LAMP primer-probe composition described in the first aspect of the present invention.

[0026] The kit is a rapid detection product based on loop-mediated isothermal amplification (LAMP) technology for detecting Staphylococcus aureus and enterotoxins entA, entB, entC, entD, and entE. It can quickly and accurately identify whether Staphylococcus aureus is present in the sample and identify the enterotoxin typing of Staphylococcus aureus. Therefore, the kit also includes other conventional reagents required for LAMP technology.

[0027] In some embodiments of the present invention, the other conventional reagents include Bst polymerase, RNase HII enzyme, reverse transcriptase, dNTPs, ultrapure water, buffer solution, and magnesium ions.

[0028] In some embodiments of the present invention, the kit further includes sample lysis buffer, sample diluent, and sample DNA extraction reagent / kit, which can be purchased commercially or prepared by the user.

[0029] In one specific embodiment of the present invention, the sample lysis buffer contains a surfactant, which is composed of one or more of NP-40, Triton X-100, PEG 6000, Tween-20, Tween-80, and sodium dodecyl sulfate (SDS). The mass concentration of each surfactant component in the sample lysis buffer is between 0.2% and 4.3%.

[0030] In some embodiments of the present invention, the LAMP primer-probe composition in the kit is packaged separately from other conventional reagents (Bst polymerase, RNase HII enzyme, reverse transcriptase, dNTPs, ultrapure water, buffer, magnesium ions).

[0031] In a preferred embodiment of the present invention, the LAMP primer-probe composition in the kit, along with other conventional reagents (Bst polymerase, RNase HII enzyme, reverse transcriptase, dNTPs, ultrapure water, buffer solution, magnesium ions), is stored in a vacuum-packed aluminum foil bag in the form of lyophilized beads. Specifically, the lyophilized beads are prepared by the following method:

[0032] S1) Prepare a buffer solution containing Bst polymerase, RNase HII enzyme, reverse transcriptase and dNTPs, add LAMP primer and probe composition to form LAMP amplification reaction solution;

[0033] S2) The LAMP amplification reaction solution is dropped into liquid nitrogen to form a sphere;

[0034] S3) Place the product in a freeze-drying device and freeze-dry overnight to form freeze-dried beads.

[0035] In the LAMP amplification reaction solution described in step S1), the final concentration of the primers or probes shown in SEQ ID NO: 1-2, 5-7, 10-12, 15-17, 20-22, 25-27, and 30 is 0.1-0.2 μM, and the final concentration of the primers shown in SEQ ID NO: 3-4, 8-9, 13-14, 18-19, 23-24, and 28-29 is 0.3-1.0 μM.

[0036] In a specific embodiment of the present invention, the lyophilized beads of the kit are divided into two groups, namely group A and group B. Group A lyophilized beads contain the LAMP primer-probe composition shown in SEQ ID NO:1-15, which is used to detect Staphylococcus aureus and its enterotoxins entA and entB; Group B lyophilized beads contain the LAMP primer-probe composition shown in SEQ ID NO:16-30, which is used to detect Staphylococcus aureus enterotoxins entC, entD, and entE.

[0037] Furthermore, the kit also includes a positive control, which is a plasmid containing Staphylococcus aureus and enterotoxins entA, entB, entC, entD and entE genes.

[0038] Furthermore, the kit also includes a negative control, which is pure water treated with diethyl pyrocarbonate (DEPC).

[0039] Fourthly, the present invention provides a method for detecting Staphylococcus aureus and its enterotoxins based on LAMP technology, not for the purpose of disease treatment or diagnosis, characterized in that the method includes the following steps:

[0040] S1: Extract genomic DNA or RNA from the sample to be tested, reverse transcribe the RNA into cDNA, and combine the DNA and cDNA as the sample DNA.

[0041] S2: Add the sample DNA to the LAMP amplification reaction solution and amplify at 65℃ for 20-25 min;

[0042] S3: Judge the results based on the amplification curve using a multicolor fluorescence isothermal amplification instrument or a fluorescence PCR instrument.

[0043] The sample to be tested in step S1) is selected from all samples that may contain Staphylococcus aureus that need to be tested.

[0044] The LAMP amplification reaction solution described in step S2 is a mixture of buffer containing Bst polymerase, RNase HII enzyme, reverse transcriptase, dNTPs, and a LAMP primer-probe composition. Further, the LAMP amplification reaction solution also includes lyophilized beads prepared from the LAMP amplification reaction solution.

[0045] The result determination method described in step S3) is as follows:

[0046] (1) If no S-type amplification curve is observed in any of the fluorescence channels, the sample is considered negative.

[0047] (2) If the fluorescence channel for detecting Staphylococcus aureus shows an obvious S-shaped amplification curve, and other fluorescence channels do not show an S-shaped amplification curve, then the sample is judged to be positive, but there is no enterotoxin.

[0048] (3) If the fluorescence channel for detecting Staphylococcus aureus shows an obvious S-shaped amplification curve, and one or more of the other fluorescence channels also show an obvious S-shaped amplification curve, then the sample is judged to be positive and the corresponding enterotoxin is present.

[0049] This invention provides a kit for detecting Staphylococcus aureus and its enterotoxins, which has advantages such as high specificity, high sensitivity, short detection time, simple result interpretation, convenient operation, and low cost. Compared with commonly used detection methods, the isothermal amplification technology used in this invention can be carried out under isothermal conditions. Only a simple isothermal device is needed to amplify the target gene sequence. Furthermore, by introducing an RNase HII enzyme digestion system and continuously screening and optimizing the target gene sequence and LAMP amplification primers, a triple LAMP reaction detection method is finally achieved in the same system. This method is highly suitable for rapid and convenient detection by grassroots food safety testing departments, and can be fully applied even in environments with relatively insufficient molecular biology expertise and skills, making it of great significance for food safety testing. Attached Figure Description

[0050] Figure 1 Amplification curve of a sample that is positive for Staphylococcus aureus and has an enterotoxin type of entB;

[0051] Figure 2 Staphylococcus aureus gene sensitivity detection diagram;

[0052] Figure 3 Amplification curve of a sample that is positive for Staphylococcus aureus and has an enterotoxin type of entA;

[0053] Figure 4 Amplification curve of a sample that is positive for Staphylococcus aureus and has an enterotoxin type of entB;

[0054] Figure 5 Amplification curve of a sample that is positive for Staphylococcus aureus and has an enterotoxin type of entC;

[0055] Figure 6 Amplification curve of a sample that is positive for Staphylococcus aureus and has an enterotoxin type of entD;

[0056] Figure 7 Amplification curve of a sample that is positive for Staphylococcus aureus and has an enterotoxin type of entE;

[0057] Figure 8 Table 2 shows the amplification curves of the primer-probe combination for the sample;

[0058] Figure 9 Table 3 shows the amplification curves of the primer-probe combination for the sample. Detailed Implementation

[0059] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0060] Example 1: Design and preparation of LAMP primer-probe composition

[0061] Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technique based on strand substitution autocyclic reactions. This technique uses four specific primers designed for six regions of the target gene sequence (including upstream and downstream outer primers F3 and B3, and upstream and downstream inner primers FIP and BIP, where FIP consists of F1C and F2, and BIP consists of B1C and B2). Using DNA polymerase with strand substitution activity, the nucleic acid amplification reaction can be completed under isothermal conditions of 60-65℃.

[0062] LAMP primer design is crucial for accurate detection. The LAMP primer combinations provided in this invention are determined through the following methods: screening target gene sequences; performing BLAST comparisons on the similarity of the target gene sequences; designing multiple primer pairs for six regions of the target gene using online software (F3c, F2c, and F1c regions at the 3' end of the target gene, and B1, B2, and B3 regions at the 5' end of the target gene); manually modifying these primers; and selecting 2-3 sets of LAMP primers with the theoretically best parameters and highest success rate for experimental verification, ultimately selecting the LAMP primers with the best experimental results. Based on the above, to achieve simultaneous amplification of multiplex LAMP in the same system, this invention introduces a RNase HII restriction enzyme digestion system and designs fluorescence to make the detection results more accurately visualized.

[0063] After screening and optimization, the LAMP primer-probe combination for detecting Staphylococcus aureus and its enterotoxins is shown in the table below:

[0064] Table 1. Sequence information of LAMP primer combinations

[0065] .

[0066] The " / " in the table above is just a separator and does not mean "or"; the "r" in "rG", "rC", and "rA" represents ribonucleic acid and is used to distinguish it from deoxyribonucleotides "G", "C", and "A".

[0067] Example 2: Preparation of a kit based on LAMP primer and probe composition

[0068] The kit includes the following components:

[0069] (1) Freeze-dried beads;

[0070] (2) Sample lysis buffer, sample diluent, negative control, and positive control;

[0071] (3) Packaging boxes that separate and group these reagent bottles or tubes.

[0072] The freeze-dried beads were prepared by the following method:

[0073] S1) Prepare a buffer solution containing Bst polymerase, RNase HII enzyme, reverse transcriptase and dNTPs, add the LAMP primers and probes shown in SEQ ID NO:1-15 of Table 1, and mix to form a LAMP amplification reaction solution; in the LAMP amplification reaction solution, the final concentration of the primers shown in SEQ ID NO:1-2, 6-7, 11-12 is 0.1-0.2 μM, the final concentration of the primers shown in SEQ ID NO:3-4, 8-9, 13-14 is 0.3-1.0 μM, and the final concentration of the probes shown in SEQ ID NO:5, 10, 15 is 0.1-0.2 μM.

[0074] S2) The LAMP amplification reaction solution is dropped into liquid nitrogen to form spheres, each drop being 25 μl;

[0075] S3) Place it in a freeze-drying device and freeze-dry overnight to form freeze-dried beads A.

[0076] The LAMP amplification reaction solution was prepared using the same method. The final concentration of the primers shown in SEQ ID NO:16-17, 21-22, and 26-27 was 0.1-0.2 μM, the final concentration of the primers shown in SEQ ID NO:18-19, 23-24, and 28-29 was 0.3-1.0 μM, and the final concentration of the probes shown in SEQ ID NO:20, 25, and 30 was 0.1-0.2 μM. The solutions were then lyophilized to form lyophilized beads B.

[0077] Example 3: Method for detecting Staphylococcus aureus based on LAMP technology

[0078] The testing procedure is as follows:

[0079] S1: In our routine foodborne disease surveillance work, we collected suspected fecal samples and used PCR to screen for samples that were positive for Staphylococcus aureus and had an enterotoxin type of entB (barcode number 23240806000499).

[0080] S2: Place the sample into a plastic tube containing the sample lysis buffer, break the swab, close the tube cap, and shake vigorously for 20 seconds.

[0081] S3: Take out one drop and add it to the plastic tube containing the diluent. Shake for 20 seconds to mix.

[0082] S4: Take one drop from the diluent or use a pipette to draw 25 μl and add it to reaction tube A (containing lyophilized bead A) and reaction tube B (containing lyophilized bead B), respectively, and mix it with the lyophilized beads (prepared in Example 2) in the tubes. Add 25 μl of the negative and positive quality control samples directly.

[0083] S5: Place the reaction tube on a shaker and shake for 10 seconds. After a short centrifugation, place it in a dedicated fluorescence device for amplification (fluorescence isothermal amplification instrument or fluorescence PCR instrument).

[0084] S6: Program settings: 65℃, 30-second FAM fluorescence reading, 40 cycles, total duration 20 minutes;

[0085] S7: After amplification, carefully remove the reaction tube, wrap it with gloves, tie a knot and discard it to avoid aerosol contamination;

[0086] S8: Result Interpretation: Group A lyophilized beads showed obvious S-shaped amplification curves in the FAM and Cy5 channels, such as... Figure 1 As shown, the test sample was positive for Staphylococcus aureus, and the enterotoxin type was entB, which is consistent with the PCR test results.

[0087] Example 4 Sensitivity Detection

[0088] A certain amount of plasmid containing gene fragments of Staphylococcus aureus and five enterotoxins (entA, entB, entC, entD, and entE) was dissolved in an appropriate amount of ultrapure water. The OD value was measured, and the result was expressed using the formula OD... 260 The concentration was calculated at ×50µg / ml, and the number of copies per milliliter was calculated based on the molar mass of the plasmid. Then, it was sequentially diluted with ultrapure water to a concentration of 1×10⁻⁵µg / ml. 3 copies / ml, 3×10 2 Copy / ml, 1×10 2 copies / ml, 3×10 1 Copy / ml.

[0089] Add 25µl of plasmid template of various gradients to the reaction tubes, with three replicates for each gradient. The reaction tubes, along with the negative control, are incubated at 65℃ for 30 seconds (FAM, ROX, and Cy5 fluorescence are read separately) for 40 cycles on a Hongshi SLAN-96S real-time PCR instrument.

[0090] Staphylococcus aureus and six genes (entA, entB, entC, entD, and entE) of five enterotoxins, 1×10⁻⁶. 3 copies / ml, 3×10 2 Copy / ml, 1×10 2 All replicates at these three concentrations (copy / ml) were positive, 3 × 10⁻⁶. 1 The results for both copies / ml and the negative control were negative. Therefore, the detection sensitivity for Staphylococcus aureus and the five enterotoxin genes can reach 1×10⁻⁶. 2 Copy / ml. The sensitivity test results for the Staphylococcus aureus gene are as follows: Figure 2As shown, the results for the other 5 genes are the same as those for the other 5 genes. Figure 2 Similarities are not provided here to save space.

[0091] Example 5 Specificity Detection

[0092] Samples 1 (barcode 21240812015199) were positive for Staphylococcus aureus and had an enterotoxin type of entA; sample 2 (barcode 22240719006299) was positive for Staphylococcus aureus and had an enterotoxin type of entB; sample 3 (barcode 22240805012199) was positive for Staphylococcus aureus and had an enterotoxin type of entC; sample 4 (barcode 22240805000299) was positive for Staphylococcus aureus and had an enterotoxin type of entD; and sample 5 (barcode 21240812015799) was positive for Staphylococcus aureus and had an enterotoxin type of entE. These samples were added to reaction tubes containing lyophilized beads according to the method provided in Example 3 for LAMP amplification, and the amplification curves were detected.

[0093] The amplification curve of sample 1 is as follows: Figure 3 As shown, the amplification curve of sample 2 is as follows: Figure 4 The amplification curve of sample 3 is as follows: Figure 5 The amplification curve of sample 4 is as follows: Figure 6 The amplification curve of sample 5 is as follows: Figure 7 As can be seen from the above results, the LAMP primer and probe composition provided by this invention can amplify Staphylococcus aureus and the corresponding enterotoxin genes, showing positive curves. For other non-corresponding enterotoxin genes, the curves show negative results, indicating that the specificity meets the requirements.

[0094] Example 6: Screening process of LAMP primers and probes

[0095] The LAMP primers for detecting Staphylococcus aureus and its enterotoxins provided by this invention are prepared by the following method: First, the target gene sequence is determined; then, LAMP primers are designed using primer design software. Multiple primer pairs can be obtained for each target gene. After theoretical parameter screening and manual modification, we finally select 2-3 sets of LAMP primers (for each target gene) for experimental verification. Since the purpose of this invention is to overcome the multiplex LAMP reaction in the same tube, preventing mutual interference between LAMP primers targeting different target genes is the most difficult problem encountered in the experiment. Therefore, we need to perform base substitutions and trials on the sequence of each set of primers, repeatedly testing the amplification effect of different primers to obtain a multiplex amplification system without mutual interference.

[0096] Since this invention relates to a maximum of triple amplification systems, we exemplarily provide a set of amplification interference scenarios that occur in reaction tube A. The LAMP primer-probe combinations for Staphylococcus aureus, enterotoxin entA, and entB are shown in the table below:

[0097] Table 2 Sequence information of LAMP primer combinations

[0098]

[0099] Following the method provided in Example 3, sample 2 (barcode 22240719006299) that was Staphylococcus aureus positive and had an enterotoxin type of entB was taken. DNA was extracted from the sample and added to a reaction tube containing the primers and probes shown in Table 2 for LAMP amplification. The results are as follows: Figure 8 As shown, the entA gene was amplified, which was far from the expected result, indicating that there was a specificity problem in the primer and probe design in Table 2.

[0100] Based on Table 2, the inventors made multiple adjustments to the primers for Staphylococcus aureus, enterotoxin entA, and enterotoxin entB, resulting in the primer probes shown in Table 3.

[0101] Table 3 Sequence information of LAMP primer combinations

[0102] .

[0103] Following the method provided in Example 3, sample 2 (barcode 22240719006299) that was Staphylococcus aureus positive and had an enterotoxin type of entB was taken. DNA was extracted from the sample and added to a reaction tube containing the primers and probes shown in Table 3 for LAMP amplification. The results are as follows: Figure 9 As shown, no gene exhibited an amplification curve. After analysis, we believe the primers have been optimally adjusted. Figure 9 The result was most likely due to the probe. The inventors further optimized the probe, obtaining the LAMP primer-probe combination shown in Table 1, which ultimately enabled the correct detection of Staphylococcus aureus-positive samples with enterotoxin type entB.

[0104] The above optimization process illustrates that even with theoretically optimal parameters, primers designed using software for LAMP amplification, capable of achieving target gene-specific amplification, can still experience interference in multiplex LAMP amplification systems. Therefore, manual optimization of primers and probes based on software design results is essential, representing the creative effort of technical personnel. Only through this process can specific amplification of multiple target genes be achieved within the same system.

[0105] The above specific embodiments are merely illustrative of the invention and do not represent a limitation thereof. Those skilled in the art will recognize that other variations of the specific structure of this invention are possible.

Claims

1. A LAMP primer-probe composition, characterized in that, The primer-probe composition consists of the primers and probes shown in i)-vi): i) LAMP primers and probes for detecting Staphylococcus aureus, wherein the nucleotide sequences of the primers are shown in SEQ ID NO:1-SEQ ID NO:4 and the nucleotide sequences of the probes are shown in SEQ ID NO:5; ii) LAMP primers and probes for detecting Staphylococcus aureus enterotoxin entA, wherein the nucleotide sequences of the primers are shown in SEQ ID NO:6-SEQ ID NO:9 and the nucleotide sequences of the probes are shown in SEQ ID NO:10; iii) LAMP primers and probes for detecting Staphylococcus aureus enterotoxin entB, wherein the nucleotide sequences of the primers are shown in SEQ ID NO:11-SEQ ID NO:14 and the nucleotide sequences of the probes are shown in SEQ ID NO:15; iv) LAMP primers and probes for detecting Staphylococcus aureus enterotoxin entC, wherein the nucleotide sequences of the primers are shown in SEQ ID NO:16-SEQ ID NO:19 and the nucleotide sequences of the probes are shown in SEQ ID NO:20; v) LAMP primers and probes for detecting Staphylococcus aureus enterotoxin entD, wherein the nucleotide sequences of the primers are shown in SEQ ID NO:21-SEQ ID NO:24 and the nucleotide sequences of the probes are shown in SEQ ID NO:25; vi) LAMP primers and probes for detecting Staphylococcus aureus enterotoxin entE, wherein the nucleotide sequences of the primers are shown in SEQ ID NO:26-SEQ ID NO:29 and the nucleotide sequences of the probes are shown in SEQ ID NO:30; The probe is labeled with a fluorescent reporter group at its 5' end and a fluorescent quencher group at its 3' end; The primers and probes shown in i)-iii) are in the same reaction system, and the 5' ends of the probes described in SEQ ID NO:5, SEQ ID NO:10 and SEQ ID NO:15 are labeled with FAM, ROX and Cy5, respectively; the primers and probes shown in iv)-vi) are in the same reaction system, and the 5' ends of the probes described in SEQ ID NO:20, SEQ ID NO:25 and SEQ ID NO:30 are labeled with FAM, ROX and Cy5, respectively.

2. The use of the LAMP primer-probe composition of claim 1 in the preparation of products for detecting and / or assisting in the detection of Staphylococcus aureus and enterotoxins, wherein the Staphylococcus aureus enterotoxin is a combination of enterotoxin entA, enterotoxin entB, enterotoxin entC, enterotoxin entD and enterotoxin entE.

3. The application according to claim 2, characterized in that, The products used for detecting Staphylococcus aureus and enterotoxins include reagents, kits, test strips, membrane strips, chips, or detection platforms.

4. A reagent kit, characterized in that, The kit includes the LAMP primer and probe composition of claim 1.

5. The reagent kit according to claim 4, characterized in that, The kit also includes other standard reagents required for LAMP technology, including Bst polymerase, RNase HII enzyme, reverse transcriptase, dNTPs, ultrapure water, buffer, and magnesium ions.

6. The reagent kit according to claim 5, characterized in that, The LAMP primer-probe composition in the kit is packaged separately from other conventional reagents, or stored as lyophilized beads in a vacuum-sealed aluminum foil bag; the lyophilized beads are prepared by the following method: S1) Prepare a buffer solution containing Bst polymerase, RNase HII enzyme, reverse transcriptase and dNTPs, and add the LAMP primer and probe composition according to claim 1 to form a LAMP amplification reaction solution; S2) The LAMP amplification reaction solution is dropped into liquid nitrogen to form a sphere; S3) Place the product in a freeze-drying device and freeze-dry overnight to form freeze-dried beads.

7. The reagent kit according to claim 6, characterized in that, The lyophilized beads in the kit are divided into two groups, namely group A and group B. Group A lyophilized beads contain the LAMP primer-probe composition shown in SEQ ID NO:1-15, which is used to detect Staphylococcus aureus and its enterotoxins entA and entB. Group B lyophilized beads contain the LAMP primer-probe composition shown in SEQ ID NO:16-30, which is used to detect Staphylococcus aureus enterotoxins entC, entD, and entE.

8. A method for detecting Staphylococcus aureus and its enterotoxins based on LAMP technology, not for the purpose of disease treatment or diagnosis, characterized in that, The method includes the following steps: S1: Extract genomic DNA or RNA from the sample to be tested, reverse transcribe the RNA into cDNA, and combine the DNA and cDNA as the sample DNA. S2: Add the sample DNA to the LAMP amplification reaction solution and amplify at 65℃ for 20-25 min; S3: Judge the results based on the amplification curve using a multicolor fluorescence isothermal amplification instrument or a fluorescence PCR instrument; The sample to be tested in step S1) is selected from all samples that may contain Staphylococcus aureus that need to be tested; The LAMP amplification reaction solution described in step S2 is a mixture of a buffer containing Bst polymerase, RNase HII, reverse transcriptase and dNTPs and the LAMP primer and probe composition described in claim 1.

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