Primer probe for identification of shigella-like bacterium and shigella based on lamp technology and its application

Abstract

The application discloses a primer probe for identifying the parapathogenic shigella and shigella based on the LAMP technology and application thereof, and belongs to the technical field of molecular diagnosis.The LAMP primer probe provided by the application has high specificity and sensitivity, can simultaneously perform five LAMP reactions in one tube, does not interfere with each other, achieves the purpose of accurate detection, meets the requirements of rapid and convenient detection of primary detection departments, and is crucial for rapidly distinguishing infectious pathogens, blocking disease transmission, implementing public health monitoring and early warning of epidemic situation.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostic technology and relates to a primer probe based on LAMP technology for identifying Shigella-like bacteria and Shigella, and its application. Background Technology

[0002] Shigella is a Gram-negative, short, non-motile, non-spore-forming bacillus belonging to the Enterobacteriaceae family. It primarily parasitizes the intestines of humans and primates and is the main pathogen causing bacterial dysentery in humans. Based on antigenic structure, it is divided into four groups: Group A (Shigella dysenteriae) is the most virulent, causing typical dysentery and easily leading to toxic shock; Group B (Shigella flexneri) is the predominant circulating strain (accounting for 60%-70%), easily progressing to chronic infection, and has a complex antigenic structure; Group C (Shigella boydii) is less common, with a prolonged course after infection; Group D (Shigella sonnei) is the only group that slowly ferments lactose, with milder symptoms but a high likelihood of becoming a carrier. Typical symptoms after infection include high fever (39-40℃), bloody and purulent stools, tenesmus, and severe cases can lead to toxic encephalopathy or shock (more common in children), with a mortality rate of approximately 1%-5%.

[0003] *Shigella spp.* is the only species in the genus *Shigella* of the family Vibrioceae, and is a Gram-negative, short rod-shaped bacterium. This bacterium causes intestinal infection by secreting cholera-like toxins and adhesins, leading to watery diarrhea (3-10 times daily), abdominal pain, and fever (up to 39°C). In severe cases, it can cause mucoid bloody stools or sepsis. *Shigella spp.* is very similar to *Shigella* in morphology, staining, transmission routes, and the symptoms it causes, and even their antigenic structures are quite similar, making cross-reactivity in serological tests highly likely and leading to misdiagnosis. This poses risks to epidemic prevention and control and clinical medication. Therefore, developing a technology for rapid identification of *Shigella spp.* and *Shigella* is of significant practical importance for reducing clinical misdiagnosis, standardizing treatment protocols, and implementing public health monitoring and early warning of epidemics.

[0004] Loop-mediated isothermal amplification (LAMP) is a novel isothermal nucleic acid amplification method that has been developed in recent years. Compared with PCR, LAMP 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 PCR 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 LAMP technology a perfect match for the needs of grassroots testing departments for speed and convenience.

[0005] Patent document CN102827928A discloses a rapid diagnostic method for *Shigella spp.* based on LAMP technology. However, this method only detects *Shigella spp.* and does not simultaneously identify *Shigella*. Furthermore, the results are presented using turbidity and colorimetry, which introduces many human interference factors, leading to poor method stability. Similarly, patent document US20240309466A1 discloses a method for detecting enterotoxigenic *Escherichia coli* and *Shigella* using LAMP technology. This technology still cannot differentiate between *Shigella spp.* and *Shigella*. Secondly, based on currently disclosed LAMP-based methods, it can be observed that because LAMP detection methods mainly include electrophoresis, turbidity detection, or colorimetric detection, each reaction system must be amplified separately, making it impossible to perform multiplex nucleic acid amplification reactions simultaneously in a single tube. This deficiency significantly reduces the detection efficiency of grassroots testing departments.

[0006] Based on this, the present invention provides a method for rapid detection of Shigella-like bacteria and Shigella based on LAMP technology. The method can not only simultaneously identify Shigella-like bacteria and four types of Shigella, including Shigella flexneri, Shigella boydii, Shigella dysenteriae, and Shigella sonnei, but also achieve a fivefold LAMP amplification reaction in one tube. This method well meets the needs of grassroots testing departments for speed and convenience, and is crucial for rapidly distinguishing infectious pathogens, blocking disease transmission, and implementing public health monitoring and early warning of epidemics. Summary of the Invention

[0007] The purpose of this invention is to provide a set of primers and probes based on LAMP technology that can specifically identify *Shigella spp.* and four other *Shigella* species. These primers and probes can simultaneously perform a five-fold LAMP reaction in a single tube without interference, achieving accurate detection. Furthermore, this invention provides a kit prepared based on the aforementioned LAMP primers and probes. For ease of use, the active ingredients in the kit are stored in lyophilized beads. This kit offers advantages such as low cost, convenient operation, short detection time, and accurate and visualized result interpretation.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] In a first aspect, the present invention provides a set of LAMP primers and probes, characterized in that the primers and probes are composed of the primers and probes shown in i)-v):

[0010] i) LAMP primers and probes for detecting Shigella-like bacteria, wherein the nucleotide sequences of the primers are shown in SEQ ID NO:1-SEQ ID NO:4 and the nucleotide sequence of the probe is shown in SEQ ID NO:5;

[0011] ii) LAMP primers and probes for detecting Shigella flexneri, 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;

[0012] iii) LAMP primers and probes for detecting Shigella boydii, 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;

[0013] iv) LAMP primers and probes for detecting Shigella dysenteriae, 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;

[0014] v) LAMP primers and probes for detecting Shigella sonnei, 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.

[0015] Furthermore, the probe is labeled with a fluorescent reporter group at its 5' end and a fluorescent quencher group at its 3' end.

[0016] The fluorescent reporter group is selected from FAM, ROX, Cy5, TET, JOE, Cy3, Cy5.5, Quasar705, HEX or FITC.

[0017] The fluorescence quenching group is selected from BHQ1, BHQ2, BHQ3, TAMRA, or MGB.

[0018] In this invention, the 5' ends of the probes in the same reaction system are labeled with different fluorescent reporter groups, thus ensuring that the fluorescence signals of the same reaction system do not interfere with each other. "Signal non-interference" means that the fluorescent groups used by the probes are different and do not affect each other's detection; that is, different channels can be used for detection. For example, this invention can realize a fivefold LAMP reaction in the same system, where five probes can be labeled with FAM, HEX, ROX, Cy5, and Quasar705, respectively. These groups have different absorbance values, allowing selection of different channels without signal interference between the signals.

[0019] Secondly, the present invention provides the application of the LAMP primer probe described in the first aspect of the present invention in the preparation of a product for identifying Shigella-like enomonas and four Shigella species, wherein the four Shigella species are Shigella flexneri, Shigella boydii, Shigella dysenteriae, and Shigella sonnei.

[0020] The product forms for detecting Shigella spp. and four Shigella species include, but are not limited to, reagents, kits, test strips, membrane strips, chips, or detection platforms.

[0021] Thirdly, the present invention provides a kit, characterized in that the kit includes the LAMP primers and probes described in the first aspect of the present invention.

[0022] The kit is a rapid detection product based on LAMP technology for detecting or identifying Shigella morphogenetica, Shigella flexneri, Shigella boydii, Shigella dysenteriae, and Shigella sonnei. It can quickly and accurately detect whether the sample contains Shigella morphogenetica or the above four Shigella species. It is easy to operate, has a short detection time, and the detection results can be visualized through fluorescence signals.

[0023] Furthermore, the kit also includes standard reagents required for LAMP technology, such as Bst polymerase, RNase HII enzyme, reverse transcriptase, dNTPs, ultrapure water, buffer solution, and magnesium ions.

[0024] In some embodiments of the present invention, the kit further includes sample lysis buffer, sample diluent, and sample nucleic acid extraction reagent / kit, which can be purchased commercially or prepared by the user.

[0025] In some embodiments of the present invention, the LAMP primers and probes in the kit are packaged separately from the conventional reagents required for LAMP technology (Bst polymerase, RNase HII enzyme, reverse transcriptase, dNTPs, ultrapure water, buffer, magnesium ions).

[0026] In a preferred embodiment of the present invention, the LAMP primers and probes in the kit, along with conventional reagents required for LAMP technology (Bst polymerase, RNase HII enzyme, reverse transcriptase, dNTPs, ultrapure water, buffer solution, magnesium ions), are stored in a vacuum-packed aluminum foil bag in the form of lyophilized beads. Specifically, the lyophilized beads are prepared by the following method:

[0027] S1) Prepare a buffer solution containing Bst polymerase, RNase HII enzyme, reverse transcriptase and dNTPs, add LAMP primers and probes to form the LAMP amplification reaction solution;

[0028] S2) The LAMP amplification reaction solution is dropped into liquid nitrogen to form a sphere;

[0029] S3) Place the product in a freeze-drying device and freeze-dry overnight to form freeze-dried beads.

[0030] In the LAMP amplification reaction solution described in step S1), the final concentration of the primers shown in SEQ ID NO: 1-2, 6-7, 11-12, 16-17, and 21-22 is 0.1-0.2 μM, the final concentration of the primers shown in SEQ ID NO: 3-4, 8-9, 13-14, 18-19, and 23-24 is 0.3-1.0 μM, and the final concentration of the probes shown in SEQ ID NO: 5, 10, 15, 20, and 25 is 0.1-0.2 μM.

[0031] Furthermore, the kit also includes a positive control, which is a plasmid containing gene fragments of *Shigella spp.*, *Shigella flexneri*, *Shigella boydii*, *Shigella dysenteriae*, and *Shigella sonnei*.

[0032] Furthermore, the kit also includes a negative control, which is pure water treated with diethyl pyrocarbonate (DEPC).

[0033] Fourthly, the present invention provides a method for identifying *Shigella*-like bacteria and four Shigella species based on LAMP technology, not for the purpose of disease treatment or diagnosis, characterized in that the method includes the following steps:

[0034] 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.

[0035] S2: Add LAMP amplification reaction solution to the sample DNA and amplify at 65℃ for 20-25 min;

[0036] S3: Judge the results based on the amplification curve using a multicolor fluorescence isothermal amplification instrument or a fluorescence PCR instrument.

[0037] The sample to be tested in step S1 is selected from all samples that may contain Shigella-like bacteria and four types of Shigella, such as blood, feces, saliva, sputum, oropharyngeal swabs, and nasopharyngeal swabs.

[0038] The LAMP amplification reaction solution described in step S2 is a mixture of buffer solution containing Bst polymerase, RNase HII enzyme, reverse transcriptase, dNTPs, and LAMP primers and probes. In some specific embodiments of the present invention, the LAMP amplification reaction solution may also be lyophilized beads prepared from the LAMP amplification reaction solution.

[0039] The result determination method described in step S3 is as follows:

[0040] (1) If no S-type amplification curve is observed in any of the fluorescence channels, the sample is considered negative.

[0041] (2) If an S-shaped curve appears in the fluorescence channel corresponding to Shigella or the four Shigella species, the corresponding pathogen is considered positive, meaning that the corresponding pathogen is present in the sample.

[0042] The technical solution provided by this invention has the following advantages:

[0043] This invention provides a kit for detecting *Shigella* spp. and four Shigella species, offering advantages such as high specificity, high sensitivity, short detection time, simple result interpretation, convenient operation, and low cost. Compared to commonly used detection methods, the isothermal amplification technology employed in this invention can be performed under isothermal conditions. Only a simple isothermal device is needed to amplify the target gene sequence. Furthermore, by introducing an RNase HII restriction enzyme system and continuously screening and optimizing the target gene sequence and LAMP amplification primers, a five-fold LAMP reaction detection method is ultimately achieved in the same system. This method is highly suitable for rapid and convenient detection by grassroots food safety testing departments and is of great significance for grassroots pathogen screening, epidemiological tracing, and the implementation of public health monitoring and early warning of epidemics. Attached Figure Description

[0044] Figure 1 Table 1 shows the amplification curves of Shiga-like nematodes samples using LAMP primers and probes.

[0045] Figure 2 Table 2 shows the amplification curves of Shiga-like nematodes samples using LAMP primers and probes.

[0046] Figure 3 Table 2 shows the amplification curves of Shigella flexneri samples using LAMP primers and probes.

[0047] Figure 4 Table 3 shows the amplification curves of Shigella flexneri samples using LAMP primers and probes.

[0048] Figure 5 Table 3 shows the Shigella boulardii samples using LAMP primers and probes.

[0049] Figure 6 Table 3 shows the Shigella dysenteriae samples using LAMP primers and probes.

[0050] Figure 7 Table 3 shows the Shigella sonnei samples with LAMP primers and probes.

[0051] Figure 8 Sensitivity detection curve for Shigella flexneri.

[0052] Figure 9 The specificity detection curves for the LAMP primers and probes shown in Table 3 are presented. Detailed Implementation

[0053] 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.

[0054] Example 1: Design and preparation of LAMP primers and probes

[0055] LAMP reaction is a novel nucleic acid amplification technology based on strand substitution autocyclic reaction. This technology designs four specific primers (including upstream and downstream outer primers F3 and B3 and upstream and downstream inner primers FIP and BIP) for six regions of the target gene sequence. Using DNA polymerase with strand substitution activity, the nucleic acid amplification reaction can be completed under isothermal conditions of 60-65℃.

[0056] The feasibility, specificity, and sensitivity of LAMP detection methods hinge on the design of LAMP primers and probes. Key factors to consider and balance in LAMP primer design include primer distance, Tm value of primer regions, end stability of primer regions, GC content, and secondary structure. This invention screens LAMP primers based on the following strategy: 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 pairs; and selecting 2-3 sets of LAMP primers with theoretically best parameters and highest success rates for experimental verification, thus identifying the optimal LAMP primers. Building upon this, to achieve simultaneous amplification of multiple LAMP sequences in the same system, this invention introduces a RNase HII restriction enzyme digestion system and designs fluorescence to quantify the detection results more accurately.

[0057] Currently, primer design largely relies on software. However, software-designed primers may not be able to efficiently and specifically amplify target genes, especially when multiple (specifically five) LAMP amplification systems are placed in the same tube, as shown in this invention. Avoiding mutual interference between the amplification systems is a core technical problem that technicians urgently need to solve. The following are some examples of LAMP primer and probe screening in this invention:

[0058] The table below shows the LAMP primers and probes obtained by manual modification and screening based on the software design of this invention:

[0059] Table 1. Sequence information of LAMP primers and probes

[0060]

[0061] Following the method provided in Example 3, sample A (barcode 25061263) containing *Shigella* was taken, and LAMP amplification reaction solution containing the LAMP primers and probes shown in Table 1 was added. LAMP amplification was then performed in a reaction tube, and the results are as follows: Figure 1 The expected FAM channel did not amplify; instead, the HEX channel for detecting Shigella flexneri did. This result indicates that the primers for Shigella flexneri have poor specificity and interfere with the LAMP primers or probes for detecting Shigella-like bacteria. Based on this, the inventors adjusted the LAMP primers and probes for both Shigella-like bacteria and Shigella flexneri, obtaining the optimized LAMP primers and probes shown in Table 2.

[0062] Table 2 Sequence information of LAMP primers and probes

[0063]

[0064] As above, following the method provided in Example 3, sample A (barcode 25061263) containing *Shigella* was taken, and LAMP amplification reaction solution containing the LAMP primers and probes shown in Table 2 was added. LAMP amplification was then performed in the reaction tube, and the results are as follows: Figure 2 The FAM channel showed a clear S-shaped amplification curve, while the other channels showed no amplification, indicating that the primers and probes shown in Table 2 can accurately detect *Shigella flexneri*. Next, using the same detection method, sample B (barcode 25062233) of *Shigella flexneri* was detected with the LAMP primers and probes described in Table 2, and the results are as follows... Figure 3 As shown, other channels showed no amplification, but the expected HEX channel only showed a small amplification, indicating that the primers for Shigella flexneri were interfered with by other primers. Based on this, the inventors adjusted the primers and probes for Shigella flexneri to obtain the LAMP primers and probes shown in Table 3.

[0065] Table 3 Sequence information of LAMP primers and probes

[0066]

[0067] In Tables 1-3, the " / " is merely a separator and does not mean "or"; the "r" in "rG", "rC", "rA" and "rU" represents ribonucleic acid, used to distinguish it from deoxyribonucleotides "G", "C", "A" and "T".

[0068] Sample B (barcode 25062233) containing the LAMP primers and probes described in Table 3 was further tested for Shigella flexneri, and the results are as follows: Figure 4As shown, the HEX channel exhibited a distinct S-shaped curve, while other channels showed no amplification. Using the same method, samples of *Shigella boydii* C (barcode 25101207), *Shigella dysenteriae* D (barcode 25070305), and *Shigella sonnei* E (barcode 25063359) were detected using LAMP primers and probes as described in Table 3. The amplification curves are shown below. Figure 5-7 As shown, the ROX, Cy5, and Quasar705 channels showed S-shaped curves, while other channels showed no amplification. This indicates that the LAMP primers and probes provided in Table 3 can accurately identify Shigella spp., Shigella flexneri, Shigella boydii, Shigella dysenteriae, and Shigella sonnei.

[0069] The above optimization process illustrates that even if the theoretical parameters of the LAMP amplification primers designed by the software are of high quality and can achieve target gene-specific amplification, mutual interference can still occur in the five-fold LAMP amplification system described in this invention. Therefore, it is essential to perform manual optimization based on the actual detection problems that occur on the basis of the software design results. This optimization and screening is precisely the creative work of the technicians.

[0070] Example 2: Preparation of a kit based on LAMP primer and probe composition

[0071] The kit includes the following components:

[0072] (1) Freeze-dried beads;

[0073] (2) Sample lysis buffer, sample diluent, negative control, and positive control;

[0074] (3) Packaging boxes that separate and group these reagent bottles or tubes.

[0075] The freeze-dried beads were prepared by the following method:

[0076] 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:1-25 in Table 3, 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, 16-17, 21-22 is 0.15 μM, the final concentration of the primers shown in SEQ ID NO:3-4, 8-9, 13-14, 18-19, 23-24 is 0.75 μM, and the final concentration of the probes shown in SEQ ID NO:5, 10, 15, 20, 25 is 0.1 μM.

[0077] S2) The LAMP amplification reaction solution is dropped into liquid nitrogen to form spheres, each drop being 25 μl;

[0078] S3) Place the product in a freeze-drying device and freeze-dry overnight to form freeze-dried beads.

[0079] Example 3: A method for identifying Shigella-like bacteria and Shigella based on LAMP technology

[0080] The testing procedure is as follows:

[0081] S1: Collect suspicious fecal samples, put the samples into a plastic tube containing sample lysis buffer, break the swab, close the tube cap, and shake vigorously for 20 seconds.

[0082] S2: Take out one drop and add it to the plastic tube containing the diluent. Shake for 20 seconds to mix.

[0083] S3: Take one drop from the diluent or pipette 25 μl and add it to the reaction tube to mix with the lyophilized beads (prepared in Preparation Example 2) in the tube. Add 25 μl of the negative and positive control samples directly.

[0084] S4: 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).

[0085] S5: Program settings: 65℃, 30-second FAM fluorescence reading, 40 cycles, total duration 20 minutes;

[0086] S6: After amplification, carefully remove the reaction tube, wrap it with gloves, tie a knot and discard it to avoid aerosol contamination;

[0087] S8: Result determination: 1) If there is no S-shaped amplification curve in all fluorescence channels, the sample is determined to be negative; 2) If a certain fluorescence channel shows an obvious S-shaped amplification curve and other fluorescence channels show no amplification, the sample in the corresponding channel is determined to be positive.

[0088] Example 4 Sensitivity Detection

[0089] A certain amount of purified plasmids containing gene fragments from *Shigella*-like bacteria and four Shigella species were taken, and their OD values ​​were measured 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.

[0090] 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, HEX, ROX, Cy5, and Quasar 705 fluorescence readings are performed separately) for 40 cycles on a Hongshi SLAN-96S real-time PCR instrument.

[0091] Five genes totaling 1×10 from Shigella-like phylloxera and four Shigella species. 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 copies / ml and negative control were negative. Therefore, the detection sensitivity for *Shigella spp.* and four *Shigella* genes can all reach 1×10⁻⁶. 2 Copy / ml. The sensitivity test results for Shigella flexneri genes are as follows: Figure 8 As shown, the gene sensitivity results of the other four bacteria are similar to those of... Figure 2 Similarities are not provided here to save space.

[0092] Example 5 Specificity Detection

[0093] Because *Escherichia coli*, *Klebsiella pneumoniae*, and the target strain *Shigella* of this invention share high homology, false positive results may occur. Therefore, this experiment used sample F (barcode 25062255) containing *Escherichia coli* and sample G (barcode 25062227) containing *Klebsiella pneumoniae* for specificity verification. LAMP amplification was performed in a reaction tube containing lyophilized beads (containing the LAMP primers and probes described in Table 3) according to the method provided in Example 3, and the amplification curve was detected. The amplification curve is shown below. Figure 9 As shown, all gene amplification curves were negative, indicating that the specificity met the requirements.

[0094] Based on the above verifications, this invention successfully obtained a set of LAMP primers and probes that can identify Shigella-like bacteria and four Shigella species. These primers and probes can not only achieve fivefold LAMP reactions in the same reaction tube, but also have high sensitivity and good specificity, meeting the needs for rapid, convenient, real-time, and in-situ detection.

[0095] 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 set of LAMP primers and probes, characterized in that, The primer probe consists of the primer probes shown in i)-v): i) LAMP primers and probes for detecting Shigella-like bacteria, wherein the nucleotide sequences of the primers are shown in SEQ ID NO:1-SEQ ID NO:4 and the nucleotide sequence of the probe is shown in SEQ ID NO:5; ii) LAMP primers and probes for detecting Shigella flexneri, 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 Shigella boydii, 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 Shigella dysenteriae, 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 Shigella sonnei, 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.

2. The LAMP primer probe according to claim 1, characterized in that, The probe has a fluorescent reporter group labeled at its 5' end, and the fluorescent reporter group labeled at the 5' end is different for each probe. The probe also has a fluorescent quencher group labeled at its 3' end. The fluorescent reporter group is selected from FAM, ROX, Cy5, TET, JOE, Cy3, Cy5.5, Quasar705, HEX or FITC. The fluorescent quencher group is selected from BHQ1, BHQ2, BHQ3, TAMRA or MGB.

3. The use of the LAMP primer probe according to claim 1 or 2 in the preparation of a product for identifying Shigella-like bacteria and four Shigella species, wherein the four Shigella species are Shigella flexneri, Shigella boydii, Shigella dysenteriae, and Shigella sonnei.

4. The application according to claim 3, characterized in that, The products used for detecting Shigella spp. and four Shigella species include reagents, kits, test strips, membrane strips, chips, or detection platforms.

5. A reagent kit, characterized in that, The kit includes the LAMP primers and probes as described in claim 1 or 2.

6. The reagent kit according to claim 5, characterized in that, The kit also includes Bst polymerase, RNaseHII enzyme, reverse transcriptase, dNTPs, ultrapure water, buffer solution, and magnesium ions.

7. The reagent kit according to claim 6, characterized in that, The kit also includes sample lysis buffer, sample diluent, and sample nucleic acid extraction reagent / kit.

8. The reagent kit according to claim 6, characterized in that, The LAMP primers and probes in the kit, along with Bst polymerase, RNase HII enzyme, reverse transcriptase, dNTPs, ultrapure water, buffer solution, and magnesium ions, are stored in vacuum-packed aluminum foil bags as lyophilized beads. These lyophilized beads are prepared using the following method: S1) Prepare a buffer solution containing Bst polymerase, RNase HII enzyme, reverse transcriptase and dNTPs, add LAMP primers and probes to form the 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.

9. The reagent kit according to claim 5, characterized in that, The kit also includes a positive control and a negative control.

10. A method for identifying *Shigella*-like bacteria and four Shigella species 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 LAMP amplification reaction solution to the sample DNA 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 LAMP amplification reaction solution described in step S2 is a mixture of a buffer containing Bst polymerase, RNase HII enzyme, reverse transcriptase, dNTPs, and the LAMP primers and probes described in claim 1 or 2.

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