An RPA-CRISPR / Cas12a paracasei zhang detection reagent kit, a detection method and application

Abstract

This invention discloses an RPA-CRISPR / Cas12a *Lactobacillus paracasei* Zhang detection kit, detection method, and application, belonging to the field of rapid microbial detection technology. The *Lactobacillus paracasei* Zhang detection method based on RPA-CRISPR / Cas12a can be applied to the rapid detection of *Lactobacillus paracasei* Zhang in real samples. This method combines engineered crRNA with RPA-specific amplification and crRNA-specific sequence recognition, giving the detection method excellent specificity and sensitivity. The entire fluorescence detection platform is operated at 37℃, and the results can be observed visually using either fluorescence or blue light excitation, depending on available conditions, making it suitable for on-site use.

Description

Technical Field

[0001] This invention relates to the field of rapid microbial detection technology, and in particular to an RPA-CRISPR / Cas12a Lactobacillus paracasei Zhang detection kit, detection method, and application. Background Technology

[0002] Probiotics, as a class of live microorganisms with significant probiotic functions, can colonize the host's gut when ingested in sufficient quantities, exerting health benefits such as anti-oxidation, anti-inflammation, regulation of gut microbiota balance, and assistance in improving metabolic diseases. They have extremely high application value in the fields of medicine and food processing. With in-depth research, probiotics have expanded from their initial medical research field to a wide range of applications in fermented dairy products, meat products, baked goods, and other scenarios, becoming one of the core categories with the greatest market potential in the probiotic industry. However, the continuous enrichment of product types and the ongoing expansion of application scenarios have sparked controversies regarding the authenticity of product labels, posing a serious challenge to industry quality supervision. Taking probiotics widely used in the food industry (such as *Lactobacillus paracasei*) as an example, these strains are often added as core fermentation strains to products such as active lactic acid bacteria beverages, fermented milk, and cheese. However, commercially available probiotic products commonly have discrepancies between the actual strains and those stated on the label, posing a risk of substituting closely related species or incorrectly labeling strains. This seriously affects the accuracy and credibility of product labeling and also restricts the standardized development of the probiotic industry. Lactobacillus paracasei Zhang is a probiotic isolated from fermented mare's milk. This strain exhibits excellent gastrointestinal tolerance and intestinal colonization ability. It can exert multiple probiotic effects, such as immune regulation, intestinal barrier protection, and organ protection, by reshaping the intestinal flora structure and regulating metabolic homeostasis. Its genomic characteristics and functional properties are significantly different from other Lactobacillus paracasei strains. Therefore, establishing a precise detection technology for Lactobacillus paracasei Zhang at the probiotic strain level is of key significance for verifying the authenticity of product ingredients, strengthening quality control, and protecting consumer rights.

[0003] Currently, routine detection methods for probiotics mainly rely on traditional culture methods and 16S rRNA gene sequencing. Traditional culture methods depend on colony morphology and biochemical characteristics for identification, which are time-consuming (usually 48-72 hours) and have low sensitivity (LOD > 10). 2Problems such as insufficient resolution of CFU / mL and closely related strains within the same genus make it difficult to distinguish between different probiotic strains within the same genus. Although 16S rRNA gene sequencing technology is widely used for bacterial identification, the 16S rRNA gene sequences of most probiotic strains within the same genus are highly homologous, enabling identification only at the genus level or some species level, failing to achieve precise differentiation at the strain level, and thus failing to meet the demand for precise detection at the probiotic strain level. In recent years, molecular diagnostic technologies based on the CRISPR-Cas system have attracted much attention due to their high specificity and signal amplification capabilities. Taking the Cas12a protein as an example, after activation through crRNA-mediated target recognition, it can exhibit non-specific single-stranded DNA (ssDNA) trans-cleavage activity. When combined with recombinase polymerase isothermal amplification (RPA) technology, it can achieve rapid detection of nucleic acids at room temperature and can achieve precise differentiation based on strain-specific target sequences. The combined use of RPA amplification and CRISPR technology can effectively improve the specificity and sensitivity of detection, eliminating the need for precise temperature control equipment and simplifying the detection process. However, such technologies are mostly focused on the quantitative detection of foodborne pathogens or can only achieve species-level detection of probiotics. Bottlenecks remain in the areas of quality control of compound probiotic products and the precise differentiation of different functional probiotic strains—that is, rapid detection at the strain level. Furthermore, the complex environment in complex food matrices can easily interfere with the detection signal, leading to inaccurate detection or decreased sensitivity, further limiting the practical application of existing technologies. Therefore, developing a probiotic strain-level detection technology that is adaptable to complex food matrices and combines high sensitivity with ease of operation is of great significance for the quality control of probiotic products, the screening of functional strains, and the standardization of detection. Summary of the Invention

[0004] The purpose of this invention is to provide an RPA-CRISPR / Cas12a Lactobacillus paracasei Zhang detection kit, detection method, and application to solve the problems existing in the prior art. The Lactobacillus paracasei Zhang detection method designed based on RPA-CRISPR / Cas12a can be applied to the rapid detection of Lactobacillus paracasei Zhang in actual samples.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides an RPA-CRISPR / Cas12a Lactobacillus paracasei Zhang detection kit, the kit comprising an RPA reaction system and a CRISPR / Cas12a system; The RPA reaction system includes an upstream primer and a downstream primer. The CRISPR / Cas12a system contains crRNA, Cas12a protein, and ssDNA; The sequence of the upstream primer for RPA is shown in SEQ ID NO.1, and the sequence of the downstream primer for RPA is shown in SEQ ID NO.2; The sequence of the crRNA is shown in any one of SEQ ID NO.3-SEQ ID NO.5.

[0006] Optionally, the sequence of the crRNA is shown in SEQ ID NO.4.

[0007] The present invention also provides the application of the aforementioned detection kit in the detection of Lactobacillus paracasei Zhang.

[0008] This invention also provides a method for detecting Lactobacillus paracasei Zhang using RPA-CRISPR / Cas12a, comprising the following steps: RPA amplification of DNA samples was performed using upstream and downstream RPA primers; simultaneously, a CRISPR / Cas12a system was prepared to react concurrently with RPA amplification. The RPA amplification product was mixed with the CRISPR / Cas12a system and the reaction continued. After the reaction was completed, the fluorescence signal was collected. The presence or absence of the fluorescence signal was used to determine whether the sample was Lactobacillus paracasei Zhang. The sequence of the upstream primer for RPA is shown in SEQ ID NO.1, and the sequence of the downstream primer for RPA is shown in SEQ ID NO.2; The CRISPR / Cas12a system contains crRNA, Cas12a protein, and ssDNA; The sequence of the crRNA is shown in any one of SEQ ID NO.3-SEQ ID NO.5.

[0009] Optionally, the RPA amplification reaction conditions are 37℃ for 20 min; the RPA amplification reaction system consists of RPA lyophilized particles, 19.5 μL ddH2O, 1 μL upstream RPA primer, 1 μL downstream RPA primer, 1 μL DNA sample to be tested, and 2.5 μL magnesium acetate solution.

[0010] Optionally, the CRISPR / Cas12a system consists of 0.5 μL Cas12a protein, 2 μL 10×HOLMESbuffer, 1 μL crRNA, 2.0 μL RPA amplification product, 1 μL ssDNA, and 13.5 μL DEPC water.

[0011] Optionally, the sequence of the crRNA is shown in SEQ ID NO.4.

[0012] Optionally, the continued reaction is carried out at 37°C for 20 min.

[0013] Optionally, if a fluorescence signal is present, the sample is determined to be / contains *Lactobacillus paracasei* Zhang; if no fluorescence signal is present, the sample is determined not to be / does not contain *Lactobacillus paracasei* Zhang.

[0014] The present invention discloses the following technical effects: The target strain of Lactobacillus paracasei used in this invention is Lactobacillus paracasei Zhang. Its specific gene was obtained through comparative genomic analysis, and RPA primers and specific crRNA were designed for this gene to form an RPA-CRISPR detection system.

[0015] The detection method established in this invention is universal and can be extended to the detection of targets of other strains by changing the primer sequence and crRNA sequence.

[0016] The room temperature detection process established in this invention completes the entire process from nucleic acid amplification to signal output within 60 minutes. The detection sensitivity is significantly better than that of PCR-CRISPR and qPCR instrument detection schemes that require constant temperature and condition switching.

[0017] In summary, the present invention provides a method for detecting *Lactobacillus paracasei* Zhang based on RPA-CRISPR / Cas12a, which can be applied to the rapid detection of *Lactobacillus paracasei* Zhang in actual samples. This method combines RPA-specific amplification with crRNA-specific sequence recognition, giving it excellent specificity and sensitivity. The entire fluorescence detection platform is operated at 37°C, and the results can be observed visually using either fluorescence or blue light excitation, depending on available conditions, making it suitable for on-site use. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 A schematic diagram illustrating the principle and flow of the Lactobacillus paracasei detection method provided by this invention; Figure 2 Graphs showing component absence and system activation verification; A: Fluorescence control results for system absence; B: Visualized fluorescence results; Figure 3Figure A shows the results of validating the optimal reaction temperature for RPA amplification; A: Gel electrophoresis results at different reaction temperatures; B: Gray values ​​corresponding to Figure A. Figure 4 Optimization of CRISPR / Cas12a system components: A: Optimization of the Cas12a / crRNA addition ratio; B: Optimization of the amount of RPA amplification products added; C: Optimization of the final concentration of ssDNA in the system; D: Results of crRNA cleavage efficiency for different combinations. Figure 5 Figure A shows the results of specific identification of strains; A: Validation results of fluorescence detection for different strains; 1: *Lactobacillus paracasei* Zhang, 2: *Lactobacillus paracasei* K56, 3: *Lactobacillus paracasei* AR1117, 4: *Lactobacillus paracasei* AR1119, 5: *Lactobacillus paracasei* AR1541, 6: *Lactobacillus paracasei* AR1568, 7: *Lactobacillus paracasei* LC2W; B: Visual detection results for different strains. Figure 6 To demonstrate the fluorescence recognition and corresponding visualization results of this detection method under mixed bacterial conditions; 1: Lactobacillus paracasei K56, 2: Mixture of Lactobacillus paracasei K56 and Zhang, 3: Lactobacillus paracasei AR1117, 4: Mixture of Lactobacillus paracasei AR1117 and Zhang, 5: Lactobacillus paracasei AR1119, 6: Mixture of Lactobacillus paracasei AR1119 and Zhang, 7: Lactobacillus paracasei AR1541, 8: Mixture of Lactobacillus paracasei AR1541 and Zhang, 9: Lactobacillus paracasei AR1568, 10: Mixture of Lactobacillus paracasei AR1568 and Zhang, 11: Lactobacillus paracasei LC2W, 12: Mixture of Lactobacillus paracasei LC2W and Zhang; Figure 7 The results were obtained by detecting the target strain at different bacterial concentrations using the CRISPR / Cas12a system. Figure 8 The graphs show the linear fit between fluorescence intensity and the logarithms of different concentrations of *Lactobacillus paracasei* Zhang in actual samples; A: Linear relationship between fluorescence intensity and bacterial concentration in standard bacterial solutions; B: Linear relationship between fluorescence intensity and bacterial concentration in milk samples. Note: Compared with the control group, This indicates that P < 0.05. This indicates that P < 0.01. P < 0.001, and ns indicates no significant difference. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Unless otherwise specified, all reagents used in the following examples were obtained commercially.

[0026] The RPA lyophilized particles and magnesium acetate solution used in the following examples were purchased from Suzhou Keer Life Science Technology Co., Ltd., and the Cas12a (Lbcas12a) protein and ssDNA probe were purchased from Shanghai Tulougang Biotechnology Co., Ltd. The ssDNA (catalog number 31101) is a single-stranded DNA oligonucleotide probe, labeled with a FAM fluorescent reporter group at the 5' end and a fluorescence quencher group at the 3' end. An automated nucleic acid extractor was purchased from Nanjing Novizan Biotechnology Co., Ltd., and primer and crRNA synthesis were outsourced to Shanghai Jierui Biotechnology Co., Ltd.

[0027] This invention innovatively constructs a highly efficient CRISPR / Cas12a-based strain-level detection system for *Lactobacillus paracasei* (*Lactobacillus paracasei* Zhang), combining it with an optimized RPA amplification system to achieve targeted activation of the amplification products into the CRISPR system. This system can complete detection within 60 minutes under isothermal conditions of 37℃, with a detection limit of 1 CFU / mL for *Lactobacillus paracasei* samples. This combined RPA amplification and CRISPR system detection solves the problems of insufficient specificity, low sensitivity, and cumbersome operation associated with traditional detection methods.

[0028] Example 1: Construction of a method for detecting Lactobacillus paracasei based on RPA-CRISPR / Cas12a 1. RPA primer design and specific crRNA design Targeting the unique gene sequence of the *Lactobacillus paracasei* strain (*Lactobacillus paracasei* Zhang), RPA primers (sequences shown in SEQ ID NO. 1-2) were designed using Premier 6. The primer length was 28-35 nt, and the GC content was 40-60%. All generated sequences were aligned to the NCBI database using BLAST to ensure efficient recognition and specific amplification of the target DNA. Specific crRNAs (sequences shown in SEQ ID NO. 3-5) were designed and generated online using Eezassay's website (www.ezassay.com) and aligned to the NCBI database to ensure specificity.

[0029] RPA upstream primer (SEQ ID NO.1): 5'-AAGATTATCGCCAACTTAAACGGGTACACCC-3'; RPA downstream primer (SEQ ID NO.2): 5'-CACCGATTCGAACTAAACGGATCACTGGATT-3'; crRNA1 (SEQ ID NO.3): 5'-UAAUUUCUACUAAGUGUAGUAUGCAAGCCUUAGAACAGCU-3'; crRNA2 (SEQ ID NO.4): 5'-UAAUUUCUACUAAGUGUAGAUUUAGACGGGAAAGUGGAUAU-3'; crRNA3 (SEQ ID NO. 5): 5'-UAAUUUCUACUAAGUGUAGAUUAUGCCCCAUAAGACUAAUC-3'.

[0030] 2. Construction of a quantitative detection method for Lactobacillus paracasei strain Zhang based on RPA-CRISPR / Cas12a Figure 1 The diagram illustrates the principle and flow of the *Lactobacillus paracasei* Zhang detection method provided by this invention. By integrating bacterial genomic DNA extraction, RPA amplification, and Cas12a / crRNA cleavage analysis, a rapid, sensitive, and simple method is developed to detect the target *Lactobacillus paracasei* strain Zhang in complex systems. When the target DNA encounters Cas12a in the system, crRNA guides Cas12a to specifically recognize and cleave the target nucleic acid sequence, activating the trans-cleavage activity of Cas12a. This causes Cas12a to non-specifically cleave the ssDNA probe in the system, separating the fluorescent and quenching groups labeled on the ssDNA and releasing a fluorescent signal. This allows for visual detection under blue light, and the intensity of the fluorescent signal in the reaction system can be measured simultaneously. The specific reaction steps are as follows: S1, RPA reaction system: RPA lyophilized particles, 19.5 μL ddH2O, 1 μL RPA upstream primer (10 μM), 1 μL RPA downstream primer (10 μM), 1 μL target DNA and 2.5 μL magnesium acetate solution were added to the PCR tube in sequence. S2. The prepared RPA reaction system was isothermally amplified at 37℃ for 20 min. After amplification, 25 μL of Tris phenol chloroform mixture was added to the system for purification, and the mixture was centrifuged for 5 min (at this time, the supernatant can be taken for agarose gel electrophoresis to verify the specificity of the primers). S3. To establish a reaction system based on the combined detection of RPA and CRISPR / Cas12a, this invention prepared a CRISPR / Cas12a reaction system with a total volume of 20 μL. The reaction system consisted of the following: 0.5 μL Cas12a protein (10 μM), 2 μL 10×HOLMES buffer, 1 μL crRNA, 2.0 μL RPA amplification product, 1 μL ssDNA, and 13.5 μL DEPC water. All components were thoroughly mixed and incubated at 37°C for 20 min to achieve specific recognition of the target sequence and signal reading.

[0031] S4. After the above reaction is completed, visual detection and observation are performed under blue light irradiation, and fluorescence detection is performed under 480 nm excitation light to collect fluorescence signals. At the same time, systems lacking Cas12a, crRNA, target amplification products and ssDNA are set up as fluorescence control systems.

[0032] The results are as follows Figure 2As shown, strong fluorescence was only detected in a complete reaction system containing the target amplification product, the Cas12a-crRNA ribonucleoprotein complex, and the fluorescent ssDNA reporter factor, demonstrating successful system activation. Furthermore, clear fluorescence was observed using gel imaging and blue light illumination. These results indicate that the RPA-CRISPR / Cas12a-based strain-level detection method performs as expected and enables subsequent quantitative analysis of *Lactobacillus paracasei* Zhang, achieving rapid strain detection within 60 minutes, effectively improving detection efficiency.

[0033] Example 2: Optimization of Method Conditions for Detecting Strains using the High-Performance RPA-CRISPR / Cas12a System To achieve the best detection results, the RPA reaction temperature and CRISPR system components were optimized.

[0034] RPA system optimization: The RPA reaction system parameters are basically the same as in Example 1, except that the RPA amplification temperatures were set to 35℃, 37℃, 39℃, 41℃, and 43℃, respectively. The results are as follows: Figure 3 As shown, the yield of the target product reaches its maximum when RPA amplification is carried out at 37°C.

[0035] For the CRISPR system, the composition is optimized to obtain the most suitable reaction conditions.

[0036] The Cas12a / crRNA addition ratio was optimized: the parameters were basically the same as those in Example 1 RPA reaction system, except that the Cas12a / crRNA addition ratios were 0.5; 1; 1.5; 2; 2.5, with sterile water as the control group (NTC). The results are as follows... Figure 4 As shown in Figure A, when the Cas12a / crRNA system was added at a ratio of 0.5 and 1, there was no significant difference in fluorescence intensity. However, when the Cas12a / crRNA system was added at a ratio of 1.5, the fluorescence intensity decreased significantly. Furthermore, when the Cas12a / crRNA system was added at ratios of 2 and 2.5, the fluorescence intensity was not significantly different from that at a ratio of 1.5. This indicates that as the amount of Cas12a protein increases, excess Cas12a will compete with the Cas12a-crRNA complex for crRNA binding, disrupting the stability of the binary complex and thus reducing target recognition efficiency. Therefore, a Cas12a / crRNA system addition ratio of 0.5 is the most suitable.

[0037] Optimization of RPA amplification product addition: The RPA reaction system parameters were basically the same as in Example 1, except that the addition amounts of RPA amplification products were set to 1; 2; 4; 6; 8 (μL), with sterile water as the control group (NTC). Results are as follows... Figure 4 As shown in Figure B, there is a significant difference in fluorescence intensity when the amount of RPA amplification product added is 1 μL and 2 μL; when the amount of RPA amplification product added is 4 μL, 6 μL and 8 μL, the fluorescence intensity is almost unchanged. Considering the cost-effectiveness, the amount of 2 μL is the most suitable.

[0038] The final concentration of ssDNA in the system was optimized: the parameters were basically the same as those in Example 1 RPA reaction system, except that the final concentration of ssDNA in the system was set to 125 nM; 250 nM; 500 nM; 750 nM; 1000 nM, with sterile water as the control group (NTC). The results are as follows: Figure 4 As shown in Figure C, the higher the final concentration of ssDNA in the system, the higher the fluorescence intensity. When the final concentration of ssDNA in the system is 500 nM, there is a significant difference compared to the system with a final concentration of 250 nM. However, for systems with a final concentration of 750 nM and higher, although the fluorescence intensity increases with the increase in final concentration of ssDNA, there is no significant difference compared to the final concentration of 500 nM. Considering the cost-effectiveness, choosing a final concentration of 500 nM of ssDNA is the most suitable.

[0039] The selection of crRNA was optimized by using crRNA1-crRNA3 or a combination of two of them (1:1). The results are as follows: Figure 4 As shown in Figure D, all three crRNAs can activate the CRISPR system. The single crRNA2 and the combination of crRNA1+crRNA3 (1:1) showed the strongest fluorescence signal, and there was no significant difference in cleavage efficiency. Considering cost and simplicity, single crRNA2 was selected as the best crRNA for the CRISPR system.

[0040] Example 3 Performance evaluation of RPA-CRISPR / Cas12a detection method for Lactobacillus paracasei Zhang 1. Specificity assessment of RPA-CRISPR / Cas12a detection method for detecting Lactobacillus paracasei The specificity of the detection method was evaluated using *Lactobacillus paracasei* Zhang, *Lactobacillus paracasei* K56, *Lactobacillus paracasei* AR1117, *Lactobacillus paracasei* AR1119, *Lactobacillus paracasei* AR1541, *Lactobacillus paracasei* AR1568, and *Lactobacillus paracasei* LC2W (strain sources are shown in Table 1).

[0041] Table 1. Strains and their sources used Using sterile water as a blank control, the test was performed under conditions where only the target DNA of the bacterial strain was altered. The results are as follows: Figure 5 As shown, NTC is the control group, and 1-7 are Lactobacillus paracasei Zhang (Zhang), Lactobacillus paracasei K56 (K56), Lactobacillus paracasei AR1117 (AR1117), Lactobacillus paracasei AR1119 (AR1119), Lactobacillus paracasei AR1541 (AR1541), Lactobacillus paracasei AR1568 (AR1568), and Lactobacillus paracasei LC2W (LC2W), respectively. When only Lactobacillus paracasei Zhang is present, there is a strong fluorescence signal response, while the fluorescence signal intensity of other groups is almost negligible.

[0042] In a mixed bacterial environment, genomic DNA was extracted from different bacteria and the fluorescence intensity was measured using this system (see...). Figure 6 The samples were: NTC (control group), and samples 1-12, in order: *Lactobacillus paracasei* K56, a mixture of *Lactobacillus paracasei* K56 and Zhang, *Lactobacillus paracasei* AR1117, a mixture of *Lactobacillus paracasei* AR1117 and Zhang, *Lactobacillus paracasei* AR1119, a mixture of *Lactobacillus paracasei* AR1119 and Zhang, *Lactobacillus paracasei* AR1541, a mixture of *Lactobacillus paracasei* AR1541 and Zhang, *Lactobacillus paracasei* AR1568, a mixture of *Lactobacillus paracasei* AR1568 and Zhang, *Lactobacillus paracasei* LC2W, and a mixture of *Lactobacillus paracasei* LC2W and Zhang. The results showed that a strong fluorescence signal could only be collected when the genomic DNA of the target strain (*Lactobacillus paracasei* Zhang) was present, indicating that the method provided by this invention has good specificity and anti-interference ability, attributed to the specificity of the RPA amplification primers and the precise recognition ability of crRNA.

[0043] 2. Investigation of the sensitivity of the RPA-CRISPR / Cas12a system for detecting *Lactobacillus paracasei* Zhang at the strain level. Sensitivity tests were conducted under optimal conditions, with the concentrations of the test bacterial solutions set to 0 (NTC) and 10, respectively. 0 10 1 10 2 10 3 10 4 10 5 10 6 CFU / mL. A graph showing the relationship between bacterial concentration and fluorescence intensity was plotted by measuring fluorescence intensity, as shown below. Figure 7As shown, the fluorescence intensity gradually increases with increasing bacterial concentration. This indicates that with increasing bacterial concentration, more amplification products are produced, activating the trans-cleavage activity of Cas12a. Furthermore, at 10... 1 ~10 6 Within the CFU / mL range, fluorescence intensity showed a good linear relationship with the logarithm of bacterial concentration. Quantitative PCR revealed a detection limit of up to 1 CFU / mL, and the linear equation was obtained as follows: Figure 8 As shown in Figure A, this demonstrates that the method for detecting *Lactobacillus paracasei* strains provided by this invention has good sensitivity and can meet practical detection requirements.

[0044] Example 4: Study on the application of RPA-CRISPR / Cas12a detection method at the strain level in milk sample detection. The concentration of *Lactobacillus paracasei* Zhang in milk was determined using the method provided in this invention, employing fluorescence detection and spiked recovery. All samples were measured three times and the average value was taken. The concentration to be measured was set to 10... 2 10 3 10 4 10 5 10 6 Linear regression analysis of CFU / mL with the logarithm of fluorescence intensity and colony forming units yielded the following linear equation: Figure 8 As shown in Figure B, the spiked recovery rate of *Lactobacillus paracasei* Zhang in actual samples ranged from 94.24% to 115.38%. Calculations showed that the detection limit in milk could reach 1.15 CFU / mL, indicating that the detection method of the present invention can accurately determine the concentration of *Lactobacillus paracasei* Zhang in actual samples.

[0045] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A detection kit for Lactobacillus paracasei Zhang using RPA-CRISPR / Cas12a, characterized in that, The kit includes an RPA reaction system and a CRISPR / Cas12a system; The RPA reaction system includes an upstream primer and a downstream primer. The CRISPR / Cas12a system contains crRNA, Cas12a protein, and ssDNA; The sequence of the upstream primer for RPA is shown in SEQ ID NO.1, and the sequence of the downstream primer for RPA is shown in SEQ ID NO.2; The sequence of the crRNA is shown in any one of SEQ ID NO.3-SEQ ID NO.

5.

2. The detection kit as described in claim 1, characterized in that, The sequence of the crRNA is shown in SEQ ID NO.

4.

3. The application of the detection kit as described in claim 1 or 2 in the detection of Lactobacillus paracasei Zhang.

4. A method for detecting Lactobacillus paracasei Zhang using RPA-CRISPR / Cas12a, characterized in that, Includes the following steps: RPA amplification of DNA samples was performed using upstream and downstream RPA primers; simultaneously, a CRISPR / Cas12a system was prepared to react concurrently with RPA amplification. The RPA amplification product was mixed with the CRISPR / Cas12a system and the reaction continued. After the reaction was completed, the fluorescence signal was collected. The presence or absence of the fluorescence signal was used to determine whether the sample was Lactobacillus paracasei Zhang. The sequence of the upstream primer for RPA is shown in SEQ ID NO.1, and the sequence of the downstream primer for RPA is shown in SEQ ID NO.2; The CRISPR / Cas12a system contains crRNA, Cas12a protein, and ssDNA; The sequence of the crRNA is shown in any one of SEQ ID NO.3-SEQ ID NO.

5.

5. The detection method as described in claim 4, characterized in that, The reaction conditions for RPA amplification were 37℃ for 20 min; the reaction system for RPA amplification consisted of RPA lyophilized particles, 19.5 μL ddH2O, 1 μL upstream primer, 1 μL downstream primer, 1 μL DNA sample to be tested, and 2.5 μL magnesium acetate solution.

6. The detection method as described in claim 4, characterized in that, The CRISPR / Cas12a system consisted of 0.5 μL Cas12a protein, 2 μL 10×HOLMES buffer, 1 μL crRNA, 2.0 μL RPA amplification product, 1 μL ssDNA, and 13.5 μL DEPC water.

7. The detection method as described in claim 4, characterized in that, The sequence of the crRNA is shown in SEQ ID NO.

4.

8. The detection method as described in claim 5, characterized in that, The conditions for continuing the reaction were 37°C for 20 minutes.

9. The detection method as described in claim 5, characterized in that, If a fluorescent signal is present, the sample is determined to be / contains *Lactobacillus paracasei* Zhang; if no fluorescent signal is present, the sample is determined not to be / does not contain *Lactobacillus paracasei* Zhang.

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