Qualitative detection method and quantitative detection method of salmonella based on RPA-CRISPR / Cas12a

Abstract

The invention discloses a qualitative detection method and a quantitative detection method of salmonella based on RPA-CRISPR / Cas12a (recombinase polymerase amplification-CRISPR / CRISPR / Cas12a). The qualitative detection method comprises the following steps: reacting a mixed system at least comprising an RPA reagent, a salmonella upstream primer, a salmonella downstream primer, a CRISPR / Cas12a system and a to-be-detected sample to obtain a reaction product of the to-be-detected sample; and detecting the reaction product of the to-be-detected sample by adopting a fluorescence detection method or an electrochemical test paper detection method, and qualitatively detecting the salmonella in the to-be-detected sample according to an absorption peak at 500-520nm of a fluorescence spectrum or a T line and a current value of electrochemical test paper. According to the salmonella detection method provided by the invention, potential pollution risks in a traditional step-by-step reaction process are avoided, and meanwhile, the detection method shows high stability, relatively high specificity, excellent sensitivity and excellent field detection capability.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology detection, and specifically relates to a qualitative detection method and a quantitative detection method of Salmonella based on RPA-CRISPR / Cas12a. Background Art

[0002] Disease outbreaks caused by pathogens have become a major challenge that needs to be addressed in the field of global public health. These pathogens usually quietly invade the human body through unclean food, causing abdominal cramps, fever, diarrhea, and even life-threatening serious consequences. Salmonella has become one of the important "culprits" in the field of food contamination due to its unique harmfulness and prevalence. Every year in Europe, Salmonella causes nearly 9,000 people to become ill; in the United States, it causes 26,500 people to be hospitalized; in China, it is also the main cause of foodborne diseases. Given its extremely low infection dose and widespread prevalence, official agencies around the world continue to formulate strict standards for the limit of Salmonella residues. It is worth noting that the Chinese national standard clearly stipulates that the amount of Salmonella residues in pre-packaged products must be strictly controlled below 0 CFU / mL. Therefore, it is particularly important and urgent to develop an efficient and reliable Salmonella detection technology.

[0003] At present, a large number of researchers have successfully developed a series of highly sensitive and specific detection platforms by integrating CRISPR / Cas type V and type VI systems with isothermal amplification technology. These platforms can accurately identify DNA and RNA viruses at aM-level sensitivity, and their performance is comparable to that of qPCR technology. However, most CRISPR-based detection methods require two independent operation steps: isothermal amplification and CRISPR / Cas cleavage, which undoubtedly increases the complexity of the operation and increases the probability of contamination caused by step-by-step reactions, limiting the widespread promotion and application of this technology. Although integrating isothermal amplification and CRISPR / Cas cleavage into a single test tube reaction is of great significance, it does face severe challenges. In a one-tube reaction, after the nonspecific trans-cleavage activity of the Cas enzyme is activated, it may cleave the primers that did not react in the isothermal amplification, thereby weakening the amplification efficiency and affecting the sensitivity of the detection. Summary of the invention

[0004] The main purpose of the present invention is to provide a qualitative detection method and a quantitative detection method of Salmonella based on RPA-CRISPR / Cas12a to overcome the shortcomings of the prior art.

[0005] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0006] The embodiment of the present invention provides a qualitative detection method for Salmonella based on RPA-CRISPR / Cas12a, which includes:

[0007] reacting a mixed system comprising at least an RPA reagent, a Salmonella upstream primer, a Salmonella downstream primer, a CRISPR / Cas12a system, and a sample to be tested to obtain a reaction product of the sample to be tested;

[0008] Furthermore, the reaction product of the sample to be tested is detected by fluorescence detection method or electrochemical test paper detection method, and the qualitative detection of Salmonella in the sample to be tested is achieved according to the absorption peak at 500-520nm of the fluorescence spectrum or the T line and current value of the electrochemical test paper.

[0009] The embodiment of the present invention also provides a quantitative detection method of Salmonella based on RPA-CRISPR / Cas12a, which comprises:

[0010] reacting a mixed system comprising at least an RPA reagent, a Salmonella upstream primer, a Salmonella downstream primer, a CRISPR / Cas12a system, and a sample to be tested to obtain a reaction product of the sample to be tested;

[0011] And, the reaction product of the sample to be tested is evenly mixed with a buffer solution, and the electrochemical test paper is used for detection for at least 10 minutes, and the catalytic solution is dripped on the T line of the test paper after the reaction for 2 minutes, and then the current value Y is detected, and then the Salmonella concentration X in the sample to be tested is calculated based on the standard curve of Salmonella concentration X and current value Y.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] (1) Compared with the traditional RPA and CRISPR / Cas12a step-by-step reaction mode, the one-tube RPA-CRISPR / Cas12a reaction system developed in the present invention not only perfectly inherits the high sensitivity and excellent specificity of the original method, but also has a significant improvement in the convenience of operation and greatly reduces the complexity of the operation. More importantly, this design effectively reduces the risk of contamination caused by opening the lid, thereby ensuring the purity of the experimental process and the reliability of the results;

[0014] (2) The electrochemical test paper provided by the present invention has high sensitivity and strong specificity, can be carried around, is easy to operate, and does not require reliance on professional technicians. The combination of test paper and electrochemistry solves the problem of low sensitivity and inability to accurately quantify with traditional test paper;

[0015] (3) The redox reaction of the electrochemical test paper provided by the present invention is highly dependent on the presence of AuNPs (gold nanoparticles). This innovative design cleverly avoids the reliance on expensive biological enzymes. Through this unique mechanism, the test paper can simultaneously achieve the output of visualization and electrochemical dual-modal signals, which not only greatly broadens the scope of application of detection, but also ensures that effective detection can be performed in different regions and under different conditions, fully meeting the needs of diversification and flexibility.

[0016] (4) The one-tube RPA-CRISPR / Cas12a electrochemical test paper detection method developed by the present invention greatly improves the detection sensitivity, and the detection limit can be as low as 3.84 CFU / mL; in addition, the method is accurate and practical for the detection of actual samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 This is a schematic diagram of the detection principle of a one-tube RPA-CRISPR / Cas12a electrochemical test paper in Example 2 of the present invention;

[0019] Figure 2A-2B This is a schematic diagram of the detection results of fluorescence detection of a one-tube RPA-CRISPR / Cas12a reaction system and electrochemical test paper in Example 3 of the present invention;

[0020] Figure 3A-3C This is a schematic diagram of the linear detection results of a tube-type RPA-CRISPR / Cas12a electrochemical test paper in Example 4 of the present invention;

[0021] Figure 4A-4B This is a schematic diagram of the specific detection results of a tube-type RPA-CRISPR / Cas12a electrochemical test paper in Example 5 of the present invention;

[0022] Figure 5A-Figure 5B This is a schematic diagram of the results of detecting actual samples using a tube-type RPA-CRISPR / Cas12a electrochemical test paper in Example 6 of the present invention. DETAILED DESCRIPTION

[0023] In view of the defects of the prior art, the inventor of this case has proposed the technical solution of the present invention after long-term research and extensive practice. The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Specifically, as one aspect of the technical solution of the present invention, a qualitative detection method of Salmonella based on RPA-CRISPR / Cas12a includes:

[0025] reacting a mixed system comprising at least an RPA reagent, a Salmonella upstream primer, a Salmonella downstream primer, a CRISPR / Cas12a system, and a sample to be tested to obtain a reaction product of the sample to be tested;

[0026] Furthermore, the reaction product of the sample to be tested is detected by fluorescence detection method or electrochemical test paper detection method, and the qualitative detection of Salmonella in the sample to be tested is achieved according to the absorption peak at 500-520nm of the fluorescence spectrum or the T line and current value of the electrochemical test paper.

[0027] In some preferred embodiments, the Salmonella upstream primer has a sequence as shown in SEQ ID NO.1.

[0028] In some preferred embodiments, the Salmonella downstream primer has a sequence as shown in SEQ ID NO.2.

[0029] In some preferred embodiments, the qualitative detection method specifically comprises:

[0030] The RPA reagent, H2O, the upstream primer of Salmonella, and the downstream primer of Salmonella are evenly mixed, and then the CRISPR / Cas12a system and the sample to be tested are added, mixed, and reacted at 29-45°C for 30-90 minutes to obtain the reaction product of the sample to be tested; wherein the CRISPR / Cas12a system includes: 1×NEBuffer 1, 0.04μM Cas12a, 0.12μM crRNA, 0.6μM FAM-ssDNA-BHQ and 10mM magnesium acetate, the crRNA has the sequence shown in SEQ ID NO.3, and the FAM-ssDNA-BHQ has the sequence shown in SEQ ID NO.4;

[0031] And, the reaction product of the sample to be tested is mixed with 1×PBS, and fluorescence spectrum detection is performed. When the obtained fluorescence spectrum shows an absorption peak at 500-520nm, the sample to be tested contains Salmonella; when the obtained fluorescence spectrum does not show an absorption peak at 500-520nm, the sample to be tested does not contain Salmonella.

[0032] In some preferred embodiments, the qualitative detection method specifically comprises:

[0033] The RPA reagent, H20, the upstream primer of Salmonella, and the downstream primer of Salmonella are evenly mixed, and then the CRISPR / Cas12a system and the sample to be tested are added, mixed, and reacted at 29-45°C for 30-90 minutes to obtain the reaction product of the sample to be tested; wherein the CRISPR / Cas12a system includes: 1×NEBuffer 1, 0.04μM Cas12a, 0.12μM crRNA, 8nMFITC-ssDNA-Bio and 10mM magnesium acetate, the crRNA has the sequence shown in SEQ ID NO.3, and the FITC-ssDNA-Bio has the sequence shown in SEQ ID NO.5;

[0034] And, the reaction product of the sample to be tested is evenly mixed with a buffer solution, and an electrochemical test paper is used for detection for at least 10 minutes. When the electrochemical test paper simultaneously presents a C line and a T line, and a current value detected after 2 minutes of adding a catalytic solution to the T line of the test paper after the reaction is greater than 3.5 μA, the sample to be tested does not contain Salmonella; when the electrochemical test paper only presents a C line, and a current value detected after 2 minutes of adding a catalytic solution to the T line of the test paper after the reaction is less than 3.5 μA, the sample to be tested contains Salmonella.

[0035] Furthermore, the electrochemical test paper includes a base plate, a screen-printed electrode, an NC membrane, a sample pad, a conjugation pad and a water absorbent pad. The screen-printed electrode is located between the base plate and the NC membrane. Streptavidin is sprayed on the NC membrane as a T line, and a sheep anti-mouse secondary antibody is sprayed as a C line. The T line is precisely fitted to the working electrode area of ​​the screen-printed electrode for detecting the current signal generated by the catalytic reaction on the T line. AuNPs-FITC-Ab (a complex of colloidal gold coupled to FITC antibody) is dripped on the conjugation pad.

[0036] Furthermore, the catalytic solution is a 5×PBS solution containing potassium ferrocyanide and a borocyanide complex.

[0037] In some preferred embodiments, the detection method of Salmonella based on RPA-CRISPR / Cas12a comprises:

[0038] Extract the DNA of Salmonella from actual samples; design efficient upstream and downstream primers to accelerate the amplification process of RPA; establish a one-tube RPA-CRISPR / Cas12a equilibrium reaction system by optimizing the key parameters of RPA and CRISPR / Cas12a system, and obtain the reaction product after the reaction;

[0039] Identification by fluorescence detection or electrochemical test paper detection:

[0040] Fluorescence detection method: add 75 μL 1×PBS to the reaction product, mix well, and detect with a fluorescence spectrometer. If an absorption peak appears near 520 nm, it is judged as positive, indicating that the sample contains Salmonella; if no absorption peak appears, it is judged as negative, indicating that the sample does not contain Salmonella;

[0041] Electrochemical test paper detection method: Take 1.5 μL of the reaction product and add it to 48.5 μL of buffer solution, mix well, and detect it with electrochemical test paper. After 10 minutes, if the test paper shows C and T lines at the same time, and the catalytic solution is added to the T line of the test paper after the reaction, and a larger current value is detected after 2 minutes, it is judged as negative, indicating that the tested sample does not contain Salmonella; if the test paper only shows one C line, and the catalytic solution is added to the T line of the test paper after the reaction, and a smaller current value is detected after 2 minutes, it is judged as positive, indicating that the tested sample contains Salmonella.

[0042] Preferably, the upstream primer (F) is as shown in SEQ ID NO.1; the downstream primer (R) is as shown in SEQ ID NO.2.

[0043] Preferably, the reaction product suitable for fluorescence detection is RPA-Cas12a-FQ, and the method of obtaining is as follows: add 20μLDCEP H2O and 5μL 10μM F and R to a tube of RPA reagent lyophilized powder (basic nucleic acid amplification kit provided by Xianda Gene Technology Co., Ltd.), mix well, and divide into two tubes, each with a volume of 15μL, recorded as system A, and then add the necessary components of the CRISPR / Cas12a system to the system A, including: 1×NEBuffer 1, 0.04μM Cas12a, 0.12μMcrRNA, 0.6μM FAM-ssDNA-BHQ and 10mM magnesium acetate, and finally add 1μL template to the detection system. The system is placed at 41°C for 90min, and the reaction product is RPA-Cas12a-FQ; the sequence of the FAM-ssDNA-BHQ is shown in SEQID NO.4.

[0044] As a preferred method, the reaction product suitable for electrochemical test paper detection is RPA-Cas12a-FB, and the method of obtaining is as follows: 20 μL DCEP H2O and 5 μL 10 μM F and R are added to a tube of RPA reagent lyophilized powder (basic nucleic acid amplification kit provided by Xianda Gene Technology Co., Ltd.), mixed, and divided into two tubes, each with a volume of 15 μL, recorded as system A, and then the necessary components of the CRISPR / Cas12a system are added to the system A, including: 1×NEBuffer 1, 0.04 μM Cas12a, 0.12 μM crRNA, 8nM FITC-ssDNA-Bio and 10mM magnesium acetate, and finally 1 μL template is added to the detection system. The system is placed at 41 ° C for 90 min, and the reaction product is RPA-Cas12a-FB; the sequence of the FITC-ssDNA-Bio is shown in SEQ ID NO.5.

[0045] Preferably, the electrochemical test paper comprises a bottom plate, a screen-printed electrode, an NC membrane, a sample pad, a conjugation pad and a water-absorbing pad in sequence, the screen-printed electrode is between the bottom plate and the NC membrane, the NC membrane is sprayed with streptavidin as a T line, and a goat anti-mouse secondary antibody is sprayed as a C line, the T line is precisely fitted with the working electrode area of ​​the screen-printed electrode, and is used to detect the current signal generated by the catalytic reaction on the T line, and AuNPs-FITC-Ab is dripped on the conjugation pad. The NC membrane with conductive properties without a liner is used, and the model is AE99.

[0046] Preferably, the buffer solution comprises: 10 mM pH 7.4 Tris-HCl, 5 mM MgCl2, 5 mM KCl, 2×SSC, 1% Tween-20, 0.5% PEG20000, 1 mM CTAB, and 1% BSA.

[0047] Preferably, the catalytic solution is 5 mM potassium ferrocyanide (Fe 3+ ) and 5 mM borazone complex (AB) in 5× PBS.

[0048] The present invention requires the presence of AuNPs to cause the reaction. 100 μL of the catalytic solution is dropped on the T line of the test paper after the reaction and the three-electrode area of ​​the screen-printed electrode, and the reaction is carried out for 2 minutes. Under negative conditions, a large number of AuNPs are captured on the T line of the test paper, and the AB in the catalytic solution converts Fe 3+ Reduction to Fe 2+ At this time, there is a large amount of electron transfer, and the current value is high after electrochemical detection; under positive conditions, AuNPs are not captured on the T line of the test paper, and AB in the catalytic solution cannot transfer Fe without the presence of AuNPs. 3+ Reduction to Fe2+ At this time, there is no electron transfer, and the current value is low after electrochemical detection.

[0049] Another aspect of an embodiment of the present invention further provides a quantitative detection method for Salmonella based on RPA-CRISPR / Cas12a, comprising:

[0050] reacting a mixed system comprising at least an RPA reagent, a Salmonella upstream primer, a Salmonella downstream primer, a CRISPR / Cas12a system, and a sample to be tested to obtain a reaction product of the sample to be tested;

[0051] And, the reaction product of the sample to be tested is evenly mixed with a buffer solution, and the electrochemical test paper is used for detection for at least 10 minutes, and the catalytic solution is dripped on the T line of the test paper after the reaction for 2 minutes, and then the current value Y is detected, and then the Salmonella concentration X in the sample to be tested is calculated based on the standard curve of Salmonella concentration X and current value Y.

[0052] In some preferred embodiments, the Salmonella upstream primer has a sequence as shown in SEQ ID NO.1.

[0053] In some preferred embodiments, the Salmonella downstream primer has a sequence as shown in SEQ ID NO.2.

[0054] In some preferred embodiments, the quantitative detection method specifically comprises:

[0055] The RPA reagent, H20, Salmonella upstream primer, and Salmonella downstream primer are evenly mixed, and then the CRISPR / Cas12a system and the sample to be tested are added, mixed, and reacted at 29-45°C for 30-90 minutes to obtain a reaction product of the sample to be tested; wherein the CRISPR / Cas12a system includes: 1×NEBuffer 1, 0.04μM Cas12a, 0.12μM crRNA, 8nMFITC-ssDNA-Bio and 10mM magnesium acetate, the crRNA has a sequence as shown in SEQ ID NO.3, and the FITC-ssDNA-Bio has a sequence as shown in SEQ ID NO.5.

[0056] In some preferred embodiments, the quantitative detection method specifically comprises:

[0057] Prepare Salmonella concentrations of 0 and 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 107 CFU / mL, and testing the series of standard solutions using the same testing conditions as those for testing the sample to be tested to obtain a current value Y of the standard solution;

[0058] A standard curve of the Salmonella concentration X and the current value Y of the standard solution is determined.

[0059] In some preferred embodiments, the electrochemical test paper includes a base plate, a screen-printed electrode, a NC membrane, a sample pad, a conjugate pad and a water absorbent pad. The screen-printed electrode is located between the base plate and the NC membrane. Streptavidin is sprayed on the NC membrane as a T line, and goat anti-mouse secondary antibody is sprayed as a C line. The T line is precisely fitted to the working electrode area of ​​the screen-printed electrode for detecting the current signal generated by the catalytic reaction on the T line. AuNPs-FITC-Ab is dripped on the conjugate pad.

[0060] In some preferred embodiments, the buffer solution comprises: 10 mM pH 7.4 Tris-HCl, 5 mM MgCl2, 5 mM KCl, 2×SSC, 1% Tween-20, 0.5% PEG20000, 1 mM CTAB and 1% BSA;

[0061] In some preferred embodiments, the catalytic solution is a 5×PBS solution containing potassium ferrocyanide and borazine complex.

[0062] In some preferred embodiments, the detection range X of the Salmonella concentration of the quantitative detection method is 10 0 -10 6 CFU / mL.

[0063] The Salmonella detection method provided by the present invention avoids the potential contamination risk in the traditional step-by-step reaction process, and the electrochemical test paper has the dual advantages of convenience and accurate quantification; at the same time, the detection method exhibits strong stability, strong specificity, excellent sensitivity and outstanding on-site detection capabilities, and has great potential for application in the fields of food safety monitoring and disease prevention and control.

[0064] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and drawings. This embodiment is implemented on the premise of the technical solution of the invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0065] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies.

[0066] Example 1

[0067] Design of one-tube RPA-CRISPR / Cas12a primers and probes for detection of Salmonella

[0068] (1) Design of efficient and specific primers for Salmonella: Referring to the Salmonella timY gene sequence published in GenBank (Gene No.: CP149283.1), according to the RPA primer design principles, specific primers were designed using Premier 6.0 software: forward primer F (SEQ ID NO.1) and reverse primer R (SEQ ID NO.2). The upstream and downstream primers for Salmonella were synthesized by Shanghai Bioengineering Co., Ltd. The primer information is as follows:

[0069] SEQ ID NO.1: 5'-ACCCATTTGTATTGGTTGTTACGGCTATTTTG-3'

[0070] SEQ ID NO.2: 5'-GCGGCTGCTCGCCTTTGCTGGTTTTAGGTTTG-3'

[0071] (2) Design and sequence specificity analysis of crRNA: The 177bp amplicon generated by F / R amplification in the RPA reaction was used as the target sequence. According to the PAM sequence of LbCas12a, which is 5'-TTTV-3' (where V is an arbitrary nucleotide), the PAM sequence in the target sequence was searched, and the 20 to 23 nucleotides after the PAM sequence and located in the middle region outside the RPA primer were selected. The sequence specificity was compared and analyzed using NCBI online BLAST to confirm whether the sequence only exists in Salmonella. A common sequence fragment recognized by LbCas12a was added before the 5' end of 20 nucleotides. crRNA (SEQ ID NO.3) was synthesized by Bio-Tech Co., Ltd. The crRNA information is as follows:

[0072] SEQ ID NO.3:

[0073] 5'-AAUUUCUACUCUUGUAGAUUAAAUAGAAGAGUACGCUUAAAAC-3'

[0074] (3) Design of ssDNA reporter probe: Based on the trans-cleavage activity of CRISPR-Cas12a, ssDNA is non-specifically cut to design the ssDNA reporter probes required for the fluorescence detection system and the electrochemical test paper detection system. Since the LbCas12a protein has better cleavage activity for adenine (A) and thymine (T), the ssDNA sequence is designed to be TTATT. The ssDNA of the fluorescence detection system is labeled with FAM at the 5' end and BHQ at the 3' end, denoted as FAM-ssDNA-BHQ. The FAM-ssDNA-BHQ reporter probe was synthesized by Shanghai Bioengineering Co., Ltd. The ssDNA of the electrochemical test paper detection system is labeled with FTIC at the 5' end and Bio at the 3' end, denoted as FITC-ssDNA-Bio. The FITC-ssDNA-Bio reporter probe was synthesized by General Bioengineering Co., Ltd. The information of the two reporter probes is as follows:

[0075] SEQ ID NO.4: 5'-FAM-TTATT-BHQ-3',

[0076] SEQ ID NO. 5: 5'-FITC-TTATT-Bio-3'.

[0077] Example 2

[0078] Figure 1 The core principle of detecting Salmonella based on a one-tube RPA-CRISPR / Cas12a electrochemical test paper technology is elaborated in detail. This technology cleverly integrates the RPA and CRISPR / Cas12a systems into a single reaction tube. When the sample does not contain Salmonella, the RPA reaction cannot generate the target double-stranded DNA, and thus cannot match the specific crRNA, resulting in the inactivation of the trans-cleavage activity of Cas12a. Therefore, the single-stranded reporter probe (5'-FITC-TTATT-Bio-3') is not cut. At this time, when the electrochemical test paper is used for detection, the complete single-stranded reporter probe can form an AuNPs-FITC-Ab-5'-FITC-TTATT-Bio-3'-SAV sandwich structure with the gold pad conjugate (AuNPs-FITC-Ab) and the streptavidin (SAV) on the T line of the test paper, causing the test paper to show both the T line and the C line. Furthermore, after the catalytic solution was added to the T line of the test paper, the T line captured a large number of gold nanoparticles, which promoted the occurrence of redox reactions, thereby detecting a higher current value.

[0079] On the contrary, if Salmonella is present in the sample, the RPA reaction will specifically produce target double-stranded DNA, which will specifically bind to crRNA, activate the trans-cleavage activity of Cas12a, and then non-specifically cut the single-stranded reporter probe. At this time, during the electrochemical test paper detection, since the single-stranded reporter probe has been cut, the above sandwich structure can no longer be formed, and the test paper only shows the C line. After the catalytic solution is added to the T line, the redox reaction fails to occur due to the lack of assistance from gold nanoparticles, so the detected current value is significantly reduced, and this result is judged to be positive.

[0080] It is worth noting that the device used to detect the redox current on the T line is a screen-printed electrode, which is cleverly assembled between the nitrocellulose (NC) membrane and the base plate to ensure a close fit with the test paper T line, thereby accurately detecting the electrochemical signal generated by the redox reaction on the T line.

[0081] Example 3

[0082] Establishment of a one-tube RPA-CRISPR / Cas12a method for detecting Salmonella

[0083] (1) Fluorescence detection method

[0084] Reaction system and detection process: (total volume is 25 μL): add 20 μL DCEPH2O and 5 μL 10 μM F and R to a tube of RPA reagent lyophilized powder, mix well, and divide equally into two tubes, each with a volume of 15 μL, recorded as system A, then add the necessary components of the CRISPR / Cas12a system to system A, including: 1×NEBuffer 1, 0.04 μM Cas12a, 0.12 μM crRNA, 0.6 μM FAM-ssDNA-BHQ and 10 mM magnesium acetate, and finally add 1 μL template (negative 0 CFU / mL, positive 10 4 CFU / mL). The system was placed at 41°C for 90 min. After the reaction, 75 μL of 1×PBS was added to the reaction product, mixed well, and detected by fluorescence spectrometer. The detection results were as follows: Figure 2A shown.

[0085] (2) Electrochemical test paper detection method

[0086] Reaction system and detection process: (total volume is 25 μL): add 20 μL DCEPH2O and 5 μL 10 μM F and R to a tube of RPA reagent lyophilized powder, mix well, and divide equally into two tubes, each with a volume of 15 μL, recorded as system A, then add the necessary components of the CRISPR / Cas12a system to system A, including: 1×NEBuffer 1, 0.04 μM Cas12a, 0.12 μM crRNA, 8nM FITC-ssDNA-Bio and 10mM magnesium acetate, and finally add 1 μL template (negative 0 CFU / mL, positive 10 4 CFU / mL). The system was placed at 41°C for 90 minutes. After the reaction, 1.5 μL of the reaction product was added to 48.5 μL of buffer solution, mixed evenly, and tested with electrochemical test paper. After 10 minutes, 100 μL of catalytic solution was added to the T line of the test paper after the reaction. After 2 minutes, the current value on the T line was detected. The test results are as follows: Figure 2B The catalytic solution is 5 mM potassium ferrocyanide (Fe 3+ ) and 5 mM borazoane complex (AB) in 5× PBS solution. The catalysis can only occur in the presence of AuNPs.

[0087] Example 4

[0088] Sensitivity evaluation of one-tube RPA-CRISPR / Cas12a electrochemical test strips for detection of Salmonella

[0089] The DNA template of Salmonella was diluted in a series of precise steps, with the concentration gradient set at 0, 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 CFU / mL, a one-tube RPA-CRISPR / Cas12a reaction was performed under optimal conditions, and finally the detection sensitivity was evaluated using an electrochemical test paper sensor. Figure 3A-3C As shown, Figure 3A The visual detection results of the electrochemical test paper are intuitively displayed, where the visual detection limit can reach 10 1 CFU / mL. Figure 3B It presents Figure 3A The time current generated by the redox reaction on the T line of the electrochemical test paper shows that the detection limit can be as low as 10 0 CFU / mL, that is, the level of single colony forming units. In addition, Figure 3CA linear relationship model between the current value and the corresponding Salmonella concentration was established. 0 -10 5 Within the concentration range of CFU / mL, there is a good linear correlation between the two. 2 The value was as high as 0.985, which further verified the accuracy and reliability of this method in the quantitative detection of Salmonella concentration.

[0090] Example 5

[0091] Specificity evaluation of one-tube RPA-CRISPR / Cas12a electrochemical test strips for detection of Salmonella

[0092] In order to verify the specificity of the established method, a series of high concentrations of common foodborne pathogens were tested under optimized conditions, including Enterobacter sakazakii, Staphylococcus aureus, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Listeria monocytogenes and Escherichia coli. Figure 4A-4B Shown are the test results. Figure 4A The results of the electrochemical test strips for different pathogens are presented. It was observed that even when the concentration of other non-target pathogens was 100 times that of Salmonella, the T line of the test strip showed no signs of disappearance, and its intensity remained consistent with that of the blank control group. Only when Salmonella was present did the intensity of the T line of the test strip significantly weaken. Figure 4B The figure shows the current value generated by the redox reaction on the T line of the electrochemical test paper. It can be clearly seen from the figure that under the influence of other non-target pathogens, the current value remains at a high level; and only in the presence of Salmonella does the current value drop significantly. The above results strongly prove that this method has strong specificity and can ensure that the test results are not interfered by other target bacteria, thereby improving the accuracy and reliability of the test.

[0093] Example 6

[0094] Performance evaluation of one-tube RPA-CRISPR / Cas12a electrochemical test strips for detection of Salmonella in milk

[0095] After comprehensively evaluating the performance indicators of the one-tube RPA-CRISPR / Cas12a electrochemical test strip sensor, the sensor's detection performance for Salmonella in milk samples was further verified. Specifically, milk samples that were confirmed to be uncontaminated with Salmonella by qPCR technology were selected, and then a series of known concentrations of Salmonella were added to them. These samples were tested and analyzed under uniform and strict experimental conditions. The results are as follows Figure 5A-Figure 5B shown. Figure 5AThe visual detection results and time-current detection results of the electrochemical test paper are intuitively displayed. Through observation, it can be clearly found that the visual detection limit of the electrochemical test paper can reach 10 2 CFU / mL, while the electrochemical detection method is more sensitive, with a detection limit as low as 10 0 CFU / mL, the detection sensitivity is increased by 100 times compared with the method of using test paper alone, showing significant superiority. Figure 5B The linear relationship model between the current value and the concentration of Salmonella in milk samples was further constructed. The results showed that when the concentration of Salmonella was between 10 0 -10 6 Within the range of CFU / mL, the current value and concentration showed a highly consistent linear correlation. 2 The value is as high as 0.984. This result not only strongly confirms the accuracy and reliability of this method in detecting Salmonella concentration in actual milk samples, but also provides a solid theoretical and experimental foundation for our subsequent application and research.

[0096] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0097] It should be understood that the technical solution of the present invention is not limited to the above-mentioned specific implementation cases. Any technical deformation made according to the technical solution of the present invention without departing from the scope of protection of the purpose of the present invention and the claims shall fall within the protection scope of the present invention.

Claims

1. A qualitative detection method for Salmonella based on RPA-CRISPR / Cas12a, characterized in that: include: reacting a mixed system comprising at least an RPA reagent, a Salmonella upstream primer, a Salmonella downstream primer, a CRISPR / Cas12a system, and a sample to be tested to obtain a reaction product of the sample to be tested; Furthermore, the reaction product of the sample to be tested is detected by fluorescence detection method or electrochemical test paper detection method, and the qualitative detection of Salmonella in the sample to be tested is achieved according to the absorption peak at 500-520nm of the fluorescence spectrum or the T line and current value of the electrochemical test paper.

2. The qualitative detection method according to claim 1, characterized in that: The Salmonella upstream primer has a sequence as shown in SEQ ID NO.1; And / or, the Salmonella downstream primer has a sequence as shown in SEQ ID NO.

2.

3. The qualitative detection method according to claim 1, characterized in that: Specifically include: The RPA reagent, H2O, the upstream primer of Salmonella, and the downstream primer of Salmonella are evenly mixed, and then the CRISPR / Cas12a system and the sample to be tested are added, mixed, and reacted at 29-45°C for 30-90 minutes to obtain a reaction product of the sample to be tested; wherein the CRISPR / Cas12a system includes: 1×NEBuffer 1, 0.04μM Cas12a, 0.12μM crRNA, 0.6μM FAM-ssDNA-BHQ and 10mM magnesium acetate, the crRNA has a sequence as shown in SEQ ID NO.3, and the FAM-ssDNA-BHQ has a sequence as shown in SEQ ID NO.4; And, the reaction product of the sample to be tested is mixed with 1×PBS, and fluorescence spectrum detection is performed. When the obtained fluorescence spectrum shows an absorption peak at 500-520nm, the sample to be tested contains Salmonella; when the obtained fluorescence spectrum does not show an absorption peak at 500-520nm, the sample to be tested does not contain Salmonella.

4. The qualitative detection method according to claim 1, characterized in that: Specifically include: The RPA reagent, H2O, the upstream primer of Salmonella, and the downstream primer of Salmonella are evenly mixed, and then the CRISPR / Cas12a system and the sample to be tested are added, mixed, and reacted at 29-45°C for 30-90 minutes to obtain the reaction product of the sample to be tested; wherein the CRISPR / Cas12a system includes: 1×NEBuffer 1, 0.04μM Cas12a, 0.12μM crRNA, 8nM FITC-ssDNA-Bio and 10mM magnesium acetate, the crRNA has the sequence shown in SEQ ID NO.3, and the FITC-ssDNA-Bio has the sequence shown in SEQ ID NO.5; And, the reaction product of the sample to be tested is evenly mixed with a buffer solution, and an electrochemical test paper is used for detection for at least 10 minutes. When the electrochemical test paper simultaneously presents a C line and a T line, and a current value detected after 2 minutes of adding a catalytic solution to the T line of the test paper after the reaction is greater than 3.5 μA, the sample to be tested does not contain Salmonella; when the electrochemical test paper only presents a C line, and a current value detected after 2 minutes of adding a catalytic solution to the T line of the test paper after the reaction is less than 3.5 μA, the sample to be tested contains Salmonella.

5. The qualitative detection method according to claim 4, characterized in that: The electrochemical test paper comprises a bottom plate, a screen-printed electrode, a NC membrane, a sample pad, a conjugation pad and a water-absorbing pad. The screen-printed electrode is located between the bottom plate and the NC membrane. Streptavidin is sprayed on the NC membrane as a T line, and goat anti-mouse secondary antibody is sprayed as a C line. The T line is precisely fitted with the working electrode area of ​​the screen-printed electrode to detect the current signal generated by the catalytic reaction on the T line. AuNPs-FITC-Ab is dripped on the conjugation pad. And / or, the catalytic solution is a 5×PBS solution containing potassium ferrocyanide and a borazine complex.

6. A quantitative detection method for Salmonella based on RPA-CRISPR / Cas12a, characterized in that: include: reacting a mixed system comprising at least an RPA reagent, a Salmonella upstream primer, a Salmonella downstream primer, a CRISPR / Cas12a system, and a sample to be tested to obtain a reaction product of the sample to be tested; And, the reaction product of the sample to be tested is evenly mixed with a buffer solution, and the electrochemical test paper is used for detection for at least 10 minutes, and the catalytic solution is dripped on the T line of the test paper after the reaction for 2 minutes, and then the current value Y is detected, and then the Salmonella concentration X in the sample to be tested is calculated based on the standard curve of Salmonella concentration X and current value Y.

7. The quantitative detection method according to claim 6, characterized in that: The Salmonella upstream primer has a sequence as shown in SEQ ID NO.1; And / or, the Salmonella downstream primer has a sequence as shown in SEQ ID NO.

2.

8. The quantitative detection method according to claim 6, characterized in that: Specifically include: The RPA reagent, H2O, the upstream primer of Salmonella, and the downstream primer of Salmonella are evenly mixed, and then the CRISPR / Cas12a system and the sample to be tested are added, mixed, and reacted at 29-45°C for 30-90 minutes to obtain the reaction product of the sample to be tested; wherein the CRISPR / Cas12a system includes: 1xNEBuffer 1, 0.04μM Cas12a, 0.12μM crRNA, 8nM FITC-ssDNA-Bio and 10mM magnesium acetate, the crRNA has the sequence shown in SEQ ID NO.3, and the FITC-ssDNA-Bio has the sequence shown in SEQ ID NO.5; And / or, the quantitative detection method specifically comprises: Prepare Salmonella concentrations of 0 and 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 CFU / mL, and testing the series of standard solutions using the same testing conditions as those for testing the sample to be tested to obtain a current value Y of the standard solution; A standard curve of the Salmonella concentration X and the current value Y of the standard solution is determined.

9. The quantitative detection method according to claim 6, characterized in that: The electrochemical test paper comprises a bottom plate, a screen-printed electrode, a NC membrane, a sample pad, a conjugation pad and a water-absorbing pad. The screen-printed electrode is located between the bottom plate and the NC membrane. Streptavidin is sprayed on the NC membrane as a T line, and goat anti-mouse secondary antibody is sprayed as a C line. The T line is precisely fitted with the working electrode area of ​​the screen-printed electrode to detect the current signal generated by the catalytic reaction on the T line. AuNPs-FITC-Ab is dripped on the conjugation pad. And / or, the buffer solution comprises: 10 mM pH 7.4 Tris-HCl, 5 mM MgCl2, 5 mM KCl, 2×SSC, 1% Tween-20, 0.5% PEG20000, 1 mM CTAB and 1% BSA; And / or, the catalytic solution is a 5×PBS solution containing potassium ferrocyanide and a borazine complex.

10. The quantitative detection method according to claim 6, characterized in that: The detection range X of the Salmonella concentration in the quantitative detection method is 10 0 -10 6 CFU / mL.

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