Amplification-free microbial molecule detection method and detection kit
Through the amplification-free microbial molecular detection method based on Craspase/SNLuc-Csx30, the problems of long detection cycle, insufficient sensitivity and professional equipment in the prior art are solved, and high sensitivity and high specific pathogen detection without nucleic acid amplification are achieved, which is suitable for rapid and on-site detection.
Patent Information
- Application Number
- CN202510186963.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-20
- Publication Date
- 2025-05-23
AI Technical Summary
The existing pathogen detection technology has problems such as long detection cycle, insufficient sensitivity, requiring professional equipment and operators, being easily contaminated and having high time cost, making it difficult to meet the needs of fast, accurate and on-site testing.
Using Craspase/SNLuc-Csx30-free amplification detection method, the Craspase protein nucleic acid complex, SNLuc-Csx30 protein and Furimazine molecule is used to detect pathogens without nucleic acid amplification, and the output signal is amplified by bioluminescence to simplify operation and result reading.
It realizes amplification-free detection without nucleic acid amplification. The detection process is simple, specific, high sensitivity, and the minimum detection limit can reach 1ng/μL. It is suitable for on-site instant detection, reducing time and cost.
Smart Images
Figure CN120026088A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of pathogen detection, and in particular to an amplification-free microbial molecular detection method and a detection kit based on Craspase / SNLuc-Csx30. Background Art
[0002] In the public health defense system, accurate detection of pathogens constitutes a key technical guarantee. Accurate and rapid detection of pathogens can provide key evidence for clinical diagnosis and achieve targeted treatment. At the same time, in food safety supervision, it can prevent contaminated food from entering the market and ensure food safety.
[0003] Traditional detection methods in the past, such as culture methods, are classic but have a long detection cycle and are difficult to meet the urgent needs of rapid diagnosis at present; although the immunological method has a faster detection speed, it has shortcomings in sensitivity and is difficult to accurately capture trace pathogens. Against this background, molecular detection technology has developed rapidly. Nucleic acid amplification technology represented by polymerase chain reaction (PCR) can efficiently amplify extremely trace amounts of nucleic acids to detectable levels, greatly improving the sensitivity of detection. However, nucleic acid amplification has strict requirements on experimental conditions and requires professional laboratory equipment and strictly trained operators, which limits its application in primary medical institutions and on-site rapid testing. Secondly, the amplification process is susceptible to contamination, resulting in false positive results and affecting the accuracy of diagnosis. Furthermore, the amplification time is relatively long, and it usually takes several hours from sample processing to obtaining results, and the time cost is too high.
[0004] Therefore, in order to provide more solid and efficient technical support for public health security, it is urgent to develop a new molecular diagnostic method that does not require nucleic acid amplification and can achieve on-site instant detection. Summary of the invention
[0005] In order to solve the problems in the background technology, the present invention provides a non-amplification microbial molecular detection method and detection kit based on Craspase / SNLuc-Csx30. The kit does not need to amplify the pathogen nucleic acid when detecting pathogens, and can cope with trace detection.
[0006] To achieve the above object, the first technical solution adopted by the present invention is:
[0007] An amplification-free microbial molecular detection method and detection kit based on Craspase / SNLuc-Csx30, comprising: Craspase protein-nucleic acid complex, SNLuc-Csx30 protein, and Furimazine molecule;
[0008] The Craspase protein-nucleic acid complex is composed of Cas7-11 protein, crRNA and Csx29 protein. The crRNA sequence is the guide RNA of the Cas7-11 protein. The crRNA sequence includes a fixed sequence and a binding sequence. The binding sequence is a 25nt sequence that is complementary to the RNA of the pathogen. The binding sequence is located at 3' of the fixed sequence, and its sequence is shown in SEQ ID NO.1.
[0009] SNLuc-Csx30 protein is a hydrolysis substrate of Csx29 protein;
[0010] Furimazine molecule is the catalytic substrate of SNLuc-Csx30 protein.
[0011] Preferably, the molar ratio of Craspase protein-nucleic acid complex, SNLuc-Csx30 protein, and Furimazine molecule is 1:1:1000; the RNA to be tested is 10 -9 ~10 -4 g / μL.
[0012] Preferably, the 5×Craspase buffer comprises 200 mM Tris-HCl at pH 7.5, 30 mM MgCl 2 , 500 mM NaCl, 50% glycerol, 5 mM DTT.
[0013] Preferably, the 5×SNLuc-Csx30 buffer comprises 100 mM Tris-HCl at pH 7.5, 25 mM MgCl 2 , 750 mM KCl, 50% glycerol, 5 mM DTT, and 5 mg / mL BSA.
[0014] The second technical solution adopted by the present invention is:
[0015] A non-diagnostic method for detecting a microbial molecule based on Craspase / SNLuc-Csx30 without amplification is performed using the non-diagnostic method for detecting a microbial molecule based on Craspase / SNLuc-Csx30 without amplification described in any one of the first technical solutions, comprising the following steps:
[0016] Provide Craspase protein-nucleic acid complex, SNLuc-Csx30 protein and Furimazine molecule;
[0017] The Craspase protein-nucleic acid complex and SNLuc-Csx30 protein were mixed in 5×Craspase buffer, incubated at 4°C for 5 minutes, and then the RNA to be tested was added, and incubated at 37°C for 30-60 minutes to obtain a first mixed solution;
[0018] Furimazine was added to 5×SNLuc-Csx30 buffer to obtain a second mixed solution;
[0019] uniformly mixing the first mixed solution and the second mixed solution to obtain a third mixed solution;
[0020] Determining the peak luminescence intensity of the third mixed solution;
[0021] The presence of pathogens and the content of pathogens when pathogens are present are determined according to the peak value of the luminescence intensity of the third mixed solution.
[0022] Compared with the prior art, the present invention has the following beneficial effects:
[0023] 1) During the detection process of the kit of the present invention, there is no need to amplify the nucleic acid of the pathogen, and amplification-free isothermal detection can be performed.
[0024] 2) During the detection process of the kit of the present invention, the output signal is amplified by bioluminescence, which simplifies the operation and result reading.
[0025] 3) The detection method of the present invention has high specificity and is designed to detect pathogenic bacteria RNA, and will hardly produce a positive reaction to other non-target pathogens.
[0026] 4) The detection method of the present invention has high sensitivity, and the minimum detection limit can be as low as 1 ng / μL. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 It is a schematic diagram of the principle of the method for detecting pathogenic bacteria nucleic acid provided by the present invention; the green hook-shaped figure represents the Cas7-11 protein; the blue irregular curved shape represents the Csx29 protein; the red line is the crRNA; the blue line is the pathogenic bacteria RNA; the green irregular ellipse and the blue irregular figure connected by the black line represent SNLuc-Csx30; the gray sphere represents the uncatalyzed Furimazine molecule; the bright blue sphere represents the catalytically generated Furimamide molecule;
[0028] Figure 2 It is the standard curve drawn in Example 2;
[0029] Figure 3 This is the blind test result of wheat stripe rust in Example 3;
[0030] Figure 4 These are the test results of samples infected with wheat stripe rust at different stages in Example 4. DETAILED DESCRIPTION
[0031] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments.It should be understood that these descriptions are exemplary only, and are not intended to limit the scope of the present invention.In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.Unspecified conditions in the embodiments are carried out according to the conditions of normal conditions or manufacturer recommendations.Reagents used or instruments that do not specify manufacturers are conventional products that can be purchased and obtained commercially.
[0032] The principle of the present invention for detecting pathogenic nucleic acid is: a crRNA that can be complementary to the RNA sequence corresponding to the pathogenic ITS sequence is designed. Craspase is a protein-nucleic acid complex composed of Cas7-11 protein, crRNA and Csx29 protein, which mediates the allosteric regulation mechanism by binding to pathogenic RNA to exert the protease function. In the resting and crRNA binding state, the Csx29 protease activity is inhibited, and when the pathogenic RNA containing the unmatched original spacer flanking sequence (PFS) induces the conformational change of the Csx29 domain, the gap between Cas7-11 and Csx29 widens, allowing the Csx30 protein to enter the binding site, and finally triggers the cutting of the Csx30 protein. Split NanoLuc is a 19kDa split luciferase, divided into two fragments, Large Bit and Small Bit. We fused Large Bit and Small Bit to the N-terminus and C-terminus of Csx30 protein, respectively, so that the two fragments can be close to each other and bind to restore luciferase activity, catalyzing the Furimazine molecule to produce a high bioluminescent signal. After the pathogen RNA is recognized, the SNLuc-Csx30 (Split NanoLuc-Csx30) fusion protein will be cut by the activated Craspase, and Large Bit and Small Bit will also separate, unable to continue to maintain the catalytic active center, and the bioluminescent signal will be significantly reduced. Therefore, we can visualize the infection of pathogenic microorganisms through the strength and difference of the bioluminescent signal.
[0033] It should be noted that the RNA of the pathogen to be detected is also referred to as Target RNA below, which is obtained by extracting and diluting the enriched pathogen.
[0034] The first embodiment of the present invention provides a non-amplification microbial molecular detection kit based on Craspase / SNLuc-Csx30, comprising: Craspase protein-nucleic acid complex, SNLuc-Csx30 protein, and Furimazine molecule;
[0035] The Craspase protein-nucleic acid complex is composed of Cas7-11 protein, crRNA and Csx29 protein. The crRNA sequence is the guide RNA of the Cas7-11 protein. The crRNA sequence includes a fixed sequence and a binding sequence. The binding sequence is a 25nt sequence that is complementary to the RNA of the pathogen. The binding sequence is located at 3' of the fixed sequence, and its sequence is shown in SEQ ID NO.1.
[0036] SNLuc-Csx30 protein is a hydrolysis substrate of Csx29 protein;
[0037] Furimazine molecule is the catalytic substrate of SNLuc-Csx30 protein.
[0038] Understandably, the sequence of crRNA varies depending on the pathogen to be detected. Different pathogens contain specific RNA fragments with different genetic information, and the binding site of crRNA is designed based on these specific RNA fragments. The sequence of crRNA is expressed as; 5'-fixed sequence-binding sequence-3'.
[0039] The detection kit of the present invention is suitable for the detection of pathogenic bacteria, and the crRNA sequence is shown in SEQ ID NO.1.
[0040] It should be noted that the process of Furimazine molecules being catalyzed by SNLuc-Csx30 is bioluminescence, which does not require an excitation wavelength to produce bright signals, and the emission wavelength is 400-550nM.
[0041] The second embodiment of the present invention provides a non-diagnostic method for detecting a microbial molecule based on Craspase / SNLuc-Csx30 without amplification, using the Craspase / SNLuc-Csx30-based microbial molecule detection kit described in any one of the first embodiments for detection, comprising the following steps:
[0042] Provide Craspase protein-nucleic acid complex, SNLuc-Csx30 protein and Furimazine molecule;
[0043] The Craspase protein-nucleic acid complex and SNLuc-Csx30 protein were mixed in 5×Craspase buffer, incubated at 4°C for 5 minutes, and then the RNA to be tested was added, and incubated at 37°C for 30-60 minutes to obtain a first mixed solution;
[0044] Furimazine was added to 5×SNLuc-Csx30 buffer to obtain a second mixed solution;
[0045] uniformly mixing the first mixed solution and the second mixed solution to obtain a third mixed solution;
[0046] Determining the peak luminescence intensity of the third mixed solution;
[0047] The presence of pathogens and the content of pathogens when pathogens are present are determined according to the peak value of the luminescence intensity of the third mixed solution.
[0048] In some embodiments, the amplification-free microbial molecular detection method based on Craspase / SNLuc-Csx30 comprises the following steps:
[0049] 1) Craspase and SNLuc-Csx30 were mixed in 5× Craspase buffer with a pH of 7.5 and ultrapure water at a final concentration of 10 nM, incubated at 4°C for 5 min, and then the RNA to be tested was added at 10 -9 ~10 -4 g / μL, incubate at 37°C for 30-60 min to obtain a first mixed solution;
[0050] 2) Adding Furimazine to a final concentration of 10 uM into 5×SNLuc-Csx30 buffer to obtain a second mixed solution;
[0051] 3) uniformly mixing the first mixed solution and the second mixed solution to obtain a third mixed solution;
[0052] 4) Take 30 μL of the third mixed solution in step 3) into a black 384-well plate and read the luminescent signal.
[0053] The standard curve is obtained by adding different amounts of pathogen RNA standards (i.e., Target RNA) and reading the luminescence signal. The luminescence peak value tested by this method using actual sample RNA can be substituted into the standard curve to calculate the mass concentration of the corresponding pathogen, providing a reference for the detection of pathogens.
[0054] Preferably, the 5×Craspase buffer in step 1) is composed of 200 mM Tris-HCl at pH 7.5, 30 mM MgCl 2 , 500 mM NaCl, 50% glycerol, 5 mM DTT.
[0055] Preferably, the 5×SNLuc-Csx30 buffer in step 2) is composed of 100 mM Tris-HCl at pH 7.5, 25 mM MgCl 2 , 750 mM KCl, 50% glycerol, 5 mM DTT, and 5 mg / mL BSA.
[0056] Preferably, the third mixed solution in step 3) is obtained by mixing the first mixed solution and the second mixed solution in a ratio of 2:1. Specifically, the first mixed solution and the second mixed solution are 20 μL and 10 μL, respectively.
[0057] In order to make the technical solution of the present invention clearer, a non-amplification microbial molecular detection method, a detection kit, and a detection effect based on Craspase / SNLuc-Csx30 are described in detail through multiple specific embodiments below.
[0058] The sequences involved in the following examples are shown in Table 1. The Target RNA sequence is a sequence selected from the RNA sequence corresponding to the ITS sequence of wheat stripe rust. The crRNA sequence is the guide RNA of the Cas7-11 protein, and the crRNA sequence includes a fixed sequence and a binding sequence, and the binding sequence is a 25nt sequence that is complementary to the Target RNA of wheat stripe rust.
[0059] Table 1 Related sequences
[0060]
[0061] Example 1 Preparation of RNA from wheat stripe rust
[0062] In this embodiment, the steps for preparing the RNA solution of wheat stripe rust are as follows:
[0063] Target RNA was extracted using TRIzol reagent. 1 mL of the solution was added to the fungus and incubated at room temperature for 5 min. Then, 200 μL of chloroform was added, shaken for 15 s, and centrifuged at 12,000 × g for 15 min at 4 °C. The aqueous phase was transferred to a 1.5 mL centrifuge tube. Then, 500 μL of isoamyl alcohol was added to the tube, incubated at room temperature for 10 min, and centrifuged at 12,000 × g for 5 min at 4 °C. Washed with 1 mL of 70% ethanol, resuspended in 50 μL of H 2 In O.
[0064] Example 2 Drawing a standard curve
[0065] In this embodiment, the standard curve solution is drawn, and the steps are as follows:
[0066] 1) Prepare wheat stripe rust RNA standard solution
[0067] Use RNA solvent to prepare 2 μL of wheat stripe rust RNA standard solutions with concentrations of 0 ng / μL, 1 ng / μL, 10 ng / μL, 100 ng / μL, 1 μg / μL, 20 μg / μL, 40 μ / μL, 60 μg / μL, 80 μg / μL, and 100 μg / μL respectively.
[0068] 2) Draw a standard curve
[0069] ① Take 4 μL of 5× Craspase buffer with a pH of 7.5 and add 10 μL of ddHO 2 O, 2 μL Craspase and 2 μL SNLuc-Csx30 were added to the mixture, and the mixture was incubated at 4° C. for 5 min. Then, 2 μL RNA solvent was added, and the mixture was incubated at 37° C. for 30 to 60 min to obtain a first mixed solution.
[0070] ② Take 4 μL of Furimazine and add 6 μL of 5×SNLuc-Csx30 buffer with a pH value of 7.5 and shake well to obtain the second mixed solution;
[0071] ③ Evenly mix the first mixed solution and the second mixed solution to obtain a third mixed solution; take 30 μL of the second mixed solution, measure its luminescence intensity, and record the peak luminescence intensity corresponding to the wheat stripe rust RNA standard solution;
[0072] ④ Use wheat stripe rust RNA standard solutions with wheat stripe rust RNA concentrations of 0 ng / μL, 1 ng / μL, 10 ng / μL, 100 ng / μL, 1 μg / μL, 20 μg / μL, 40 μ / μL, 60 μg / μL, 80 μg / μL, and 100 μg / μL to replace the RNA solvent in step ①, repeat steps ①-③ to obtain a series of luminescence intensity peaks of wheat stripe rust RNA standard solutions, and draw a standard curve with the mass concentration of wheat stripe rust RNA as the abscissa and the luminescence intensity peak corresponding to the wheat stripe rust RNA standard solution as the ordinate. The regression equation of the fitted standard curve is y=-6778.88x+65994.49, R 2 =0.98, where y represents the luminescence intensity and x represents the RNA concentration of wheat stripe rust. The standard curve is as follows Figure 2 shown.
[0073] Example 3 Blind test of actual samples
[0074] In this embodiment, wheat leaves were ground to extract RNA and tested, and the steps were as follows:
[0075] 1) Take a number of 40 portions of wheat leaves from different sources, freeze the leaves (cut into about 2-3 cm in length) and make them into powders, and prepare wheat stripe rust RNA according to Example 1, which are successively recorded as sample solutions 1# to 40#.
[0076] 2) Wheat sample solution detection
[0077] ① Obtained by qPCR, where 1#-20# are negative sample solutions not infected with wheat stripe rust, and 21#-40# are positive samples infected with wheat stripe rust. The numbers of these 40 samples are hidden and then the order is shuffled;
[0078] ② Take 4 μL of 5× Craspase buffer with a pH of 7.5 and add 10 μL of ddHO 2 O, add 2 μL Craspase and 2 μL SNLuc-Csx30, mix, incubate at 4°C for 5 min, add 2 μL RNA solvent, incubate at 37°C for 30-60 min, to obtain a first mixed solution;
[0079] ③ Take 4 μL of Furimazine and add 6 μL of 5×SNLuc-Csx30 buffer with a pH value of 7.5 and shake well to obtain the second mixed solution;
[0080] ④ Evenly mix the first mixed solution and the second mixed solution to obtain a third mixed solution; take 30 μL of the second mixed solution, measure its luminescence intensity, and record the peak luminescence intensity corresponding to the wheat stripe rust RNA sample solution;
[0081] ⑤ Use wheat stripe rust RNA 1#~40# sample solutions to replace the RNA solvent in step ②, repeat steps ②-④, and obtain a series of luminescence intensity peaks of wheat stripe rust RNA sample solutions. Substitute the data into the standard curve and calculate the content of wheat stripe rust in 1#~40# sample solutions. Figure 3 As shown, if the luminescence data value is lower, the smaller it is than the luminescence signal value of the added RNA solvent, it means that the wheat stripe rust RNA content in the wheat sample solution is higher; if the data value is higher, the closer it is to the luminescence signal value of the added RNA solvent, it means that the wheat stripe rust RNA content in the wheat leaves is lower.
[0082] Example 4 Detection of samples infected with wheat stripe rust at different stages
[0083] In this example, wheat leaves infected with wheat stripe rust at different stages were ground to extract RNA and tested, and the steps were as follows:
[0084] 1) Wheat leaves infected with wheat stripe rust at different stages were taken, the leaves (cut into about 2-3 cm in length) were frozen and made into powder, and wheat stripe rust RNA was prepared according to Example 1, and sample solutions 1# to 14# were recorded according to the number of days of infection.
[0085] 2) Wheat sample solution detection
[0086] ① Obtained by qPCR, where 1#-3# are negative sample solutions that cannot detect wheat stripe rust, and 3#-14# are positive samples that can detect wheat stripe rust infection. As the number of infection days increases, the detected wheat stripe rust content is higher. Hide the numbers of these 10 samples and then shuffle the order;
[0087] ② Take 4 μL of 5× Craspase buffer with a pH of 7.5 and add 10 μL of ddHO 2 O, add 2 μL Craspase and 2 μL SNLuc-Csx30, mix, incubate at 4°C for 5 min, add 2 μL RNA solvent, incubate at 37°C for 30-60 min, to obtain a first mixed solution;
[0088] ③ Take 4 μL of Furimazine and add 6 μL of 5×SNLuc-Csx30 buffer with a pH value of 7.5 and shake well to obtain the second mixed solution;
[0089] ④ Evenly mix the first mixed solution and the second mixed solution to obtain a third mixed solution; take 30 μL of the second mixed solution, measure its luminescence intensity, and record the peak luminescence intensity corresponding to the wheat stripe rust RNA sample solution;
[0090] ⑤ Use wheat stripe rust RNA 1#~10# sample solutions to replace the RNA solvent in step ②, repeat steps ②-④, and obtain a series of luminescence intensity peaks of wheat stripe rust RNA sample solutions. Substitute the data into the standard curve and calculate the content of wheat stripe rust in 1#~10# sample solutions. Figure 4 As shown, the luminescence signal in the 1#-3# sample solution did not decrease significantly, indicating that the content of wheat stripe rust was too low to be detected. The luminescence signal in the 4# sample solution decreased significantly, indicating that a small amount of wheat stripe rust was detected on the fourth day. The luminescence signal in the 4#-6# samples gradually decreased, indicating that the content of wheat stripe rust in the sample solution was gradually increasing.
[0091] In summary, the pathogen RNA detection method of the present invention has high sensitivity and specificity, can be used to detect pathogens, and is expected to be used for actual pathogen infection detection, with good application prospects.
[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A non-amplification microbial molecular detection kit based on Craspase / SNLuc-Csx30, characterized in that: Contains: Craspase protein-nucleic acid complex, SNLuc-Csx30 protein, Furimazine molecule; The Craspase protein-nucleic acid complex is composed of Cas7-11 protein, crRNA and Csx29 protein. The crRNA sequence is the guide RNA of the Cas7-11 protein. The crRNA sequence includes a fixed sequence and a binding sequence. The binding sequence is a 25nt sequence that is complementary to the RNA of the pathogen. The binding sequence is located at 3' of the fixed sequence, and its sequence is shown in SEQ ID NO.
1. SNLuc-Csx30 protein is a hydrolysis substrate of Csx29 protein; Furimazine molecule is the catalytic substrate of SNLuc-Csx30 protein.
2. The amplification-free microbial molecular detection kit based on Craspase / SNLuc-Csx30 according to claim 1, characterized in that: The molar ratio of Craspase protein-nucleic acid complex, SNLuc-Csx30 protein, and Furimazine molecule was 1:1:1000; the RNA to be tested was 10 -9 ~10 -4 g / μL.
3. The amplification-free microbial molecular detection kit based on Craspase / SNLuc-Csx30 according to claim 1, characterized in that: Contains 5×Craspase buffer, consisting of 200 mM Tris-HCl, 30 mM MgCl2, 500 mM NaCl, 50% glycerol, and 5 mM DTT at pH 7.
5.
4. The amplification-free microbial molecular detection kit based on Craspase / SNLuc-Csx30 according to claim 1, characterized in that: Contains 5×SNLuc-Csx30 buffer, composed of 100 mM Tris-HCl, pH=7.5, 25 mM MgCl2, 750 mM KCl, 50% glycerol, 5 mM DTT, 5 mg / mL BSA.
5. A non-diagnostic microbial molecular detection kit based on Craspase / SNLuc-Csx30, characterized in that: The detection is performed using a non-amplification microbial molecular detection kit based on CraspaseSNLuc-Csx30 as described in any one of claims 1 to 3, comprising the following steps: Provide Craspase protein-nucleic acid complex, SNLuc-Csx30 protein and Furimazine molecule; The Craspase protein-nucleic acid complex and SNLuc-Csx30 protein were mixed in 5×Craspase buffer, incubated at 4°C for 5 minutes, and then the RNA to be tested was added, and incubated at 37°C for 30-60 minutes to obtain a first mixed solution; Furimazine was added to 5×SNLuc-Csx30 buffer to obtain a second mixed solution; uniformly mixing the first mixed solution and the second mixed solution to obtain a third mixed solution; Determining the peak luminescence intensity of the third mixed solution; The presence of pathogens and the content of pathogens when pathogens are present are determined based on the peak luminescence intensity of the third mixed solution.