Sequence combination for detecting total amount of bacteria based on crisper / cas12a system and application

Abstract

The present application relates to the technical field of microorganism detection, and in particular to a sequence combination for detecting total amount of bacteria based on a CRISPR / Cas12a system and application, the sequence combination comprising at least one of crRNAs shown in SEQ ID NO: 1-3.The present application also provides a corresponding detection method, which realizes the detection of total amount of bacteria based on the CRISPR / Cas12a system for the first time, and provides a good technical means for the quantitative detection of total amount of bacteria.The detection method based on the CRISPR / Cas12a system provided by the present application is completed under isothermal conditions, does not require professional instruments and equipment, can be quickly and simply detected, and has good sensitivity.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, specifically to a sequence combination and its application for detecting total bacterial count based on the CRISPR / Cas12a system. Background Technology

[0002] With the increasing demands for microbial contamination control in fields such as environmental monitoring, food safety, and clinical diagnosis, rapid and accurate detection of total bacterial count has become a common industry requirement. Traditional culture methods are limited by their long processing time (≥24 h), low sensitivity (≥10² CFU / mL), and the "missed detection" of unculturable bacteria, making it difficult to meet the needs of rapid on-site screening. Although polymerase chain reaction (PCR) and its derivative technologies have improved in terms of sensitivity, they rely on temperature-controlled cycling instruments, and the false positive rate caused by non-specific primer amplification remains high, with a single detection cycle still ≥2 h.

[0003] In recent years, the CRISPR / Cas12a collateral cleavage system has been proven to amplify single-copy-level nucleic acid signals under isothermal conditions. However, existing research has largely focused on the qualitative detection of single or a few pathogenic bacteria, lacking a universal quantitative detection solution for total bacterial count. Therefore, how to construct a rapid method for total bacterial count detection based on the high specificity of Cas12a recognition and collateral cleavage activity, which requires no culture, requires no temperature variation, and is resistant to interference from complex sample backgrounds, has become a pressing technical challenge in the field of microbial detection. Summary of the Invention

[0004] The purpose of this invention is to provide a sequence combination and its application for detecting total bacterial count based on the CRISPR / Cas12a system, which has good sensitivity when used to detect total bacterial count.

[0005] Therefore, in a first aspect, the present invention provides a sequence combination for detecting total bacterial count based on a CRISPR / Cas12a system, comprising at least one of the following crRNAs: crRNA1, the sequence of which is shown in SEQ ID NO: 1; crRNA2, the sequence of which is shown in SEQ ID NO: 2 crRNA3, the sequence of which is shown in SEQ ID NO: 3.

[0006] In some embodiments, the sequence combination includes two or more of crRNA1, crRNA2, and crRNA3.

[0007] In some embodiments, the sequence combination further includes a signaling reporter molecule.

[0008] In some embodiments, the signal reporting molecule is labeled with a fluorescent reporter group and a fluorescent quencher group at both ends.

[0009] In some embodiments, the signal reporting molecule includes ssDNAreporter-FAM for detecting bacteria using a quantitative real-time RCP instrument, having the nucleotide sequence SEQ ID NO: 7 (TTATT) and labeled with FAM at the 5' end and BHQ1 at the 3' end.

[0010] In some embodiments, the nucleotide sequence of the signal reporter molecule includes SEQ ID NO: 8, and is labeled FAM at the 5' end and BHQ1 at the 3' end.

[0011] A second aspect of the present invention provides a kit for detecting total bacterial count, comprising a CRISPR / Cas12a detection reagent and the sequence combination described in the first aspect of the present invention.

[0012] In some embodiments, the CRISPR / Cas12a detection reagent includes a Cas12a enzyme, such as the LbCas12a enzyme.

[0013] A third aspect of the invention provides the application of the sequence combination or the kit for detecting total bacterial count in the detection or classification of total bacteria.

[0014] A fourth aspect of the present invention provides a method for detecting total bacterial count, comprising the following steps: Step 1: Provide the sample to be tested; Step 2: Extract genomic DNA from the bacteria in the sample to be tested, as a template for testing; Step 3: Mix the template to be tested obtained in Step 2 with a reaction system containing a CRISPR / Cas12a system, wherein the system contains the Cas12a protein and the sequence combination described in the first aspect of the present invention; Step 4: Incubate the reaction system to form the Cas12a / crRNA complex. If the target DNA is present, the trans-cleavage activity of Cas12a will be activated. Step 5: Detect the products of the reaction system to determine whether bacteria are present in the sample to be tested.

[0015] In some embodiments, the sequence combination includes a signal reporter molecule, and in step 5, the signal of the signal reporter molecule in the reaction system is detected; if the signal is present, it indicates the presence of bacteria.

[0016] In some embodiments, the reaction system includes the following components: Cleavage Buffer, LbCas12a enzyme, crRNA, signal reporter molecule, and template to be tested.

[0017] In some embodiments, the concentration of the LbCas12a enzyme in the reaction system is 25 nM to 300 nM, optionally 25 nM to 200 nM; and / or, The concentration of the crRNA is 50 nM to 200 nM, optionally 50 nM to 75 nM; and / or, The concentration of the signal reporting molecule is 100 nM to 1000 nM, and can be selected as 250 nM to 500 nM.

[0018] In some embodiments, the molar ratio of the LbCas12a enzyme to the crRNA is 1:(1~2).

[0019] In most cases, a molar ratio of LbCas12a enzyme to crRNA of approximately 1:1 is sufficient for detection. In some embodiments, when the crRNA utilization efficiency is low, the molar ratio of crRNA can be appropriately increased to ensure complete cleavage of the target dsDNA in the template to be tested. For example, the molar ratio of LbCas12a enzyme to crRNA can be 1:(1.25–2).

[0020] In some embodiments, the incubation conditions include reacting at 37°C ± 2°C for 15 min to 80 min.

[0021] In some embodiments, the bacteria that the method can detect include at least one of the genera Arthrobacter, Bacillus, and Animalococcus.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes CRISPR / Cas12a detection technology to achieve rapid and visual detection of bacteria. This technology is simple to operate, can be performed under isothermal conditions, requires no expensive instruments, and has a short detection time. The sequence combination provided by this invention exhibits good sensitivity when used to detect total bacterial count and shows promising application prospects. Attached Figure Description

[0023] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. In the drawings: Figure 1 Results of bacterial colony morphology and species identification; In this figure, A and D are Arthrobacter and Agrobacterium, respectively, and B, C, E, F, and G belong to Bacillus. Figure 2 Detection results for different strains using different primers and CRISPR / Cas12a; Among them, AF represents the fluorescence signal detection results of different strains of primer pairs uni1 to uni6; bac1 represents Arthrobacter, bac2, bac4, bac6, bac7 and bac9 represent Bacillus, and bac5 represents Planococcus. Figure 3 The detection results (fluorescence intensity is the result of sample fluorescence intensity minus baseline) are optimized for each component in the CRISPR / Cas12a detection system. The bar charts corresponding to the same horizontal axis are, from left to right: long probe sample group, long probe blank group, short probe sample group, and short probe blank group. Figure 4 The sensitivity test results of the CRISPR / Cas12a detection system when using uni1 and uni2; Among them, A and B are the fluorescence signal detection results of the CRISPR / Cas12a system for the CRISPR / Cas12a system with the CRISPR / Cas12a system based on the CRISPR / Cas12a DNA dilution gradient of primers uni1 to uni2 (the fluorescence intensity is the result after subtracting the baseline from the sample fluorescence intensity). Figure 5 The sensitivity detection results of the CRISPR / Cas12a detection system when using uni3 (fluorescence intensity is the result of sample fluorescence intensity minus baseline) are shown in a set of bars on the same horizontal axis, from left to right: long probe sample group, long probe blank group, short probe sample group, and short probe blank group. Figure 6 The graph shows the change in fluorescence intensity over reaction time when the CRISPR / Cas12a detection system of this invention is used for detection. Detailed Implementation

[0024] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

[0025] Example 1 This embodiment provides a CRISPR-Cas12a detection system and tests the universality of the designed crRNA sequence.

[0026] 1. Design of universal bacterial crRNA 16S rRNA gene sequences of eubacteria (including Gram-positive bacteria, Gram-negative bacteria, and actinomycetes) were downloaded from NCBI. Multiple alignments of the downloaded sequences were performed using MEGA, and the sequences were also compared with the dataset provided in the literature “Wang Y, Qian PY. Conservative Fragments in Bacterial 16S rRNA Genes and Primer Design for 16SRibosomal DNA Amplicons in Metagenomic Studies [J]. PLOS ONE, 2009, 4(10):e7401.”. Conserved regions were identified from the alignment results, and six crRNA sequences were designed. The specific crRNA sequences are shown in Table 1.

[0027] Table 1

[0028] II. Screening crRNA primer sequences Bacterial culture, purification, and DNA extraction: Air samples were collected from the air environment for 5 minutes using a staged air sampler. Bacteria were then cultured and purified using solid culture media until only one type of colony appeared on the media, followed by a second purification. The final purified solid culture medium is shown below. Figure 1 As shown. After purification, the bacteria were cultured in liquid medium to expand their size. The resulting bacterial culture was then prepared into a bacterial suspension. Genomic DNA was extracted using the kit to obtain the DNA sample of the bacteria to be tested, isolated from the environment. Specific procedures were performed according to the kit instructions.

[0029] Primer screening: The extracted bacterial DNA samples were added to the CRISPR / Cas12a system. The CRISPR / Cas12a fluorescence detection system is shown in Table 2. The crRNAs were prepared using the primer sequences shown in Table 1 to evaluate the universality of different crRNAs. The reagents used were TOLOBIO's LbCas12a nuclease and matching reagents.

[0030] Table 2 CRISPR / Cas12a fluorescence detection reaction system

[0031] In use, the CRISPR / Cas12a fluorescence detection system was mixed and reacted at 37℃ for 80 min, and the fluorescence signal was read. The results are shown in [Figure number missing]. Figure 2 .

[0032] Depend on Figure 2It was found that the fluorescence signals of the seven strains measured by the CRISPR / Cas12a system corresponding to different crRNA primers were all higher than those of the corresponding control group, indicating that all six crRNA primers could detect different bacterial species. Among them, the fluorescence signal intensity of uni1, uni2, and uni3 was more significant than that of uni4, uni5, and uni6. Therefore, primers uni1, uni2, and uni3 were selected for subsequent experiments. Based on the fluorescence signal trends of uni1, uni2, and uni3, they were ordered according to priority, with the serial numbers 3, 1, and 2 respectively.

[0033] Example 2 This embodiment optimizes the component concentrations of the CRISPR / Cas12a detection system. In addition to using ssDNAReporter-FAM (hereinafter referred to as the short probe) as a signal reporter molecule for detection, 5'-FAM-CTCTCATTTTTTTTTTAGAGAG-BHQ1-3' (nucleotide sequence SEQ ID NO: 8, hereinafter referred to as the long probe) was also used as a signal reporter molecule for detection.

[0034] 1. LbCas12a enzyme concentration The concentration gradients of LbCas12a enzyme were set to 25 nM, 50 nM, 100 nM, 200 nM and 300 nM, with uni3 as crRNA, and CRISPR / Cas12a reaction was performed according to the detection reaction system (total volume of 30 µL) shown in Table 3.

[0035] Table 3 CRISPR / Cas12a detection reaction system

[0036] The obtained fluorescence detection results are as follows Figure 3 As shown, LbCas12a enzyme concentrations in the range of 25 nM to 300 nM all exhibit good detection performance, with the preferred concentration being 25 nM to 200 nM, and the optimal concentration being 50 nM.

[0037] 2. crRNA concentration Using uni3 as crRNA, the crRNA concentration gradients were set to 50 nM, 75 nM, 100 nM, 125 nM and 150 nM. CRISPR / Cas12a reactions were performed according to the detection reaction system (total volume of 30 µL) shown in Table 4.

[0038] Table 4 CRISPR / Cas12a detection reaction system

[0039] The obtained fluorescence detection results are as follows Figure 3 As shown, good detection results can be obtained when the crRNA concentration is between 50 nM and 150 nM. Among them, the optimal crRNA concentration for the corresponding system is 50 nM when using a long probe, and 75 nM when using a short probe.

[0040] 3. Signal reporting molecular concentration For long and short probes, concentration gradients of 100 nM, 250 nM, 500 nM, 750 nM and 1000 nM were set, respectively. uni3 was used as crRNA, and CRISPR / Cas12a reactions were performed according to the detection reaction system (total volume 30 µL) shown in Table 5.

[0041] Table 5 CRISPR / Cas12a detection reaction system

[0042] The obtained fluorescence detection results are as follows Figure 3 As shown, good detection results can be achieved when the concentration of the signal reporting molecule is between 100 nM and 1000 nM. Among them, the optimal concentration is 250 nM when using a long probe and 500 nM when using a short probe.

[0043] 4. Different crRNA combinations Seven different crRNA combinations were set up, as shown in Table 6. A checkmark indicates that the combination contains the corresponding crRNA. Uni3 was used as the crRNA, and the reaction system is shown in Table 7.

[0044] Table 6 Different crRNA combinations

[0045] Table 7 Components of the CRISPR / Cas12a system

[0046] The obtained fluorescence detection results are as follows Figure 3 As shown in the figure, when using long probes, the effects of different crRNA combinations in the corresponding system, ranked from best to worst, are 7>5>6>4>1>3>2; when using short probes, the effects of different crRNA combinations in the corresponding system, ranked from best to worst, are 7>5>6>4>3>2>1. It can be seen that the detection effect is better when using combinations of two or more crRNAs simultaneously.

[0047] Example 3 This embodiment analyzes the sensitivity of the CRISPR / Cas12a detection system, using uni1 and uni2 crRNAs.

[0048] The DNA concentration of Bacillus cereus (57.6 ng / μL) was serially diluted 10-fold, for a total of 9 dilutions, at 10-fold increments. 0 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 The DNA template concentrations were 57.09 ng / µL, 5.709 ng / µL, 570.9 pg / µL, 57.09 pg / µL, 5.709 pg / µL, 0.5709 pg / µL, 57.09 fg / µL, 5.709 fg / µL, and 0.5709 fg / µL, respectively. Sensitivity was measured using the CRISPR-Cas12a detection systems corresponding to uni1 and uni2 in Table 1, and the results are shown in [Table 1]. Figure 4 .

[0049] Depend on Figure 4 It can be seen that when the dilution factor is 10... 0 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 At any time, a certain fluorescence intensity can be detected. Figure 4 In A, 10 0 10 -1 The fluorescence curves of the uni1 sample showed significant differences from those of the control group, indicating that the sensitivity of the CRISPR / Cas12a system corresponding to uni1 was 5.709 ng / μL. Figure 4 In B, 10 0 10 -1 10 -2 The corresponding fluorescence curves showed significant differences from the control group, indicating that the sensitivity of the CRISPR / Cas12a system corresponding to uni2 was 570.9 pg / μL.

[0050] Example 4 This embodiment analyzes the sensitivity of the CRISPR / Cas12a detection system, using uni3 crRNA.

[0051] The DNA concentration of Bacillus cereus (273.67 ng / µL) was serially diluted 10-fold, for a total of 8 dilutions, at 10-fold increments. 0 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 The DNA template concentrations were 273.67 ng / µL, 27.367 ng / µL, 2.737 ng / µL, 273.7 pg / µL, 27.37 pg / µL, 2.737 pg / µL, 0.2737 pg / µL, and 27.37 fg / µL, respectively. Sensitivity was measured using the CRISPR / Cas12a detection system shown in Table 7. The results are shown below. Figure 5 As shown.

[0052] Depend on Figure 5 From A, we know that the dilution factor is 10. 0 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 At any given time, they all exhibit a certain fluorescence intensity. Figure 5 In B, gradient 10 0 10 -1 The fluorescence results showed significant differences from the control group, with a gradient of 10. 0 10 -1 10 -2 10 -3 10 -4 10 -5 The difference between the fluorescence values ​​of the sample and the blank was positive, indicating that the sensitivity of the CRISPR / Cas12a system corresponding to uni3 was 2.737 pg / µL.

[0053] Depend on Figure 6 It can be seen that the fluorescence signals of the reaction system are clearly distinguishable after 15 min of reaction, indicating that the signals can be distinguished after a reaction time of 15 min.

[0054] It should be noted that the sensitive DNA concentration of the bacteria used in this embodiment is lower than the DNA concentration of actual environmental samples. Therefore, the above results indicate that the detection system provided by this invention has high sensitivity and can meet the needs of practical detection.

[0055] The foregoing description of specific exemplary embodiments of the present invention is not intended to limit the invention to the precise forms disclosed. The exemplary embodiments were chosen and described to explain the specific principles of the invention and its practical applications, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments and various different choices and modifications of the invention. However, the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A sequence combination for detecting total bacterial count based on the CRISPR / Cas12a system, characterized in that, Including at least one of the following crRNAs, crRNA1, the sequence of which is shown in SEQ ID NO: 1; crRNA2, the sequence of which is shown in SEQ ID NO: 2 crRNA3, the sequence of which is shown in SEQ ID NO:

3.

2. The sequence combination as described in claim 1, characterized in that, This includes combinations of two or more of crRNA1, crRNA2, and crRNA3.

3. The sequence combination as described in claim 2, characterized in that, The sequence combination also includes a signal reporter molecule; Preferably, the signal reporter molecule is labeled with a fluorescent reporter group and a fluorescent quencher group at both ends.

4. The sequence combination as described in claim 2 or 3, characterized in that, The signal reporter molecule includes an ssDNA reporter-FAM for the detection of bacteria using a quantitative real-time RCP instrument, with the nucleotide sequence SEQ ID NO: 7, and labeled with FAM at the 5' end and BHQ1 at the 3' end; or, The nucleotide sequence of the signal reporter molecule includes SEQ ID NO: 8, and is labeled with FAM at the 5' end and BHQ1 at the 3' end.

5. A kit for detecting total bacterial count, characterized in that, Includes CRISPR / Cas12a detection reagents and the sequence combinations of any one of claims 1 to 4.

6. The reagent kit as described in claim 5, characterized in that, The CRISPR / Cas12a detection reagent includes the Cas12a enzyme, preferably the LbCas12a enzyme.

7. The use of the sequence combination of any one of claims 1 to 4 or the kit of any one of claims 5 to 6 in the detection or classification of total bacteria.

8. A method for detecting bacteria, characterized in that, Includes the following steps: Step 1: Provide the sample to be tested; Step 2: Extract genomic DNA from the bacteria in the sample to be tested, as a template for testing; Step 3: Mix the template to be tested obtained in Step 2 with a reaction system containing a CRISPR / Cas12a system, wherein the system contains the Cas12a protein and the sequence combination described in the first aspect of the present invention; Step 4: Incubate the reaction system to form the Cas12a / crRNA complex. If the target DNA is present, the trans-cleavage activity of Cas12a will be activated. Step 5: Detect the products of the reaction system to determine whether bacteria are present in the sample to be tested.

9. The method as described in claim 8, characterized in that, The sequence combination includes a signal reporter molecule; in step 5, the signal of the signal reporter molecule in the reaction system is detected, and if the signal is present, it indicates the presence of bacteria.

10. The method as described in claim 8, characterized in that, The reaction system comprises the following components: Cleavage Buffer, LbCas12a enzyme, crRNA, signal reporter molecule, and template to be tested; Preferably, in the reaction system, the concentration of the LbCas12a enzyme is 25 nM to 300 nM, optionally 25 nM to 200 nM; and / or, The concentration of the crRNA is 50 nM to 200 nM, and can be selected as 50 nM to 75 nM; And / or, The concentration of the signal reporting molecule is 100 nM to 1000 nM, and can be selected as 250 nM to 500 nM; Preferably, in the reaction system, the molar ratio of the LbCas12a enzyme to the crRNA is 1:(1~2), and can be selected as 1:(1.25~2). Preferably, the incubation conditions include reacting at 37℃±2℃ for 15 min to 80 min.

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