One-pot method for detecting e. coli: clustering of regular inter-palinous repeat sequences

Abstract

This article discloses a method for detecting enteroagglutinating Escherichia coli (EAEC) in a sample. The method comprises: (i) extracting genomic DNA from an EAEC in a sample; (ii) mixing a recombinase polymerase amplification (RPA) premix containing a forward and reverse primer with a CRISPR / Cas premix containing a cluster-associated intertemporal palindromic repeat (CRISPR / Cas) protein, a CRISPR ribonucleic acid (crRNA), a fluorescent reporter, MnSO4, and dithiothreitol (DTT) to prepare a composition, wherein the crRNA specifically targets the aggR gene fragment of an EAEC; (iii) contacting the genomic DNA extracted in step (i) with the composition of step (ii) to amplify the extracted genomic DNA and initiate CRISPR detection in situ; and (iv) measuring a fluorescence signal emitted by the fluorescent reporter of the product of step (iii), wherein an increase in fluorescence signal compared to a control group indicates the presence of EAEC in the sample, wherein the control group is a sample without EAEC.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202610169339.7 (22) Application Date 2026.02.05 (30) Priority Data 63 / 758,868 2025.02.14 US (71) Applicant City University of Hong Kong Address Tat Chee Road, Kowloon Tong, Hong Kong, China (72) Inventors Lin Zhiquan Liu Xiaowei Guo Junjie Li Junhan (74) Patent Agency Beijing Ruibang Intellectual Property Agency (General Partnership) 11481 Patent Attorney Xu Dingfeng Fu Weijia (51) Int.Cl. C12Q 1 / 689 (2018.01) C12Q 1 / 6844 (2018.01) C12N 15 / 11 (2006.01) C12R 1 / 19 (2006.01) (54) Invention Title: Method for Detecting One-Pot Inter-Peripheral Palindromic Repeat Sequence Clusters in Escherichia coli (57) Abstract: This paper discloses a method for detecting enteroaggregative Escherichia coli (EAEC) in a sample. The method comprises: (i) extracting genomic DNA from an EAEC in a sample; (ii) mixing a recombinase polymerase amplification (RPA) premix containing a forward and reverse primer with a CRISPR / Cas premix containing a cluster-associated intertemporal palindromic repeat (CRISPR / Cas) protein, a CRISPR ribonucleic acid (crRNA), a fluorescent reporter, MnSO4, and dithiothreitol (DTT) to prepare a composition, wherein the crRNA specifically targets the aggR gene fragment of an EAEC; (iii) contacting the genomic DNA extracted in step (i) with the composition of step (ii) to amplify the extracted genomic DNA and initiate CRISPR detection in situ; and (iv) measuring a fluorescence signal emitted by the fluorescent reporter of the product of step (iii), wherein an increase in fluorescence signal compared to a control group indicates the presence of EAEC in the sample, wherein the control group is a sample without EAEC. Claims (2 pages), Description (11 pages), Sequence Listing (electronic publication), Drawings (3 pages) CN 122564140 A 2026.08.14 CN 1 22 56 41 40 A 1. A method for detecting enteroagglutinating Escherichia coli (EAEC) in a sample, comprising: (i) extracting genomic DNA from EAEC in the sample; (ii) mixing a recombinase polymerase amplification (RPA) premix with a cluster association of palindromic repeats (CRISPR / Cas) premix to prepare a composition, wherein the RPA...The premix contains a forward primer with sequence number 11 and a reverse primer with sequence number 12. The CRISPR / Cas premix contains a CRISPR / Cas protein, a CRISPR ribonucleic acid (crRNA), a fluorescent reporter, manganese sulfate (MnSO4), and dithiothreitol (DTT), wherein the crRNA specifically targets the aggR gene fragment of EAEC and has a ribonucleic acid sequence with sequence numbers 1 to 10; (iii) contacting the genomic DNA extracted in step (i) with the composition of step (ii) to amplify the extracted genomic DNA and initiate CRISPR detection in situ; and (iv) measuring a fluorescent signal emitted by the CRISPR product of step (iii); wherein, in step (ii), the DTT content in the CRISPR / Cas premix is ​​at least 10 times the MnSO4 content; in step (iii) The in situ amplification and detection system is performed at 39°C for 30 minutes; and in step (iv), an increase in fluorescence signal compared to the control group indicates the presence of EAEC in the sample, wherein the control group is a sample without EAEC. 2. The method of claim 1, wherein in step (ii), the RPA premix further comprises a reaction buffer, deoxynucleoside triphosphates (dNTPs), and magnesium acetate. 3. The method of claim 2, wherein the concentration of magnesium acetate in the RPA premix is ​​30 mM. 4. The method of claim 1, wherein in step (ii), the crRNA has a ribonucleic acid sequence with sequence number 5, and its concentration in the CRISPR / Cas premix is ​​approximately 0.5 μM. 5. The method of claim 1, wherein in step (ii), the CRISPR / Cas protein is Cas 9, Cas 12a, or Cas 12b. 6. The method of claim 5, wherein the CRISPR / Cas protein is Cas 12a, and its concentration in the CRISPR / Cas premix is ​​approximately 125 nM. 7. The method of claim 6, wherein in step (ii), the fluorescent reporter is an AT-rich DNA reporter with a fluorescent group at its 5' end and a quenching group at its 3' end. 8. The method of claim 7, wherein the fluorescent group is 6-carboxyfluorescein (6-FAM); and the quenching group is black hole quencher 1 (BHQ1). 9. The method of claim 8, wherein the fluorescent reporter is...10. The method of claim 9, wherein the fluorescent reporter is TTATT-5C, TTATT-2T, TTATT-5T, TTATT-10T, TTATT-20T, TTATT-5dU, TTATT-5A, TTATT-5G, TTATT-5rU, or 10C. 11. The method of claim 1, wherein in step (ii), the volume of the RPA premix is ​​10 μL, and the volume of the CRISPR / Cas premix is ​​5 μL. 12. The method of claim 1, wherein the method can detect 1 to 1 × 10⁹ copies of the EAEC aggR gene. Claims 1 / 2 pages 2 CN 122564140 A 13. The method according to claim 1, wherein the sample is any one of blood, serum, oral mucosa, body fluid, tissue, feces, bodily secretions, culture medium, soil or water. Claims 2 / 2 pages 3 CN 122564140 A One-pot method for detecting Escherichia coli palindromic repeat sequence clustering Technical Field

[0001] This invention relates to the field of pathogenic microorganism detection, and more particularly to a method for detecting pathogenic Escherichia coli (E. coli) through a palindromic repeat sequence clustering correlation (CRISPR / Cas) system. Background Art

[0002] Enteroaggregative Escherichia coli (EAEC) is frequently found in wastewater treatment plants worldwide. Some studies have even found that EAEC is the most common type of pathogenic Escherichia coli (17.8% to 46.7%). Despite wastewater treatment processes, EAEC may survive inadequate sterilization and cause infectious disease contamination of the surrounding environment through wastewater discharge. Interestingly, one report indicates that EAEC strains can survive the 55°C high-temperature sterilization step during wastewater treatment by entering a dormant state, demonstrating its versatility. Therefore, treated municipal wastewater discharges from wastewater treatment plants may become a source of EAEC contamination in receiving water bodies such as rivers and oceans. In addition, wastewater containing EAEC may also enter the surrounding environment through wastewater leaks or overflows. Furthermore, EAEC exists in bioaerosols generated from wastewater, which may contribute to the contamination of wastewater.The treatment plant workers and residents of nearby communities pose a potential health hazard. Furthermore, due to increasing water scarcity, the treated wastewater is used for agricultural irrigation, thus exposing product consumers to the risk of E. coli infection.

[0003] In view of the serious consequences of improper municipal wastewater management, there is a need in the relevant field for a monitoring system to detect EAEC in wastewater. Summary of the Invention

[0004] In view of the above needs, this disclosure aims to provide a method for detecting EAEC in samples, particularly a one-pot intertemporal palindromic repeat cluster association (CRISPR / Cas) method capable of detecting 1 to 1 × 10⁹ copies of the EAEC aggR gene in a sample. The method comprises the following steps:

[0005] (i) extracting genomic DNA from the EAEC in the sample;

[0006] (ii) mixing a recombinase polymerase amplification (RPA) premix with a CRISPR / Cas premix to prepare a composition, wherein,

[0007] the RPA premix contains a forward primer with sequence number 11 and a reverse primer with sequence number 12;

[0008] the CRISPR / Cas premix contains a CRISPR / Cas protein, a CRISPR ribonucleic acid (crRNA), a fluorescent reporter, manganese sulfate (MnSO4), and dithiothreitol (DTT), wherein the crRNA specifically targets the aggR gene fragment of EAEC and has a ribonucleic acid sequence with sequence numbers 1 to 10;

[0009] (iii) contacting the genomic DNA extracted in step (i) with that extracted in step (ii). The composition is used to amplify the extracted genomic DNA and initiate CRISPR amplification in situ; and

[0010] (iv) to measure a fluorescence signal emitted by the fluorescent reporter of the product of step (iii); wherein,

[0011] in step (ii), the DTT content in the CRISPR / Cas premix is ​​at least 10 times the MnSO4 content; Specification 1 / 11 page 4 CN 122564140 A

[0012] in step (iii), the in situ amplification and detection system is performed at 39°C for 30 minutes; and

[0013] in step (iv), an increase in fluorescence signal compared to the control group indicates the presence of EAEC in the sample, wherein the control group is a sample without EAEC.

[0014] According to a preferred embodiment of the present disclosure, in step (ii), the crRNA has a ribonucleic acid sequence with sequence number 5 and a concentration of approximately 0.5 μM in the CRISPR / Cas premix.

[0015] According to one embodiment of this disclosure, in step (ii), the RPA premix further comprises a reaction buffer, deoxynucleotide triphosphates (dNTPs), and magnesium acetate. Preferably, the concentration of magnesium acetate in the RPA premix is ​​30 mM.

[0016] Examples of CRISPR / Cas proteins suitable for this method include, but are not limited to, Cas 9, Cas 12a, Cas 12b, etc. According to a preferred embodiment of this disclosure, the CRISPR / Cas protein is Cas 12a, and its concentration in the CRISPR / Cas premix is ​​approximately 125 nM.

[0017] According to one embodiment of this disclosure, the fluorescent reporter is an AT-rich DNA reporter with a fluorescent group (e.g., 6-carboxyfluorescein (6-FAM)) at its 5' end and a quenching group (e.g., black hole quencher 1 (BHQ1)) at its 3' end. Examples of fluorescent reporters suitable for this method include, but are not limited to, TTATT, TTATT-5C, TTATT-2T, TTATT-5T, TTATT-10T, TTATT-20T, TTATT-5dU, TTATT-5A, TTATT-5G, TTATT-5rU, 10C, etc. According to a preferred embodiment of this disclosure, the fluorescent reporter is TTATT-5C, and its concentration in the CRISPR / Cas premix is ​​approximately 5 μM.

[0018] According to a preferred embodiment of the present disclosure, in step (ii), the concentrations of MnSO4 and DTT in the CRISPR / Cas premix are approximately 3 mM and 30 mM, respectively.

[0019] According to an embodiment of the present disclosure, in step (ii), the volume of the RPA premix is ​​10 μL, and the volume of the CRISPR / Cas premix is ​​5 μL.

[0020] According to an embodiment of the present disclosure, the method can detect 1 to 1 × 10⁹ copies of the EAEC aggR gene.

[0021] In all embodiments, the sample is any one of blood, serum, oral mucosa, body fluid, tissue, feces, bodily secretions, culture medium, soil, water, etc.

[0022] After referring to the following embodiments, those skilled in the art will easily understand the basic spirit and other inventive objectives of the present invention, as well as the technical means and implementation methods adopted by the present invention.

[0023] Brief Description of the Drawings: To make the above and other objects, features, advantages and embodiments of the present invention more apparent and understandable, the accompanying drawings are described below:

[0024] FIG1Figure 1 is a flowchart of 100 steps of a method for detecting EAEC in a sample, according to a preferred embodiment of the present disclosure;

[0025] Figure 2 is a linear regression analysis diagram of fluorescence readings in the one-pot system at a 30-minute endpoint, with the aggR gene copy number ranging from 1 to 1×10⁵ copies, according to an embodiment of the present disclosure;

[0026] Figure 3 is a specificity test diagram of the one-pot system for various microorganisms at a 30-minute endpoint, according to an embodiment of the present disclosure;

[0027] Figure 4A is a wastewater addition test analysis result diagram of the one-pot system at a 30-minute endpoint, according to an embodiment of the present disclosure; Specification 2 / 11 pages 5 CN 122564140 A

[0028] Figure 4B is a wastewater addition test RT-qPCR diagram of the one-pot system at a 30-minute endpoint, according to an embodiment of the present disclosure. Analysis results graph; and

[0029] Figure 4C is an analysis graph of wastewater samples collected from two wastewater treatment plants on different dates at a 30-minute endpoint, illustrated according to an embodiment of the present disclosure.

[0030] According to conventional operating practices, the various features and components in the figure are not drawn to scale, but are drawn in a way that best presents the specific features and components related to the present invention. In addition, similar components / parts are referred to by the same or similar component symbols in different figures. Detailed Description

[0031] In order to make the description of the present disclosure more detailed and complete, the following illustrative description is provided with respect to embodiments and specific examples of the present invention; however, this is not the only form of implementing or using the specific examples of the present invention. The embodiments cover features of various specific examples and method steps and their order for constructing and operating these specific examples. However, other specific examples may also be used to achieve the same or equivalent functions and order of steps.

[0032] 1. Definitions

[0033] The term "protospacer adjacent motif (PAM) site" in this document refers to a short DNA sequence that is crucial for guiding Cas proteins to the target site for cleavage. The most well-known example is SpCas9, which recognizes 5'-NGG-3', while other Cas proteins such as Cas12a and SaCas9 recognize different PAMs, such as 5'-TTTV-3' (Cas12a) or 5'-NNGRRT-3' (SaCas9).

[0034] The term "CRISPR RNA" in this document refers to..."crRNA" refers to RNA transcripts derived from CRISPR loci. Mature crRNA transcripts contain a partially conserved repetitive sequence and a spacer sequence complementary to the target DNA. crRNA binds to trans-activating CRISPR RNA (tracrRNA) to form functional guide RNA (gRNA), or may fuse to form single-stranded guide RNA (sgRNA) to guide Cas proteins (e.g., Cas9) to specific DNA sequences, thereby cleaving and inhibiting foreign genetic material, such as viruses. It should be noted that PAM is not present in the crRNA sequence, but must be located downstream of the target site in the genomic DNA.

[0035] The term "introducer" in this document refers to an oligonucleotide that, after forming a double strand with a polynucleotide template, serves as the starting point for nucleic acid synthesis and extends from its 3' end along the template to form an extended double strand. The nucleotide sequence added during the extension process is determined by the sequence of the template polynucleotide. The primer is catalyzed by DNA polymerase, RNA polymerase, or reverse transcriptase, serving as the starting point for nucleic acid synthesis.

[0036] Numerical ranges are disclosed herein, which set an upper limit and a lower limit. Unless otherwise stated, the range includes all values ​​less than or equal to the minimum value (whether the lower or upper limit), and all values ​​between the range values.

[0037] Unless the context clearly specifies otherwise, the singular nouns used herein encompass the plural form of the noun; and the plural nouns used herein also encompass the singular form of the noun.

[0038] Several experimental examples are presented below to illustrate certain aspects of the invention to facilitate practice by those skilled in the art, and these examples should not be construed as limiting the scope of the invention. It is believed that those skilled in the art, upon reading the description presented herein, can fully utilize and practice the invention without excessive interpretation. All published documents cited herein are considered part of this specification in their entirety.

[0039] 2. Method for detecting EAEC using a CRISPR / Cas system

[0040] One example of the method described in this disclosure is the use of a CRISPR / Cas system to detect the aggR gene sequence of EAEC present in a sample (e.g., wastewater) as specified in page 3 / 11 of the manual, CN 122564140 A. This method is characterized by: nucleic acid amplification and detection being performed in situ, and the sensitivity of the system being improved by adding MnSO4 and DTT to the system.

[0041] Please refer to Figure 1, which is a flowchart illustrating the steps for detecting EAEC in a sample. In summary, this method 100The method comprises four stages: Stage 1: Preparation of nucleic acid sample; Stage 2: Preparation of composition for one-pot reaction; Stage 3: Amplification of nucleic acid and initiation of CRISPR detection; and Stage 4: Detection of signal.

[0042] Stage 1: Preparation of nucleic acid sample

[0043] This method 100 begins with the extraction of genomic DNA from EAEC in the sample (step 110) to prepare a nucleic acid sample for subsequent use. Generally, EAEC in the sample is lysed to release its contents, which include DNA, proteins, etc., wherein the cell membrane / cell wall of EAEC can be destroyed by heating, surfactants, enzymes or mechanical force (e.g., grinding, ultrasonic treatment, etc.). The lysate is then purified, wherein proteins are removed by proteases (e.g., proteinase K), salts (e.g., NaCl), etc., and the nucleic acid in the lysate is precipitated with ethanol or isopropanol.

[0044] According to one embodiment of this disclosure, the sample containing EAEC is first treated with a lysis buffer (e.g., ASL buffer) and heated at 95°C for 5 minutes to increase DNA yield and remove any potential PCR inhibitors; and the DNA is extracted by proteinase K, followed by heating at 95°C for 5 minutes again. Furthermore, the extracted DNA is washed and resuspended in the buffer for subsequent use.

[0045] Examples of EAEC-containing samples that can be analyzed by the method of the present invention include, but are not limited to, blood, serum, oral mucosa, body fluids, tissues, feces, bodily secretions, culture media, soil, water, etc. According to an embodiment of this disclosure, the sample is water, more preferably, a water sample taken from a wastewater treatment plant.

[0046] Second stage: Preparation of composition for one-pot reaction

[0047] In this disclosure, in a one-pot reaction, the nucleic acid sample obtained in the first stage is amplified and detected by a CRISPR / Cas system. Therefore, this stage prepares a composition suitable for in situ amplification of genomic DNA and initiation of CRISPR detection. Specifically, the composition comprises components for isothermal amplification of the genomic DNA extracted in step 110 within a single reaction vessel, and components for initiating CRISPR to detect the amplification product. According to the embodiments disclosed herein, these components are a recombinase polymerase amplification (RPA) premix and a CRISPR / Cas premix, which are mixed to prepare the desired composition (step 120), wherein the RPA premix is ​​used for amplifying nucleic acids, and the CRISPR / Cas premix is ​​used for CRISPR detection of the amplification product (i.e., the amplified product).

[0048] According to the embodiments disclosed herein, the RPA premix contains a forward primer of sequence number 11 and a reverse primer of sequence number 12, which hybridize and anneal to the 5' and 3' ends of the complementary sequence of the target gene (i.e., the aggR gene) in the extracted genomic DNA, respectively. The hybridization product is then extended by polymerase chain reaction (PCR) to generate an amplifier, which is then further amplified and detected by a CRISPR / Cas system. Preferably, the RPA premix may further contain a reaction buffer required for isothermal amplification of nucleic acids, deoxynucleoside triphosphates (dNTPs, including dATP, dGTP, dCTP, and dTTP), and magnesium acetate. According to a preferred embodiment, the concentration of magnesium acetate in the RPA premix is ​​30 mM.

[0049] According to the embodiments disclosed herein, the CRISPR / Cas premix is ​​used to amplify and detect the amplifier generated by the RPA premix. Therefore, the CRISPR / Cas premix contains a CRISPR / Cas protein, a crRNA, a fluorescent reporter, MnSO4, and DTT, wherein the crRNA specifically targets the aggR gene fragment of EAEC and has a ribonucleic acid sequence numbered 1 to 10. According to embodiments of the present invention, any crRNA with sequence numbers 1 to 10 can guide the CRISPR / Cas protein to initiate cis-cleave of the amplified product, thereby generating a signal detectable on page 4 / 11 of the specification (CN 122564140 A). In a preferred embodiment, the crRNA has a ribonucleic acid sequence numbered 5 and is present at a concentration of approximately 0.5 μM in the CRISPR / Cas premix.

[0050] The CRISPR / Cas protein applicable to this disclosure is a Cas protein capable of recognizing any PAM site sequence selected from the group consisting of GTTC, ATTC, CTTA, CTTC, GTTG, TCTA, TCTC, CTTC, and ATTG. Examples of CRISPR / Cas proteins applicable to this disclosure include, but are not limited to, Cas9, Cas12a, and Cas12b. According to a preferred embodiment of this disclosure, the CRISPR / Cas protein is Cas12a, which can recognize the PAM site sequence of CTTC, and its concentration in the CRISPR / Cas premix is ​​approximately 125 nM.

[0051] According to an embodiment of this disclosure, the fluorescent reporter applicable to this disclosure is an AT-rich DNA reporter with a fluorescent group (e.g., 6-FAM) at its 5' end, and itsThe 3' end has a quenching group (e.g., BHQ1). Examples of fluorescent reporters suitable for this method include those previously disclosed by Lee et al. (BioChip Journal (2022) 16:463–470), the contents of which are incorporated herein by reference. Specifically, the fluorescent reporter may be any one of TTATT, TTATT-5C, TTATT-2T, TTATT-5T, TTATT-10T, TTATT-20T, TTATT-5dU, TTATT-5A, TTATT-5G, TTATT-5rU, 10C, etc. According to a preferred embodiment of this disclosure, the fluorescent reporter is TTATT-5C, and its concentration in the CRISPR / Cas premix is ​​approximately 5 μM.

[0052] According to a preferred embodiment of this disclosure, the concentrations of MnSO4 and DTT in the CRISPR / Cas premix are approximately 3 mM and 30 mM, respectively. According to a preferred embodiment of this disclosure, compared to the absence of added MnSO4 and DTT, MnSO4 and DTT can improve detection sensitivity, enabling the method to successfully detect one copy of the aggR gene.

[0053] According to an embodiment of this disclosure, in step 120, 10 μL of RPA premix and 5 μL of CRISPR / Cas premix are mixed to produce a composition for subsequent in-situ one-pot reaction.

[0054] Third Stage: Amplifying Nucleic Acids and Initiating CRISPR Detection

[0055] In this stage, the genomic DNA extracted from the EAEC sample in step 110 is contacted with the composition prepared in step 120, so that the EAEC genomic DNA, especially the EAEC aggR gene, is first amplified by PCR using the forward and reverse primers in the RPA premix. The resulting amplified product is then further amplified and detected in situ using the CRISPR / Cas system provided by the CRISPR / Cas premix, thereby generating a signal for subsequent detection (step 130). It should be noted that in step 130, both PCR amplification and CRISPR detection are performed in a single reaction vessel (or in situ) at 39°C for 30 minutes.

[0056] Fourth Stage: Detection Signal

[0057] Since the aggR gene of EAEC is labeled with a fluorescent reporter (i.e., TTATT-5C) during CRISPR / Cas amplification, the generated fluorescent signal can be used as a detection signal.

[0058] According to the embodiments of this disclosure, the fluorescent signal increases with the increase of nucleic acid copy number in the sample. According to the preferred embodiment of this disclosure, this method can detect a copy number of the aggR gene of [missing information].1 to 1 × 10⁹ copies.

[0059] The advantage of the method described above is that the amplification and detection of the product can be performed in situ (or in a single reaction vessel), and the signal can be directly read by the fluorescence signal of the electrode, thereby eliminating the need for a reporter system required in traditional CRISPR / Cas systems, such as a fluorescence resonance energy transfer (FRET) reporter system containing short DNA or RNA oligonucleotides labeled with fluorescent and quenching groups.

[0060] It should be understood that the present invention is not limited to the specific embodiments described, as various variations are possible; it should also be understood that the terminology used herein is only for describing specific embodiments and is not intended to limit the scope of the invention, as the scope of the invention is defined only by the appended claims.

[0061] Example Description 5 / 11 pages 8 CN 122564140 A

[0062] Materials and Methods

[0063] Design of RPA Primers and crRNA

[0064] The CRISPR RNA (crRNA) (i.e., guide RNA sequence) was designed based on the PAM sequence available on the RPA amplifier, which was generated via an RPA primer pair designed based on the targeted aggR gene (Gene Bank: Z18751.1). The synthetic DNA aggR gene was ordered in plastid form from Genewiz Biotechnology Co., Ltd. (Suzhou, China). The crRNA sequence used was synthesized by BGI Genomics (Shenzhen, China) and is listed in Table 1.

[0065]

[0066] Reagents for the RPA-CRISPR / Cas12a Two-Pot System

[0067] The RPA reaction was performed using TwistAmp Liquid Basic Kit reagents (TwistDX, Maidenhead, UK) according to the manufacturer's operating procedures. In short, 6 μL of the RPA premix contained 1× reaction buffer, 1× Basic E-mix, 0.48 μM each of forward and reverse primers (Genewiz Biotechnology Co., Ltd., Suzhou, China), 1× core reaction mixture, 1.84 mM dNTPs, and 14 mM magnesium acetate. Furthermore, the plastid DNA template corresponding to 1×10⁶ aggR gene copies was used as the baseline concentration for method development, as per page 6 / 11, CN 122564140 A of the specification. The reaction was carried out at 39°C in a Veriti 96-well thermal cycler (Applied Biosystems, Foster City, California, USA) for 20 minutes. Subsequently, 5 μL of the RPA product was used as a template to carry out the second step of the two-step reaction.

[0068] The CRISPR / Cas12a reaction was performed using the EnGen Lba Cas12a reagent kit (New England Biolabs, Ipswich, Massachusetts, USA) in a 20 μL volume. The reaction premix contained 1× NEB buffer 2.1, 2.5 μM fluorescent reporter, 125 nM Cas12a enzyme from Lachnospiraceae bacterium, and 0.5 μM crRNA (i.e., listed in Table 1). The fluorescent reporter used was based on the TTATT-5C reporter (FAM-TTATTCCCCC-BHQ1, sequence number: 16), which is described as previously disclosed (Lee, S. et al., BioChip Journal (2022) 16(4):463-470). The reaction was performed at 37°C for 30 minutes using a StepOnePlus™ real-time polymerase chain reaction system (Applied Biosystem, Foster City, California, USA), with the FAM reporter signal read every minute to capture its fluorescence signal.

[0069] Reagents for the RPA-CRISPR / Cas12a one-pot system

[0070] In the one-pot reaction, the above RPA premix and CRISPR / Cas12a premix were mixed evenly before the reaction started. Combinations of RPA premix and CRISPR / Cas12a premix with different volume ratios were tested. At the same time, different concentrations of primers, dNTPs and magnesium acetate in the RPA reaction, and different concentrations of fluorescent reporter and crRNA in the CRISPR / Cas12a reaction were also tested. In addition, the enhancing effects of the following chemicals on the one-pot reaction were tested: MnSO4, DTT, betaine, pullulan, poly(vinyl alcohol, PVA) (average molecular weight 30,000-70,000), and dimethyl sulfoxide (DMSO). Furthermore, the reaction temperatures of 37°C, 39°C, and 42°C were evaluated.

[0071] Real-time quantitative polymerase chain reaction (RT-qPCR)

[0072] The primer and probe sequences used are shown in Table 2. A 20 μL qPCR analysis reaction contained 1× Premix Ex Taq™ (probe qPCR) (Takara, Otsu, Japan), 0.2 μM each of the forward and reverse primers, and 0.25 μM...Fluorescently labeled probes, 1× ROX reference dye, and 2 μL DNA template were used. The thermal cycling procedure was as recommended by the manufacturer, consisting of an initial temperature of 95°C for 20 seconds, followed by 40 cycles, each consisting of 95°C for 1 second and 60°C for 20 seconds. Instruction manual, pages 7 / 11, CN 122564140 A

[0073]

[0074] Bacterial culture and extraction of genomic DNA

[0075] The bacterial strains used in this disclosure include pathogenic Escherichia coli strains ATCC 29552 (aggR-positive EAEC pathogenic type), NR-4 (enterotoxic pathogenic type), NR-22051 (enterohemorrhagic pathogenic type), non-pathogenic strains TO P1 0, Glucosamine 621H, Corynebacterium glutamicum ATCC 13032, and Pseudomonas putida ATCC 12633. These bacterial cultures were cultured overnight in 1× nutrient broth medium. For pathogenic Escherichia coli strains, 250 μL of culture medium was centrifuged, washed, and then resuspended in 250 μL of sterile deionized water. Subsequently, the strain was thermally lysed at 95°C for 10 minutes to release its genomic DNA and inactivate it, in order to facilitate operational safety. The genomic DNA of other bacterial strains was extracted using the GeneJET genomic DNA purification kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The genomic DNA of the above strains was used for specificity testing.

[0076] Wastewater Samples and Additive Tests

[0077] Wastewater samples were obtained from aeration tanks and returned activated sludge of two local municipal wastewater treatment plants in February and November 2023. The wastewater sample (0.4 mL) was centrifuged, and the resulting precipitate was resuspended in 1 mL of adenylosuccinate lyase (ASL) buffer (Qiagen, Hilden, Germany) and heated at 95°C for 5 minutes to increase DNA yield and remove potential inhibitors during genomic DNA extraction. Next, 1 mL of InhibitEX buffer from the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) was added to extract DNA, followed by a second heating at 95°C.5 minutes. Subsequent steps were performed according to the manufacturer's operating procedures, but with some modifications to the reagent volumes. Specifically, the volumes of supernatant, AL buffer, and ethanol were increased from 200 μL to 600 μL, the proteinase K volume was increased from 15 μL to 25 μL, and the final washing volume was changed from 200 μL to 50 μL. In the extraction method described on page 8 / 11 of the above genomic DNA manual, CN 122564140 A, different amounts of E. coli ATCC 29552 were added to the wastewater precipitate sample for addition testing before resuspending the wastewater sample in ASL buffer.

[0078] Comparative Example 1: RPA Two-Pot Reaction

[0079] In this comparative example, the RPA-CRISPR / Cas12a two-pot reaction was performed using crRNA with sequence number 1 to detect EAEC. This crRNA has the typical TTTC PAM site found in RPA amplifiers targeting aggR. Therefore, a two-step procedure was adopted. First, an RPA reaction was performed in a test tube at a constant temperature of 39°C for 20 minutes to pre-amplify the aggR target gene in the plasmid. Then, the product obtained from the RPA reaction was transferred to another test tube containing a CRISPR / Cas12a reagent mixture and incubated at 37°C for 30 minutes for detection. At high template concentrations (1×10⁶ to 1×10⁹ gene copy numbers), the fluorescence signal rapidly reached saturation (within 5 minutes) (data not shown). When the gene copy number was 1×10⁵, the slope of the fluorescence signal was significantly slower, resulting in a significant difference in detection time compared to 1×10⁶ gene copy numbers (data not shown). Corresponding linear regression analysis showed an R² of 0.89 for gene copy numbers ranging from 1×10² to 1×10⁷. Given that the fluorescence reading at a gene copy number of 1×10² is extremely close to the background signal noise threshold, the detection limit of this two-pot detection system is 1×10³ gene copy number.

[0080] Example 1: Establishment of a one-pot system disclosed herein

[0081] 1.1 Design and testing of crRNA for establishing a one-pot reaction

[0082] The two-pot system requires a liquid transfer step and two different operating temperatures, resulting in a cumbersome and time-consuming operation process, and also posing a risk of aerosol contamination. Furthermore, as shown in Comparative Example 1, at signal saturation, this two-pot system failed to achieve satisfactory sensitivity at high gene copy numbers, with a detection limit of 1×10³ gene copy number, and its linear regression R² is less than 0.90, making the system unsuitable for quantitative analysis.

[0083] To simplify operation and improve the sensitivity and quantitative characteristics of the detection method, this embodiment establishes a one-pot system in which the RPA isothermal amplification step and the CRISPR / Cas12a detection step are both performed in a single test tube.

[0084] For this purpose, a preliminary single-tube reaction experiment was first performed, in which 10 μL of RPA premix and 5 μL of CRISPR / Cas12a premix containing crRNA (i.e., crRNA with sequence number: 2-10) were reacted at 37°C. When using a plastic template containing aggR for testing, almost no signal output was observed. This phenomenon may be due to the over-cleavage activity of the activated CRISPR / Cas12a on the amplified product generated by RPA amplification, resulting in insufficient amplified product concentration as a target for the CRISPR / Cas12a system, and therefore, the trans-cleavage activity of the fluorescent reporter cannot be activated.

[0085] To overcome the aforementioned reaction incompatibility issues, we attempted to reduce the cleavage activity of CRISPR / Cas12a on the initiating RPA amplifications. To this end, we designed and tested nine crRNAs with sequence numbers 2-10, possessing suboptimal PAM sites (i.e., GTTC, ATTC, CTTA, CTTC, GTTG, TCTA, TCTC, CTTC, and ATTG) to ensure successful operation of the one-pot system. The results showed that the crRNA with the suboptimal CTTC PAM site (i.e., crRNA aggR4 or crRNA with sequence number 5) performed best, exhibiting the highest fluorescence readings and reaction rates (i.e., the slope of fluorescence intensity over time). Therefore, this suboptimal crRNA (i.e., sequence number 5) was used for subsequent one-pot system development.

[0086] Further evaluation was conducted to determine whether reducing the crRNA concentration could minimize the initiation cleavage activity of CRISPR / Cas12a on the RPA amplifications, thereby improving the efficiency of the one-pot system. However, the results showed that reducing the crRNA concentration actually reduced the performance of the one-pot reaction (data not shown). Next, while keeping the CRISPR / Cas12a premix volume constant at 5 μL, the RPA premix volume was adjusted to 5 μL, 15 μL, or 20 μL for system optimization. The results showed that the activity under these volume conditions could not surpass the performance previously achieved using 10 μL of RPA premix. Subsequently, the effect of CRISPR / Cas12a premix volumes of 10 μL, 15 μL, and 20 μL was further evaluated. The results showed that compared to the 5 μL used on page 9 / 11 of the previous specification (CN 122564140 A), the results were not satisfactory.Compared to the CRISPR / Cas12a premix, the one-pot reaction activity was significantly reduced at higher volumes.

[0087] Therefore, in this one-pot system, the volumes of the RPA and CRISPR / Cas12a premix were set to 10 μL and 5 μL, respectively. We also tested the effects of different concentrations of fluorescent reporters (1.25 μM, 5 μM, and 7.5 μM). The results showed that higher concentrations of the premix improved the sensitivity of the one-pot reaction system compared to the initial 2.5 μM concentration used for the CRISPR / Cas12a premix. Given that the CRISPR / Cas12a premix produced similar maximum fluorescence peaks at 5 μM and 7.5 μM fluorescent reporter concentrations, 5 μM fluorescent reporters were used in this one-pot system to reduce costs. In addition, we also tested different reaction temperatures and found that the reaction performed better at 39°C, so this temperature was adopted as the reaction temperature.

[0088] To improve the yield of the starting amplifier in the RPA reaction, the concentrations of the primer, dNTPs, and MgOAc in the RPA premix were increased. The results showed that increasing the MgOAc concentration significantly improved the one-pot system, with 30 mM being optimal, while the effects of other parameters were less significant.

[0089] 1.2 Chemical Additives

[0090] In this embodiment, chemical additives including betaine, pullulan, L-proline, MnSO4, DTT, PVA, and DMSO were tested to evaluate their impact on improving the sensitivity of the CRISPR / Cas12a reaction in this one-pot system.

[0091] The results showed that 1 mM Mn2+ or 10 mM DTT significantly improved the reaction rate of the one-pot analysis and helped improve the fluorescence reading. However, further increasing the concentration of these chemicals in the reaction did not improve the analytical sensitivity.

[0092] Further, the two chemical substances (i.e., Mn2+ and DTT) were added to the analysis at different concentrations to optimize the one-pot system. The results showed that the system containing 1 mM Mn2+ and 10 mM DTT at the final concentration exhibited the highest sensitivity.

[0093] 1.3 Detection Limit and Specificity Test

[0094] The final fine-tuned one-pot analysis established in this embodiment included the following: 10 μL of RPA premix containing 1× reaction buffer, 1× basic mixture, 0.48 μM each of forward and reverse primers, 1× core reaction mixture, 1.84 mM dNTPs and 30 mM magnesium acetate, and 5 μL of RPA premix containing 1× NEB buffer 2.1, 5 μM fluorescent reporter, and 125 nMA CRISPR / Cas12a premix of Cas12a enzyme, 0.5 μM crRNA, 3 mM MnSO4, and 30 mM DTT was mixed. The reaction was carried out at 39°C for 30 minutes.

[0095] A good linear relationship (R² = 0.92) was obtained between the fluorescence reading and the aggR gene copy number (range 1 to 1 × 10⁵ aggR gene copies), indicating that an appropriate dynamic range has been obtained, making this analysis suitable for the quantitative measurement of gene copy number, and the detection limit reached 1 aggR gene copy number (Figure 2).

[0096] Specificity tests were performed using non-pathogenic Escherichia coli strains (TOP10), EAEC strain (ATCC 29552), and two other pathogenic Escherichia coli strains (NR-4 and NR-22051). In addition, non-Escherichia coli strains, including Staphylococcus oxidans 621H, Corynebacterium glutamicum ATCC 13032, and Pseudomonas aeruginosa ATCC 12633, were also included in the evaluation. The results showed that only the EAEC strain (ATCC 29552) produced a high fluorescence reading in the one-pot detection analysis, while the other strains were all below the detection limit (Figure 3).

[0097] Example 2: Detection of EAEC in wastewater samples using the one-pot RPA-CRISPR / Cas12a system

[0098] In this example, 102 to 107 colony-forming units (CFU) of Escherichia coli ATCC 29552 were added to wastewater samples taken from a wastewater treatment plant. Subsequently, DNA was extracted from these samples and analyzed using the one-pot RPA-CRISPR / Cas12a system / method established in Example 1 or the conventional RT-qPCR method. Instruction manual, pages 10 / 11, CN 122564140 A

[0099] When using spiking samples for analysis, a good linear fit curve (R² = 0.97) was observed between CFU and fluorescence readings, with a detection limit of 10² CFU (Figure 4A). In contrast, when using RT-qPCR to detect the same wastewater spiking samples, the detection limit was 10⁴ CFU (Figure 4B). The higher detection limit of RT-qPCR compared to one-pot RPA-CRISPR / Cas12a analysis may be due to the challenging nature of the wastewater matrix itself, which contains inhibitors that hinder the RT-qPCR reaction. Even after diluting the DNA template tenfold to reduce the influence of inhibitors on RT-qPCR, this problem could not be improved. When using aggR plastids in DEPC-treated water as a template for testing, the RT-qPCR method could detect up to10 copies of the aggR gene.

[0100] To verify its practical applicability, five wastewater samples were collected from two different wastewater treatment plants and tested for the presence of EAEC. The one-pot RPA-CRISPR / Cas12a system identified EAEC in two of the wastewater samples (347 ± 300 and 611 ± 204 CFU / mL), both from the same municipal wastewater treatment plant (Plant 1), but on different sampling dates. Samples from another municipal wastewater treatment plant (Plant 2) were all below the detection limit (Figure 4C). However, using the RT-qPCR method, we could not determine the concentration of EAEC in all wastewater samples because the number of CFUs (measured using one-pot RPA-CRISPR / Cas12a analysis) was below the detection limit of the method. It should be noted that since the copy number of the aggR gene may vary between different strains, the measured CFU values ​​are only approximate. In comparison, the EAEC concentration observed in the severely polluted influent of a wastewater treatment plant in Mexico was gene copies / mL, where the AggR-activated Island C (aaiC) is a gene located within the EAEC chromosomal genome island that encodes a gene related to the type 6 secretion system.

[0101] In summary, compared with existing products on the market, such as the SSI Diagnostica Enteroaggregative E. coli PCR Kit, BioCode Gastrointestinal Pathogen Detection Kit (GPP), Biofire FilmArray Gastrointestinal (GI) Detection Kit, and Diatheva Enteroaggregative E. coli Detection Kit (MBK0084), all of which rely on PCR technology, this method provides faster and more sensitive EAEC detection. Current PCR-based products require trained personnel, long processing times, and specialized equipment, making them unsuitable for rapid on-site detection. Due to its simple setup, this method has the potential to be improved for detecting other pathogens and can be further developed into portable devices suitable for on-site and real-time detection applications.

[0102] It should be understood that the above descriptions of various embodiments are for illustrative purposes only, and those skilled in the art can make various modifications accordingly. The foregoing description, examples, and related data have provided a complete description of the structure and usage of exemplary embodiments of the present invention. Although the above description is presented with a certain degree of specificity, or reference...The present invention has been described with reference to one or more embodiments. However, those skilled in the art to which this invention pertains may make various modifications and alterations to the disclosed embodiments without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be determined by the claims in the appended patent application. Instruction Manual 11 / 11 Page 14 CN 122564140 A Figure 1 Figure 2 Instruction Manual Appendix 1 / 3 Page 15 CN 122564140 A Figure 3 Figure 4A Instruction Manual Appendix 2 / 3 Page 16 CN 122564140 A Figure 4B Figure 4C Instruction Manual Appendix 3 / 3 Page 17 CN 122564140 A ABSTRACT Disclosed herein is a method for detecting Enteroaggregative Escherichia coli (EAEC) in a sample. The method includes (i) extracting genomic DNA from EAEC in the sample; (ii) mixing a recombinase polymerase amplification (RPA) premix comprising forward and reverse primers with a CRISPR / Cas premix comprising a CRISPR / Cas protein, a CRISPR RNA (crRNA), a fluorescence reporter, MnSO4, and dithiothreitol (DTT) to produce a composition, wherein the crRNA is specific to a segment of the aggR gene of EAEC; (iii) contacting the extracted genomic DNA of step (i) with the composition of step (ii) to amplify the extracted genomic DNA and initiate CRISPR detection in situ; and (iv) measuring a fluorescence signal emitted from thefluorescence reporter of the product of step (iii), wherein an increase in the fluorescence signal compared to that of a control indicates the presence of EAEC in the sample, wherein the control is a sample free of EAEC.

Claims

1. A method for detecting enteroagglutinating Escherichia coli (EAEC) in a sample, comprising: (i) Genomic DNA was extracted from the EAEC in the sample; (ii) A composition is prepared by mixing a recombinase polymerase amplification (RPA) premix with a CRISPR / Cas premix, wherein, The RPA premix contains a forward primer with sequence number 11 and a reverse primer with sequence number 12. The CRISPR / Cas premix contains a CRISPR / Cas protein, a CRISPR ribonucleic acid (crRNA), a fluorescent reporter, manganese sulfate (MnSO4), and dithiothreitol (DTT), wherein the crRNA specifically targets the aggR gene fragment of EAEC and has a ribonucleic acid sequence with sequence number 1 to 10. (iii) Contact the genomic DNA extracted in step (i) with the composition of step (ii) to amplify the extracted genomic DNA and initiate CRISPR detection in situ; and (iv) Measure a fluorescent signal emitted by the CRISPR product from step (iii); in, In step (ii), the DTT content in the CRISPR / Cas premix is ​​at least 10 times the MnSO4 content; In step (iii), the in situ amplification and detection system is operated at 39°C for 30 minutes; and In step (iv), an increase in fluorescence signal compared to the control group indicates the presence of EAEC in the sample, where the control group is a sample without EAEC.

2. The method according to claim 1, wherein in step (ii), the RPA premix further comprises a reaction buffer, deoxynucleoside triphosphates (dNTPs), and magnesium acetate.

3. The method according to claim 2, wherein the concentration of magnesium acetate in the RPA premix is ​​30 mM.

4. The method according to claim 1, wherein in step (ii), the crRNA has a ribonucleic acid sequence with sequence number 5 and a concentration of approximately 0.5 μM in the CRISPR / Cas premix.

5. The method according to claim 1, wherein in step (ii), the CRISPR / Cas protein is Cas 9, Cas 12a, or Cas 12b.

6. The method according to claim 5, wherein the CRISPR / Cas protein is Cas 12a and its concentration in the CRISPR / Cas premix is ​​approximately 125 nM.

7. The method according to claim 6, wherein in step (ii), the fluorescent reporter is an AT-rich DNA reporter having a fluorescent group at its 5' end and a quenching group at its 3' end.

8. The method according to claim 7, wherein the fluorescent group is 6-carboxyfluorescein (6-FAM); and the quenching group is black hole quencher 1 (BHQ1).

9. The method according to claim 8, wherein the fluorescent reporter is any one of TTATT, TTATT-5C, TTATT-2T, TTATT-5T, TTATT-10T, TTATT-20T, TTATT-5dU, TTATT-5A, TTATT-5G, TTATT-5rU, or 10C.

10. The method of claim 9, wherein the fluorescent reporter is TTATT-5C and its concentration in the CRISPR / Cas premix is ​​5 μM.

11. The method according to claim 1, wherein in step (ii), the volume of the RPA premix is ​​10 μL and the volume of the CRISPR / Cas premix is ​​5 μL.

12. The method of claim 1, wherein the method is capable of detecting 1 to 1×10 9 One copy of the EAECaggR gene.

13. The method according to claim 1, wherein the sample is any one of blood, serum, oral mucosa, body fluid, tissue, feces, bodily secretions, culture medium, soil or water.

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