Nucleic acid product for detecting burkholderia gladioli and subspecies thereof in sample, kit containing same and application of nucleic acid product

Abstract

The invention discloses a nucleic acid product for detecting burkholderia gladioli and a subspecies thereof in a sample, a kit containing the nucleic acid product and application of the nucleic acid product. The nucleic acid product comprises an ssDNA (single-stranded deoxyribonucleic acid) fluorescent probe, an ssRNA (single-stranded ribonucleic acid) fluorescent probe, crRNA1, crRNA2 and a primer group. When the nucleic acid product is used for detecting purely-cultured burkholderia gladioli pseudomonas coconut venom subsp.gladioli subsp.gladioli, the detection limit can reach 100 CFU / mL.

Description

Technical Field

[0001] The present application relates to the technical field of molecular biology, and in particular to a nucleic acid product for detecting Burkholderia gladiolus and its subspecies in a sample, a kit containing the same, and uses thereof. Background Art

[0002] Burkholderia gladiolipv.cocovenenans (hereinafter referred to as Burkholderia gladiolipv.cocovenenans) is a deadly foodborne pathogen commonly found in spoiled cereal products, potato products and Tremella fuciformis. The fumonisic acid it secretes is a tricarboxylic fatty acid that inhibits the ATP / ADP exchange function of mitochondrial adenine nucleotide translocase, blocks the phosphorylation process of the respiratory chain, and thus causes death. Fumonisic acid is odorless and tasteless, so it is difficult to detect contaminated food by taste and smell, and it is heat-stable, so even if the bacteria are killed during cooking, it will not be destroyed. Therefore, it is challenging to detect this toxin during food preparation and consumption. At present, poisoning incidents caused by Burkholderia gladiolipv.cocovenenans are rare, but they still occur from time to time in areas that still maintain traditional eating habits. Since there is no specific antidote for fumonisic acid, once poisoned, the mortality rate is as high as 40%-100%. The existing national food safety standard GB4789.29-2020 "Food Microbiology Inspection of Burkholderia gladiolus (Pseudomonas cocovenenans subspecies)" has a cumbersome inspection process for this pathogen, and the inspection cycle is as long as 15 to 20 days.

[0003] Although several liquid chromatography and mass spectrometry-based methods have been developed for the detection of fumonisin, these methods are costly and time-consuming. Importantly, even if the toxin cannot be detected in foods contaminated with Burkholderia gladioli, there is still a potential risk of toxin production during the circulation of goods. Therefore, nucleic acid-based techniques, such as quantitative fluorescence amplification (qPCR) and loop-mediated isothermal amplification (LAMP), are very promising alternative methods for the detection of Burkholderia gladioli. However, the detection limit of these methods is about 10 2 CFU / mL.

[0004] The bon gene cluster is critical for the production of fumonisin. However, due to the diversity caused by the variation of the bon gene cluster and the lack of relevant research at present, such as insufficient research on the corresponding toxin production, a positive test for the bon gene cluster does not indicate whether a certain gladiolus Burkholderia can produce fumonisin. Therefore, in addition to detecting the subspecies of cocoferment, it is also necessary to widely test gladiolus Burkholderia species. Food contaminated by non-pathogenic gladiolus Burkholderia or pathogenic subspecies of cocoferment requires more effective testing procedures to classify and handle contaminated food and reduce the risk of secondary contamination.

[0005] Therefore, there is an urgent need in the art to develop new products for detecting / differentiating Burkholderia gladiolus and its subspecies in samples. Summary of the invention

[0006] Based on this, it is necessary to provide at least one nucleic acid product for detecting Burkholderia gladiolus and its subspecies in a sample, a kit containing the same, and its use.

[0007] In a first aspect of the present application, a nucleic acid product is provided, which comprises a ssDNA fluorescent probe, a ssRNA fluorescent probe, crRNA1, crRNA2 and a primer set.

[0008] In a second aspect of the present application, a kit for detecting Burkholderia gladioli and its subspecies in a sample is provided, which comprises the nucleic acid product as described in the first aspect.

[0009] In a third aspect of the present application, there is provided a use of a nucleic acid product in preparing a product for detecting Burkholderia gladiolus and its subspecies in food.

[0010] In a fourth aspect of the present application, a method for detecting Burkholderia gladiolus or its subspecies in a sample is provided.

[0011] In one embodiment of the present application, the nucleic acid product and method of the present application are used to detect pure cultured gladiolus Burkholderia cocovenenans subsp. 0 CFU / mL, the detection limit of Burkholderia gladiolus and Pseudomonas cocovenenans subsp. 2 -10 3 CFU / mL; the detection time is completed within 1 hour. In addition, the method provided by the present application can simultaneously detect and distinguish Burkholderia gladioli and Burkholderia spp., and the screening of toxin-producing strains is more sufficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0013] Figure 1This is the specific experimental result of detecting Burkholderia gladiolus in one embodiment of the present application.

[0014] Figure 2 The detection limit experimental result of Burkholderia gladiolus ATCC 33664 in one embodiment of the present application is shown.

[0015] Figure 3 The detection limit experimental result of Burkholderia gladiolus ATCC 33664 in food matrix in one embodiment of the present application. DETAILED DESCRIPTION

[0016] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present application belongs. The terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0018] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and the like, when used in other ways to mean "one or more", are also understood in the same way unless otherwise specified.

[0019] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0020] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0021] In this application, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0022] In this application, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally contain", etc. in this application, taking "optionally include" as an example, mean "may include or not include".

[0023] The terms "contain", "include" and "include" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0024] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0025] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

[0026] In the present application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0027] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0028] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0029] In a first aspect of the present application, a nucleic acid product is provided, which comprises a ssDNA fluorescent probe, a ssRNA fluorescent probe, crRNA1, crRNA2 and a primer set;

[0030] in:

[0031] The nucleic acid sequence of the crRNA1 comprises the sequence shown in SEQ ID NO: 1;

[0032] The nucleic acid sequence of the crRNA2 comprises the sequence shown in SEQ ID NO: 2;

[0033] The primer set is used to amplify one or more of the following regions:

[0034] The 16S-23S rRNA gene region 1 (3,834,843-3,835,078) of Burkholderia gladiolus subsp. cerevisiae and the bonA gene region 2 (3,011,801-3,011,986) of Pseudomonas cocovenenans subsp.

[0035] In some embodiments, the sequences of the primers in the primer set respectively include the sequences shown in SEQ ID NOs: 3 to 6.

[0036] In some embodiments, the sequence of the crRNA1 is as shown in SEQ ID NO:1.

[0037] In some embodiments, the sequence of the crRNA2 is as shown in SEQ ID NO:2.

[0038] In some embodiments, the sequences of the primers in the primer set are shown as SEQ ID NOs: 3 to 6, respectively.

[0039] In some embodiments, the sequence of the ssDNA fluorescent probe comprises the following sequence: TTATTATT.

[0040] In some embodiments, the ssRNA fluorescent probe comprises the following structure:

[0041] mArArUrGrGrCmAmArArUrGrGrCmA.

[0042] In some embodiments, the ssDNA fluorescent probe and the ssRNA fluorescent probe are each labeled with a fluorescent group that is different from each other and does not interfere with each other, wherein the fluorescent group is, for example, selected from the group consisting of FAM, HEX, TET, VIC, JOE, ROX and Texas.

[0043] In some embodiments, the ssDNA fluorescent probe and the ssRNA fluorescent probe each further carry a quencher group, such as BHQ2 or BHQ1.

[0044] In some embodiments, the fluorescent group is FAM and the quencher group is BHQ1.

[0045] In some embodiments, the fluorescent group is HEX, TET, VIC or JOE, and the quencher group is BHQ1.

[0046] In some embodiments, the fluorescent group is ROX or Texas, and the quencher group is BHQ2.

[0047] In some embodiments, the 5' end (or 3' end) of the ssDNA fluorescent probe carries a fluorescent group 1. In some embodiments, the 3' end (or 5' end) of the ssDNA fluorescent probe carries a quencher group 1.

[0048] In the present application, the fluorescent group 1 can be conventional in the art, for example, can be selected from the group consisting of FAM, HEX, TET, VIC, JOE, ROX and Texas. In some embodiments, the fluorescent group 1 is FAM or VIC.

[0049] In the present application, the quencher group 1 can be conventional in the art, for example, can be selected from the group consisting of BHQ2 and BHQ1. In some embodiments, the quencher group 1 is BHQ1.

[0050] In some embodiments, the 5' end (or 3' end) of the ssRNA fluorescent probe carries a fluorescent group 2. In some embodiments, the 3' end (or 5' end) of the ssRNA fluorescent probe carries a quencher group 2.

[0051] In the present application, the fluorescent group 2 can be conventional in the art, for example, can be selected from the group consisting of FAM, HEX, TET, VIC, JOE, ROX and Texas. In some embodiments, the fluorescent group 2 is FAM or VIC.

[0052] In the present application, the quencher group 2 can be conventional in the art, for example, can be selected from the group consisting of BHQ2 and BHQ1. In some embodiments, the quencher group 2 is BHQ1.

[0053] In some embodiments, the structure of the ssDNA fluorescent probe is as follows:

[0054] FAM-TTATTATT-BHQ1.

[0055] In some embodiments, the structure of the ssRNA fluorescent probe is as follows:

[0056] VIC-mArArUrGrGrCmAmArArUrGrGrCmA-BHQ1.

[0057] In a second aspect of the present application, a kit for detecting Burkholderia gladioli and its subspecies in a sample is provided, which comprises the nucleic acid product as described in the first aspect.

[0058] If not otherwise specified, the term "sample" in this application may be any item that may contain Burkholderia gladioli and its subspecies, such as food, contaminated water sources, etc. In some embodiments, the sample is a culture. Exemplarily, in some cases, the content of Burkholderia gladioli or its subspecies in the original sample is extremely low, lower than or even far lower than the detection limit of the detection method to be used. At this time, it is selectively necessary to culture a small amount of bacteria in the original sample. In this case, the sample is a pure culture sample.

[0059] In some embodiments, the sample is a food.

[0060] In some embodiments, the kit comprises one or more of a recombinase-mediated isothermal nucleic acid amplification (RAA) unit and a CRISPR-Cas12a / Cas13a reaction unit.

[0061] In some embodiments, the kit comprises a recombinase-mediated isothermal nucleic acid amplification (RAA) unit and a CRISPR-Cas12a / Cas13a reaction unit.

[0062] In some embodiments, the RAA unit may comprise the primer set.

[0063] In the present application, unless otherwise specified, the RAA unit may be a unit that exists independently in form. For example, it may be contained in an independent small package in a kit, or in a separate partitioned space.

[0064] In the present application, the RAA unit may exist as a complete unit. For example, it is a mixture of primers in a primer set, and may exist in the form of dry powder or liquid storage.

[0065] In the present application, the RAA unit may also include different subunits. In one embodiment of the present application, the subunits, for example, may be different primers in the primer set described above. For example, the primer with a sequence as shown in SEQ ID NO:3 is in the form of a dry powder or a liquid storage as one of the subunits. The primer with a sequence as shown in SEQ ID NO:4 is in the form of a dry powder or a liquid storage as another subunit. The primer with a sequence as shown in SEQ ID NO:5 is in the form of a dry powder or a liquid storage as another subunit. The primer with a sequence as shown in SEQ ID NO:6 is in the form of a dry powder or a liquid storage as another subunit.

[0066] In some embodiments, the CRISPR-Cas12a / Cas13a reaction unit comprises the ssDNA fluorescent probe, the ssRNA fluorescent probe, the crRNA1 and the crRNA2, and one or more of Cas12a and Cas13a.

[0067] In some embodiments, the CRISPR-Cas12a / Cas13a reaction unit comprises the ssDNA fluorescent probe, the ssRNA fluorescent probe, the crRNA1 and the crRNA2, and Cas12a and Cas13a.

[0068] Similar to the above-mentioned RAA unit, unless otherwise specified in the present application, the CRISPR-Cas12a / Cas13a reaction unit may be a unit that exists independently in form. For example, it may be included in a separate small package in the kit, or in a separate partition space.

[0069] In the present application, the CRISPR-Cas12a / Cas13a unit can be used as a complete unit, for example, it is a mixture of different components ssDNA fluorescent probe, ssRNA fluorescent probe, crRNA1, crRNA2, and Cas12a and Cas13a, and it can exist in the form of dry powder or storage solution.

[0070] In the present application, the CRISPR-Cas12a / Cas13a unit may also include different subunits. In one embodiment of the present application, the subunits may, for example, include ssDNA fluorescent probes, ssRNA fluorescent probes, crRNA1, crRNA2, Cas12a or Cas13a, respectively.

[0071] For example, the ssDNA fluorescent probe is in the form of dry powder or liquid storage as one of the subunits; the ssRNA fluorescent probe is in the form of dry powder or liquid storage as another subunit; the crRNA1 is in the form of dry powder or liquid storage as another subunit; the crRNA2 is in the form of dry powder or liquid storage as another subunit; the Cas12a is in the form of dry powder or liquid storage as another subunit; the Cas13a is in the form of dry powder or liquid storage as another subunit.

[0072] In some embodiments, the subspecies of Burkholderia gladioli detected by the kit is Burkholderia gladioli pv. cocovenenans.

[0073] In the third aspect of the present application, there is provided use of the nucleic acid product as described in the first aspect in preparing a product for detecting Burkholderia gladiolus and its subspecies in a sample.

[0074] In some embodiments, the product is selected from the group consisting of a chip and a kit.

[0075] In a fourth aspect of the present application, a method for detecting Burkholderia gladiolus or its subspecies in a sample is provided, comprising testing the sample using the nucleic acid product described in the first aspect or the kit described in the second aspect.

[0076] In some embodiments, the method comprises:

[0077] Amplify the DNA of the sample using the RAA system comprising the primer set to prepare an amplification product;

[0078] The CRISPR-Cas12a / Cas13a reaction unit is mixed with the amplification product to form a CRISPR-Cas12a / Cas13a reaction system, and reacted.

[0079] In the present application, the amplification conditions of the RAA system can be conventional in the art, as long as the DNA extracted from the sample can be effectively amplified.

[0080] In some embodiments, the amplification conditions of the RAA system are 37° C. and 30 min.

[0081] In some embodiments, the amplification of the RAA system is performed in a PCR instrument.

[0082] In some embodiments, each 50 μL of the RAA system comprises 25 μL of buffer v, primers with sequences as shown in SEQ ID NOs: 3 to 6 and a final concentration of 0.4 μM, 2 μL of DNA in the sample, and 5 μL of magnesium acetate, supplemented with ddH2O to 50 μL.

[0083] In some embodiments, the RAA system comprises 25 μL of buffer v, 10 μM primers with sequences as shown in SEQ ID NOs: 3 to 6 and a volume of 2 μL each, 10 μL of ddH2O, 2 μL of DNA in the sample, and 5 μL of magnesium acetate. In some embodiments, the RAA system is a 50 μL system.

[0084] In some embodiments, each 50 μL of the RAA system comprises 25 μL of buffer v, 10 μM primers having sequences as shown in SEQ ID NOs: 3 to 6 and each having a volume of 2 μL, 10 μL of ddH2O, 2 μL of DNA in the sample, and 5 μL of magnesium acetate.

[0085] In some embodiments, the reaction conditions of the CRISPR-Cas12a / Cas13a reaction system may be conventional in the art, such as 37° C., 30 min.

[0086] In some embodiments, the reaction of the CRISPR-Cas12a / Cas13a reaction system is carried out in a real-time fluorescence quantitative PCR instrument.

[0087] In some embodiments, each 20 μL of the CRISPR-Cas12a / Cas13a reaction system contains 2 μL of 10× lysis buffer, 4 μL of 5× trans mixture, crRNA1 with a final concentration of 0.4 μM, crRNA2 with a final concentration of 0.2 μM, ssDNA fluorescent probe with a final concentration of 0.2 μM, ssRNA fluorescent probe with a final concentration of 0.2 μM, 0.4 μL of 40×T7 RNA polymerase, Cas12a protein with a final concentration of 25 nM, Cas13a protein with a final concentration of 25 nM, 0.8 μL of RAA amplification product, and RNase-free water is supplemented to 20 μL.

[0088] In some embodiments, the CRISPR-Cas12a / Cas13a reaction system comprises 2 μL 10× lysis buffer, 4 μL 5× trans mixture, 0.8 μL crRNA1 at a concentration of 10 μM, 0.4 μL crRNA2 at a concentration of 10 μM, 0.4 μL ssDNA fluorescent probe at a concentration of 10 μM, 0.4 μL ssRNA fluorescent probe at a concentration of 10 μM, 0.4 μL 40×T7 RNA polymerase, 0.5 μL 1 μM Cas12a, 0.5 μL 1 μM Cas13a, 0.8 μL RAA amplification product, and 9.8 μL RNase-free water.

[0089] In some embodiments, the CRISPR-Cas12a / Cas13a reaction system is a 20 μL system.

[0090] In some embodiments, each 20 μL of the CRISPR-Cas12a / Cas13a reaction system contains 2 μL of 10× lysis buffer, 4 μL of 5× trans mixture, 0.8 μL of 10 μM crRNA1, 0.4 μL of 10 μM crRNA2, 0.4 μL of 10 μM ssDNA fluorescent probe, 0.4 μL of 10 μM ssRNA fluorescent probe, 0.4 μL of 40×T7 RNA polymerase, 0.5 μL of 1 μM Cas12a, 0.5 μL of 1 μM Cas13a, 0.8 μL of RAA amplification product, and 9.8 μL of RNase-free water.

[0091] In some embodiments, the method further comprises: extracting DNA from the sample before performing amplification and reaction.

[0092] Taking food samples as an example, the steps of extracting the concentrated DNA in the samples include the following sub-steps:

[0093] (1) Crush the food sample;

[0094] (2) using a deep-processed food extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract DNA from the food crushed in substep (1);

[0095] (3) Collect the DNA extracted in sub-step (2) into the same centrifugal column and elute with elution buffer.

[0096] In some embodiments, the method further comprises interpreting the results after performing amplification and reaction.

[0097] Taking the detection using the ssDNA fluorescent probe and ssRNA fluorescent probe having structures such as FAM-TTATTATT-BHQ1 and VIC-mArArUrGrGrCmAmAr ArUrGrGrCmA-BHQ1 as an example, the following interpretations are made for pure culture samples and food samples, respectively:

[0098] For pure culture samples, if the FAM fluorescence signal exceeds the threshold but the VIC fluorescence signal does not exceed the threshold, the sample is a gladiolus Burkholderia species but not a cocovenenan subspecies; if the results show that both the FAM and VIC fluorescence signals exceed the threshold, the sample is a toxin-producing gladiolus Burkholderia cerebrovenans subspecies; if the results show that both the FAM and VIC fluorescence signals do not exceed the threshold, the sample is not a gladiolus Burkholderia species.

[0099] For food samples, sample testing and blank control testing are required at the same time. If the result of FAM fluorescence signal is significant to the blank control, but VIC fluorescence signal is not significant, then the food sample contains gladiolus Burkholderia species; if the result of FAM and VIC double fluorescence signal is significant to the blank control, then the food sample contains toxin-producing gladiolus Burkholderia cocovenenans subspecies; if the result of FAM and VIC fluorescence signal is not significant to the blank control, then the sample does not contain gladiolus Burkholderia species and subspecies.

[0100] The nucleic acid product and the kit containing the same in the present application have a wide range of applications, and can not only be directly used to detect gladiolus Burkholderia, but also can be used to screen gladiolus Burkholderia, culture gladiolus Burkholderia and any other operations related to gladiolus Burkholderia. The nucleic acid product and the kit in the present application can also be used for food safety evaluation, etc.

[0101] Some examples are provided below.

[0102] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0103] Example 1

[0104] 1. Design and preparation of primers and crRNA.

[0105] The genome sequence of Burkholderia gladioli Co14 chromosome 1 (CP033430.1) was downloaded from the National Center for Biotechnology Information (NCBI). Two target regions were selected for detection: the 16S-23S rRNA gene region 1 (3,834,843-3,835,078) of Burkholderia gladioli subsp. 16S-23S rRNA gene region 2 (3,011,801-3,011,986) of Pseudomonas cocovenenans subsp. bonA gene region 2 (3,011,801-3,011,986). The recombinase-mediated amplification (RAA) primers for these two targets were designed using Primer Premier 5.0 according to the RAA primer design principles.

[0106] The amplification of region 1 is detected by crRNA1 with Cas12a, and the amplification of detection region 2 is detected by crRNA2 with Cas13a. crRNA1 was designed using CRISPRdirect software, and crRNA2 was designed using SnapGene software. Overlapping PCR was used to amplify the DNA templates for transcription of crRNA1 and crRNA2 with primers crRNA12-F / crRNA12-R and crRNA13-F / crRNA13-R. The sequences of crRNA1, crRNA2, and primers are shown in Table 1. According to the instructions, crRNA was transcribed using the yield T7RNA in vitro transcription kit, and crRNA was purified using a column RNA rapid concentration and purification kit.

[0107] The primers, fluorescent probes, common reagents, high-yield T7 RNA in vitro transcription kit and column-based RNA rapid concentration and purification kit used were all from Bio-Tech (Shanghai) Co., Ltd. The primers and probes used are shown in the following table.

[0108] Table 1 Primer and probe sequences

[0109]

[0110] 2. Preparation of Artificially Contaminated Food Samples

[0111] Burkholderia gladiolus ATCC 33664 was inoculated into potato glucose water medium, cultured at 36℃ for 24h, and then diluted with physiological saline to a McFarland turbidity of 0.5. Fresh noodles and soaked Tremella fuciformis were used as test samples, and two 5g samples were taken respectively, one of which was used as the experimental group, and 100μL of the diluted bacterial solution was added and allowed to dry naturally, and the other was used as the control group.

[0112] 3. DNA Extraction

[0113] The samples were added to 50 mL centrifuge tubes respectively, and the food matrix was broken up. DNA was extracted using a deep-processed food extraction kit according to the instructions. Finally, the samples were collected in the same centrifuge column and eluted with 50 μL elution buffer.

[0114] 4.RAA amplification and CRISPR-Cas12a / Cas13a dual detection system

[0115] The RAA amplification reaction was performed using a recombinase-assisted amplification kit (Jiangsu Qitian Gene Biotechnology Co., Ltd.). The RAA reaction system was 25 μL of buffer V, 2 μL of each primer (RAA-SF, RAA-SR, RAA-B-T7-F, RAA-BR, 10 μM), 10 μL of sterile distilled water, 2 μL of DNA, and 5 μL of magnesium acetate. The mixture was added to a freeze-dried reaction device and reacted at 37°C in a PCR instrument for 30 min. An equal volume of sterile distilled water was used instead of DNA as a blank control.

[0116] The CRISPR-Cas13a DNA detection kit was used for subsequent analysis. The CRISPR-Cas12a / Cas13a reaction system consisted of 2 μL 10× lysis buffer, 4 μL 5× trans mixture, 0.8 μL crRNA12 (10 μM), 0.4 μL crRNA13 (10 μM), 0.4 μL ssDNA (10 μM, FAM-BHQ), 0.4 μL RNA (10 μM, VIC-BHQ), 0.4 μL 40×T7 RNA polymerase, 0.5 μL Cas12a (NE B, 1 μM), 0.5 μL Cas13a (1 μM), 0.8 μL RAA amplification product, and 9.8 μL RNase-free water. The cells were incubated at 37°C for 30 min in a real-time fluorescence quantitative PCR instrument, and the fluorescence intensity of the FAM and VIC channels was monitored simultaneously, and recorded every 1 min.

[0117] 5. Judgment of test results

[0118] Quality control system: The blank control double fluorescence signal is low level. For pure culture samples, the threshold of FAM and VIC double fluorescence signal is 1500 relative fluorescence intensity. For food samples, distilled water is used to replace the extracted nucleic acid as blank control. The sample FAM and VIC double fluorescence signal is significantly increased compared with the blank control. If the quality control system is wrong, the experiment is invalid.

[0119] For pure culture samples, if the FAM fluorescence signal exceeds the threshold but the VIC fluorescence signal does not exceed the threshold, the sample is a gladiolus Burkholderia species but not a cocovenenan subspecies; if the results show that both the FAM and VIC fluorescence signals exceed the threshold, the sample is a toxin-producing gladiolus Burkholderia cerebrovenans subspecies; if the results show that both the FAM and VIC fluorescence signals do not exceed the threshold, the sample is not a gladiolus Burkholderia species.

[0120] For food samples, sample testing and blank control testing are required at the same time. If the result of FAM fluorescence signal is significant to the blank control, but VIC fluorescence signal is not significant, then the food sample contains gladiolus Burkholderia species; if the result of FAM and VIC double fluorescence signal is significant to the blank control, then the food sample contains toxin-producing gladiolus Burkholderia cocovenenans subspecies; if the result of FAM and VIC fluorescence signal is not significant to the blank control, then the sample does not contain gladiolus Burkholderia species and subspecies.

[0121] Example 2 Specific detection of gladiolus Burkholderia species and Pseudomonas cocovenenans subspecies

[0122] 1. Specificity detection

[0123] 19 strains of gladiolus Burkholderia strains ATCC 33664 (producing fumonisin) and CICC 10574 (not producing fumonisin) (see Table 2) were activated in PDB medium for 24 hours. 100 μL of bacterial culture aliquots were inoculated on PDA medium and cultured at 26 ± 1 ° C for 5 days. A single colony was picked with a sterile inoculation stick into a 1.5 mL sterile centrifuge tube containing 50 μL of sterile water, incubated at 100 ° C for 10 minutes, and then centrifuged at 12000 rpm for 10 minutes. The supernatant was used for subsequent amplification templates.

[0124] In addition, five non-target strains including Burkholderia cepacia, Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium and Staphylococcus aureus were activated and cultured for specificity experiments.

[0125] Table 2 The strains used in the experiment and the production of fumonisin

[0126] Strain number Strain source Production of fumonisin Burkholderia gladiolus ATCC 33664 Purchased from ATCC + Burkholderia gladiolus CICC 10574 Purchased from CICC - Burkholderia gladiolus CICC 25088 Purchased from CICC - Burkholderia gladiolus CICC 25089 Purchased from CICC - Burkholderia gladiolus CICC 25108 Purchased from CICC + Burkholderia gladiolus CICC 25126 Purchased from CICC + Burkholderia gladiolus CICC 25132 Purchased from CICC + Burkholderia cepacia CICC 23648 Purchased from CICC / Pseudomonas aeruginosa ATCC 15442 Purchased from ATCC / Escherichia coli ATCC 8739 Purchased from ATCC / Salmonella Typhimurium ATCC 14028 Purchased from ATCC / Staphylococcus aureus ATCC 6538 Purchased from ATCC /

[0127] 2. Method detection limit

[0128] Burkholderia gladiolus ATCC 33664 was cultured in PDB, incubated at 36°C, 220 rpm for 36 h, diluted with saline to 0.5 McFarland standard, and further diluted with buffer EB to 6 gradients ranging from 10 5 ~10 0 CFU / mL. The bacterial concentration in the diluted solution was verified by plate counting method. Incubate at 100℃ for 10min, centrifuge at 12000rpm for 10min, take the supernatant for detection, and repeat three times in parallel.

[0129] Experimental results:

[0130] Specificity test results

[0131] To further evaluate the specificity of the RAA-CRISPR-Cas12a / Cas13a method, 19 Burkholderia gladioli strains and five non-target strains were tested. The relative fluorescence intensity of the ssDNA and ssRNA reporters at 15 min in each sample was compared. Figure 1 As shown. In the 12 samples of gladiolus Burkholderia producing fumonisin, the relative fluorescence intensity of ssDNA and ssRNA reporters were significantly higher than that of non-target strains and blank controls (p<0.05). In the 7 samples of gladiolus Burkholderia strains that did not produce fumonisin, the relative fluorescence intensity of ssDNA reporter gene was significantly higher than that of non-target strains and blank controls (p<0.05), while the relative fluorescence intensity of ssRNA reporter gene had no significant difference (p>0.05). In non-target strains and blank controls, the relative fluorescence intensity of ssDNA and ssRNA reporters was maintained at a low level. The results showed that this method can specifically detect gladiolus Burkholderia, gladiolus Burkholderia cocovina and non-gladiolus Burkholderia.

[0132] Detection limit experimental results

[0133] To further evaluate the sensitivity of the RAA-CRISPR-Cas12a / Cas13a method at the bacterial level, we used Burkholderia gladiolus ATCC 33664 to test the detection limit of the method. Figure 2 As shown, 10 5 ~10 0 The relative fluorescence intensity of ssDNA and ssRNA reporter genes in the extracts with CFU / mL was significantly higher than that in the blank control (p<0.05). This indicates that the detection limit of the RAA-CRISPR-Cas12a / Cas13a method is 10 0CFU / mL.

[0134] Example 3

[0135] Detection of Burkholderia gladiolus in artificially contaminated food samples

[0136] 1. Preparation of artificially contaminated food samples and DNA extraction

[0137] The Burkholderia gladiolus subsp. cocovinifera ATCC 33664 isolated from food was inoculated into potato glucose water medium, cultured at 36°C for 24 h, and then the bacteria were collected by centrifugation and diluted with physiological saline to a McFarland turbidity of 0.5. The dilution was performed in a 10-fold gradient, and the final bacterial concentration range was 10 6 CFU / g~10 2 CFU / g. Fresh noodles and soaked Tremella fuciformis were used as test samples. Two 5g samples were taken respectively. One of them was used as the experimental group. 100μL of diluted bacterial solution was added and allowed to air dry naturally. The other was used as the control group. After the food matrix was broken, DNA was extracted using a deep-processed food extraction kit according to the instructions.

[0138] 2. RAA amplification and CRISPR-Cas12a / Cas13a dual detection

[0139] Detection was performed using the primers, probes, RAA amplification, and CRISPR-Cas12a / Cas13a dual detection system in Example 1.

[0140] Experimental results:

[0141] The experimental results are as follows Figure 3 In these samples, the detection limits of the RAA-CRISPR-Cas12a / Cas13a method were 10 2 CFU / mL and 10 3 CFU / mL. 5 ~10 2 CFU / mL bacterial concentration range, except for 10 5 CFU / mL of fresh noodles, the relative fluorescence intensity of the ssRNA reporter gene was generally much lower than that of the ssDNA reporter gene.

[0142] However, in the specificity test of Example 2, at the same bacterial concentration, the relative fluorescence intensity of the ssRNA reporter gene was not much different from that of the ssDNA reporter gene.

[0143] These results suggest that the food matrix may reduce the fluorescence intensity, which may be due to RNA degradation caused by substances released from the food matrix. Nevertheless, the RAA-CRISPR-Cas12a / Cas13a method is still sensitive for the detection of Burkholderia gladioli subsp. cocovina. When this method was used to detect plate-cultured strains, there was a large difference in the relative fluorescence intensity of ssDNA and ssRNA between positive and negative samples, indicating that this method can quickly and easily identify Burkholderia gladioli and its subsp. cocovina for colonies isolated and cultured from food.

[0144] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0145] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A nucleic acid product, It is characterized in that It comprises a ssDNA fluorescent probe, a ssRNA fluorescent probe, crRNA1, crRNA2 and a primer set; The ssDNA fluorescent probe comprises the following sequence: TTATTATT; The ssRNA fluorescent probe comprises the following structure: mArArUrGrGrCmAmArArUrGrGrCmA; The sequence of the crRNA1 is shown in SEQ ID NO: 1; The sequence of the crRNA2 is shown in SEQ ID NO: 2; The sequences of the primers in the primer set are shown in SEQ ID NOs: 3 to 6, respectively.

2. The nucleic acid product according to claim 1, It is characterized in that The ssDNA fluorescent probe and the ssRNA fluorescent probe are each labeled with a fluorescent group and are different from each other and do not interfere with each other; The fluorescent group is selected from the group consisting of FAM, HEX, TET, VIC, JOE, ROX and Texas; Optionally, the ssDNA fluorescent probe and the ssRNA fluorescent probe each further carry a quenching group; the quenching group is selected from the group consisting of BHQ2 and BHQ1; Further optionally, the fluorescent group is FAM, and the quenching group is BHQ1; The fluorescent group is HEX, TET, VIC or JOE, and the quenching group is BHQ1; or The fluorescent group is ROX or Texas, and the quenching group is BHQ2; Alternatively, the structure of the ssDNA fluorescent probe is as follows: FAM-TTATTATT-BHQ1; The structure of the ssRNA fluorescent probe is as follows: VIC-mArArUrGrGrCmAmArArUrGrGrCmA-BHQ1.

3. A kit for detecting Burkholderia gladioli and its subspecies in a sample, It is characterized in that It comprises the nucleic acid product as claimed in claim 1; Optionally, the kit comprises one or more of a recombinase-mediated isothermal nucleic acid amplification (RAA) unit and a CRISPR-Cas12a / Cas13a reaction unit; in, The RAA unit comprises the primer set; The CRISPR-Cas12a / Cas13a reaction unit comprises the ssDNA fluorescent probe, the ssRNA fluorescent probe, the crRNA1 and the crRNA2, and one or more of Cas12a and Cas13a.

4. The kit according to claim 3, It is characterized in that The subspecies of gladiolus Burkholderia detected by the kit is gladiolus Burkholderia gladiolipv. cocovenenans.

5. Use of the nucleic acid product according to claim 1 in preparing a product for detecting Burkholderia gladiolus and its subspecies in food.

6. The use according to claim 5, It is characterized in that The products include chips and test kits.

7. A method for detecting Burkholderia gladiolus or its subspecies in a sample, It is characterized in that The method comprises testing the sample using the nucleic acid product according to claim 1 or the kit according to any one of claims 2 to 4.

8. The method according to claim 7, It is characterized in that The method comprises: Amplify the DNA of the sample using the RAA system comprising the primer set to prepare an amplification product; The CRISPR-Cas12a / Cas13a reaction unit is mixed with the amplification product to form a CRISPR-Cas12a / Cas13a reaction system, and reacted.

9. The method according to claim 8, It is characterized in that The RAA system satisfies one or two of the following conditions 1) to 2): 1) Amplification conditions were 37°C, 30 min; and, 2) Each 50 μL of the RAA system contains 25 μL of buffer v, primers with sequences as shown in SEQ ID NOs: 3 to 6 and a final concentration of 0.4 μM, 2 μL of DNA in the sample, and 5 μL of magnesium acetate, supplemented with ddH 2 O to 50 μL.

10. The method according to claim 8 or 9, It is characterized in that The CRISPR-Cas12a / Cas13a reaction system satisfies one or two of the following conditions i) to ii): i) the reaction conditions are 37° C., 30 min; and, ii) Each 20 μL of the CRISPR-Cas12a / Cas13a reaction system contained 2 μL of 10× lysis buffer, 4 μL of 5× trans mixture, crRNA1 at a final concentration of 0.4 μM, crRNA2 at a final concentration of 0.2 μM, ssDNA fluorescent probe at a final concentration of 0.2 μM, ssRNA fluorescent probe at a final concentration of 0.2 μM, 0.4 μL of 40×T7 RNA polymerase, Cas12a protein at a final concentration of 25 nM, Cas13a protein at a final concentration of 25 nM, 0.8 μL of RAA amplification product, and RNase-free water was added to 20 μL.

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