Primer group, kit and detection method for rapidly detecting helvetia striata based on molecular identity card

Abstract

The invention discloses a molecular identity card-based primer group, a kit and a detection method for rapidly detecting helvetia striatus, and belongs to the technical field of molecular biology. The primer group disclosed by the invention comprises an upstream primer as shown in SEQ ID NO. 1 and a downstream primer as shown in SEQ ID NO. 2. According to the invention, a 22bp short sequence is screened out from the ITS sequence of the helvetia striata as a molecular identity card of the helvetia striata, a specific primer is designed according to the molecular identity card, a molecular identity card technology is creatively combined with a real-time fluorescent PCR dye method, and amplification products accumulated in the PCR process are detected by using a fluorescent dye SYBR Green I, so that the molecular identity card of the helvetia striata is obtained. And the identification of the helvetia strigosa can be completed within 45 minutes. The method has the advantages of convenience, rapidness, strong specificity, high sensitivity and good applicability, and can provide technical support for rapid detection of poisonous mushrooms.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a primer set, a kit and a detection method for rapid detection of Helmetosporium striata based on a molecular identification card. Background Art

[0002] Mushroom poisoning is one of the serious food safety issues. Amanita argyi contains amatoxins, which can cause acute liver damage poisoning, and its mortality rate is second only to the highly toxic mushrooms of the genus Amanita. Amanita argyi grows on rotten wood, sawdust piles or ground with high humus. Its biggest feature is that it is "small, yellowish brown, striped, and grows on trees", and it is one of the most common highly toxic mushrooms. After accidentally eating Amanita argyi poisoning, vomiting and diarrhea symptoms will appear immediately. Most patients will get better on the second day and enter a "false recovery period" of 1-2 days, followed by obvious liver damage and complications such as neurological symptoms and coagulation dysfunction. When poisoning by poisonous mushrooms occurs, the poisoned person often does not know what kind of poisonous mushroom he has eaten, and it is very likely that he cannot provide uncooked samples, which greatly increases the difficulty of subsequent identification and treatment. In order to provide symptomatic treatment, it is necessary to accurately detect the type of poisonous mushrooms. Medical staff can only detect it through the post-meal leftovers provided by the poisoned person and the vomitus of the patient. However, at present, the identification and identification of Helmetosporium striata is mainly based on morphological characteristics and DNA molecular sequences, which requires professional taxonomic knowledge and molecular biological identification conditions, and is time-consuming, which is not conducive to the timely diagnosis and treatment of poisoned patients by primary medical units. In addition, the target samples in vomitus are digested by gastric juice, and their DNA undergoes varying degrees of degradation, making it difficult to accurately detect them using existing detection methods. Therefore, rapid, accurate, and low-cost detection technology can provide important technical support for the treatment of patients poisoned by poisonous mushrooms and poisoning incidents.

[0003] Real-time fluorescence PCR achieves the purpose of detection by collecting fluorescent group signals, and is divided into two methods: dye method and probe method. In 2024, Duan et al. designed TaqMan-MGB probes and specific primers based on molecular ID technology, real-time fluorescence PCR technology and stable base difference sites, and established a rapid detection technology for lethal Amanita in the Amanita group of the genus Amanita. The probe method can increase the specificity of the reaction collection signal through the probe. The signal can only be collected when amplification occurs on the fragment bound by the probe, but the cost of synthesizing the probe is high; the dye method is economical, but its specificity is not as good as the probe method. Therefore, the development of an economical, highly applicable, highly specific and sensitive rapid identification technology for Helmetospora fasciatus is important for mushroom species identification and symptomatic treatment in mushroom poisoning incidents. Summary of the invention

[0004] The purpose of the present invention is to provide a primer set, a kit and a detection method for rapid detection of Helmetospora striata based on a molecular ID card, so as to solve the problems existing in the above-mentioned prior art. The present invention innovatively combines the molecular ID card technology with the real-time fluorescent PCR dye method, uses the molecular ID card of Helmetospora striata as a forward primer to design specific primers, and uses the fluorescent dye SYBR Green I to detect the amplification products accumulated in the PCR process, thereby achieving detection and identification of Helmetospora striata within 45 minutes. The method is convenient and fast, has strong specificity and high sensitivity, has good applicability, and can provide technical support for the rapid detection of poisonous mushrooms.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a primer set for rapid detection of Helmetosporium striata based on molecular identification card, wherein the primer set comprises an upstream primer as shown in SEQ ID NO.1 and a downstream primer as shown in SEQ ID NO.2.

[0007] The present invention also provides the use of the primer set in preparing a reagent or a kit for rapid detection of Capsulosporium striata.

[0008] The present invention also provides a reagent for rapidly detecting Helmetosporium striata based on a molecular ID card, wherein the reagent comprises the above-mentioned primer set.

[0009] The present invention also provides a kit for rapid detection of Helmetosporium striata based on molecular identification card, and the kit comprises the above primer set.

[0010] Furthermore, the kit also includes SYBR Green qPCRMix.

[0011] The present invention also provides application of the primer set, the reagent or the kit in rapid detection of Helmetosporium striatum.

[0012] The present invention also provides a method for quickly detecting Helmetosporium fasciatum, comprising the following steps:

[0013] Extracting DNA from the sample to be tested;

[0014] Using the DNA as a template, the primer set is used to perform real-time fluorescence PCR amplification;

[0015] If a fluorescent signal and an amplification curve appear in the amplification curve graph, it means that the sample to be tested contains Helmetosporium striata; if no fluorescent signal or amplification curve appears, it means that the sample to be tested does not contain Helmetosporium striata.

[0016] Furthermore, the reaction system of the real-time fluorescence PCR amplification is: 5 μL of 2×SYBR Green qPCR Mix, 0.5 μL of each of 10 μmol / L upstream and downstream primers, 1 μL of DNA template, and 3 μL of RNase-Free Water.

[0017] Furthermore, the reaction procedure of the real-time fluorescence PCR amplification is: 95°C for 2 min, 95°C for 15 s, 60°C for 30 s, and 40 cycles.

[0018] The present invention discloses the following technical effects:

[0019] The present invention screens out a short sequence of 22 bp from the ITS sequence of Helmetosporium striata as the molecular ID of Helmetosporium striata, designs specific primers based on the molecular ID, and innovatively combines the molecular ID technology with the real-time fluorescent PCR dye method, and uses the fluorescent dye SYBR Green I to detect the amplification products accumulated in the PCR process, thereby achieving the identification of Helmetosporium striata within 45 minutes, and the detection limit can reach 10-5ng. SYBR Green I, as a widely used fluorescent marker, can interact with and fuse with the minor groove in double-stranded DNA in a non-specific manner, but does not affect its effect on single-stranded DNA. If it is not adsorbed by the double-stranded DNA on the surface of the solution, SYBR Green I only produces very little fluorescence; on the contrary, once it is combined with it, it emits a very strong fluorescent signal, making the detection result more intuitive.

[0020] Compared with the dye method, the detection method of the present invention has stronger specificity; compared with the probe method, the detection method of the present invention has lower cost; it has the characteristics of strong specificity and high sensitivity, and the detection results are not affected by boiling the samples and digestion by gastric juice. It is suitable for the detection of post-meal residues and vomitus in mushroom poisoning incidents, and provides technical support for the rapid detection of poisonous mushrooms. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 This is the specific test result of Helmetosporium fasciatum;

[0023] Figure 2 This is the sensitivity test result of Helmetosporium fasciatum;

[0024] Figure 3These are the suitability test results for the Capsicum annuum. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Example 1

[0031] 1. Materials and methods

[0032] 1.1 Specimen collection and identification

[0033] The specimens used in this embodiment come from the collection of the herbarium and the field collection, and are stored in the form of dry specimens in the Fungus Herbarium of Jilin Agricultural University (FUNGARIUM OF JILINAGRICULTURALUNIVERSITY, FJAU). All specimens have been identified by morphology and ITS sequence comparison. Specimen related information is shown in Table 1.

[0034] Table 1 Specimen information

[0035]

[0036] 1.2 Extraction of genomic DNA

[0037] The genomic DNA of the target species specimens that had been accurately identified by morphology was extracted using a modified alkaline lysis method (alkaline lysis solution formula: 0.5 mol / L NaOH solution, 1% mass concentration of Triton X-100, 1% mass concentration of PVP40). The DNA concentration was detected using a NanoDrop 2000 ultra-micro spectrophotometer, and the OD was calculated. 260 / OD 280 If the ratio is close to 1.8, it indicates that the purity of the extract is good, and the qualified DNA sample is placed in a 4°C refrigerator for later use, or in a -20°C refrigerator for later use.

[0038] 1.3 PCR amplification and sequencing

[0039] ITS fragments were used for amplification and sequencing. The PCR amplification reaction system was a 25 μL system: 12.5 μL Taq PCR MasterMix (2×, blue dye), 1 μL each of 10 μM forward and reverse primers, 1 μL DNA template (10 ng / μL), and finally ddH2O was used to make up to 25 μL. Then amplification was performed in a PCR instrument (Bio-Rad T100). The ITS amplification primers were ITS1 and ITS4, and the amplification program was: 94°C pre-denaturation for 3 min, 30 cycles: 94°C denaturation for 30 s, 55°C annealing for 45 s, and 72°C extension for 1 min; after the cycle, the final extension was performed at 72°C for 10 min, and then stored at 4°C. The amplified product was detected by 1% agarose gel electrophoresis, and the PCR product with clear and bright electrophoresis bands was sent for sequencing. The sequencing primers were the same as the PCR amplification primers (sequencing was completed by Shanghai Sangon Biotech Co., Ltd.).

[0040] 1.4 Data Analysis

[0041] Sequencher (v5.4.5) software was used to perform sequence splicing and manual proofreading on the sequencing files sent back by the sequencing company to remove overlapping peak fragments in the forward and reverse primer intervals and low-quality fragments at both ends. At the same time, the reverse complementarity principle of forward and reverse sequences was used to solve a poly structure caused by the repetition of a single base to ensure the successful splicing of high-quality fragments.

[0042] 1.5 Molecular ID mining and primer design

[0043] Aliview software was used to perform multiple sequence alignment of the successfully spliced ​​ITS sequence of Helmetospora striata and 198 sequences of the same species and different species of the same genus downloaded from Genbank to find the specific sequence interval of the species sequence of Helmetospora striata, and the selected target sequence was analyzed by blastn in the BLAST tool of the NCBI website. After a large amount of sequence data analysis, a 22bp short sequence of Helmetospora striata (SEQ ID NO.1: 5'-GATGTGGTTGTCGCTGACTCTT-3') was screened out and defined as the molecular ID card of Helmetospora striata. Based on the molecular ID card of Helmetospora striata, the molecular ID card was used as one of the primers, and primer 5.0 software was used to design the primer. The primer was synthesized by Shanghai Sangon Biotech Co., Ltd. (Table 2).

[0044] Table 2 Primer information designed for RT-PCR test

[0045]

[0046] 1.6 Specificity evaluation

[0047] The specimens listed in Table 1 were used as test materials for the specificity of the real-time fluorescence PCR method for detecting the spores of the spores. The target species material (No. 1), 18 poisonous and non-toxic mushroom materials of the same genus and other genera (No. 2-19) and a blank control (NTC) with no template added with ddH2O were tested by real-time fluorescence PCR under the same conditions. The reaction system of real-time fluorescence PCR was 10μL system, see Table 3, and the reaction program was: 95℃2min, 95℃15s, 60℃30s, 40 cycles. The number of cycles was used as the horizontal axis, and the increase in the fluorescence signal of the amplification curve (ΔRn) was used as the vertical axis. If the amplification curve shows an obvious amplification curve, it means that it is positive, indicating that the target species has been detected; if there is no amplification curve, it means that it is negative, and no target species has been detected.

[0048] Table 3 PCR amplification reaction system

[0049]

[0050]

[0051] 1.7 Sensitivity evaluation

[0052] In order to test the sensitivity of the real-time fluorescence PCR method for Helmetosporium fasciatum, the initial concentration of DNA (1 ng / μL) was diluted 10 times with DNA diluent (Sangon Biotech) as shown in Table 4. In the 10 μL reaction system, the DNA contents were 1 ng, 10 -1 ng, 10 -2 ng, 10-3 ng, 10 -4 ng, 10 -5 ng and 10 -6 ng, and set up a no-template blank control (NTC) with an equal amount of ddH2O added, and perform real-time fluorescence PCR detection under the same conditions (reaction system and reaction procedure see 1.6). The cycle number is used as the horizontal axis, and the fluorescence signal increase value (ΔRn) of the amplification curve is used as the vertical axis. If the amplification curve shows an obvious amplification curve, it means it is positive, indicating that the sample at this concentration has been detected; if there is no amplification curve, it means it is negative, indicating that the sample at this concentration cannot be detected.

[0053] Table 4 RT-PCR test sensitivity test treatment

[0054]

[0055] 1.8 Applicability Evaluation

[0056] When poisoning by poisonous mushrooms occurs, the victim often does not know what kind of poisonous mushrooms he or she has eaten, and is very likely to be unable to provide uncooked samples, which greatly increases the difficulty of subsequent identification and treatment. In order to provide symptomatic treatment, it is necessary to accurately detect the type of poisonous mushrooms. Medical staff can only conduct tests based on the meal leftovers provided by the victim and the vomitus of the patient. In the suitability test, the on-site environment will be simulated to process the samples to be tested, and different times of boiling in 100°C water and digestion with gastric juice will be performed to simulate meal leftovers and vomitus.

[0057] In order to detect the applicability of the real-time fluorescence PCR method for Helmetosporium fasciatum, as shown in Table 5, the samples were boiled at 100°C in groups, cooled to room temperature, and then artificial gastric juice (USP, sterile) was added to simulate digestion in groups and kept warm in a constant temperature water bath at 37°C. Template DNA was then extracted according to the method of "1.2 Extraction of genomic DNA" for real-time fluorescence PCR detection. Real-time fluorescence PCR detection was performed under the same conditions (reaction system and reaction procedure see 1.6). The number of cycles was used as the horizontal axis, and the increase in the fluorescence signal of the amplification curve (ΔRn) was used as the vertical axis. If the amplification curve shows an obvious amplification curve, it means that it is positive, which means that the sample under the treatment condition is detected; if there is no amplification curve, it means that it is negative, which means that the sample under the treatment condition cannot be detected.

[0058] Table 5 RT-PCR test suitability test treatment

[0059]

[0060] Note: 30+ means 1% Target species + 99% Lentinula Edodes

[0061] 2. Results Analysis

[0062] 2.1 Specificity test results

[0063] Based on the molecular ID technology, specific primers for Helmetospora fasciatus were designed, and the DNA of each sample was tested by real-time fluorescence PCR to verify the specificity of the method. Figure 1 As shown, the Helmetosporium striata had a fluorescent signal and an obvious amplification curve, while the negative control and the blank control had no signal and no amplification, indicating that the method has good specificity for Helmetosporium striata.

[0064] 2.2 Sensitivity test results

[0065] The sensitivity of the method was verified by real-time fluorescence PCR detection of DNA samples of different concentrations using the designed specific primers. Figure 2 As shown, the DNA content is 10 -5 ng, all three replicates showed fluorescence signals and obvious amplification curves, indicating that the molecular detection limit of Helmetosporium fasciatum can reach 10 -5 ng. Although fluorescence signals and amplification curves appeared after the detection limit of DNA concentration, the repeatability of the amplification curve was poor, so it was not considered that DNA concentrations below the detection limit could be detected. Neither fluorescence signals nor amplification curves appeared in NTC.

[0066] 2.3 Applicability test results

[0067] The designed specific primers were used to perform real-time fluorescence PCR on samples that had been boiled and digested with artificial gastric juice, as well as a mixture of Helmetella fasciata and Lentinula edodes at a ratio of 1:99, to verify the applicability of this method. Figure 3 As shown in the figure, treatments 1-7 all had fluorescence signals and obvious amplification curves, while NTC had no fluorescence signals and amplification curves. Real-time fluorescence PCR can detect Helmetella fasciata that was boiled at 100℃ for 30 minutes and digested with artificial gastric juice for 4 hours, as well as a mixture of Helmetella fasciata and Lentinula edodes at a ratio of 1:99, indicating that this method has good applicability to Helmetella fasciata that has been boiled and digested with gastric juice.

[0068] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer set for rapid detection of Capsella striata based on molecular identification, characterized in that: The primer set includes an upstream primer as shown in SEQ ID NO.1 and a downstream primer as shown in SEQ ID NO.

2.

2. Use of the primer set according to claim 1 in preparing a reagent or a kit for rapid detection of Capsulosporium striata.

3. A reagent for rapid detection of Capsicum fasciatum based on molecular identification card, characterized in that: The reagent comprises the primer set according to claim 1.

4. A kit for rapid detection of Capsicum annuum based on molecular identification card, characterized in that: The kit comprises the primer set according to claim 1.

5. The kit according to claim 4, characterized in that The kit also includes SYBR Green qPCRMix.

6. Use of the primer set according to claim 1, the reagent according to claim 3, or the kit according to claim 4 or 5 in rapid detection of Helmetosporium striata.

7. A method for rapid detection of Helmetosporium fasciatum, characterized in that: The steps include: Extracting DNA from the sample to be tested; Using the DNA as a template, the primer set according to claim 1 is used to perform real-time fluorescent PCR amplification; If a fluorescent signal and an amplification curve appear in the amplification curve graph, it means that the sample to be tested contains Helmetosporium striata; if no fluorescent signal or amplification curve appears, it means that the sample to be tested does not contain Helmetosporium striata.

8. The method according to claim 7, characterized in that The reaction system of the real-time fluorescent PCR amplification is: 5 μL of 2×SYBR Green qPCR Mix, 0.5 μL of each of 10 μmol / L upstream and downstream primers, 1 μL of DNA template, and 3 μL of RNase-Free Water.

9. The method according to claim 7, characterized in that: The reaction program of the real-time fluorescence PCR amplification is: 95°C for 2 min, 95°C for 15 s, 60°C for 30 s, and 40 cycles.

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