CN112852992A - Primer group, kit and method for identifying Pleurotus citrinopileatus based on loop-mediated isothermal amplification technology - Google Patents
Primer group, kit and method for identifying Pleurotus citrinopileatus based on loop-mediated isothermal amplification technology Download PDFInfo
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Abstract
The invention discloses a primer group, a kit and a method for identifying Pleurotus citrinopileatus based on a loop-mediated isothermal amplification technology, and relates to the technical field of mushroom germplasm identification. The primer group disclosed by the invention comprises primers shown in SEQ ID NO. 1-5. The primer group disclosed by the invention can quickly and accurately identify or detect the Pleurotus citrinopileatus and has higher specificity and sensitivity; the invention has important significance for the prevention of mushroom poisoning, the rapid detection of poisonous mushrooms such as the Pleurotus citrinopileatus in the poisoning event and the targeted treatment and diagnosis after poisoning.
Description
Technical Field
The invention relates to the technical field of mushroom germplasm identification, in particular to a primer group, a kit and a method for identifying Pleurotus citrinopileatus based on a loop-mediated isothermal amplification technology.
Background
The mushroom is widely distributed as a large fungus in the global scope, and has high nutritive value and medicinal efficacy. Statistically, there are over 14,000 kinds of mushrooms in the world at present, and 3,800 kinds of mushrooms are known in China, about 480 of which are poisonous mushrooms. Many of the poisonous mushrooms have morphological characteristics similar to the edible mushrooms and are difficult to distinguish with the naked eye. In China, many food poisoning incidents are caused by the fact that poisonous mushrooms are eaten by mistake every year. Among them, Lyophyllum macrophyllum (Chlorophylum molybdites) is one of poisonous mushrooms which are easily eaten by mistake. The Pleurotus citrinopileatus is distributed in different areas of China all the year round and is the species of poisonous mushroom nearest to the population. Only 2019 years ago, the mushroom poisoning accident caused by 55 causes of eating the big green pleat umbrella by mistake in China occurs, and at least 133 patients are involved.
Mushroom poisoning becomes one of food safety problems which need to be solved urgently in China. For different species of poisonous mushrooms, they contain multiple toxins, which can lead to different toxic symptoms. Ingestion of the Pleurotus citrinopileatus results in severe gastrointestinal discomfort, which may last for 1-6 hours. Failure to obtain timely treatment can cause failure of multiple organ functions and even life-threatening. When a mushroom poisoning event occurs, an effective, simple and rapid detection method is required for accurate clinical treatment or forensic analysis. In the event of food poisoning by poisonous mushrooms, the mushroom sample loses its original morphological characteristics after being cooked and digested, and thus cannot be identified morphologically. Various mushroom species identification techniques have been developed, of which DNA-based molecular biology techniques are the most widely used. As the basis of the biological genetic material, the nucleic acid is not influenced by age, season, developmental stage and environmental factors, has high thermal stability, and is not interfered by a processing mode. With the development of PCR technology, various detection methods such as real-time fluorescence PCR, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), DNA barcode technology, etc. have been derived. However, these methods are expensive and time consuming and cannot be applied to rapid field tests. The simple, effective, accurate and sensitive mushroom strain identification method is easy to establish, and is beneficial to timely symptomatic treatment at the early stage of poisoning.
At present, an effective identification method for the Pleurotus citrinopileatus is lacking.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a primer group, a kit and a method for identifying Pleurotus citrinopileatus based on a loop-mediated isothermal amplification technology. The primer group, the kit and the method provided by the invention can rapidly and accurately identify or detect the Pleurotus citrinopileatus and have higher specificity and sensitivity; the invention has important significance for the prevention of mushroom poisoning, the rapid detection of poisonous mushrooms such as the Pleurotus citrinopileatus in the poisoning event and the targeted treatment and diagnosis after poisoning.
The invention is realized by the following steps:
in one aspect, the invention provides a primer set for identifying Pleurotus citrinopileatus based on loop-mediated isothermal amplification technology, wherein the primer set comprises: an outer primer pair, an inner primer pair and a loop primer; wherein, the nucleic acid sequence of the outer primer pair is shown as a primer comprising SEQ ID NO.1 and SEQ ID NO.4, the nucleic acid sequence of the inner primer pair is shown as a primer comprising SEQ ID NO.2 and SEQ ID NO.3, and the nucleic acid sequence of the loop primer is shown as SEQ ID NO. 5.
Among them, loop-mediated isothermal amplification (LAMP) is the most widely used method and has received increasing attention. The LAMP reaction can be carried out at a constant temperature of 60-65 ℃, and the exponential amplification is carried out within one hour by 109-1010The target gene is multiplied, and the reaction result can be directly obtained by observing the color change under the sunlight by naked eyes. Over a decade of development, LAMPHas been widely used in the fields of pathogenic microorganism rapid detection, clinical disease diagnosis, food hygiene inspection and the like. However, in the identification of mushroom species, there is no relevant content for the identification of Pleurotus giganteus species at present.
In this regard, the inventors of the present invention specifically analyzed the ITS sequences of various mushroom species including Pleurotus citrinopileatus species, but since the ITS sequences of these species still have a relatively high degree of homology, it is difficult to find a specific target sequence according to the existing methods. However, the inventors determined a target sequence capable of specifically distinguishing the Pleurotus citrinopileatus from other similar species from ITS sequence by creative work, and designed a LAMP primer set capable of specifically identifying the Pleurotus citrinopileatus according to the specific target sequence and creative work: SEQ ID NO. 1-5.
Experimental data of the embodiment of the invention show that the LAMP primer group provided by the invention can directly and rapidly identify the variety of the Pleurotus citrinopileatus; the specificity is high, and the number of mushroom varieties which can be distinguished reaches 43; it also has higher sensitivity, and the detection limit reaches 1 pg; the method can be applied to the prevention of mushroom poisoning and the rapid and accurate detection of whether the poisonous mushroom in the poisoning event is the Pleurotus citrinopileatus Sing, and has important significance for the targeted treatment and diagnosis after poisoning.
In another aspect, the present invention provides the use of the primer set as described above for identifying Pleurotus citrinopileatus.
Based on the detection performance of the primer set provided by the invention, a person skilled in the art can use various scenes or fields (such as poisoning events, variety identification and the like) of the primer set provided by the invention to detect or identify whether the sample to be detected contains the Pleurotus citrinopileatus component, and the primer set belongs to the protection scope of the invention regardless of the application.
In another aspect, the present invention provides a kit for identifying Pleurotus citrinopileatus, which comprises the primer set as described above.
Optionally, in some embodiments of the invention, the kit further comprises a LAMP reaction solution; the LAMP reaction solution contains: 0.58-0.62. mu.M of the outer primer pair (SEQ ID NO.1 and SEQ ID NO.4 are each 0.58-0.62. mu.M), 4.6-5.0. mu.M of the inner primer pair (SEQ ID NO.2 and SEQ ID NO.3 are each 4.6-5.0. mu.M), and 1.8-2.2. mu.M of the loop primer.
Alternatively, in some embodiments of the invention, the LAMP reaction solution contains: potassium chloride, ammonium sulfate, magnesium sulfate heptahydrate and Tween-20.
Alternatively, in some embodiments of the invention, the LAMP reaction solution contains: phenol red dye and Bst DNA polymerase.
Alternatively, in some embodiments of the invention, the LAMP reaction solution contains: NTPs.
In another aspect, the present invention provides a method for identifying Pleurotus citrinopileatus based on loop-mediated isothermal amplification, comprising: the nucleic acid template extracted from the sample to be tested is mixed with the primer set as described above to perform the LAMP reaction.
Alternatively, in some embodiments of the present invention, in the reaction system in which the LAMP reaction is carried out, the concentration of the pair of outer primers is 0.58 to 0.62 μ M; the concentration of the inner primer pair is 4.6-5.0 mu M; the concentration of the loop primer is 1.8-2.2. mu.M.
The sensitivity and specificity of the detection can be improved by appropriate concentrations of the primers, and in some embodiments, by controlling the concentration of each primer within the above-mentioned range, the sensitivity and specificity of the detection can be further improved.
Alternatively, in some embodiments of the invention, the reaction system in which the LAMP reaction is carried out contains a phenol red dye and Bst DNA polymerase.
Alternatively, in some embodiments of the invention, the reaction system in which the LAMP reaction is carried out contains potassium chloride, ammonium sulfate, magnesium sulfate heptahydrate, and Tween-20.
Alternatively, in some embodiments of the invention, the reaction system in which the LAMP reaction is carried out contains NTPs.
Alternatively, in some embodiments of the invention, the reaction temperature for performing the LAMP reaction is 61-65 ℃.
Optionally, in some embodiments of the invention, the sample to be tested is a mushroom, vomit or stool.
Based on the primer set provided by the invention, a person skilled in the art can select a moderate sample to be detected according to actual detection requirements, wherein the sample to be detected comprises but is not limited to mushrooms, vomit and feces; any sample of interest may be used as the sample to be tested according to the present invention. The person skilled in the art can extract nucleic acids as templates from the test sample based on methods routine in the art.
Alternatively, in some embodiments of the invention, the nucleic acid template is genomic DNA.
It should be noted that, in some embodiments, one skilled in the art can extract RNA from the sample to be tested as a template for the LAMP reaction, and only needs to make appropriate adjustments to the LAMP reaction. Therefore, whether genomic DNA or RNA is used as a nucleic acid template, it is within the scope of the present invention.
Optionally, in some embodiments of the invention, the method further comprises: judging the LAMP reaction result by adopting a chromogenic dye visual observation method or a real-time turbidity detection method; the judgment method of the visual observation method of the chromogenic dye comprises the following steps: after the LAMP reaction is finished, adding a color-developing agent into the amplification product of the LAMP reaction or adding a color-developing agent (such as the phenol red dye and Bst DNA polymerase) into the LAMP reaction system in advance, and judging according to the change of the color development result. The real-time turbidity detection method comprises the following steps: observing whether the amplification product is white turbid or not, and judging that the amplification product is positive if the amplification product is white turbid, namely existence of the Pleurotus citrinopileatus DNA, wherein the sample to be detected contains the Pleurotus citrinopileatus component; no white turbidity was negative, i.e.no Pleurotus citrinopileatus DNA was present.
Alternatively, in some embodiments of the invention, the method is not aimed at diagnosis or treatment of a disease.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is the result of specific detection of LAMP primer set of Pleurotus citrinopileatus according to example 1, in which: 1: lyophyllum macrophyllum (Chlorophylum molybdites); 2: bare foot proximal umbrella (Gymnopus subnudus); 3: suillus bovis (Suillus bovis); 4: naked oak mushroom (Gymnopus dryophilus); 5: russula vinosa (Panaeolus subbentatus); 6: leuconostoc rubrum (Leucomoagaricus rubrum); 7: macrolephora macrolepica (Macrolepiota dolichaulula); 8: white rhizoid umbrellas (Rhizocybe alba); 9: ceriporia aurantiaca (laccararia aurantia); 10: gristle-fold goose-cream (Amanita griseofolia); 11: lactarius subbreviceps (Lactarius subbrevipes); 12: russula variegated variety (Russula variata); 13: flammulina velutipes (Russula senecis); 14: amanita rubra (Amanita hemibalapha); 15: boletus lividans (Boletus kauffmanii); 16: boletus neobrevipitae (Tylopilus neofleus); 17: peeling puffball (Lasiosphaera fenzlii); 18: russula rosea (Lasiosphaera fenzlii); 19: butyl riboletus yiciibus; 20: russula velelovskyi (Russula velelovskyi); 21: sarcodon caeruleum (Hydnellum caeruleum); 22: tricholoma saponarium (Tricholoma saponarium); 23: hybrid silk canopy umbrella (Inocybe mixtilis); 24: arthrobacter circinelloides (Hydnellum concrescens); 25: amanita spissacea (Amanita spissacea); 26: tricholoma giganteum (Tricholoma immbricatum); 27: pleurotus eryngii (Pleurotus eryngii); 28: flammulina velutipes (Flammulina filiformis); 29: shiitake (Flammulina filiformis); 30: hypsizygus marmoreus (Hypsizygus marmoreus); 31: tricholoma olivaceoluteolum; 32: amanita citrinobulanata; 33: green mushroom macula (Russula crustose); 34: mucor mucosae (Russula crustose); 35: amanita parvipanensis (Amanita parvipanensis); 36: tricholoma lobyense, (Tricholoma albobruneum); 37: amanita verrucosa (Amanita verrucosilva); 38: tricholoma matsutake (Tricholoma matsutake); 39: amanita sapiensis (Amanita sepiiacea); 40: amanita globosa (Amanita concentrica); 41: the lateral ear (Pleurotus ostreatus); 42: schizophyllum (inocybe rimosa); 43: amanita pseudogriseoviana (Amanita pseudogriseovilinata); 44: russula sanguinea (Russula sanguinea); 0: ddH2O。
FIG. 2 provides example 1 withThe result of detecting the sensitivity of the LAMP primer group of Pleurotus citrinopileatus is shown in the figure: 1: 50 ng/. mu.L; 2: 5 ng/. mu.L; 3: 0.5 ng/. mu.L; 4: 0.05 ng/. mu.L; 5: 5 pg/. mu.L; 6: 0.5 pg/. mu.L; 7: 0.05 pg/. mu.L; 8: 5 fg/. mu.L; 9: 0.5 fg/. mu.L; 10: 0.05 fg/. mu.L; 0: ddH2O。
FIG. 3 is the result of the detection of the applicability of the LAMP primer set for Pleurotus citrinopileatus according to example 1, in which: a-boiling a sample of Pleurotus citrinopileatus; b-boiled digested Pleurotus citrinopileatus sample; 1: a Pleurotus Ostreatus; 2: 50% of Pleurotus citrinopileatus and 50% of Pleurotus eryngii; 3: 10% of Pleurotus citrinopileatus and 90% of Pleurotus eryngii; 4, 1 percent of large green-pleated umbrella and 99 percent of pleurotus eryngii; 0: ddH2O。
FIG. 4 is a result of detecting the specificity of the control LAMP primer group in comparative example 1, in which: 1: macrolephora macrolepica (Macrolepiota dolichaulula); 2: white rhizoid umbrellas (Rhizocybe alba); 3: lactarius subbreviceps (Lactarius subbrevipes); 4: boletus neobrevipitae (Tylopilus neofleus); 5: amanita sepiiacea (Amanita sepiiacea); 6: amanita conitrica (Amanita conitrica); 7: lyophyllum macrophyllum (Chlorophylum molybdites); 0: ddH2O。
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a primer group for identifying the Pleurotus citrinopileatus based on the loop-mediated isothermal amplification technology, which comprises primers shown in SEQ ID NO.1-SEQ ID NO. 5.
The design concept of the primer set provided in this example is as follows: an ITS gene sequence is selected as an amplification target to design a Lyophyllum macranthum LAMP specific primer group. Downloading 11 Pleurotus citrinopileatus ITS sequences from NCBI GenBank database, and selecting the in-seed conserved segment of Pleurotus citrinopileatus as a primer design region through homologous sequence comparison. To ensure the specificity of the primers, the inventors creatively re-subject the ITS sequence of Pleurotus giganteus species to multiple sequence alignments of other 17 Pleurotus giganteus species (C.neomastidosum, C.arizonicum, C.sphaerosporum, C.agaricoides, C.alborubesens, C.brunneum, C.demangei, C.globosum, C.horense, C.leventinum, C.lucitanicum, C.nothiorodensis, C.olivium, C.palaeotropicum, C.pseudolobosum, C.rhododes and C. subhacodes) to find a specific region as a primer binding site. The 17 species are near-source species of the Pleurotus citrinopileatus and have the closest genetic evolution relation with the Pleurotus citrinopileatus, and the 17 species are selected as background primer groups of the Pleurotus citrinopileatus to design specific primer sequences, so that the specificity of the primers can be fully ensured. Through the analysis and creative thinking of the inventor, the LAMP specific primer group capable of identifying the Pleurotus citrinopileatus is finally determined, and comprises two outer primers (C.mol-F3 and C.mol-B3), two inner primers (C.mol-FIP consisting of complementary sequences of F1c and F2; C.mol-BIP consisting of B1c and B2) and a loop primer (C.mol-LB), wherein the sequences are shown in Table 1.
TABLE 1 LAMP-specific primer sequences of Pleurotus citrinopileatus
| Primer and method for producing the same | Sequence (5 '-3') | SEQ ID NO. | |
| C.mol- | cttactcgatgggttgtcgc | 1 | |
| C.mol- | actacaagtggtgcacaggtggctcctctggagcatgtgca | 2 | |
| C.mol- | ttgaggggtctgagagagtggccgtgctttcacactgcagt | 3 | |
| C.mol- | tggccaggtagaagagagc | 4 | |
| C.mol- | gggaattctcccggatgtgagg | 5 |
Example 2
Specific detection of LAMP primer set of example 1
The method comprises the following steps: two pairs of outer primers, two pairs of inner primers and loop primers are respectively added with ddH2O was pre-diluted to 200 μ M, 50 μ M and 200 μ M, then mixed at 3: 10: 96 to obtain a primer mixture. The LAMP reaction system is 10 mu L, and comprises: mu.L LAMP premix (2X) (purchased from New England Biolabs, USA, containing 50mM potassium chloride, 10mM ammonium sulfate, 3mM magnesium sulfate heptahydrate, Tween-200.1%, 100. mu.M phenol red dye, 0.8. mu.L NTPs (10mM), 0.1. mu.L Bst 2.0 WarmStart)TMDNA polymerase (8U/. mu.L)), 1.09. mu.L of the primer mixture (final inner primer concentration of 4.8. mu.M ((4.8. mu.M for each of SEQ ID NO.2 and SEQ ID NO. 3)), final outer primer concentration of 0.6. mu.M (0.6. mu.M for each of SEQ ID NO.1 and SEQ ID NO. 4), and final loop primer concentration of 2. mu.M), 2. mu.L of the DNA template, and made up to 10. mu.L with water. The LAMP reaction is carried out in a PCR thermal cycler at a constant temperature of 65 ℃ for about 45 min. And after the reaction stage is finished, placing the reaction tube on white paper, and judging a reaction result according to the color change. As the pH indicator phenol red in the reaction system is taken as a dye, Bst enzyme plays a polymerization role along with the progress of LAMP amplification, and the reverse reaction is changedThe amount of protons in the reaction solution further changes the pH value, so that the reaction solution changes from pink to yellow. If DNA of Pleurotus citrinopileatus is present in the reaction system, the color of the reaction system changes from pink to yellow.
To examine the specificity of the LAMP primer set of example 1, this example was provided with a total of one positive control (Pleurotus citrinopileatus), 43 negative controls (mushrooms with a morphology similar to that of Pleurotus citrinopileatus and common poisonous and edible mushrooms) and one blank control (ddH)2O). The extraction of DNA was carried out using DNA secure novel plant genome DNA extraction kit (TIAGEN, China).
The result is shown in figure 1, tube 1 is a positive control tube, the reaction solution changes from pink to yellow, the other 43 negative controls and tube 0 blank controls do not change color, the reaction solution remains pink, and no cross reaction is detected; the result shows that the LAMP primer provided in example 1 only amplifies DNA of Pleurotus citrinopileatus, and does not amplify DNA of mushrooms with similar morphology and common poisonous and edible mushrooms non-specifically, and the result shows that the LAMP primer set provided in example 1 has high specificity, can effectively identify Pleurotus citrinopileatus and distinguish the Pleurotus citrinopileatus from other 43 mushrooms.
Example 3
Sensitivity detection of LAMP primer set of example 1
The method comprises the following steps: referring to the method of example 2, genomic DNA of Pleurotus citrinopileatus was diluted 10-fold in a series from 100ng to 0.1fg, and the color change of the reaction mixture after the reaction was completed was observed.
As a result, as shown in FIG. 2, the sensitivity is the minimum amount of DNA that can be detected, and the reaction solution in tubes 2-6 turns from pink to yellow, and the detection limit is 1pg (the amount of DNA added in the reaction system is 2. mu.L), which is far lower than many conventional detection methods, and is sufficient to meet the actual detection requirement, and has higher sensitivity. In the reaction tube (reaction tube 1) containing 100ng of the template DNA, although the color was changed, a pale pink color remained, indicating that an excessively high concentration of the DNA template had an inhibitory effect on the LAMP reaction. This is due to the complex matrix of mushrooms, some containing large amounts of polysaccharides and polyphenols. After a long period of time, polyphenols may be oxidized and irreversibly bind proteins and nucleic acids, forming a macromolecular complex. Many sugars affect the activity of the polymerase and inhibit the amplification reaction of LAMP. Therefore, during the application, attention should be paid to control the content of the DNA template in the reaction system.
Example 4
Suitability detection of the LAMP primer set of example 1.
To further verify the applicability of the LAMP primer set of example 1, this example simulates the mushroom processing and human digestion processes, respectively. The boiled and digested Pleurotus citrinopileatus and Pleurotus citrinopileatus mixture were tested by the LAMP method of reference example 2.
The method comprises the following steps:
(1) mixing Pleurotus citrinopileatus and Pleurotus eryngii according to the mass ratio of 1: 1. 1: 10 and 1: 100, mixing to prepare a mixture of the green Chinese ruffles; then decocting the mixture of Pleurotus Citrinopileatus Sing and Pleurotus Citrinopileatus Sing in water at 100 deg.C for 10 min. DNA extraction and LAMP reaction were carried out by the method of example 2, and the color change of the reaction solution after the reaction was completed was observed.
(2) The artificial gastric juice consisted of 0.05g potassium chloride, 0.42g sodium chloride and 0.32g pepsin, made to volume with 100mL water, then adjusted to pH 2.0 with 2mol/L hydrochloric acid. Incubating mixture of boiled Pleurotus Citrinopileatus Sing and Pleurotus Citrinopileatus Sing (mass ratio of Pleurotus Citrinopileatus Sing to Pleurotus eryngii 1: 1, 1: 10 and 1: 100) in artificial gastric juice at 37 deg.C for 4 h. DNA extraction and LAMP reaction were carried out by the method of example 2, and the color change of the reaction solution after the reaction was completed was observed.
The results are shown in FIG. 3.
It is considered that in practical use, mushrooms are usually processed in advance before they are consumed. In addition, in clinical practice, it is often desirable to detect vomit or feces from patients with mushroom poisoning. The poisonous mushrooms are eaten by mistake and enter the stomach, and the digestive juice will further degrade the DNA. Therefore, in order to evaluate the feasibility of the LAMP primer set of example 1, this example performed LAMP detection in boiled and digested mushroom samples. As a result, as shown in FIG. 3, the reaction solution in reaction tubes Nos. 1 to 4 changed from pink to yellow, and the blank control remained pink, indicating that the LAMP primer set of example 1 can detect the target species, i.e., Pleurotus citrinopileatus, from the single and mixed mushroom samples (content of Pleurotus citrinopileatus is as low as 1%) after water boiling and digestion. The LAMP primer group and the LAMP detection method thereof provided by the embodiment 1 of the invention have higher practicability.
Comparative example 1
A control LAMP primer group was set, and the primer sequences thereof are shown in Table 2 below.
TABLE 2
The reaction system and reaction conditions in the control LAMP primer group example 2 were used to detect DNA of different varieties of mushrooms, and the results are shown in FIG. 4. It can be seen that the specificity of the control LAMP primer group is poor, and other non-target species except the Pleurotus citrinopileatus (No. 1, 2, 3, 4 and 6 tubes are changed from pink to yellow, namely Pleurotus citrinopileatus, Clitocybe albus, Lactaria jujuba, Tylophora neobrevifolia and Amyda ringgolensis) can be amplified; the LAMP primer group of the embodiment 1 of the invention does not amplify the species and shows stronger specificity.
In summary, the embodiment of the invention provides a primer group and a method for a Pleurotus citrinopileatus based on a loop-mediated isothermal amplification detection method, the method can complete the specificity identification of the Pleurotus citrinopileatus within 45 minutes, and the detection result can be directly observed by naked eyes and can be used for on-site rapid detection. The above experimental results confirm the high specificity and sensitivity of the LAMP primer set provided in the examples and can be successfully used for detection of target species in boiled and digested mushroom mixes (content of Pleurotus citrinopileatus is as low as 1%). The results show that the LAMP primer group and the detection method of the Lyophyllum decastes provided by the embodiment of the invention have the characteristics of accuracy, sensitivity, rapidness, low cost and visualization, and can be used for clinical treatment and forensic analysis.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
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<213> Artificial sequence
<400> 4
<210> 5
<211> 22
<212> DNA
<213> Artificial sequence
<400> 5
gggaattctc ccggatgtga gg 22
<210> 6
<211> 19
<212> DNA
<213> Artificial sequence
<400> 6
<210> 7
<211> 44
<212> DNA
<213> Artificial sequence
<400> 7
aggtgcgttc aaagattcga tgcgaaatgc gataagtaat gtga 44
<210> 8
<211> 38
<212> DNA
<213> Artificial sequence
<400> 8
gcgctccttg gtattccgag agctggagga gttgagaa 38
<210> 9
<211> 19
<212> DNA
<213> Artificial sequence
<400> 9
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence
<400> 10
Claims (10)
1. A primer group for identifying Pleurotus citrinopileatus based on a loop-mediated isothermal amplification technology is characterized by comprising the following components in parts by weight: an outer primer pair, an inner primer pair and a loop primer; wherein, the outer primer pair comprises primers with nucleic acid sequences shown as SEQ ID NO.1 and SEQ ID NO.4, the inner primer pair comprises primers with nucleic acid sequences shown as SEQ ID NO.2 and SEQ ID NO.3, and the nucleic acid sequence of the loop primer is shown as SEQ ID NO. 5.
2. Use of the primer set of claim 1 for identifying an umbrella of Pleurotus citrinopileatus.
3. A kit for identifying an Pleurotus citrinopileatus, comprising the primer set according to claim 1.
4. The kit of claim 3, wherein the kit comprises a LAMP reaction solution; the LAMP reaction solution contains: 0.58-0.62. mu.M of the outer primer pair, 4.6-5.0. mu.M of the inner primer pair and 1.8-2.2. mu.M of the loop primer.
5. A method for identifying Pleurotus citrinopileatus based on loop-mediated isothermal amplification technology is characterized by comprising the following steps: mixing a nucleic acid template extracted from a sample to be tested with the primer set according to claim 1, and performing LAMP reaction.
6. The method according to claim 5, wherein in the reaction system in which the LAMP reaction is carried out, the concentration of the pair of outer primers is 0.58 to 0.62. mu.M; the concentration of the inner primer pair is 4.6-5.0 mu M; the concentration of the loop primer is 1.8-2.2. mu.M.
7. The method of claim 6, wherein the LAMP reaction is carried out in a reaction system containing a phenol red dye and Bst DNA polymerase.
8. The method according to any one of claims 5 to 7, wherein the LAMP reaction is carried out at a reaction temperature of 61 to 65 ℃.
9. The method according to any one of claims 5 to 7, wherein the sample to be tested is a mushroom, vomit or feces.
10. The method of any one of claims 5 to 7, wherein the nucleic acid template is genomic DNA.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111575397A (en) * | 2020-06-01 | 2020-08-25 | 中国检验检疫科学研究院 | Toxic amanita species identification method based on DNA micro-barcode technology and application |
| CN113943828A (en) * | 2021-10-20 | 2022-01-18 | 湖南师范大学 | Primer composition, kit and method for rapidly detecting toxic mushroom russula japonica based on LAMP (loop-mediated isothermal amplification) |
| CN114058726A (en) * | 2021-10-29 | 2022-02-18 | 中国检验检疫科学研究院 | Primer group, kit and method for identifying poisonous mushroom ringscale amantake |
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| CN116287392A (en) * | 2023-02-28 | 2023-06-23 | 江苏农林职业技术学院 | LAMP primer composition and application thereof |
| CN116287395A (en) * | 2023-02-07 | 2023-06-23 | 中国检验检疫科学研究院 | Kit and method for molecular identification of toxic mushrooms |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102251055A (en) * | 2011-08-22 | 2011-11-23 | 山东省眼科研究所 | Primers and kit for detecting fusarium on basis of loop-mediated isothermal amplification technology |
| CN105648038A (en) * | 2014-08-21 | 2016-06-08 | 黄耀江 | LAMP (loop-mediated isothermal amplification) universal primer for detecting aspergillus toxin-producing fungi and kit containing primer |
| CN110172527A (en) * | 2019-06-20 | 2019-08-27 | 南京林业大学 | A kind of LAMP detection primer of S.trachelii, detection kit and its application |
| WO2021009185A1 (en) * | 2019-07-15 | 2021-01-21 | National University Of Ireland, Galway | Primers for isothermal amplification |
-
2021
- 2021-02-18 CN CN202110190526.0A patent/CN112852992A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102251055A (en) * | 2011-08-22 | 2011-11-23 | 山东省眼科研究所 | Primers and kit for detecting fusarium on basis of loop-mediated isothermal amplification technology |
| CN105648038A (en) * | 2014-08-21 | 2016-06-08 | 黄耀江 | LAMP (loop-mediated isothermal amplification) universal primer for detecting aspergillus toxin-producing fungi and kit containing primer |
| CN110172527A (en) * | 2019-06-20 | 2019-08-27 | 南京林业大学 | A kind of LAMP detection primer of S.trachelii, detection kit and its application |
| WO2021009185A1 (en) * | 2019-07-15 | 2021-01-21 | National University Of Ireland, Galway | Primers for isothermal amplification |
Non-Patent Citations (3)
| Title |
|---|
| NCBI: "Chlorophyllum molybdites voucher Mushroom Observer 329988 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence，MT357082.1", 《NCBI》 * |
| 何正蜜: "剧毒鹅膏等温扩增检测及鹅膏毒素基因多样性研究", 《中国优秀博硕士学位论文全文数据库（博士） 基础科学辑》 * |
| 马丹，等: "环介导等温扩增法检测转基因玉米MON88017品系", 《检验检疫学刊》 * |
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|---|---|---|---|---|
| CN111575397A (en) * | 2020-06-01 | 2020-08-25 | 中国检验检疫科学研究院 | Toxic amanita species identification method based on DNA micro-barcode technology and application |
| CN111575397B (en) * | 2020-06-01 | 2022-05-03 | 中国检验检疫科学研究院 | Toxic amanita species identification method based on DNA micro-barcode technology and application |
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