CN114317796B - LAMP primer composition for detecting pythium admittedly and detection method thereof - Google Patents
LAMP primer composition for detecting pythium admittedly and detection method thereof Download PDFInfo
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Abstract
The invention discloses an LAMP primer composition for detecting pythium admittedly and a detection method thereof. The primer composition for detecting pythium admittedly is composed of a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3, a loop primer LF and an LB. The LAMP reaction program is as follows: the solid Pythium irregulare existing in a sample to be detected can be quickly, conveniently, efficiently, highly specifically and sensitively detected by isothermal amplification for 60min at 63 ℃, a complex instrument is not needed, and the field detection of the solid Pythium irregulare can be better met.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to an LAMP primer composition for rapidly detecting pythium admittedly and a visual detection method thereof.
Background
Pythium induciscens belongs to the kingdom of trichomonadaceae (traminipila), the phylum oomyceta (Oomycota), the class Oomycetes (oomyces), the order Peronosporales (Peronosporales), the family Pythiaceae (Pythiaceae), the genus Pythium (Pythium), which can infect tobacco, soybean and carrot to cause root rot, rot in seedling stage, etc., causing serious economic loss in tobacco, soybean and carrot production. At present, no effective control measures are available for the disease, and strengthening quarantine and preventing the spread of pathogenic bacteria are the most effective measures for controlling the disease. Therefore, epidemic situation monitoring is enhanced, a rapid detection method is established, and basis is provided for disease risk and research decision, so that loss caused by pythium admittedly can be reduced.
At present, few detection technologies are available for pythium admittedly. The traditional detection method mainly adopts a plate separation method and a bait method and carries out identification according to the morphology of the pythium admittedly. The method is time-consuming and labor-consuming, needs abundant and professional pathogenic bacteria identification experience, is difficult to distinguish pythium admittedly from similar species according to morphological characteristics, and cannot meet the requirements on field production. With the development of molecular biology, particularly the popularization of PCR technology, more and more molecular biology technologies are applied to the detection of Pythium admittedly. However, these detection techniques are often required to be performed in laboratories with specialized instruments, reagents and strict environmental conditions, the instruments and the reagents are expensive and have high operation requirements, the process is complex, the detection time is still long, the requirement of rapid detection cannot be met, and the detection techniques are not suitable for being used and popularized in basic level environments.
Loop-mediated isothermal amplification (LAMP) is a new nucleic acid amplification technology developed in recent years, and has the advantages of simple operation, rapidness, high specificity, low cost and the like, so that the Loop-mediated isothermal amplification technology becomes a new nucleic acid amplification technology capable of replacing common PCR. It designs 4 specific primers aiming at 6 regions of a target gene, causes self-circulation strand displacement reaction under the action of Bst large fragment polymerase, and generates white magnesium pyrophosphate precipitate as a byproduct while synthesizing a large amount of target DNA within 60-65 ℃ within 60min. The color development dyes such as SYBR Green, hydroxy Naphthol Blue (HNB) and the like are added into the reaction system, and the judgment can be carried out by observing the color change by naked eyes. The LAMP amplification process depends on 6 independent areas for identifying the target sequence, so the reaction specificity is very strong, the nucleic acid amplification process is carried out under the constant temperature condition, a common water bath or equipment with a stable heat source can meet the reaction requirement, and the detection cost is greatly reduced. At present, the technology is widely applied to the rapid detection of fungi, bacteria, viruses and oomycetes, but the detection of the pythium admittedly has not been reported at home and abroad.
The invention designs a specific LAMP primer composition of the pythium admittedly by taking an ITS gene as a detection target sequence, and establishes an LAMP rapid detection method of the pythium admittedly on the basis.
Disclosure of Invention
The invention aims to provide an LAMP detection primer composition for rapidly detecting pythium admittedly and a molecular detection method thereof, aiming at the problems that the biological detection method for pythium admittedly needs a long period, wastes time and labor, is complicated and has poor specificity in the prior art and the PCR detection technology needs a thermocycler and cannot rapidly detect the pythium admittedly.
The purpose of the invention can be realized by the following technical scheme:
the LAMP primer composition for detecting pythium admittedly is composed of a forward inner primer FIP shown as SEQ ID N0.1, a reverse inner primer BIP shown as SEQ ID N0.2, a forward outer primer F3 shown as SEQ ID NO.3, a reverse outer primer B3 shown as SEQ ID N0.4, a forward loop primer LF shown as SEQ ID N0.5 and a reverse loop primer LB shown as SEQ ID N0.6.
The primer composition is applied to the detection of pythium admittedly, and the detection is the detection for the purpose of non-disease diagnosis.
The primer composition disclosed by the invention is applied to preparation of a LAMP detection kit for pythium admittedly.
An LAMP kit for detecting pythium admittedly, which contains the primer composition of the invention.
As a preferable preference of the invention, the LAMP kit for detecting pythium admittedly also comprises 10 × ThermoPol Buffer, mgS0 4 dNTPs, betaine, bst DNA polymerase, hydroxynaphthol blue.
The kit disclosed by the invention is applied to the detection of pythium admittedly, and the detection is the detection for the purpose of non-disease diagnosis.
The LAMP detection method of pythium admittedly is characterized in that DNA of a microorganism to be detected is extracted, a DNA solution is taken as a reaction template, LAMP reaction is carried out by using the primer composition, then the color change of an amplification product is observed, if the color is sky blue, the detection result is positive, pythium admittedly exists, if the color is purple, the detection result is negative, and the pythium admittedly does not exist.
As a preferable aspect of the present invention, the LAMP reaction system: 2.5 μ L10 × ThermoPol Buffer,8 mmol. L -1 MgS0 4 ，1.2mmol·L -1 dNTPs, inner primers FIP and BIP each 1.6. Mu. Mol. L -1 0.4. Mu. Mol. L for each of the outer primers F3 and B3 -1 Loop primers LF and LB each 0.8. Mu. Mol. L -1 ，0.8μmol·L -1 Betaine, 8 U.mu.L -1 Bst DNA polymerase， 180mmol·L -1 Hydroxynaphthol blue, 2. Mu.L template DNA, for a total of 26. Mu.L.
As a preferable aspect of the present invention, the program of the LAMP reaction is: amplifying for 60min at 63 ℃.
The method for detecting pythium admittedly provided by the invention takes the extracted DNA as a template, and the LAMP primer composition is utilized to carry out LAMP reaction; hydroxylnaphtol blue (HNB) belongs to one of metal ion indicators. HNB being Mg 2+ The color of the titrant changes along with the change of the pH of the solution, so that the titrant can be used for monitoring Mg in a LAMP reaction system 2+ The change in concentration and the pH of the solution act as a color indicator. HNB is added into the reaction liquid before the reaction, the reaction system is purple, and Mg is generated in the reaction process 2+ Binding with the by-product of the LAMP reaction produces a large amount of precipitate, mg in solution 2+ The concentration is reduced, the pH value is changed, and the color of the HNB is changed from purple to sky blue. Therefore, the presence or absence of Pythium admittedly is judged by the color change of the reaction system after the reaction is finished: sky blue indicates that the detection is positive and pythium admittedly exists; purple indicates that the test result is negative and pythium admittedly does not exist.
Compared with the prior art, the invention has the advantages and positive effects that:
(1) The practicability is good. The gel electrophoresis of the products by the common PCR reaction is easy to cause product diffusion, which is a main source of laboratory pollution; ethidium Bromide (EB) has a great toxicity and can accumulate carcinogenesis; the long-term observation of the ultraviolet lamp can also cause certain damage to the experimenters. The LAMP reaction is only carried out in a constant-temperature water bath, and whether the target strain exists or not is judged by observing the color change of the solution after the reaction is finished, so that the application value of the LAMP reaction in the field is improved.
(2) And (5) performing isothermal amplification. Unlike PCR method which needs thermal cycling, the LAMP method is independent of a thermal cycler, can be carried out as long as a stable heat source is available, and greatly expands the application range of LAMP.
(3) The accuracy is high: because the traditional method for detecting the pythium admittedly is only used for identification according to morphological characteristics, the growth of the pythium admittedly is influenced by temperature, the morphology is unstable, and meanwhile, the growth of the pythium admittedly is influenced by similar species, so that the accurate identification is difficult. According to the genome sequence of the pythium admittedly, the genome sequence of the pythium admittedly is compared with the genome sequences of other pythium admittedly by using Blast software, a section of sequence specific to the pythium admittedly is selected, and a specific LAMP primer is designed. The LAMP reaction specifically recognizes 6 independent regions on the target sequence by 4 primers (FIP, BIP, F3, B3), and the specificity is relatively high (Table 1). In addition, the forward loop primer LF and the reverse loop primer LB can improve the reaction rate, and together with other four primers, the method can rapidly carry out pythium admittedly-detection under the condition of ensuring the reaction accuracy. The LAMP reaction specifically recognizes 6 independent regions on the target sequence through 4 primers, and the specificity and the sensitivity are higher compared with 2 independent regions of the target sequence recognized by PCR primers.
(4) The sensitivity is high: the LAMP detection method of pythium admittedly provided by the invention has very high sensitivity, and can reach 100fgDNA, which indicates that the detection method can accurately and rapidly detect pythium admittedly under the condition of low DNA concentration.
For a better understanding and practice, the present invention is described in detail below with reference to the accompanying drawings.
Drawings
FIG. 1 shows the nucleotide sequence and primer positions of the T.admittedly ITS gene.
FIG. 2 shows that the primers designed by the present invention can be used to perform LAMP amplification detection on DNA samples of different Pythium fastidiosum strains and other strains, and the results show that: different strains of pythium admittedly show sky blue positive reaction
FIG. 3 LAMP amplification detection of DNA samples of other bacterial strains using primers designed according to the present invention
The results show that: the solutions in the reaction tubes of the other test bacterial strains all gave a purple negative reaction (FIG. 3).
FIG. 4 sensitivity of LAMP detection of Pythium admittedly:
amplifying genome DNA with different concentrations by LAMP; the reaction system contained 10ng, 1ng, 100pg, 10pg, lpg, 100fg, and L0fg DNA, respectively, in 26. Mu.L (FIG. 4).
Detailed Description
In order to make the objects and technical solutions of the present invention more apparent, the present invention is further described by specific examples, not by way of example only.
Example 1: detection of Pythium admittedly by LAMP
1. LAMP primer composition for detecting pythium admittedly: a forward inner primer FIP shown as SEQ ID N0.1, a reverse inner primer BIP shown as SEQ ID N0.2, a forward outer primer F3 shown as SEQ ID NO.3, a reverse outer primer B3 shown as SEQ ID N0.4, a forward loop primer LF shown as SEQ ID N0.5 and a reverse loop primer LB shown as SEQ ID N0.6.
2. The detection method comprises the following steps: extracting the DNA of the microorganism to be detected, taking a DNA solution as a reaction template, adding a detection solution to carry out LAMP reaction, wherein the LAMP reaction system for detecting the pythium admittedly is as follows: 2.5 μ L10 × ThermoPol Buffer,8 mmol. L - 1 MgS0 4 ，1.2 mmol·L -1 dNTPs, inner primers FIP and BIP each 1.6. Mu. Mol. L -1 0.4. Mu. Mol. L for each of the outer primers F3 and B3 -1 Loop primers LF and LB each 0.8. Mu. Mol. L -1 ，0.8μmol·L -1 Betaine, 8 U.mu.L -1 Bst DNA polymerase，180mmol·L -1 Hydroxy naphthol blue, 2 mu L template DNA, and the total amount of the system is 26 mu L; the LAMP reaction program is as follows: amplifying at 63 deg.C for 60min, and observing the color change of the amplified product if it appears dayBlue indicates that the detection result is positive and pythium admittedly exists, and purple indicates that the detection result is negative and pythium admittedly does not exist.
3. In order to verify the specificity of the LAMP method, 7 different strains of Pythium admittedly and other 22 strains of fungi and oomycetes were used as test materials (Table 2), and the LAMP detection results show that: only the reaction tube solution using Pythium admittedly as a template shows a sky-blue positive reaction (FIG. 2), and other reference oomycetes, fungal strains and negative controls all show a purple negative reaction (FIG. 3).
TABLE 1
TABLE 2
Example 2: sensitivity of Pythium fastigiatum LAMP detection
To determine the sensitivity of the LAMP detection method, the extracted DNA of Pythium admittedly was measured with a spectrophotometer and diluted in a 10-fold gradient to a mass concentration of 10 ng. Mu.L -1 、1ng·μL -1 、100pg·μL -1 、10pg·μL -1 、 1pg·μL -1 、100fg·μL -1 And 10 fg. Mu.L -1 Separately, 2. Mu.L of each was used as a template for LAMP reaction. The reaction program was 63 ℃ for 60min. HNB coloration results show that: when the DNA concentration of the pythium admittedly reaches 100 fg. Mu.L -1 The solution in the reaction tube became sky blue in color (FIG. 4).
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Sequence listing
<110> Jiangsu academy of agriculture, forestry, and occupational technology
<120> LAMP primer composition for detecting pythium admittedly and detection method thereof
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Claims (8)
1. The LAMP primer composition for detecting pythium admittedly is characterized by comprising a forward inner primer FIP shown as SEQ ID No.1, a reverse inner primer BIP shown as SEQ ID No.2, a forward outer primer F3 shown as SEQ ID No.3, a reverse outer primer B3 shown as SEQ ID No.4, a forward loop primer LF shown as SEQ ID No.5 and a reverse loop primer LB shown as SEQ ID No. 6.
2. The LAMP primer composition of claim 1, which is used for detecting pythium admittedly, wherein the detection is for the purpose of non-disease diagnosis.
3. The LAMP primer composition of claim 1, which is applied to preparation of a kit for detecting pythium admittedly.
4. A LAMP kit for detecting Pythium admittedly, characterized in that the kit contains the LAMP primer composition of claim 1.
5. The LAMP kit for detecting Pythium admittedly according to claim 4, wherein the kit further comprises ThermoPol Buffer, mgS04, dNTPs, betaine, bst DNA polymerase, hydroxynaphthol blue.
6. The use of the LAMP kit of claim 4 or 5 for the detection of P.admittedly, said detection being for non-disease diagnosis purposes.
7. An LAMP detection method of pythium admittedly, characterized by extracting the DNA of the microorganism to be detected, taking the DNA solution as a reaction template, carrying out LAMP reaction by using the primer composition of claim 1, then observing the color change of the amplified product, if the color is sky blue, the detection result is positive, pythium admittedly exists, if the color is purple, the detection result is negative, the pythium admittedly does not exist; the LAMP reaction system comprises the following steps: 2.5. Mu.L of 10 × ThermoPol Buffer,8 mmol.L -1 MgS0 4 ，1.2 mmol·L -1 dNTPs, inner primers FIP and BIP are each 1.6. Mu. Mol. L -1 0.4. Mu. Mol. L for each of the outer primers F3 and B3 -1 Loop primers LF and LB each 0.8. Mu. Mol. L -1 ，0.8 μmol·L -1 Betaine, 8 U.mu.L -1 Bst DNA polymerase，180mmol·L -1 Hydroxynaphthol blue, 2. Mu.L template DNA, totaling 26. Mu.L.
8. The LAMP detection method according to claim 7, characterized in that the program of the LAMP reaction is: amplifying for 60min at 63 ℃.
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Application publication date: 20220412 Assignee: Jiangsu Haili Instrument Co.,Ltd. Assignor: JIANGSU POLYTECHNIC College OF AGRICULTURE AND FORESTRY Contract record no.: X2023980040132 Denomination of invention: A LAMP primer composition and detection method for detecting persistent Pythium Granted publication date: 20221125 License type: Common License Record date: 20230821 |
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Application publication date: 20220412 Assignee: Jurong Mushroom Technology Co.,Ltd. Assignor: JIANGSU POLYTECHNIC College OF AGRICULTURE AND FORESTRY Contract record no.: X2023980050203 Denomination of invention: A LAMP primer combination and detection method for detecting stubborn rot mold Granted publication date: 20221125 License type: Common License Record date: 20231207 |