CN114107539A - LAMP primer composition for detecting Pythium delavayi and application thereof - Google Patents
LAMP primer composition for detecting Pythium delavayi and application thereof Download PDFInfo
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Abstract
The invention discloses a LAMP primer composition for detecting Pythium delicicum and application thereof. The primer composition for detecting the Pythium delavayi consists of a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3 and a reverse loop primer LB. The LAMP reaction program is as follows: the method can be used for amplifying for 60min under the isothermal condition of 63 ℃, can quickly, conveniently, efficiently, specifically and sensitively detect the presence of the Pythium delavayi in a sample to be detected, does not need a complex instrument, and can better meet the field detection of the Pythium delavayi.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to an LAMP primer composition for detecting Pythium delicicum and application thereof.
Background
Pythium delicicum (Pythium deliense) belongs to the kingdom of the Pimenta (Straminipila), the phylum Oomycota (Oomycota), the class Oomycota (Oomycotes), the order Peronospora (Peronospora), the family Pythiaceae (Pythiaceae), the genus Pythium (Pythium). The Pythium delicicum can infect plants such as tobacco, soybean, mung bean, ginger and the like as an important plant pathogen, has a wide host range, causes symptoms such as plant seedling rot, root rot, damping-off, dwarfing and the like, and causes serious economic loss in the production of infected plants. 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 a basis is provided for disease risk and research decision, so that the loss caused by the Pythium dorelii is reduced.
Currently, few detection techniques are available for Pythium delavayi. 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 delicicum. The method is time-consuming and labor-consuming, needs abundant and professional pathogenic bacteria identification experience, is difficult to distinguish the Pythium delavayi 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 delavayi. 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 60 min. 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 delicicum is not reported at home and abroad.
The selection of target genes is one of important factors of LAMP detection, the source of primers designed by the LAMP detection is mainly an intertranscriptional spacer (ITS), but enough sites are not available for distinguishing similar species, so that a new detection target is developed to become a hot spot for detection.
The invention designs a specific LAMP primer composition of the Pythium dorsalis by taking Phosphoglycokinase (PGK) gene as a detection target sequence, and establishes an LAMP rapid detection method of the Pythium dorsalis on the basis.
Disclosure of Invention
The invention aims to provide an LAMP detection primer composition for rapidly detecting the Pythium delicicum and a molecular detection method thereof, aiming at the problems that the biological detection method of the Pythium delicicum in the prior art needs long period, wastes time and labor, is complicated and has poor specificity and the PCR detection technology needs a thermal cycler and cannot rapidly detect the Pythium delicicum.
The purpose of the invention can be realized by the following technical scheme:
the LAMP primer composition for detecting the Pythium delavayi consists 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 and a reverse loop primer LB shown as SEQ ID N0.5 (shown in a figure 1).
The primer composition disclosed by the invention is applied to detection of the Pythium delicicum.
The primer composition disclosed by the invention is applied to preparation of a LAMP detection kit for the Pythium delicicum.
An LAMP kit for detecting the Pythium delicicum contains the primer composition.
The LAMP kit for detecting the Pythium delavayi also comprises the following components: 10 XThermoPol Buffer, MgS04dNTPs, betaine, Bst DNA polymerase, hydroxynaphthol blue.
The kit disclosed by the invention is applied to detection of the Pythium delicicum.
The LAMP detection method of the Pythium debaryanum is characterized in that DNA of a microorganism to be detected is extracted, a DNA solution is taken as a reaction template, the DNA solution is added into a detection solution in an LAMP kit to carry out LAMP reaction, then the color change of an amplification product is observed, if the color change is sky blue, the detection result is positive, the Pythium debaryanum exists, if the color change is purple, the detection result is negative, and the Pythium debaryanum does not exist.
Preferably, the LAMP reaction system comprises: 2.5. mu.L of 10 × ThermoPol Buffer, 8 mmol.L-1MgS04，1.2mmol·L-1dNTPs, inner primers FIP and BIP each 1.6. mu. mol. L-10.4. mu. mol. L for each of the outer primers F3 and B3-1，0.8μmol·L-1Loop primer LB, 0.8. mu. mol. L-1Betaine, 8 U.mu.L-1Bst DNA polymerase，180mmol·L-1Hydroxynaphthol blue, 2. mu.L of template DNA, sterile water to 26. mu.L.
As a preferred aspect of the present invention, the LAMP reaction is performed by the following steps: amplifying for 60min at 63 ℃.
The method for detecting the Pythium delicicum of the inventionPerforming LAMP reaction by using the LAMP primer composition by using the extracted DNA as a template; hydroxylnaphtol blue (HNB) belongs to one of metal ion indicators. HNB being Mg2+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 system2+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 process2+Binding with the by-product of the LAMP reaction produces a large amount of precipitate, Mg in solution2+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 delicicum is judged by the color change of the reaction system after the completion of the reaction: sky blue indicates positive detection, presence of Pythium delicicum; purple indicates that the test result is negative and no Pythium delicicum exists.
Compared with the prior art, the invention has the advantages and positive effects that:
the invention discovers that a new target gene is used for LAMP detection of the Pythium delavayi, and the LAMP primer composition is designed for detection of the Pythium delavayi, and has the advantages of strong specificity, high accuracy and good sensitivity when the gene is opportunistic and particularly the specific target region on the gene.
(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) is extremely toxic 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: the traditional method for detecting the Pythium delavayi is only used for identifying according to morphological characteristics, but the growth of the Pythium delavayi is influenced by temperature, is unstable in morphology and is influenced by similar species, and therefore the method is difficult to accurately identify. According to the genome sequence of the Pythium delavayi, the genome sequence of the Pythium delavayi is compared with the genome sequences of other Pythium ultimum by using Blast software, and a specific end sequence of the Pythium delavayi is selected and a specific LAMP primer is designed. The LAMP reaction specifically recognizes 6 independent regions on the target sequence (Table 1) by 4 primers (FIP, BIP, F3, B3), and the specificity is relatively high. In addition, the reverse loop primer LB can improve the reaction rate, and together with other four primers, the method can rapidly carry out the detection of the Pythium delemar under the condition of ensuring the reaction accuracy. The LAMP reaction specifically recognizes 6 independent regions on the target sequence through 4 primers, and has higher specificity and sensitivity compared with 2 independent regions of the target sequence recognized by PCR primers.
(4) The sensitivity is high: the LAMP detection method of the Pythium delavayi established by the invention has very high sensitivity and can reach 100fgDNA, which indicates that the detection method can accurately and rapidly detect the Pythium delavayi under the condition of lower DNA concentration.
For a better understanding and practice, the invention is described in detail below with reference to the accompanying drawings.
Drawings
FIG. 1 shows the nucleotide sequence and primer positions of Pythium delavayi PGK gene.
FIG. 2 FIG. 3 shows the specificity verification of the LAMP primer for detecting Pythium delbrueckii:
the primers designed by the invention are used for carrying out LAMP amplification detection on DNA samples of different strains of the Pythium delbrueckii and other bacterial strains, the color change of a reaction solution is observed after the constant temperature amplification is carried out in a water bath at 63 ℃ for 60min, and the result shows that: different strains of the Derrilla germanica showed sky-blue positive reaction (figure 2), and solutions in reaction tubes of other strains of the test bacteria showed purple negative reaction (figure 3).
FIG. 4 sensitivity of LAMP detection of Pythium delbrueckii:
amplifying genome DNA with different concentrations by LAMP; the reaction system of 26. mu.L contained amplification results of 10ng, 1ng, 100pg, 10pg, lpg, 100fg, and L0fg DNAs, respectively (FIG. 4).
Detailed Description
In order to make the purpose and technical solution of the present invention more clear, the present invention further describes specific examples, but not only these examples.
Example 1: detection of Pythium delbrueckii by LAMP
1. LAMP primer composition for detecting Pythium delicicum: 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 and a reverse loop primer LB shown as SEQ ID N0.5 (FIG. 1).
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 the reaction solution containing the primers to carry out LAMP reaction, wherein the LAMP reaction system is as follows: 2.5. mu.L of 10 × ThermoPol Buffer, 8 mmol.L- 1MgS04，1.2mmol·L-1dNTPs, inner primers FIP and BIP each 1.6. mu. mol. L-10.4. mu. mol. L for each of the outer primers F3 and B3-1，0.8μmol·L-1Loop primer LB, 0.8. mu. mol. L-1Betaine, 8 U.mu.L-1Bst DNA polymerase，180mmol·L-1Hydroxy naphthol blue, 2 mu L template DNA and sterile water to 26 mu L; the procedure for the LAMP reaction was: and (3) carrying out reaction amplification at 63 ℃ for 60min, then observing the color change of an amplification product, wherein if the color is sky blue, the detection result is positive, the Pythium delemar exists, and if the color is purple, the detection result is negative, the Pythium delemar does not exist.
3. In order to verify the specificity of the LAMP method, 4 different strains of Dryopteris delbrueckii and other 18 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 delavayi as a template shows a sky blue positive reaction (FIG. 2), and other tested oomycetes, fungal strains and negative controls all show a purple negative reaction (FIG. 3).
TABLE 1
TABLE 2
Example 2: sensitivity of Pythium delavayi LAMP detection
To determine the sensitivity of the LAMP detection method, the concentration of the extracted DNA of Pythium delicicum was measured with a spectrophotometer, and the DNA was diluted in a 10-fold gradient so that the mass concentration was 10 ng. mu.L in this order-1、1ng·μL-1、100pg·μL-1、10pg·μL-1、1pg·μL-1、100fg·μL-1And 10 fg. mu.L-1Separately, 2. mu.L of each was used as a template for LAMP reaction. The reaction program was 63 ℃ for 60 min. HNB coloration results show that: when the DNA concentration of Pythium delavayi reaches 100 pg. mu.L-1The solution in the reaction tube became sky blue (FIG. 4).
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed 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
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Claims (9)
1. The LAMP primer composition for detecting the Pythium delavayi 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 and a reverse loop primer LB shown as SEQ ID No. 5.
2. The LAMP primer composition of claim 1, for use in detection of Pythium delicicum, which is a detection for non-disease diagnostic purposes.
3. The application of the LAMP primer composition of claim 1 in preparation of a kit for detecting Pythium delavayi.
4. A LAMP kit for detecting Pythium delavayi, characterized in that the kit contains the LAMP primer composition of claim 1.
5. The LAMP kit for detecting Pythium delemar according to claim 4, wherein the kit further comprises 10 XThermoPol Buffer, MgS04dNTPs, betaine, Bst DNA polymerase and hydroxyl naphthol blue.
6. The use of the LAMP kit of claim 4 or 5 for the detection of Pythium delicicum, which is a detection for non-disease diagnostic purposes.
7. An LAMP detection method of Pythium delicicum 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 according to claim 1, then color change of an amplification product is observed, if the color of the amplification product is sky blue, the detection result is positive, Pythium delicicum exists, if the color of the amplification product is purple, the detection result is negative, and Pythium delicicum does not exist.
8. The LAMP detection method according to claim 7, wherein the LAMP reaction system is 2.5. mu.L 10 XThermoPol Buffer, 8 mmol.L-1MgS04，1.2mmol·L-1dNTPs, inner primers FIP and BIP each 1.6. mu. mol. L-10.4. mu. mol. L for each of the outer primers F3 and B3-1，0.8μmol·L-1Loop primer LB, 0.8. mu. mol. L-1Betaine, 8 U.mu.L- 1Bst DNA polymerase，180mmol·L-1Hydroxynaphthol blue, 2. mu.L of template DNA, sterile water to 26. mu.L.
9. The LAMP detection method according to claim 7 or 8, characterized in that the program of the LAMP reaction is: amplifying for 60min at 63 ℃.
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Citations (3)
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| CN106222263A (en) * | 2016-07-30 | 2016-12-14 | 南京农业大学 | A kind of LAMP primer composition thing, test kit and detection method thereof for detecting Pythium ultimum bacterium |
| CN108060257A (en) * | 2018-01-17 | 2018-05-22 | 南京农业大学 | It is a kind of that strong male rotten mould Primer composition and its detection method are detected based on loop-mediated isothermal amplification technique |
| US20200362424A1 (en) * | 2018-02-05 | 2020-11-19 | Syngenta Participations Ag | Methods for detecting fungi in turf grass with a lamp assay having novel primer sets |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106222263A (en) * | 2016-07-30 | 2016-12-14 | 南京农业大学 | A kind of LAMP primer composition thing, test kit and detection method thereof for detecting Pythium ultimum bacterium |
| CN108060257A (en) * | 2018-01-17 | 2018-05-22 | 南京农业大学 | It is a kind of that strong male rotten mould Primer composition and its detection method are detected based on loop-mediated isothermal amplification technique |
| US20200362424A1 (en) * | 2018-02-05 | 2020-11-19 | Syngenta Participations Ag | Methods for detecting fungi in turf grass with a lamp assay having novel primer sets |
Non-Patent Citations (3)
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| ZIN MAR HTUN等: "Loop-mediated Isothermal Amplification (LAMP) for Identification of Pythium insidiosum", 《INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES》 * |
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| 赵媛媛: "强雄腐霉的LAMP检测和瓜果腐霉的RPA检测方法的建立", 《中国优秀博硕士学位论文全文数据库(硕士)基础科学辑》 * |
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Application publication date: 20220301 Assignee: Nanjing Jitian Biotechnology Co.,Ltd. Assignor: JIANGSU POLYTECHNIC College OF AGRICULTURE AND FORESTRY Contract record no.: X2022980024868 Denomination of invention: LAMP primer composition for detection of Pythium delhi and its application Granted publication date: 20220708 License type: Common License Record date: 20221209 |