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Lamp primer composition and application for detecting tomato infestans

A technology of tomato late blight bacteria and primer composition, which is applied in the directions of biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, and can solve the problems of low accuracy, long detection time, long time consumption, etc., and achieve results. Reliable, sensitive and specific results

Active Publication Date: 2020-07-17
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The object of the present invention is to provide the LAMP primer composition and application for detecting tomato infestans. In the prior art, the detection and identification of tomato infestans are mainly based on morphological characteristics, and the method is time-consuming, cumbersome and empirical. , low accuracy, it is difficult to timely monitor the occurrence of diseases and control the spread and prevalence of pathogenic bacteria, and the existing PCR molecular detection needs to rely on expensive instruments such as amplifiers, and the detection time is long. A new molecular detection method for infestans infestans, LAMP detection of infestans infestans in tomato, short detection cycle, high accuracy, high sensitivity, and the detection results can be observed with naked eyes

Method used

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  • Lamp primer composition and application for detecting tomato infestans
  • Lamp primer composition and application for detecting tomato infestans
  • Lamp primer composition and application for detecting tomato infestans

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1: Design of specific primer composition and primer specificity verification for loop-mediated isothermal amplification (LAMP) detection of tomato infestans

[0030] 1. Extraction of genomic DNA of the tested strains

[0031] The genomic DNA of the tested strains (Table 1) was extracted by the CTAB method. The specific method is as follows: Take a small amount of mycelium powder in a 1.5mL centrifuge tube (it is better that the mycelium powder just covers the bottom of the semicircle), add 900 µL of 2% CTAB ( cetyltrimethylammonium bromide) extract (2% CTAB; 100 mmol / L Tris-HCl, pH 8.0; 20 mmol / L EDTA, pH 8.0; 1.4 mol / LNaCl) and 90 µL SDS (ten Sodium dialkylbenzene sulfonate) [Note: CTAB, SDS needs to be preheated at 60°C], use a shaker to shake and mix, 60°C water bath for 1h (DNA is released into the buffer), 12000 r min -1 Centrifuge for 15 min; take 700 µL of the supernatant, add an equal volume of phenol, chloroform, isoamyl alcohol mixture (volume ratio 2...

Embodiment 2

[0042] Example 2: Detection Sensitivity of Loop-Mediated Isothermal Amplification (LAMP) for Tomato Infestans Infestans

[0043] 1. Preparation of genomic DNA at different concentrations

[0044] Dilute the genome DNA of Phytophthora infestans with sterile ultrapure water, and prepare a series concentration of 10 times order of magnitude for later use;

[0045] 2. Sensitivity determination and result observation of LAMP detection method

[0046] Genomic DNA of Phytophthora infestans at different concentrations was used as a template, and the outer primers F3 / B3 and inner primers FIP / BIP were used for LAMP amplification. The LAMP detection reaction system was 25 μL, including 5 μM outer primers F3 and 1.0 μL each of B3, 1.0 μL each of 40 μM inner primers FIP and BIP, LAMP reaction mixture [40 mM Tris-HCl, 20 mM (NH 4 ) 2 SO 4 , 20 mM KCl, 16 mM MgSO 4 , 1.6 mol / L Betaine (Betaine), 2.0 mM dNTPs, 0.2% Trion X-100] 12.5 μL, 8 U Bst 1.0 μL of polymerase, 1.0 μL of DNA templat...

Embodiment 3

[0049] Example 3: LAMP detection of tomato infestans in pathogenic tissue

[0050] Sample collection: Collect tomato late blight symptoms and healthy leaves from Zhouning, Sanming, and Liancheng in Fujian and bring them back to the laboratory for later use;

[0051] Plant tissue DNA extraction: DNA was extracted by NaOH rapid cleavage method, the specific process is as follows: add 10µL 0.5 mol / L NaOH to each mg of plant tissue, fully grind the tissue into a paste in a mortar and transfer it to a 1.5mL centrifuge tube centrifuge at 12,000 rpm for 6 min, take 5 µL of the supernatant and add 495 µL 0.1 mol / L Tris-HCl (pH=8.0) to mix well, and take 1.0 µL as a PCR template for amplification.

[0052] LAMP amplification detection and observation: Using the above-mentioned extracted DNA as a template, use the outer primer F3 / B3 and inner primer FIP / BIP for LAMP amplification. The LAMP detection reaction system is 25 μL, including 5 μM outer primer F3 and 1.0 μM each for B3. μL, 1....

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Abstract

The invention discloses an LAMP (Loop-mediated Isothermal Amplification) primer combination for detecting tomato phytophthora infestans and application. The LAMP primer combination for detecting the tomato phytophthora infestans consists of four specific primers, and the primer combination disclosed by the invention is applied in the LAMP detection of the tomato phytophthora infestans. A detection system disclosed by the invention is incubated under the isothermal condition of 64 DEG C for 1 hour, SYBR green I developer is adopted for detection by development, and with whether green fluorescence is observed as a standard, a detection result is judged. The LAMP primer combination has the advantages of high accuracy, high specificity, convenience in operation and high practicability, does not need complex instruments, realizes isothermal amplification, provides a novel molecular detection technique for the tomato phytophthora infestans, can be used in rapid, sensitive and accurate detection in the early stage or infection incubation of the tomato phytophthora infestans in production practice, and provides a reliable technical and theoretical basis for the prevention and treatment of the tomato phytophthora infestans.

Description

technical field [0001] The invention belongs to the technical field of detection, identification and prevention of crop diseases, and specifically relates to a loop-mediated isothermal amplification (LAMP) detection primer composition of tomato infestans and its application, which can be used for rapid, sensitive and specific molecular detection of tomato infestans It can also be used for early diagnosis of tomato late blight and monitoring and identification of pathogens. Background technique [0002] tomato ( Lycopersicon esculentum Miller) alias tomato, foreign persimmon, belongs to Solanaceae ( Solanaceae ) Tomato ( Lycopersicon ). Tomatoes are rich in nutrients. They are not only cooked and raw vegetables, but also fruits, and are very popular among the masses. According to research, each person can eat 50-100 g of fresh tomatoes per day, which can meet the human body's needs for several vitamins and minerals. Tomatoes are also rich in antioxidants, which can pre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2531/119C12Q2563/107C12Q2537/1376
Inventor 兰成忠姚锦爱阮宏椿吴玮
Owner INST OF PLANT PROTECTION FAAS
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