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A LAMP detection primer set of broccoli stem rot fungus and a rapid detection method thereof

A technology for detecting primers and primer sets, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of low accuracy, long detection time, long time consumption, etc., to achieve strong specificity, reliable results, Sensitive effect

Inactive Publication Date: 2019-01-11
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to detect and identify Jianlan stem rot pathogen mainly based on morphological characteristics in the prior art, the method takes a long time, the procedure is cumbersome, the experience is strong, the accuracy is low, and it is difficult to monitor and control the occurrence of the disease in time The problem of the spread and prevalence of pathogenic bacteria, and the PCR molecular detection needs to rely on expensive instruments such as amplification instruments, and the detection time is long. A new rapid molecular detection method for Jianlan stem rot is provided, and the LAMP rapid detection method for Jianlan stem rot is provided. Detection, short detection cycle, high accuracy, high sensitivity, visual inspection results

Method used

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  • A LAMP detection primer set of broccoli stem rot fungus and a rapid detection method thereof
  • A LAMP detection primer set of broccoli stem rot fungus and a rapid detection method thereof
  • A LAMP detection primer set of broccoli stem rot fungus and a rapid detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Design of specific primer set for LAMP detection of Jianlan stem rot fungus and detection and verification of primer specificity

[0032] 1. Extract the genomic DNA of the sample to be tested

[0033] When used to detect pure cultures of pathogenic bacteria, the CTAB method was used to extract genomic DNA. The specific method was as follows: Take a small amount of mycelium powder in a 1.5 mL centrifuge tube (it is better that the mycelium powder just covers the bottom of the semicircle), add 900 µL 2 % CTAB (cetyltrimethylammonium bromide) extract (which contains: mass fraction 2% CTAB; final concentration 100 mmol L -1 Tris-HCl, pH8.0; final concentration 20 mmol·L -1 EDTA, pH8.0; 1.4 mol L -1 NaCl) and 90 µL pH7.2, SDS (sodium dodecylbenzenesulfonate) with a mass fraction of 20% [Note: CTAB, SDS needs to be preheated at 60°C], use a shaker to shake and mix, and bathe at 60°C for 1h ( DNA released into the buffer), 12000 r min -1 Centrifuge for 15 min;...

Embodiment 2

[0046] Embodiment 2: Determination of detection sensitivity of Jianlan stem rot pathogen LAMP

[0047] 1. Preparation of Genomic DNA at Different Concentrations

[0048] Genomic DNA of Phytophthora spp. was diluted with sterile double-distilled water, and prepared into a series of 10-fold concentrations for future use.

[0049] 2. Determination of LAMP detection sensitivity and observation of results

[0050] Genomic DNA of Phytophthora japonicus at different concentrations was used as a template, and the outer primer F3 / B3 and inner primer FIP / BIP were used for LAMP amplification. The LAMP detection reaction system was 25 μL, including the following final concentration components: 20 mM Tris-HCl , 10 mM (NH 4 ) 2 SO 4 , 6.0 mM MgSO 4 , 50 mM KCl, 0.8 mM betaine, 1.4 mmol·L -1 dNTPs, 8U Bst DNA polymerase, 1.6 μmol L -1 FIP and BIP, 0.2 μmol L -1 For F3 and B3, 1 μL template DNA (10 ng·μL -1 , 1 ng·μL -1 , 100 pg·μL -1 , 10 pg·μL -1 , 1 pg·μL -1 , 100 fg·μL -1 ,...

Embodiment 3

[0053] Example 3: LAMP Rapid Detection of Jianlan Stem Rot Bacteria in Diseased Tissues

[0054] 1. Sample Collection

[0055] Plants with typical symptoms of Jianlan stem rot disease and healthy plants were collected from Zhao'an and Nanjing in Zhangzhou, Fujian and brought back to the laboratory for future use.

[0056] 2. Plant tissue DNA extraction

[0057] DNA was extracted by NaOH rapid lysis method, the specific process is as follows: add 10 µL of 0.5 mol L to each mg of plant tissue -1 NaOH, fully grind the tissue into a paste in a mortar, transfer it to a 1.5 mL centrifuge tube, centrifuge at 12,000 rpm for 6 min, take 5 µl of the supernatant and add 495 µL of 0.1 mol·L -1 Mix Tris-HCl (pH=8.0) evenly, and take 1.0 µL (50-100 ng) as a PCR template for amplification.

[0058] 3. LAMP rapid detection and observation

[0059] Using the extracted DNA as a template, LAMP amplification was performed using the outer primer F3 / B3 and the inner primer FIP / BIP. The LAMP d...

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Abstract

The invention provides a LAMP detection primer set of broccoli stem rot fungus and a rapid detection method thereof. The LAMP primer set was composed of one pair of outer primers F3 / B3 and one pair ofinner primers FIP / BIP. The sequences of F3 / B3 and FIP / BIP primers were as follows: SEQ ID 1-4. Comprises the following steps of: 1) extracting DNA of a sample to be tested; 2) LAMP amplification; 3)detect that amplification product by using a fluorescent dye SYBR Green I for color development or agarose gel electrophoresis of the amplification result. So that that invention can quickly, Convenient, high efficiency, high specificity, high sensitivity detection of broccoli stem rot pathogen, does not require complex and expensive instruments, can better meet the rapid detection of broccoli stem rot pathogen in the field, can be widely used in grass roots units of early disease diagnosis and disease pathogen monitoring, identification, for the control of broccoli stem rot disease to providereliable technical and theoretical basis.

Description

technical field [0001] The invention belongs to the technical field of disease detection, identification and prevention of horticultural crops, and in particular relates to a LAMP detection primer set of Jianlan stem rot and a rapid detection method thereof, which can be used for rapid, sensitive and specific molecular detection of Jianlan stem rot, and Jianlan stem Early diagnosis of rot and monitoring and identification of pathogens. Background technique [0002] Stem rot is the most serious worldwide soil-borne disease in Jianlan production. It is known as "orchid cancer" and "orchid killer". The pathogenic bacteria is Fusarium oxysporum ( Fusarium oxysporum ). Since it was first reported in Penang, Malaysia in 2010, it has generally occurred in orchid growing areas around the world. The disease is a systemic disease that is transmitted by soil, invades from roots or rhizomes or wounds, and parasitizes in vascular bundles. It is one of the diseases that are difficult to...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/77
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 姚锦爱余德亿黄鹏陈南川
Owner INST OF PLANT PROTECTION FAAS
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