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Loop-mediated isothermal amp lification (LAMP) primer for rapidly detecting meloidogyne javanica, application of LAMP primer and detection method of LAMP primer

A Java root-knot nematode technology and a detection method, which are applied in biochemical equipment and methods, recombinant DNA technology, and microbial assay/inspection, etc., can solve the problems of long time, complicated detection process, time-consuming and the like, and achieve convenient and efficient detection. Detect quick, easy-to-use effects

Inactive Publication Date: 2019-11-08
INST OF BAST FIBER CROPS CHINESE ACADEMY OF AGRI SCI
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  • Application Information

AI Technical Summary

Problems solved by technology

Morphological identification is mainly based on the 2nd instar larvae, male head, tail, and female perineum patterns. However, due to the large number of root-knot nematode species, the morphological characteristics of each species are very similar among species. There are large variations within the species, and it is difficult to accurately identify through morphological characteristics, and this method is time-consuming and laborious, requiring professional skills and rich experience in nematode morphological identification, and it is difficult to achieve the purpose of rapid diagnosis and detection
Although the PCR detection method overcomes the defects of morphological identification to a certain extent, it requires expensive instruments and equipment such as PCR instrument, gel electrophoresis and imaging system, and the detection process is complicated and takes a long time, which is not conducive to rapid on-site detection and popularization at the grassroots level application
[0004] There is currently no LAMP method for rapid visual detection of M. javanica in soil by directly extracting soil DNA and without opening the lid after the reaction is complete.

Method used

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  • Loop-mediated isothermal amp lification (LAMP) primer for rapidly detecting meloidogyne javanica, application of LAMP primer and detection method of LAMP primer
  • Loop-mediated isothermal amp lification (LAMP) primer for rapidly detecting meloidogyne javanica, application of LAMP primer and detection method of LAMP primer
  • Loop-mediated isothermal amp lification (LAMP) primer for rapidly detecting meloidogyne javanica, application of LAMP primer and detection method of LAMP primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1 Meloidogyne javanica SCAR marker sequence amplification

[0071] (1) extraction of root-knot nematode DNA:

[0072] Under a stereomicroscope, pick a single nematode with a pick needle and put it into a tube containing 8 μL ddH 2 O and 1 μL 10×PCRbuffer (Mg 2+ free) in a 0.2mL PCR tube; freeze the PCR tube in liquid nitrogen for 1min, then place it at 85°C for 2min, repeat 5 times; add 1μL 1mg / mL proteinase K to the PCR tube, and incubate at 56°C for 15min , and heated at 95°C for 10 minutes to obtain a DNA extract. The aforementioned DNA extraction solution can be directly used in LAMP and PCR amplification reactions.

[0073] (2) Amplification of the SCAR marker sequence of M. javanica:

[0074] MJ-F (5'-ACGCTAGAATTCGACCCTGG-3') and MJ-R (5'-GGTACCAGAAGCAGCCATGC-3') were used to amplify the SCAR marker sequence of M. javanica in the DNA extract ( As shown in SEQ ID No.1).

[0075] The PCR amplification reaction uses a 25 μL system:

[0076]

[0...

Embodiment 2

[0079] Example 2 Establishment of method for detecting root-knot nematode javanica by LAMP technology

[0080] 2.1 LAMP primer design

[0081] According to the sequencing results of the SCAR marker sequence of Meloidogyne javanica, multiple sets of primer sets were designed using the online primer design software PrimerExplorer V5, and the following three sets of LAMP primers were obtained through preliminary screening, including Mj-F3 / Mj-B3 / Mj-FIP / Mj-BIP, Mj-F3-A / Mj-B3-A / Mj-FIP-A / Mj-BIP-A and Mj-F3-B / Mj-B3-B / Mj-FIP-B / Mj-BIP- b. The primers were synthesized by Hunan Branch of Beijing Qingke Biotechnology Co., Ltd.

[0082] The three sets of designed primer sets and their sequences are as follows:

[0083] Forward outer primer Mj-F3 (SEQ ID No.2): 5'-AAGTTGGACGGTTTTCCGG-3';

[0084] Reverse outer primer Mj-B3 (SEQ ID No.3): 5'-TTTACAAAGGGGGCAGTTCC-3';

[0085] Forward internal primer Mj-FIP (SEQ ID No.4): 5'-TCCAGACGATTCTGTGCTAGTTAAAGCTGTTCCCATGTTTTAAGC-3';

[0086] Reve...

Embodiment 3

[0117] Example 3 Meloidogyne javanica LAMP Specific Detection

[0118] Collect root-knot nematode java, root-knot nematode incognita, root-knot nematode peanut, root-knot nematode northern, root-knot nematode elephant ear, root-knot nematode graminaceae, citrus semi-puncture nematode, short-bodied coffee nematode, rice root-knot nematode, The DNA of Cryptoderma sinensis and C. gardiensis were extracted as templates, and LAMP detection was performed according to the method in Example 2, so as to verify the specificity of the detection method of LAMP for Meloidogyne javanica. Table 1 shows the types and sources of plant nematode populations in each sample.

[0119] Table 1 Types and sources of tested plant nematode populations

[0120] serial number Group type host plant sampling location 1 Meloidogyne javanica industrial hemp Yunnan Xishuangbanna Dai Autonomous Prefecture 2 Meloidogyne javanica (M.javanica) Notoginseng Yunnan Pu'er 3 M...

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Abstract

The invention relates to the technical field of plant pathogen detection, in particular to a loop-mediated isothermal amp lification (LAMP) primer for rapidly detecting meloidogyne javanica, application of the LAMP primer and a detection method of the LAMP primer. The LAMP primer comprises a forward outer primer Mj-F3, a reverse outer primer Mj-B3, a forward inner primer Mj-FIP and a reverse innerprimer Mj-BIP; and primer nucleotide sequences are sequentially shown as SEQ ID NO.2-5. A LAMP detection method is built based on the LAMP primer, can be applied to rapid detection and identificationof the meloidogyne javanica in nematode samples, plant root tissue samples and soil samples and has the advantages of high specificity, high sensitivity, simple instrument equipment, simple operation, fastness and high efficiency, visual determination of a result and the like, and high popularization and application value is achieved in aspects such as field rapid detection of the meloidogyne javanica and early diagnosis and guide scientific drug use of the meloidogyne javanica.

Description

technical field [0001] The invention relates to the technical field of plant pathogen detection, in particular to a LAMP primer for rapid detection of root-knot nematode javanica and its application and detection method. Background technique [0002] Root-knot nematode (Meloidogyne spp.) is a worldwide obligate endoparasitic nematode that threatens agricultural production. It can infect more than 3,000 species of plants in 114 families, and its damage can cause at least 5% of world crop production reduction every year. . At present, there are more than 100 kinds of root-knot nematodes reported in the world, among which M. The range of hosts can infect vegetables, fruit trees, medicinal materials, flowers, forest trees and other plants, causing major economic losses to my country's agricultural production. Rapid and accurate identification and detection of root-knot nematode species is very important for the field detection and monitoring of root-knot nematode diseases and t...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6888C12Q1/6844
CPCC12Q1/6844C12Q1/6888C12Q2531/119C12Q2563/107
Inventor 宋志强梅时勇杨喜爱张晓伟肖清明肖爱平冷娟
Owner INST OF BAST FIBER CROPS CHINESE ACADEMY OF AGRI SCI
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