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LAMP primer group for fast detecting and identifying aphelenchoides ritzemabosi and detection method thereof

A detection method, the technology of chrysanthemum leaves, is applied in the direction of biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., which can solve the problems of narrow detection range, complicated technology, high detection cost, etc., and achieve high accuracy and improved High amplification speed and high sensitivity

Active Publication Date: 2017-03-22
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of high detection cost, narrow detection range, complex technology and long time in the prior art, and provide a method for rapid detection and identification and early diagnosis of chrysanthemum nematode A new, accurate, sensitive, stable and intuitive LAMP detection method for judging results. The method of the present invention is simple and convenient to operate and has strong practicability, and provides an effective method for the rapid detection and identification of chrysanthemum leaf blight nematodes in port and transportation quarantine and production site monitoring. means

Method used

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  • LAMP primer group for fast detecting and identifying aphelenchoides ritzemabosi and detection method thereof
  • LAMP primer group for fast detecting and identifying aphelenchoides ritzemabosi and detection method thereof
  • LAMP primer group for fast detecting and identifying aphelenchoides ritzemabosi and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Design and detection of LAMP primers for Nematode chrysanthemum chrysanthemum

[0056] 1. LAMP primer design

[0057] Download the 18S ribosomal RNA gene sequence of Nematode chrysanthemum chrysanthemum from NCBI (GenBank accession number: EU186067.1). Through sequence comparison and downloading of multiple similar 18S ribosomal RNA gene sequences of other nematodes, multiple alignments were used to find specific sequence fragments in the 18S sequence fragments, and multiple sets of LAMP primer sets for the detection of Nematode chrysanthemum were designed and obtained. Each includes a pair of outer primers, a pair of inner primers and a loop primer.

[0058] After preliminary detection and verification (the method is as follows), a set of optimal LAMP primers was finally screened, and its sequence is as follows:

[0059] Outer primer pair:

[0060] F3 (shown in SEQ ID NO.1): 5'-TGTTGAACCGTTCGGGGT-3'

[0061] B3 (shown in SEQ ID NO.2): 5'-TGTTTCAGCCGACAAAA...

Embodiment 2

[0080] Example 2 Specificity and Stability Detection of LAMP Primers of Nematode chrysanthemum Nematode chrysanthemum

[0081] 1. Sample Preparation

[0082] The chrysanthemum leaf nematodes preserved in our laboratory were used as the test target nematodes, and the banana perforator nematodes, pine wood nematodes, rice root nematodes, root-knot nematodes and incognita isolated from our laboratory were used as controls. nematode, testing the specificity of the LAMP primers for Nematode chrysanthemum.

[0083] The stability of LAMP-specific primers for Nematode chrysanthemum was tested by using the populations of Nematode chrysanthemum cultured under different conditions, single female adult, single male adult, single larva and single egg preserved in our laboratory.

[0084] The extraction of a single worm DNA template, and the LAMP amplification reaction system and reaction conditions are the same as in Example 1, and the results are detected by electrophoresis and fluoresce...

Embodiment 3

[0091] Example 3 Sensitivity detection of LAMP primers for Nematode chrysanthemum

[0092] 1. Sample preparation and detection method

[0093] Adopt the method for embodiment 1 to extract the single worm DNA of chrysanthemum leaf blight nematode, DNA concentration is pressed 10 0 、10 -1 、10 -2 、10 -3 、10 -4 and 10 -5 The gradient dilution was used as a template, and the LAMP primer set in Example 1 was used to perform LAMP amplification according to the reaction system and reaction conditions in Example 1, and each treatment was repeated three times. The LAMP amplification products were detected by the above-mentioned electrophoresis method and fluorescent dye method, respectively.

[0094] 2. Test results

[0095] The LAMP amplification reaction was carried out using the DNA of a single nematode chrysanthemum chrysanthemum nematode in gradient dilution as a template, and the products after the reaction were subjected to electrophoresis and fluorescent dye detection res...

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Abstract

The invention discloses an LAMP primer group for fast detecting and identifying aphelenchoides ritzemabosi and a detection method thereof. The primer group comprises a pair of outer primers F3 / B3, a pair of inner primers FIP / BIP and a loop primer LF; and the sequences of the primers are respectively and sequentially shown as SEQ ID NO.1 to 5. An LAMP detection method of the aphelenchoides ritzemabosi is built by using the LAMP primer group. An amplification product can be detected by an electrophoresis method or fluorochrome method; when stepped stripes occur in electrophoresis detection or when green fluorescence can be observed after the fluorescence dye addition, the result proves that the aphelenchoides ritzemabosi is contained in a sample to be inspected. The detection method can be used for detecting and identifying individuals of aphelenchoides ritzemabosi in different insect states (female insects, male insects, larvae or ova); the aphelenchoides ritzemabosi can be directly detected from various-nematoid mixed samples and plant tissue samples; the detection sensitivity reaches 1 / 100 single insect DNA; and the LAMP primer group and the detection method are particularly applicable to port and transfer quarantine inspection and production place monitoring.

Description

technical field [0001] The invention belongs to the technical field of molecular detection of plant diseases, and in particular relates to a LAMP primer set for rapid detection and identification of Nematode chrysanthemum leaf nematode and a detection method thereof. Background technique [0002] Aphelenchoides ritzemabosi (Schwartz, 1911) Steiner&Buhrer, 1932, also known as chrysanthemum ritzemabosi or axillary bud nematode, is a plant pathogenic nematode that parasitizes aboveground parts of plants and causes damage. Together, A. besseyi and A. fragariae are called foliar nematodes. Chrysanthemum nematode has a wide range of hosts, and can parasitize more than 200 kinds of plants, including ornamental plants, vegetables, small fruit plants and other wild plants. The most typical host is chrysanthemum (Dendranthema morifolium). Chrysanthemum leaf blight nematode harms host plants, which will reduce or lose its economic value, and seriously cause plant death. Chrysanthemum...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 谢辉王东伟白宗师赵立荣韩玉春徐春玲
Owner SOUTH CHINA AGRI UNIV
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