Loop mediated isothermal amplification (LAMP) detection method for meloidogyne hapla and application of method

A detection method and technology for root-knot nematodes, which are applied in the directions of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of limiting the popularization and application of PCR detection methods, long time, etc., and achieve short detection time, simple operation steps, Simple to use effects

Inactive Publication Date: 2012-07-18
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PCR detection requires expensive professional instruments such as PCR instrument, gel electrophoresis and imaging system (ultraviolet instrument) and molecular biology reagents, and requires professional laboratory personnel in molecular biology to operate. The above detection can only be detected under laboratory conditions. , it takes a long time, which limits the popularization and application of PCR detection method in production

Method used

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  • Loop mediated isothermal amplification (LAMP) detection method for meloidogyne hapla and application of method
  • Loop mediated isothermal amplification (LAMP) detection method for meloidogyne hapla and application of method
  • Loop mediated isothermal amplification (LAMP) detection method for meloidogyne hapla and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 Extraction, rDNA-ITS amplification and sequence analysis of the root-knot nematode DNA of northern root-knot nematode

[0088] 1.1 Extraction of DNA from M. northernis

[0089] Pick a single-headed root-knot nematode and put it in a container containing 10μdddH 2 Add 8 μl of WLB solution and 2 μl of proteinase K solution to a 0.2ml centrifuge tube of O, centrifuge in a jog, put it into liquid nitrogen, then put it into a 37°C water bath, put it into liquid nitrogen after it melts, repeat 6 ~7 times, and then frozen at -80°C for 30min. Then the centrifuge tube was taken out, incubated at 65° C. for 90 minutes, and reacted at 95° C. for 10 minutes. After treatment, it was directly used as a nematode DNA template for LAMP and PCR reactions.

[0090] 1.2 Amplification and sequence analysis of rDNA-ITS of M. borealis

[0091] The general primers rDNA1 (5'-TTGATTACGTCCCTGCCCTTT-3') and rDNA2 (5'-TTTCACTCGCCGTTACTAAGG-3') of the ITS region were used to amplify the...

Embodiment 2

[0092] Embodiment 2LAMP technology detects the establishment of the method for root-knot nematode borealis

[0093] 2.1 LAMP primer design

[0094] According to the sequencing results of M. northernis ITS sequence, design and screen the following LAMP primers and probes (such as figure 1 shown), the five primers include two outer primers (F3 and B3), two inner primers (FIP and BIP) and one loop primer (MELB), FIP consists of F1c-F2, BIP consists of B1c-B2, F1c and B1c are the complementary sequences of F1 and B1, respectively. Primers and probes were handed over to Shanghai Bioengineering Technology Service Co., Ltd. for synthetic labeling. The sequence is as follows:

[0095] ①MHF3: 5’-GAATATGAGGTGACATGTTAGG-3’

[0096] ②MHB3: 5'-TCAATGTTTCTGCAGTTCG-3'

[0097] ③MHFIP: 5’-TGAAAAAAATATTGCTGGCGTCCACCTAATCGGGTTTAAG

[0098] ACT-3'

[0099] ④ MHBIP: 5’-TCTATCCTTATCGGTGGATCACTCCACAAATTATCGCAGTT

[0100] AGCT-3'

[0101] ⑤ MHLB: 5'-GGCTCGTGGATCCATGAAGAACG-3'

[0102] ⑥MH-...

Embodiment 3

[0108] Embodiment 3 LAMP method detects different geographic populations of root-knot nematode

[0109] Two kinds of root-knot nematodes collected in our laboratory were tested by LAMP. After mixing the primer mixture and the reaction buffer mixture evenly, add 1 μl of template DNA, and proceed according to the reaction conditions in 2.3. After the reaction, add 2 μl of prepared color After the agent is mixed, observe the color change (such as image 3 Shown in A), green fluorescence can be seen in the tube with the DNA of M. northernis, and the negative control is reddish brown. Get 2 μ l of the product and electrophoresis on 2% agarose gel, stain with EB, observe and take pictures under ultraviolet light (such as image 3 Shown in B), the characteristic ladder-shaped band of LAMP can be seen in the 1-2 swimming lanes, and the negative control has no amplification product. 5 μL of FITC-labeled probe was added to the reaction product, incubated at 63° C. for 5 min, and coole...

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Abstract

The invention relates to a loop mediated isothermal amplification (LAMP) detection method for meloidogyne hapla and application of the method and belongs to the field of biological technology. 5 LAMP primers (namely, MHF3, MHB3, MHFIP, MHBIP and MHLB) are designed in the sequence conservative region according to the cloning sequencing meloidogyne hapla ITS sequence; the 5' end of the probe MH-FITC-Probe is marked by FITC; and the 5'end of the MHFIP primer is marked by the biotin. The DNA of the meloidogyne hapla is extracted, isothermal amplification is performed by the mixture of the specific primers, reaction buffer liquor and reaction enzyme, and the amplification product is subjected to color developing detection, so that the meloidogyne hapla can be detected quickly. The detection method is high in specificity, high in sensitivity, low in cost, and simple and convenient to operate, and has a high application value in the aspects of on-site quick detection on the meloidogyne haplaand early diagnosis on the meloidogyne hapla disease.

Description

technical field [0001] The invention relates to a rapid detection method for LAMP of root-knot nematode northern, belonging to the field of biotechnology. Background technique [0002] Root-knot nematode (Meloidogyne spp.) belongs to plant root obligate endoparasitic nematodes, and is one of the important plant pathogenic nematodes distributed worldwide that threaten global agricultural production. There are more than 90 species of root-knot nematodes that have been recorded so far, including M. incognita, M. javanica, M. arenaria and M. hapla) are the four most common and most important root-knot nematodes. [0003] In my country, these four kinds of root-knot nematodes occur in most provinces (regions), infecting more than a hundred kinds of crops, causing the host to reduce production by 15-25% all the year round, sometimes reaching more than 70% (Xu Jianhua, Wei Dawei, Zhan Yuding He Cheng Hurui. The types and occurrence of greenhouse vegetable parasitic nematodes in Ji...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 彭德良何旭峰彭焕
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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