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A kind of loop-mediated isothermal amplification primer composition and application thereof for detecting glyospora anthracnose

A technology of glenospora anthracnose and primer composition, which is applied in the direction of microorganisms, recombinant DNA technology, and methods based on microorganisms, and can solve the problems of few recognition sites of target gene segments, low detection specificity and sensitivity, and cumbersome detection process, etc. problems, to achieve the effect of easy popularization and application, short detection cycle and high sensitivity

Active Publication Date: 2017-01-04
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The known ITS target for molecular detection of G. anthracnose can not meet the needs of LAMP primer design. The existing molecular detection technology based on common PCR for this pathogen is relatively backward, and it takes a long time to detect the target gene segment. Few points, high false detection rate, low detection specificity and sensitivity, cumbersome detection process and other defects

Method used

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  • A kind of loop-mediated isothermal amplification primer composition and application thereof for detecting glyospora anthracnose
  • A kind of loop-mediated isothermal amplification primer composition and application thereof for detecting glyospora anthracnose
  • A kind of loop-mediated isothermal amplification primer composition and application thereof for detecting glyospora anthracnose

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1 A kind of LAMP detection kit that is used to detect glyospora anthracnose bacteria

[0060] Kit reaction system: 1mL detection solution + dye SYBR Green I;

[0061] 1mL detection solution includes: 0.8uM forward inner primer GS-FIP, 0.8uM reverse inner primer GS-BIP, 0.1uM forward outer primer GS-F3, 0.1uM reverse outer primer GS-B3, 0.1uM loop primer GS -LF, 0.1uM loop primer GS-LB, 1.4mM dNTPs, 8mM MgSO 4 , 0.8M Betaine, 0.8M Tris-HCl (pH 8.8), 0.4mM KCl, 0.4mM (NH4) 2 SO 4 , 4% Triton X-100, 8U·μL -1 Bst DNA polymerase, add ultrapure water to prepare 1ml detection solution, and the shelf life is 1 year.

Embodiment 2

[0062] Embodiment 2 The universality investigation of the kit of the present invention

[0063] Select the standard strain of Gluespora anthracnose and DNA from different hosts (Table 2) as templates, take 4 μl of the DNA solution, add 21 μL of the detection solution in Example 1 to carry out the LAMP reaction, and the reaction program is: 64°C 70min; after amplification, add 0.25 μL of dye SYBR Green I as a reaction indicator, and observe the color change of the reaction tube after the reaction. Under normal light, the sample containing G. anthracnose should turn yellow-green under normal light, and the negative control should be orange under normal light. see results figure 2 ,Depend on figure 2 Visible standard G. sojae strain, G. sojae strain and other host specialized types of G. sojae are all positive; Intraspecies versatility.

Embodiment 3

[0064] Embodiment 3 The kit specificity investigation of the present invention

[0065] The standard strain of Glycospora anthracnose, its close species Pleurotus anthracnose and A. oxysporum, and other pathogenic bacteria of different genera (P. Coccus, Cercoides chrysanthemum, Alternaria, Fusarium oxysporum, Aspergillus oryzae, Bisporium oxysporum, Ascococcus phasei, Botrytis spp., Phomopsis rot, Soybean stem brown rot) (Table 2) Take 4 μl of DNA solution as a template, add 21 μL of the detection solution in Example 1 to carry out LAMP reaction, the reaction program is: 64 ° C for 70 min; after amplification, add 0.25 μL of dye SYBR Green I as a reaction indicator, and observe the reaction after reaction Tube color changes. Anthracnose glyosporium is yellow-green, other samples are orange, see image 3 and Figure 4 .

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Abstract

The invention discloses LAMP (loop-mediated isothermal amplification) primer composition for detecting colletotrichum gloeosporioides and an application of the LAMP primer composition. A GS (glutamine synthetase) gene is used as a target gene, primers which have high specificity and high sensitivity and can be used for LAMP detection of the colletotrichum gloeosporioides are designed and screened out, and the LAMP primer composition consists of a forward outer primer GS-F3 shown in SEQ ID NO.2, a reverse outer primer GS-B3 shown in SEQ ID NO.3, a forward inner primer GS-FIP shown in SEQ ID NO.4, a reverse inner primer GS-BIP shown in SEQ ID NO.5, a loop primer GS-LF shown in SEQ ID NO.6 and a loop primer GS-LB shown in SEQ ID NO.7. The primer composition is mainly used for quickly detecting the colletotrichum gloeosporioides, and only 2 hours are spent on detection each time.

Description

technical field [0001] The invention belongs to the field of biological detection, and relates to a loop-mediated isothermal amplification primer composition for detecting glyospora anthracnose and an application thereof. Background technique [0002] C.gloeosporioides has a variety of hosts, including more than 2,200 species of plants, causing anthracnose and causing serious economic losses to agricultural production [1] . When G. glyospora anthracnose infects soybean plants, the stems, pods, leaves and petioles of soybeans can all be damaged, and the quality of soybean seeds can be reduced. The lesions on the stems of affected plants are dark gray, with irregular black spots; the lesions on the pods are irregular, brown to off-white, with small black spots densely arranged in rings; the lesions on the leaves are brown, with obvious depressions, Usually arranged in a ring pattern [2] . The conidia of G. anthracnose are produced on the sticky conidia disk. The conidia ar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 郑小波王帅帅陆辰晨田擎张海峰王源超
Owner NANJING AGRICULTURAL UNIVERSITY
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