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LAMP primer group for distinguishing types of toxins produced by gibberellic disease infected wheat and application of LAMP primer group

A primer set and scab technology, applied in the field of genetic engineering, to achieve the effects of simple detection, improved sensitivity, and short reaction time

Active Publication Date: 2015-06-03
南京微测生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its use in toxin classification has not been reported

Method used

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  • LAMP primer group for distinguishing types of toxins produced by gibberellic disease infected wheat and application of LAMP primer group
  • LAMP primer group for distinguishing types of toxins produced by gibberellic disease infected wheat and application of LAMP primer group
  • LAMP primer group for distinguishing types of toxins produced by gibberellic disease infected wheat and application of LAMP primer group

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Kit A is a LAMP for detecting the type of DON toxin produced by Fusarium rubella wheat, which includes:

[0023] SEQ ID No.1, forward internal primer DON-FIP:

[0024] 5'-TCAACTACCGGCTGCTAGTAGATAGTGGATACGTCTTCCAGGA-3'

[0025] SEQ ID No.2, reverse internal primer DON-BIP:

[0026] 5'-CCTGCCATGTAGAGGTGAGGAGCAGGGGAAGCACAATTGAGATC-3'

[0027] SEQ ID No.3, forward outer primer DON-F3:

[0028] 5'-ACAGTTATTCCAAACAGTTCG-3'

[0029] SEQ ID No.4, reverse outer primer DON-B3:

[0030] 5'-TGACAAGTCCGGTCGCAC-3'

[0031]In the prepared detection solution: 1.6 μM forward inner primer DON-FIP, 1.6 μM reverse inner primer DON-BIP, 0.2 μM forward outer primer DON-F3, 0.2 μM reverse outer primer DON-B3, 1.6 mM dNTPs , 1M betaine, 20mM Tris-HCl (pH8.8), 10mM KCl, 10mM (NH4) 2 SO 4 , 4mM MgSO 4 , 0.1% Triton X-100, 8U Bst DNA polymerase 500 units.

Embodiment 2

[0033] Kit B detects LAMP of the NIV toxin type produced by Fusarium rubella wheat, which includes:

[0034] SEQ ID No.5, forward internal primer NIV-FIP:

[0035] 5'-CTATCTGCAGAACTCGAATTGTCCCATATCACTCACATATCCAT-3'

[0036] SEQ ID No.6, reverse inner primer NIV-BIP:

[0037] 5'-ACATCGTCCAGGATGTAGTCTAGCAGGCTCAAGGTAAAGCG-3'

[0038] SEQ ID No.7, forward outer primer NIV-F3:

[0039] 5'-CACCTTGATGTGGTTGCC-3'

[0040] SEQ ID No.8, reverse outer primer NIV-B3:

[0041] 5'-GCATACATAGTACTGGCGAT-3'

[0042] In the prepared detection solution: 1.6 μM forward inner primer NIV-FIP, 1.6 μM reverse inner primer NIV-BIP, 0.2 μM forward outer primer NIV-F3, 0.2 μM reverse outer primer NIV-B3, 1.6 mM dNTPs, 1M betaine, 20mM Tris-HCl (pH8.8), 10mM KCl, 10mM (NH4) 2 SO 4 , 4mM MgSO 4 , 0.1% Triton X-100, 8U Bst DNA polymerase 500 units.

Embodiment 3

[0043] Example 3 The sensitivity of Kit A to the DON toxin type produced by Fusarium tritici was verified in a LAMP reaction.

[0044] In order to verify the sensitivity of the LAMP method, select the known DON toxin-producing strain JS05, measure the concentration (1 μg / ul) with a spectrophotometer, and perform 10-fold dilution as a template, and take 1 μl of diluted DNA at each concentration solution, add 24 μl of the detection solution prepared in Example 1 to carry out LAMP reaction, the reaction conditions are 64°C, 60min, 80°C, 10min; The amplification results of 100ng, 10ng, 1ng, 100pg, and 10pg DNA have obvious diffuse bands. Lanes 6-7 are the amplification results containing 1pg and 100fg DNA. They have no bands with the negative control water. The electrophoresis results show that LMAP The sensitivity of the reaction reaches 10ng; after the reaction, add the dye SYBR Green and observe the color change, among which tubes 1-5 are the amplification results containing ...

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Abstract

The invention relates to a LAMP primer set for distinguishing toxin-producing types of scab-susceptible wheat, which consists of LAMP primers for detecting DON-producing toxin and detecting NIV-producing toxin, wherein: the LAMP primer for detecting DON-producing toxin is: SEQ ID No. The forward inner primer shown in 1, the reverse inner primer shown in SEQ ID No.2, the forward outer primer shown in SEQ ID No.3, the reverse outer primer shown in SEQ ID No.4; The LAMP primer of NIV toxin is: forward primer shown in SEQ ID No.5, reverse inner primer shown in SEQ ID No.6, forward outer primer shown in SEQ ID No.7, SEQ ID No. .8 Reverse outer primer as indicated. In the application, kit A containing LAMP primers for detecting DON toxin and kit B for detecting LAMP primers for NIV toxin were used to perform parallel LAMP amplification on the extracted DNA of Fusarium graminearum, and the amplification results were visually Differentiation of toxin-producing types in scab-susceptible wheat.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a LAMP primer set for distinguishing toxin-producing types of scab-susceptible wheat and its application. Background technique [0002] Fusarium head blight (or scab) caused by Fusarium is an important disease that harms various cereal crops such as wheat, barley, oats, and corn. , causing disease during the seed filling maturity stage, which not only causes yield reduction, but also the toxin produced by Fusarium remains and accumulates in the crop grain, affecting the yield and quality of grain. In my country, Fusarium graminearum Schwabe is the main pathogen of head blight, which produces type B trichothecenes, mainly including deoxynivalenol (DON for short) and nivalenol Nivalenol (NIV for short). Moreover, these two toxins are highly harmful to humans and animals, causing symptoms such as vomiting, diarrhea, and dizziness in humans and animals. Therefore, accurate and rapid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/77
CPCC12Q1/6844C12Q2531/119
Inventor 邢宇俊仇剑波史建荣徐剑宏吴季荣
Owner 南京微测生物科技有限公司