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LAMP-HNB (loop-mediated isothermal amplification-hydroxy naphthol blue) primer group, kit and method for detecting leptosphaeria maculans

A technology of LAMP-HNB and basal canker, which is applied in the field of LAMP-HNB primer set for detection of canker canker bacterium, can solve the problems of high detection cost, expensive detection equipment, and long time consumption, so as to improve the detection efficiency and shorten the detection time , the effect of high sensitivity

Inactive Publication Date: 2018-01-30
湛江出入境检验检疫局检验检疫技术中心
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0003] Currently, there are two main methods for detection of canker sores in rapeseed, fluorescent quantitative PCR and common PCR. For professional operators, the applicability is limited, and the real-time fluorescent RT-PCR technology requires expensive detection equipment, and the detection cost is high, so it is not suitable for the promotion and use of grassroots and small laboratories

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  • LAMP-HNB (loop-mediated isothermal amplification-hydroxy naphthol blue) primer group, kit and method for detecting leptosphaeria maculans
  • LAMP-HNB (loop-mediated isothermal amplification-hydroxy naphthol blue) primer group, kit and method for detecting leptosphaeria maculans
  • LAMP-HNB (loop-mediated isothermal amplification-hydroxy naphthol blue) primer group, kit and method for detecting leptosphaeria maculans

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Effect test

Embodiment 1

[0058] This embodiment provides a LAMP-HNB primer set for detecting rape stem canker and its application.

[0059] 1. Design of LAMP-HNB primer set

[0060] According to the ITS sequence of the canola stem canker bacteria (GenBank ID: KT225526.1), a LAMP-HNB primer set was designed, including a pair of outer primers: F3 and B3, and a pair of inner primers: FIP and BIP. The specific sequence is as follows:

[0061] F3: 5'-GTTCGAGCGTCATTTGTACC-3' (SEQ ID NO. 1);

[0062] B3: 5'-AGTTCAGCGGGTATCCCTA-3' (SEQ ID NO. 2);

[0063] FIP: 5'-TGCCGGCTGCCAATTGTTTCCTCTGCTTGGTGTTGGGT-3' (SEQ ID NO.3);

[0064] BIP: 5'-GCAGCACATTTTGCGCCTCTCTGATCCGAGGTCAAGAGC-3' (SEQ ID NO. 4)

[0065] The above primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd. .

[0066] Experiments have proved that the combination of the above-mentioned primer pairs has strong specificity, high sensitivity and good reproducibility. However, the combination of several LAMP-HNB primer pairs randomly selected by primer...

Embodiment 2

[0069] This embodiment provides a kit and its application. The kit includes:

[0070] (1) The LAMP-HNB primer set in Example 1;

[0071] (2) HNB with the article number H991331 from Sigma;

[0072] (3) 10× Isothermal Amplification Reaction Buffer, Bst DNA Polymerase, MgSO, Item No. M0538S from NEB Company 4 ;

[0073] (4) The reagents in the DNA extraction kit with article number DP305 from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0074] Experiments have proved that the combined use of the reagents (1), (2), (3) and (4) above has a superior effect, which is embodied in the characteristics of time saving, economy, high reproducibility, high sensitivity and strong specificity. However, the combination of the LAMP-HNB primer set of (1) of the present invention and several other randomly selected reagents has more or less various defects such as low specificity, low sensitivity and poor reproducibility.

[0075] The above-mentioned kit can be applied to detect the rape stem cank...

Embodiment 3

[0077] This embodiment provides a method for detecting rape stem canker, for example, detecting whether there is rape stem canker or the nucleic acid of rape stem canker in the sample to be tested. This method uses the LAMP- in Example 1 HNB primer set and the kit of Example 2.

[0078] The aforementioned sample to be tested may be a sample of plant origin or a sample of non-plant origin. The nucleic acid can be a DNA, RNA, cDNA or cDNA fragment, or a reagent containing DNA, cDNA, RNA or cDNA fragment, or a plasmid.

[0079] When the sample contains RNA, the sample needs to be reverse-transcribed and converted into DNA, cDNA or cDNA fragments for detection.

[0080] The above method includes the following steps:

[0081] 1. DNA extraction

[0082] Follow the instructions of the kit (plant genomic DNA extraction kit, item number DP305, Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract the genomic DNA of rape stem canker pathogen, and prepare a concentration of 100ng / μL rap...

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Abstract

The invention provides an LAMP-HNB (loop-mediated isothermal amplification-hydroxy naphthol blue) primer group and application of the LAMP-HNB primer group to leptosphaeria maculans detection or preparation of a leptosphaeria maculans detection product. The invention also provides a kit for detecting the leptosphaeria maculans and application of the kit. On the basis, the invention further provides a method for detecting the leptosphaeria maculans. Through being proved by experiments, the LAMP-HNB primer group provided by the invention has the advantages of high specificity, high sensitivity and short detection time. The leptosphaeria maculans detection method built on the basis of the LAMP-HNB primer group has the advantages that whether the leptosphaeria maculans exists or not or whetherthe nucleic acid of the leptosphaeria maculans exists or not can be cheaply, sensitively, simply, conveniently and fast detected; the detection efficiency is greatly improved; a more effective methodis provided for the detection of the leptosphaeria maculans; in addition, the guidance significance is realized on entry and export relevant plants and product inspection and quarantine.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a LAMP-HNB primer set, a kit and a method for detecting rape stem canker. Background technique [0002] Rapeseed stem canker is one of the most serious fungal diseases in rape. Its pathogen is Leptosphaeria maculans, which is a bacterium of the genus Leptosphaeria maculans, which is highly pathogenic and can cause ulceration at the base of rape stems. In severe cases, it can lead to a 30%-50% reduction in rapeseed production, or even higher. Rapeseed stem canker pathogens have been found in major rape producing countries such as Australia, Canada, the United States, and Europe, but have not been reported in China, and belong to China's imported plant quarantine pests. In recent years, my country’s entry-exit inspection and quarantine agencies have repeatedly intercepted the stem canker pathogen L. maculans from imported rapeseeds from Australia, Ukraine and Canada. The climat...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6848C12Q1/04C12N15/11
Inventor 龙阳马新华袁俊杰卢乃会杨卓瑜张娜
Owner 湛江出入境检验检疫局检验检疫技术中心
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