RT-LAMP kit and method for rapid detection of sugarcane streak mosaic virus

A RT-LAMP, mosaic virus technology, applied in microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as difficulty in accurately diagnosing whether infection or carrying SCSMV, etc., to reduce experimental costs, Strong specificity, convenient and fast operation

Active Publication Date: 2018-08-17
SOUTH CHINA AGRI UNIV
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  • Abstract
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Problems solved by technology

[0004] Although the RT-LAMP technology has been applied to the detection of SCSMV at present, it is generally difficult to accurately detect the sugarcane seedlings or sugarcane stems without obvious symptoms because the early symptoms of SCSMV are not obvious or the symptoms are similar to those of sugarcane mosaic virus and sorghum mosaic virus. Diagnose whether they are infected with or carry SCSMV

Method used

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  • RT-LAMP kit and method for rapid detection of sugarcane streak mosaic virus
  • RT-LAMP kit and method for rapid detection of sugarcane streak mosaic virus
  • RT-LAMP kit and method for rapid detection of sugarcane streak mosaic virus

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Embodiment 1

[0034] An RT-LAMP kit for rapidly detecting sugarcane streak mosaic virus, comprising a set of RT-LAMP primers for detecting sugarcane streak mosaic virus, an RT-LAMP reaction system, a negative control solution and a positive control solution.

[0035] Wherein, the RT-LAMP primer set for detecting sugarcane streak mosaic virus is composed of a pair of outer primers F3, B3 and a pair of inner primers FIP, BIP; the nucleotide sequences of F3, B3, FIP, and BIP are sequentially As shown in SEQ ID NO:1~SEQ ID NO:4. The final concentrations of the outer primer pair and the inner primer pair are 0.2 mM and 1.6 mM, respectively.

[0036] The RT-LAMP reaction system included 12.5 μL reaction premix and 6 μL ultrapure water. The negative control solution is the total nucleic acid of sugarcane leaves without SCSMV, and the positive control solution is the total nucleic acid of sugarcane leaves containing SCSMV.

[0037] The reaction condition of the kit is to react at 65°C for 1 hour ...

Embodiment 2

[0055] The performance test of embodiment 2 kit

[0056] 1. Specific detection

[0057] According to the detection method in Example 1, the SCSMV positive control, the SCSMV negative control, and the sugarcane streak mosaic virus, sugarcane mosaic virus, sorghum mosaic virus, sugarcane yellow leaf virus, and perennial root dwarfing were respectively tested using the kit of Example 1. The samples of pathogenic bacteria, sugarcane baculovirus and ultrapure water were detected, and the reaction conditions were all in a water bath at 65°C for 1h, and stored at 4°C.

[0058] The result is as image 3 Shown, only have two sample detection results that contain SCSMV positive control and sugarcane line mosaic virus to be yellow (positive reaction), all the other are red (negative reaction), show that the specificity of primer provided by the present invention is strong to SCSMV, High accuracy.

[0059] 2. Sensitivity detection

[0060] According to the detection method in Example ...

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Abstract

The invention belongs to the technical field of plant virus detection, and specifically discloses a RT-LAMP kit and method for rapid detection of a sugarcane streak mosaic virus. The kit comprises a RT-LAMP primer group for detecting the sugarcane streak mosaic virus. The RT-LAMP primer group comprises a pair of outer primers (F3, B3) and a pair of inner primers (FIP, BIP). The nucleotide sequences of F3, B3, FIP, and BIP are represented by SEQ ID No.1-No.4. The detection method comprises following steps: extracting total nucleic acids of a sample to be detected, carrying out RT-LAMP reactionsby using the abovementioned kit, and determining whether the sample is infected by the sugarcane streak mosaic virus or not by a fluorescent staining method. The provided kit has the advantages of high specificity and high sensitivity and can accurately identify plants that are infected by the sugarcane streak mosaic virus; the operation of the detection method is simple and convenient; and the kit is suitable for rapid field detection of sugarcane streak mosaic virus.

Description

technical field [0001] The invention relates to the technical field of plant virus detection, in particular to an RT-LAMP kit and method for rapidly detecting sugarcane line mosaic virus. Background technique [0002] Sugarcane streak mosaic virus ( Sugarcane streak mosaic virus , SCSMV) is one of the main pathogens causing sugarcane mosaic disease, which has been reported in many sugarcane areas in the world, seriously threatening the development of sugarcane industry. The symptoms of SCSMV are mainly manifested as chlorosis of leaves, irregular mosaic of yellow and green, stripes or mottles, of different sizes, covering the whole leaf. SCSMV can be transmitted mechanically or by vegetative propagation of infected plants. Therefore, strengthening the introduction and quarantine to prevent the virus from spreading with the seedlings over long distances is the main method to control sugarcane line mosaic virus. [0003] At present, the detection methods of SCSMV mainly inc...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/94
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2521/107
Inventor 沈万宽王凯莉邓权清蒋艳陈健文
Owner SOUTH CHINA AGRI UNIV
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