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RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) detection method, RT-LAMP primer group and kit for mulberry vein banding virus as well as application

A technology of RT-LAMP and detection kits, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., which can solve the problems of long reaction time, low sensitivity, false positives, etc., and achieve simple operation , high sensitivity and strong specificity

Active Publication Date: 2017-09-08
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since Mulberry band virus is a newly discovered virus infecting mulberry trees in recent years, a systematic detection method has not yet been established, and it is necessary to develop a rapid and accurate MVBV detection method to meet the needs of the silkworm industry
[0004] The current methods for detecting plant viruses are mainly reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The reaction time is long; ELISA needs to prepare high-quality virus serum, the detection time is also long, and the sensitivity is lower than that of molecular biology methods
In addition, among members of the genus Tospovirus, if the homology of the N protein is high, and the amino acid level consistency reaches 50%, there will be cross-reactions in ELISA detection, resulting in false positive results

Method used

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  • RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) detection method, RT-LAMP primer group and kit for mulberry vein banding virus as well as application
  • RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) detection method, RT-LAMP primer group and kit for mulberry vein banding virus as well as application
  • RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) detection method, RT-LAMP primer group and kit for mulberry vein banding virus as well as application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of MVBV RT-LAMP Detection Kit

[0028] 1.1 Reagents

[0029] Primers were synthesized by Beijing Aoke Dingsheng Biotechnology Co., Ltd.; plant total RNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; Bst DNA polymerase and its reaction buffer were purchased from New England Biolabs (NEB), AMV Reverse transcriptase was purchased from Promega, nucleic acid fluorescent dye SYBR Green I (10 000 ×) was purchased from Shanghai Suo Laibao Biotechnology Co., Ltd., Betaine was purchased from Sangon Bioengineering (Shanghai) Co., Ltd., and dNTPs were purchased from Bao Biological Engineering ( Dalian) Co., Ltd. (Takara). The primer set was stored in a -20°C refrigerator, and other reagents were stored in accordance with the conditions described in the corresponding product instructions.

[0030] 1.2 Assembly of the kit

[0031] The kit includes RT-LAMP primer set, RT-LAMP reaction solution, enzyme solution and nuclei...

Embodiment 2

[0038] Example 2 RT-LAMP detection method detects MVBV and its specificity

[0039] Three samples were tested, including 1 symptomatic mulberry leaf, 1 tobacco sample infected by Tomato Ring Spot Virus (TZSV), and 1 Shuigui banana infected by Hippeastrum chlorotic ring spot virus.

[0040] Adopt the kit of embodiment 1 to detect, detection method comprises the following steps:

[0041] (1) Extract the total RNA of the sample to be tested: use the total RNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract the total RNA of the sample to be tested.

[0042] (2) Establish RT-LAMP reaction system: establish a 25 μL reaction system in a thin-walled PCR tube: RT-LAMP reaction solution 12.5 μL, enzyme solution 1 μL, outer primers MVBV-F3 and MVBV-B3 each 0.5 μL (20 μM), 2.0 μL (20 μM) of inner primers MVBV-FIP and MVBV-BIP, 2 μL of total RNA template obtained in step 1, plus RNase Free H 2 0 to 25.0 μL; the total RNA of mulberry leaves containing MVBV ...

Embodiment 3

[0046] Example 3 Sensitivity test of MVBV RT-LAMP detection method

[0047] The total RNA of the mulberry diseased leaf tissue containing MVBV extracted according to Example 2 was measured by a micro-spectrophotometer, and a nucleic acid template with a total RNA concentration of 10 ng / μL was taken for detection. It was diluted according to the 10-fold ratio and used as a template. A total of 7 gradients of 10ng / μL, 1ng / μL, 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL and 10fg / μL were set up. Perform RT-LAMP detection and conventional RT-PCR detection respectively, and the steps of RT-LAMP detection are the same as (2)-(4) in Example 2.

[0048] RT-PCR detection: The corresponding total RNA was used as a template, and the RT-PCR reaction amplification system was carried out according to the instructions of the one-step RT-PCR kit from Novizyme Biotechnology Co., Ltd. The reaction system consisted of: 2×One Step Mix 25μL , OneStep Enzyme Mix 2.5 μL, upstream primer MVBV-N-F (10 μM) 1 μ...

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Abstract

The invention discloses a RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) detection primer group for a mulberry vein banding virus. The RT-LAMP detection primer group comprises an outer primer pair MVBV-F3 and MVBV-B3 and an inner primer pair MVBV-FIP and MVBV-BIP. Therefore, an RT-LAMP detection method and a kit for the mulberry vein banding virus are invented. The RT-LAMP detection primer group, the RT-LAMP detection method and the kit for detecting the mulberry vein banding virus, disclosed by the invention, have the characteristics of high specificity, simple operation and high sensitivity; no special instrument is needed; a result can be judged by observing color change with naked eyes; no electrophoresis, ultraviolet observation and other steps are needed; the RT-LAMP detection primer group, the RT-LAMP detection method and the kit are suitable for mulberry sapling production and detection of MVBV on field mulberry, can be applied to early diagnosis of the mulberry vein banding virus, epidemiology research and the like, and are suitable for carrying out MVBV detection in basic units.

Description

technical field [0001] The invention belongs to the field of detection of Morusia japonica virus, and in particular relates to an RT-LAMP detection method of Morusia japonica virus, a primer set, a kit and an application thereof. Background technique [0002] Driven by the country's implementation of the "East Mulberry Moving West" industrial structure adjustment strategy and the industrial transfer of the eastern region, Guangxi's silkworm industry has developed rapidly since the "Tenth Five-Year Plan". Since 2005, Guangxi's silkworm cocoon production has ranked first in the country for 12 consecutive years. First. The rapid development of sericulture has made important contributions to the increase of farmers' income in Guangxi, the increase of agricultural efficiency, the great development of county economy and the construction of new countryside. However, mulberry virus disease occurs seriously in silkworm areas in Guangxi, with an incidence rate of about 40%, and the i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2563/107
Inventor 蒙姣荣吴凡陈保善李界秋李杨秀
Owner GUANGXI UNIV
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