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LAMP-LFD fast kit for silkworm BmBDV and detection method

A LAMP-LFD, kit technology, applied in the directions of microorganism-based methods, microorganism determination/inspection, biochemical equipment and methods, etc., to achieve the effect of simple and fast DNA extraction method, simple operation steps, and safety to people and the environment

Active Publication Date: 2017-06-30
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The kit has strong specificity, is easy and fast to operate, overcomes the problem that existing detection methods cannot be directly applied in production, and is suitable for the screening of silkworm BmBDV

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] materials needed:

[0039] The samples infected with silkworm BmBDV virus came from our laboratory (Institute of Life Sciences, Jiangsu University); the primers and probes used were synthesized by Shanghai Sangon Biotechnology Company; the large fragment of Bst DNA polymerase was purchased from New England Biolabs Company; dNTP, purchased from promega company; betaine, magnesium sulfate, etc. were purchased from sigma company.

[0040] S1. Rapid extraction of DNA from silkworm BmBDV samples:

[0041] Take 20-30 mg whole silkworm silkworm or silkworm excrement, or 20 µL silkworm hemolymph, add 100 µL tissue lysate to mix, incubate at 37-55 °C for 25-30 min, denature at 95 °C for 5 min, and take 5 µL as a template.

[0042] S2. Bombyx mori BmBDV virus LAMP constant temperature amplification:

[0043] (1) According to the number of samples to be tested, prepare a corresponding amount of pre-reaction solution (1.6mM BmBDV-FIP and BmBDV-BIP, 0.2mM BmBDV-F3, BmBDV-B3, BmBDV...

Embodiment 2

[0054] materials needed:

[0055] The samples infected with silkworm BmBDV virus, silkworm densovirus BmDNV, silkworm nuclear polyhedrosis virus BmNPV, and Bombyx mori N. b. were collected from the Life Science Research Institute of Jiangsu University; the primers and probes used were provided by Shanghai Sangong Biotechnology Co., Ltd. Synthesis; large fragments of Bst DNA polymerase were purchased from New England Biolabs; dNTP, purchased from promega; betaine, magnesium sulfate, etc. were purchased from sigma.

[0056] Bombyx mori bipartite densovirus (BmBDV), Bombyx mori densovirus (BmDNV), Bombyx mori nuclear polyhedrosis virus (BmNPV) and Bombyx mori microsporidia (N. b.) were obtained from the Institute of Life Sciences, Jiangsu University.

[0057] S1. Rapid extraction of sample DNA:

[0058] Take 20-30 mg of silkworm whole silkworm or silkworm excrement infected with different pathogens, or 20 μL of silkworm hemolymph, add 100 μL of tissue lysate to mix, incubate at ...

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Abstract

The invention relates to a LAMP-LFD fast kit for silkworm BmBDV and a detection method, and belongs to the technical field of virus epidemic disease diagnosis, a primer and a probe related in the kit are designed according to a conservative region of a silkworm BmBDV virus sequence published in GenBank, BmBDV virus DNA is extracted, then detected by a BmBDVLAMP reaction system established by the method, at the end of the reaction, nucleic acid rapid detection test paper is used for judging results, the results show that the BmBDV virus DNA is highly specifically amplified after the reaction at 62 DEG C of 30min, compared with commonly used detection techniques of BmBDV virus in the prior art, the LAMP-LFD fast kit has the characteristics of being simple and rapid, high in sensitivity and good in specificity, and is suitable for technical personnel in a production line and silkworm raisers to use in the field and beneficial to the diagnosis and prevention of the virus disease.

Description

technical field [0001] The invention relates to a rapid LAMP-LFD kit and detection method for silkworm BmBDV, belonging to the technical field of virus epidemic diagnosis. Background technique [0002] Bombyx mori Bidensovirus (BmBDV) belongs to the Parvoviridae densoviridae subfamily. The virus has a high lethality rate and a long duration of infection, causing chronic silkworm disease. One of the important reasons. Studies have shown that BmBDV is a non-enveloped spherical virus particle with a diameter of about 20-24nm, containing two linear single-stranded DNA molecules, the size of which is about 6.5kb (VD1) and 6kb (VD2), respectively, and its sequence has been determined and listed on GeneBank accession (accession numbers DQ017268 and DQ017269). At present, the detection method of the virus is mainly the conventional PCR detection method, but in the actual detection work, the existing detection technology has the following problems: one is that the sensitivity is no...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2565/625
Inventor 吕鹏姚勤林锋潘晔陈克平张春霞李承军张亚慧
Owner JIANGSU UNIV
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