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A kind of virus detection kit and detection method

A detection kit and the technology of the kit are applied in the field of microbial detection, which can solve the problems of low sensitivity, complicated operation and low stability, and achieve the effects of low false positive rate, simple operation and simple identification.

Active Publication Date: 2017-09-22
安徽济人药业股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the traditional indicator plant detection method of citrus schizoid virus is stable and reliable, the detection cycle is long and requires supporting facilities such as temperature room and net room; methods such as polyacrylamide gel electrophoresis and molecular hybridization can stably detect plants that have been grafted and propagated. CEVd on indicator plants, however, due to the low content of viroids in host plants in the field in spring and winter, the stability of direct sampling detection is not high; although there have been molecular biology detection reports using RT-PCR method to detect CEVd, there are still Problems such as cumbersome operation, large sampling, and low sensitivity

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  • A kind of virus detection kit and detection method
  • A kind of virus detection kit and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1 The preparation of citrus split peel viroid RT-LAMP detection kit

[0019] 1.1 Reagents

[0020] Primers were synthesized by Tiangen Biochemical Technology (Beijing) Co., Ltd.; Bst DNA polymerase and 10×ThermoPol reaction buffer were purchased from NEB; AMV reverse transcriptase was purchased from Promega; SYBR Green I and TRIZOL Reagent were purchased from Invitrogen; Reagents needed were purchased from Sigma.

[0021] 1.2 Preparation of the kit:

[0022] Reaction buffer: including 2mM dNTP, 10×ThermoPol reaction buffer, 0.6mM betaine and 6mM Mg 2+ ;

[0023] Primer: outer primer F3, its nucleotide sequence is as shown in SEQ ID NO:1; Outer primer B3, its nucleotide sequence is as shown in SEQ ID NO:2; Internal primer FIP, its nucleotide sequence is as shown in SEQ ID As shown in NO:3, the inner primer BIP has a nucleotide sequence as shown in SEQ ID NO:4. The concentration of the two outer primers is 0.2 μmol / μl, and the concentration of the two inne...

Embodiment 2

[0029] Example 2 Citrus split peel viroid specific detection

[0030] 2.1 RT-LAMP specific detection

[0031] Navel orange and citrus plants infected with CEVd, 1 plant of citrus plants infected with potato tuber viroid, and 1 plant of sweet orange without virus were provided by Citrus Research Institute of Chinese Academy of Agricultural Sciences. Graft the single buds and budless branches on the CEVd-infected plants to the lower part of the main trunk of Jincheng rootstock, and then graft the dark willow orange buds to the upper part of the rootstock to form a combination of rootstock and ear to propagate the virus. Four preserved seedlings were bred from each infected plant, and the healthy sweet orange without inoculation was used as a negative control.

[0032] The preserved seedlings were grafted onto the indicator plant Etrog citron, and the growth symptoms of the index plants were observed, and the preserved seedlings were confirmed to be infected with CEVd by the ind...

Embodiment 3

[0045] Example 3 Sensitivity detection of citrus split peel virus

[0046] 3.1 Sensitivity detection of RT-LAMP

[0047] The RNA samples of preserved seedlings whose identification results were positive in Example 2 were taken, and diluted into 100 fg-100 ng of 7 gradients with a 10-fold concentration serial dilution method.

[0048] The RT-LAMP reaction and result interpretation steps are the same as steps 2-4 in Example 2.

[0049] 3.2 Test results

[0050] Except that the PCR tube with a DNA concentration of 100fg shows orange, the PCR tubes with other concentrations all show green, indicating that the detection method of the present invention has a minimum detection limit of 1pg DNA, and the sensitivity is very high.

[0051]

[0052]

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Abstract

The invention relates to a citrus exocortis viroid RT-LAMP (reverse transcription-loop-mediated isothermal amplification) detection kit which comprises 4 specific primers, a reaction buffer solution, an AMV reverse transcriptase, a Bst DNA polymerase and a nucleic acid dye. The invention also relates to a citrus exocortis viroid RT-LAMP detection method. The kit and detection method can be used for accurately detecting or identifying citrus exocortis viroid, have the advantages of high specificity, high sensitivity, favorable repetitiveness and the like, and are suitable for directly detecting the viroid carrying condition of field orange varieties.

Description

technical field [0001] The invention relates to the field of microorganism detection, in particular, the invention relates to a citrus split peel viroid RT-LAMP detection kit and a detection method. Background technique [0002] Citrus is the largest fruit in the world. Its annual international trade volume is second only to wheat and corn. It is the third largest agricultural product in international trade and occupies a pivotal position in the national economy of various countries. However, citrus is easily infected by various viruses, viroids and some prokaryotes such as parasitic bacteria during the growth process, resulting in low yield, poor quality, and serious plant weakness or even death. There are more than 80 kinds of citrus virus diseases and similar virus diseases reported in the world at present, which have a great impact on citrus production. [0003] Citrus split skin disease is an important worldwide citrus disease caused by Citrus exocortis viroid (CEVd), ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6844C12Q1/70C12Q2521/107
Inventor 朱强曹勇徐文龙孙永城王权
Owner 安徽济人药业股份有限公司