RT-LAMP detection primers for Cyprinid Nidovirus HL39 (CyNV-HL39) and application thereof

A RT-LAMP, coronavirus technology, applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problem of no detection method, and achieve simple result judgment, good sensitivity and good specificity Effect

Active Publication Date: 2020-04-24
武汉海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2017, the applicant published the relevant research results of carp coronavirus. At present, there is no public report on Cypri inid Nidovirus (CyNV), and there is no effective detection method

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] A carp coronavirus HL39, its isolation process is as follows:

[0018] 1) Take out and grind the virus-carrying carp liver, brain, spleen, kidney and other tissues, dilute the medium 100 times, centrifuge at 2000rpm, take the supernatant and filter, inoculate into grass carp ovary cell line (GCO) Monolayer, cultivated at 25°C for 7 days, resulting in cytopathic effect (CPE), it can be seen that GCO cells shrink and become round, the refractive index increases, some cells begin to fall off, and the cell monolayer begins to rupture. Medium: M199, 10% fetal bovine serum, pH 7.0~7.2;

[0019] 2) Collect diseased cells and conduct morphological identification using transmission electron microscopy: Morphological characteristics of carp coronavirus HL39: the virus is rod-shaped, with obvious coronal-shaped spine structures around it, which is consistent with the typical morphology of coronaviruses.

[0020] 3) The virus is collected, and its whole genome is sequenced, and th...

Embodiment 2

[0023] The RT-LAMP detection primers designed based on the difference between the full sequence of carp coronavirus HL39 (shown in SEQ ID NO.1) and the full sequence of other viruses are:

[0024] F3: 5'-TGCCCACCAAGACGACTA-3';

[0025] B3: 5'-TTTGCGCTAGCGCGATTC-3';

[0026] FIP: 5'-CGTTCCAAACGTCTTGGGTCAAGAGTGACGTAACTGCTACTG-3';

[0027] BIP: 5'-ACTCACTTCTGCTCCTTGCGACGCAGAGTGTAAGAGGTTTTTCCA-3'.

Embodiment 3

[0029] Utilize the RT-LAMP primer of carp coronavirus HL39 to detect CyNV-HL39, the method steps are as follows:

[0030] 1), the acquisition of viral RNA:

[0031] use or Total RNA was extracted by LS reagent or column adsorption and elution method.

[0032] 2), RT-LAMP amplification:

[0033] Use 25 μL reaction system: internal primers FIP and BIP each 0.8 μM, external primers F3 and B3 each 0.1 μM, dNTPs1 mM, Betaine0.5M, MgSO 4 5mM, Bst DNA polymerase 8U, AMV reverse transcriptase 5U, template RNA 5μL, 10×ThermoPolReaction Buffer 2.5μL, choose RT-LAMP reaction at 64℃ and 60min, then inactivate at 80℃ for 2min.

[0034] 3) To judge the test results, one of the following three methods is adopted:

[0035] A. When the amplification reaction occurs, the pyrophosphate ions precipitated from the dNTPs and the magnesium ions in the reaction solution form a white magnesium pyrophosphate precipitate. Judging by the naked eye: if the detection tube is obviously turbid, it is...

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PUM

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Abstract

The invention belongs to the technical field of virus molecule detection, and particularly relates to RT-LAMP detection primers for CyNV-HL39 and application thereof. The RT-LAMP detection primers designed for the CyNV-HL39 with an accession number of CCTCC NO: V201988, and comprise an outer primer F3, an outer primer B3, an inner primer FIP and an inner primer BIP. A kit prepared by using the primers in the invention has the characteristics of simplicity, convenience, quickness, high specificity and sensitivity when used for detecting the CyNV, can accurately detect the CyNV-HL39 in a samplewithin 2 hours by only using one water bath kettle or metal bath, and is very suitable for on-site quick detection of the CyNV-HL39.

Description

technical field [0001] The invention belongs to the technical field of virus molecular detection, and in particular relates to an RT-LAMP detection primer and application of carp coronavirus HL39. Background technique [0002] The order Nidovirales was first proposed by the International Committee on Taxonomy of Viruses (ICTV) in 1996. The viruses of this order have a wide range of hosts and can cause aquatic animals such as mammals, birds, reptiles, fish and shrimp, etc. many serious diseases. The Coronaviridae family of the order Viridae is a class of linear single-stranded positive-sense RNA viruses, which were first isolated from chickens in 1937. Almeida et al. conducted morphological studies on these viruses, and found that the envelopes of these viruses had The spines in the shape of the corona, so it is proposed to name this type of virus as a coronavirus [1]. In 1975, the International Committee on Virus Nomenclature and Classification officially named the Coronav...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2521/107Y02A50/30
Inventor 陈孝宇郑剑王振华曾宪东
Owner 武汉海关技术中心
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