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Coronavirus HL39 of carp, and RT-PCR detection primers and application thereof

A coronavirus and HL39 technology, applied in the direction of viruses, antiviral agents, viruses/phages, etc., can solve the problems of no detection methods, etc., and achieve the effect of ensuring high specificity, fast and simple methods, and improving detection efficiency

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 relevant research results on carp coronavirus. At present, there is no public report on Cyprinid Nidovirus (CyNV), and there is no effective detection method

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] A carp coronavirus HL39, its isolation process is as follows:

[0025] 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

[0026] 2) Collect the diseased cells, and use transmission electron microscopy for morphological identification.

[0027] 3) The virus is collected, and its whole genome is sequenced, and the sequence is shown in SEQ ID NO.1.

[0028] Morphological characteristics of carp coronavirus: The virus is rod-shaped, surrounded by obvious coronal-shaped spine structures, which is consistent with the...

Embodiment 2

[0030] The 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:

[0031] The present invention is illustrated by taking ordinary PCR as an example. Other primers designed based on the differential sequence, fluorescent quantitative primers, LAMP primers, digital PCR primers, etc., can also complete the detection of carp coronavirus HL39.

[0032] A carp coronavirus HL39 RT-PCR detection primer, the primers are F: 5'-CCTCTTGACCCAAGACGGTT-3', R: 5'-CCGGTGGTTGAGAGCTTGTA-3'.

Embodiment 3

[0034] Utilize carp coronavirus HL39 RT-PCR to detect primers to the detection of carp coronavirus HL39, the method comprises:

[0035] 1. Extraction of total RNA from carp tissues or infected cells and synthesis of cDNA: using Extract the total RNA of the sample to be tested. Configure the reverse transcription reaction system: 3 μL of total RNA, 1 μL of Oligo(dT)18 primer, 8 μL of sterilized double-distilled water, mix well at 65°C for 5 minutes, and immediately put it on ice; add 4 μl of 5×Reaction Buffer, 1 μl of RNase inhibitor, 2 μL of 5mM dNTPs, Add 1 μL of M-MLV reverse transcriptase to the reaction solution in an ice bath, mix well, store at 42°C for 1 hour and 95°C for 5 minutes, and store the obtained cDNA template at -20°C for later use.

[0036] 2. Polymerase chain reaction amplification:

[0037] Use 25μl reaction system: Take 0.5μL of a pair of primers F and R with a concentration of 40μM and add them to PCR reaction tubes, then add 2μL of 2.5mM dNTPs, 25mM M...

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PUM

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Abstract

The invention belongs to the field of virus detection and specifically discloses a coronavirus HL39 of carp, and RT-PCR detection primers and application thereof. The coronavirus is separated from carp for the first time in the invention and is Cyprinid Nidovirus (CyNV) HL39. The CyNV is preserved in China Center for Type Culture Collection, with an accession number being CCTCC NO: V201988. The primers, namely F: 5'-CCTCTCTTGACCCAAGACGGTT-3' and R: 5 '-CCGGTGTTGAGAGCTTGTA-3', are designed for the sequence of CyNV; and primers can detect the CyNV, and are good in specificity and high in sensitivity.

Description

technical field [0001] The invention belongs to the technical field of virus molecular detection, and in particular relates to a carp coronavirus HL39 and RT-PCR detection primers and applications. 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 multiple diseases in aquatic animals such as mammals, birds, reptiles, and fish and shrimp. a serious disease. 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 spikes in the shape of the corona, so it is proposed to name this kind of virus as coronavirus [1] . In 1975, the International Committee on Virus Nomenclature and Classification officially ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12Q1/70C12N15/11A61P31/14C12R1/93
CPCA61P31/14C12N7/00C12N2770/20021C12N2770/20034C12Q1/701
Inventor 陈孝宇周旋陈鑫徐家文
Owner 武汉海关技术中心
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