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A kind of detection primer and application of crucian carp coronavirus hb93 and rt-pcr

A technology of RT-PCR and coronavirus, applied in the direction of application, virus, antiviral agent, etc., can solve the problem of no detection method, etc., and achieve the effect of ensuring high specificity, improving detection efficiency, and fast and simple method

Active Publication Date: 2018-01-09
HUBEI INSPECTION & QUARANTINE TECH CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no public report on Crucian Carp Bafinivirus (CCBV), and there is no effective detection method

Method used

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  • A kind of detection primer and application of crucian carp coronavirus hb93 and rt-pcr
  • A kind of detection primer and application of crucian carp coronavirus hb93 and rt-pcr

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] A crucian carp coronavirus HB93, its isolation process is as follows:

[0041] 1) Take out and grind crucian carp liver, brain, spleen, kidney and other tissues carrying the virus, dilute the culture medium by 100 times, centrifuge at 2000rpm, take the supernatant and filter, and inoculate into grass carp gonad cells (Grass carp ovary cell line, GCO) The monolayer was cultured at 25°C for 7 days, resulting in cytopathic effect (CPE). It can be seen that the GCO cells shrink and become rounded, the refractive index increases, some cells begin to fall off, and the cell monolayer begins to rupture.

[0042] Medium: M199, 10% fetal bovine serum, pH 7.0-7.2

[0043] 2) Collect the diseased cells, and use transmission electron microscopy for morphological identification.

[0044] 3) The virus is collected, and its whole genome is sequenced, and the sequence is shown in SEQ ID NO.1.

[0045]Morphological characteristics of crucian carp coronavirus: The virus is rod-shaped, s...

Embodiment 2

[0047] Primers designed based on the difference between the full sequence of crucian carp coronavirus HB93 (shown in SEQ ID NO.1) and the full sequence of other viruses:

[0048] The present invention takes ordinary PCR as an example for illustration, and other primers designed based on the differential sequence, fluorescent quantitative primers, LAMP primers, digital PCR primers, etc., can also complete the detection of crucian carp coronavirus HB93.

[0049] A crucian carp coronavirus HB93 RT-PCR detection primer, including F1: 5'-CAAAGTAAACCCTGGAGA-3', R1: 5'-ATCAATGATGGTGCAGTT-3'; F2: 5'-TGAAACAATCCAAGAAGA-3', R2: 5'-ACAAGTTATAGCCGGTGT-3 '.

Embodiment 3

[0051] Utilize crucian carp coronavirus HB93RT-PCR to detect primers to the detection of crucian carp coronavirus HB93, the method comprises:

[0052] 1. Extraction of total RNA from crucian carp tissue 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 sterile 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 dN TPs , M-MLV reverse transcriptase 1μL was added to the reaction solution in ice bath, after mixing, 42°C for 1h, 95°C for 5min, and the obtained cDNA template was stored at -20°C for later use.

[0053] 2. Polymerase chain reaction amplification:

[0054] Use 25μl reaction system: Take 0.5μL of two pairs of primers F1, R1 or F2, R2 with a concentration of 40μM and add them to two PCR reaction tubes, then add 2μL...

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Abstract

The invention belongs to the field of virus detection and discloses a crucian carp bafinivirus HB93, RT-PCR detection primers and applications of the bafinivirus and the primers. The crucian carp bafinivirus is separated from crucian for the first time by the inventor, and is the crucian carp bafinivirus HB93. The primers are designed for the sequence of the virus and include F1 that is 5'-CAAAGTAAACCCTGGAGA-3', R1 that is 5'-ATCAATGATGGTGCAGTT-3', F2 that is 5'-TGAAACAATCCAAGAAGA-3' and R2 that is 5'-ACAAGTTATAGCCGGTGT-3. The primers can be used for detecting the crucian carp bafinivirus, and have high specificity and high sensitivity.

Description

technical field [0001] The invention belongs to the technical field of virus molecular detection, and in particular relates to a crucian carp coronavirus HB93 and RT-PCR detection primers and applications. technical background [0002] The International Committee on Taxonomy of Viruses (ICTV) published the latest ninth report on virus classification in 2012, and newly established coronaviruses in Coronaviridae (Coronavi ridea) in Nidovirales. Subfamily (Coronavirinea) and Torovirinae (Torovirinae). [0003] Coronaviridae is a class of linear single-stranded positive-sense RNA viruses that 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 corona-like spines. Therefore, it is proposed to name this kind of virus as coronavirus [1] . In 1975, the International Committee on Virus Nomenclature and Classification officially named the Coronaviridae. During 2002-2003, atypi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12N15/11C12N15/50C12Q1/70C12Q1/68A61K39/215A61P31/14C12R1/93
CPCA61K39/00C12N7/00C12N2770/20021C12N2770/20034C12Q1/701
Inventor 陈孝宇梁昊郭少飞陈鑫
Owner HUBEI INSPECTION & QUARANTINE TECH CENT
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