Kit used for pig Delta coronavirus detection and detection method thereof

A coronavirus and kit technology, applied in the field of genetic engineering, can solve the problems of long reaction time and low sensitivity, and achieve the effects of high sensitivity, simple operation and good application prospects

Inactive Publication Date: 2017-07-21
SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to overcome the defects of low sensitivity and long reaction time that the detection of PDCoV basically adopts common RT-PCR and nested RT-PCR methods, and provide a method for detecting porcine Delta coronavirus kits, primer pairs and detection methods

Method used

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  • Kit used for pig Delta coronavirus detection and detection method thereof
  • Kit used for pig Delta coronavirus detection and detection method thereof
  • Kit used for pig Delta coronavirus detection and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] The design of embodiment 1 specific primer and probe

[0067] Design a pair of specific primers and probes for the conserved sequences of PDCoV in GenBank, as shown in Table 1 below, and synthesize specific fluorescent RT-PCR primers and probes according to the following sequences. with DEPC-H 2 O dilute the primers to 10 μM and store at -20°C for later use.

[0068] Table 1 Primer and Probe Combinations

[0069]

[0070] Wherein the nucleotide sequence of F1 is as shown in SEQ ID NO.1 in the sequence listing, the nucleotide sequence of R1 is as shown in SEQ ID NO.2 in the sequence listing, and the nucleotide sequence of P1 is as shown in SEQ ID NO in the sequence listing .3 shown.

Embodiment 2

[0071] The establishment and optimization of embodiment 2 reaction system

[0072] 1. Sample preparation: construct a plasmid containing the target amplification region as a positive control for PDCoV detection; positive samples of PEDV vaccine strain, TGEV vaccine strain, rotavirus vaccine strain, Jieshen virus, Kobu virus, Escherichia coli ( Standard strain ATCC 25922) (taken from Shanghai Animal Disease Prevention and Control Center Shanghai Veterinary Disease Diagnosis Center Laboratory) as a specific reference; with deionized water as a negative control, Trizol was used to extract the positive control and specific reference respectively RNA, negative control for use.

[0073] 2. Screening of primer probes: detect the RNA of the above-mentioned positive control and negative control respectively with the primer pair probes designed in Example 1, and through repeated trials, screen out the best primer probes with good specificity, sensitivity and repeatability. Needle combi...

Embodiment 3

[0081] The establishment of embodiment 3 standard curve

[0082] After measuring the concentration of PDCoV positive plasmid DNA with a spectrophotometer, use DEPC-H 2 O Make a 10-fold serial dilution of the plasmid standard so that the copy number in each 5 μL detection volume is 1.3×10 7 , 1.3×10 6 , 1.3×10 5 , 1.3×10 4 , 1.3×10 3 copy / μL, set 3 repetitions, and perform amplification. After the reaction, use ABI7500 fluorescence quantitative PCR analysis software to automatically obtain the standard curve. The results show:

[0083] The fluorescent RT-PCR method established with combination 1 of primers (F1 and R1) and probe P1 showed a typical S-type amplification curve with obvious exponential region, and the initial template concentration and Ct value of the standard showed a good correlation. Linear range, slope close to -3.04, correlation coefficient r 2 is 0.994 (see figure 1 , 2 ).

[0084] The sample can be accurately quantified according to the Ct value and ...

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Abstract

The invention discloses a kit used for pig Delta coronavirus detection and a detection method thereof. The kit comprises an RT-PCR reaction system and a primer probe mixed liquid. The primer probe mixed liquid comprises a primer pair and a probe. The nucleotide sequence of the primer pair is as shown in SEQ ID NO.1 and SEQ ID NO.2. The nucleotide sequence of the probe is as shown in SEQ ID NO.3. According to the method, a Taqman probe method is adopted, amplified reaction and product detection are performed in a closed tube state, and false positive caused by pollution to amplified products is avoided; the probe is higher in hybrid specificity and sensitivity, follow-up processing is not required after PCR, and the kit is simpler and faster to operate, safe and free of pollution; an effect of detecting a trace amount of PDCoV genomes in a sample is good, and the kit can be used for PDCoV laboratory detection and molecular epidemiological investigation.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a kit and a detection method for detecting porcine Delta coronavirus (PDCoV). Background technique [0002] Porcine deltacoronavirus (porcine deltacoronavirus, PDCoV) belongs to Coronaviridae (Coronaviridae) Coronaviridae subfamily (Coronavirinae) Dingcoronavirus genus, first obtained from dead birds, chickens, and mammals by Woo et al. of the University of Hong Kong in 2012 More than 50 coronavirus-positive samples were detected in 7,140 samples of the National Institutes of Health, seven of which were newly discovered deltacoronaviruses (Woo, Patrick C.Y. et al. "Discovery of SevenNovel Mammalian and Avian Coronaviruses in the Genus DeltacoronavirusSupports Bat Coronaviruses as the Gene Source of Alphacoronavirus and Betacoronavirus and Avian Coronaviruses as the Gene Source of Gammacoronavirus and Deltacoronavirus.” Journal of Virology 86.7(2012):3995–4008.PMC.Web....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/701C12Q2563/107C12Q2561/101C12Q2521/107Y02A50/30
Inventor 杨德全鞠厚斌刘佩红周锦萍王建刘健葛菲菲李鑫杨显超邓波葛杰白艳艳
Owner SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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