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Indirect ELISA (Enzyme-Linked Immunosorbent Assay) detection kit for detecting porcine coronavirus D

A detection kit and coronavirus technology, applied in the biological field, can solve the difficulty of serological research and other problems, and achieve the effect of low cost, strong specificity and high expression

Pending Publication Date: 2022-05-13
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, in a recent study (Lednicky, J.A., et al., (2021). Independent infections of porcine deltacoronavirus among Haitian children. Nature, 600 (7887), 133-137.) found that in three children with acute Porcine D-coronavirus was found in plasma samples from children with febrile illness. This study only identified symptomatic children as acute PDCoV infection. Further serological studies are needed to determine the frequency of such infections in the general population. However, due to the possibility of cross-reaction of the virus with human endemic coronaviruses, serological research is difficult

Method used

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  • Indirect ELISA (Enzyme-Linked Immunosorbent Assay) detection kit for detecting porcine coronavirus D
  • Indirect ELISA (Enzyme-Linked Immunosorbent Assay) detection kit for detecting porcine coronavirus D
  • Indirect ELISA (Enzyme-Linked Immunosorbent Assay) detection kit for detecting porcine coronavirus D

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Experimental program
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preparation example Construction

[0030] The present invention also provides a method for preparing the above-mentioned expression vector, including:

[0031] Using the PDCoV-NS6 gene (SEQ ID NO.1) as a template, PDCoV-NS6-F (5'-CCGGAATTCATGTGCAACTGCCAT-3') and PDCoV-NS6-R (5'-TGCTCTAGATTAATTTAATTCATCTTC-3') were used as primers for PDCoV-NS6- The NS6 gene was amplified by PCR to obtain a PDCoV-NS6 gene containing EcoR I and Xba I restriction sites at both ends, and the PDCoV-NS6 gene containing EcoR I and Xba I restriction sites was cloned into the prokaryotic expression vector pCold I The EcoR I / Xba I site obtained the pCold I-PDCoV-NS6 prokaryotic expression vector expressing porcine D-coronavirus NS6 protein.

[0032] In the present invention, the method for preparing PDCoV-NS6 protein using the above-mentioned expression vector includes: transforming the recombinant expression plasmid into the expression host Escherichia coli BL21, and collecting the bacterial cell precipitate, and obtaining the PDCoV-NS6...

Embodiment 1

[0045] The construction of embodiment 1 prokaryotic expression system and the preparation of PDCoV-NS6 protein

[0046] 1. Construction of NS6 gene expression vector

[0047] 1.1 Primer design

[0048] According to the NS6 gene sequence of porcine D-coronavirus strain (Genbank Accession Number: MW196362.1), use DNAstar software to design primers to amplify the NS6 fragment. The sequence of the upstream primer PDCoV-NS6-F is 5'-CCGGAATTCATGTGCAACTGCCAT-3', and the downstream primer The sequence of PDCoV-NS6-R is 5'-TGCTCTAGATTAATTTAATTCATCTTC-3'. Primers were synthesized by Shangya Biological Co., Ltd.

[0049] 1.2 Construction of pCold I-PDCoV-NS6 recombinant plasmid

[0050] The artificially synthesized PDCoV-NS6 gene fragment and the pCold I vector were digested with EcoR I and Xba I, respectively, and ligated to construct the pCold I-PDCoV-NS6 expression plasmid. will identify the correct recombinant plasmid (eg figure 1 shown) were sent to Shangya Biotech Co., Ltd. fo...

Embodiment 2

[0073] The operating procedure of embodiment 2 indirect ELISA method

[0074] (1) Antigen coating

[0075] Dilute the PDCoV-NS6 protein provided in Example 1 of the present invention as an antigen to 50ng / well / 100μL with coating buffer, then add it to the microtiter plate, take it out at 4°C overnight (12h-14h), and discard the well Inner liquid, wash 3 times with PBST, 3min each time, pat dry;

[0076] (2) closed

[0077] Add blocking solution (150 μL / well) to the microtiter plate and incubate at 37° C. for 120 min. Take it out, discard the blocking solution, wash with PBST 3 times, each time for 3 minutes, and pat dry;

[0078] (3) Sample addition

[0079] The sample was diluted 1:100 with blocking solution, added to the microtiter plate, 100 μL / well, and incubated at 37°C for 30 minutes. After taking it out, discard the liquid in the well, wash with PBST 3 times, each time for 3 minutes, and pat dry;

[0080] (4) Add secondary antibody

[0081] The secondary antibody...

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Abstract

The invention discloses an indirect ELISA (Enzyme-Linked Immunosorbent Assay) detection kit for detecting porcine coronavirus D. The ELISA detection kit established by using the NS6 protein of the PDCoV as the coating antigen can accurately detect the porcine coronavirus D antibody in a clinical sample. Meanwhile, as the NS6 protein serves as a specific auxiliary protein of the porcine D-type coronavirus, cross reaction with other coronaviruses does not occur, and the NS6 protein can be used for antibody detection after the PDCoV is infected to human or other animals from pigs. The ELISA kit established by the protein has the characteristics of strong specificity, high sensitivity and good repeatability, provides a new choice for rapid diagnosis and immune monitoring of PDCoV, and has important significance in prevention, control and purification of the disease.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an indirect ELISA detection kit for detecting porcine D-coronavirus. Background technique [0002] Deltacoronavirus (Deltacoronavirus) belongs to Coronaviridae (Coronaviridae) subfamily (Coronavirinae), is a new class of coronaviruses discovered in recent years. In 2012, (Patrick C.Y.Woo, et al., Y.(2012).Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and delavian coronaviruses as the genesource of gammavirus.jammavirus. Virol, 86(7).) found porcine deltacoronavirus (Porcine deltacoronavirus, PDCoV) in feces collected from live livestock and poultry market for the first time. PDCoV can infect pigs of all ages, causing symptoms such as diarrhea, vomiting and dehydration similar to porcine epidemic diarrhea and porcine transmissible gastroenteritis. The morbi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/68G01N33/58G01N33/543
CPCG01N33/56983G01N33/6854G01N33/581G01N33/54393G01N2333/165G01N2469/20
Inventor 黄耀伟李亚丽王斌史芳舒
Owner ZHEJIANG UNIV
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