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Detection agent of bovine rotavirus and bovine coronavirus, preparation and application methods of detection agent

A bovine rotavirus and coronavirus technology, applied in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc. Reduce detection cost and detection time, with strong specificity and good repeatability

Inactive Publication Date: 2012-03-07
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the sensitivity and specificity are high, it is time-consuming and laborious; although the ELISA method has high sensitivity, its disadvantage is that the specificity is poor when the detection sample is contaminated; and the above-mentioned method can only detect one pathogen in one test. Co-infection of these two pathogens cannot be carried out

Method used

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  • Detection agent of bovine rotavirus and bovine coronavirus, preparation and application methods of detection agent
  • Detection agent of bovine rotavirus and bovine coronavirus, preparation and application methods of detection agent
  • Detection agent of bovine rotavirus and bovine coronavirus, preparation and application methods of detection agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] refer to Figure 1 to Figure 5 .

[0051] 1. Selection of BRV VP7 gene and BCoV N gene amplified target sequences

[0052] Download the published BRV VP7 gene (GenBank accession numbers: U14999, AB044293, EU835945, AF545859, AF039524, FJ206084, FJ206082, FJ206068, AB077054, AB077057, U01054, DQ487202 gene sequences) and B6Co , DQ811784, AF058944, AF058942, U00735, AF220295, AB354579, EF424618, EF424617, EF424616, EF424620, DQ915164, AF391542, AF391541), after converting it into a file with the extension . Conserved sequences with gene variation, insertion, and deletion were used as target sequences for amplification of VP7 gene and N gene, respectively. The target sequence (519 bp) of bovine rotavirus VP7 gene amplification is: 5; the target sequence (879 bp) of bovine coronavirus N gene amplification is: 6.

[0053] 2. Design and synthesis of specific primers

[0054] The target sequences of the screened BRV VP7 gene and BCoV N gene were input into the primer premi...

Embodiment 2

[0078] Compared with embodiment 1, the difference of this embodiment is:

[0079] 1. Combined RT-PCR reagent standardization

[0080] According to the above test results, RT and PCR reagents were standardized according to the optimization of reaction conditions.

[0081] 1.1 The standard reagent composition of RT reaction is as follows:

[0082] Element Dosage 5×RT Buffer 2.0 μL RNase-free distilled water 4.0 μL dNTP Mixture (10 mmol / L) 2.0 μL RNase Inhibitor (40U / μl) 1.0 μL Reverse Transcriptase (200 U / μL) 1.0 μL P2 (20 pmol / μl) 1.0 μL P4 (20 pmol / μl) 1.0 μL Total RNA (≤ 1μg) 6.0 μL Total 20.0 μL

[0083] 1.2 PCR reaction standardization reagent composition is:

[0084] Element Dosage 10×RT-PCR Buffer (Mg 2+ Free) 5.0 μL MgCl 2 (25mmol / L) 4.0 μL dNTP (10mmol / L each) 4.0 μL Taq plus (5U / μL) 0.3 μL RT product 2.0~6.0 μL P1 (10 pmol / μL) 1....

Embodiment 3

[0096] Compared with Example 2, the difference of this example is that according to the double RT-PCR reaction conditions optimized in Example 2, bovine viral diarrhea virus nucleic acid samples, bovine infectious rhinotracheitis virus nucleic acid samples, PBS, Nucleic acid samples of foot-and-mouth disease virus, Escherichia coli and Salmonella were amplified by RT-PCR, and the amplification results were all negative, confirming that the established dual-PCR detection method for bovine rotavirus and coronavirus diarrhea is highly specific sex, see image 3 shown.

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Abstract

The invention discloses a detection agent of bovine rotavirus and bovine coronavirus, which comprises two pairs of specific primers capable of amplifying a bovine rotavirus VP7 gene and a bovine coronavirus N gene. The invention also discloses a preparation method, which comprises the following steps of: selecting conserved sequences of the bovine rotavirus VP7 gene and the bovine coronavirus N gene; and designing and synthesizing the specific primers. The invention also discloses an application method which comprises the steps of: processing a sample; extracting RNA in an excrement sample; carrying out a jointed RT-PCR (Reverse Transcriptase Polymerase chain Reaction); and judging a result. Through the jointed RT-PCR, the detection agent can be used for rapidly and effectively detecting the BRV and the BCoV simultaneously so that the detection cost and the detection time are greatly reduced, has better specificity and sensitivity, and has the advantages of saving time and saving labor.

Description

technical field [0001] The present invention relates to a kind of molecular detection technology that can quickly detect whether infected by bovine rotavirus and bovine coronavirus, especially provides molecular detection that can not only be used to detect bovine rotavirus and bovine coronavirus respectively, but also can be used for The preparation and application of a molecular detection reagent for simultaneously detecting the above two viruses belong to the technical field of animal epidemic detection. Background technique [0002] Bovine diarrheal disease is one of the most common diseases in the cattle industry. The main clinical features are loose, soft or watery stools, vomiting, dehydration and weight loss. For calves, diarrhea can lead to poor growth and death of newborn calves, and is also known as the killer of newborn calves. Fatal diarrhea mostly affects calves within 2 weeks after birth, with a morbidity rate of up to 80% and a case fatality rate of up to 70...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCY02A50/30
Inventor 孟庆玲乔军陈创夫张再超蔡扩军田振中王俊伟王为升杨丽红
Owner SHIHEZI UNIVERSITY
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