Primer and method for identifying bovine rotavirus and enterotoxigenic escherichia coli

A technology for enterotoxic Escherichia coli and bovine rotavirus, applied in the field of visual multiple fluorescent LAMP detection, can solve problems such as economic loss, difficulty in distinguishing, and easy misdiagnosis

Active Publication Date: 2018-02-16
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It is difficult to distinguish ETEC from BRV only based on clinical symptoms, and these two bacteria and viruses are often mixed with infection, and it ...

Method used

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  • Primer and method for identifying bovine rotavirus and enterotoxigenic escherichia coli
  • Primer and method for identifying bovine rotavirus and enterotoxigenic escherichia coli
  • Primer and method for identifying bovine rotavirus and enterotoxigenic escherichia coli

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Embodiment 1

[0074] Example 1. Visual multiple fluorescent LAMP detection of bovine rotavirus (BRV) and enterotoxigenic Escherichia coli (ETEC)

[0075] (1) Primer design

[0076]Download the VP6 gene of BRV and the LTⅠ, STa gene sequences of ETEC that have been registered on Genebank, use MEGA5.0 to conduct conservation and homology analysis, select well-conserved regions, and use primer5.0 software to design 3 sets of LAMP-specific primers. Each set includes 4 primers: outer primers F3 and B3, inner primers FIP (F1c+F2) and BIP (B1c+B2), the 5' end of each inner primer is labeled with a fluorescent group: BRV-FIP labeled FITC fluorescence, in Yellow-green at a wavelength of 520nm, ETEC-LTⅠ-FIP, ETEC-STa-FIP labeled with CY5.5 fluorescence, and red at a wavelength of 694nm. Primers were synthesized by Dalian Bao Biological Company. The specific nucleotide sequences of the primers are shown in Table 1.

[0077] Table 1

[0078]

[0079] (2) Template preparation

[0080] Take 250 μ...

Embodiment 2

[0089] Embodiment 2, verification experiment of method

[0090] (1) Specificity experiment

[0091] 1) Strains and strains

[0092] 3 strains of enterotoxigenic Escherichia coli (ETEC), 8 strains of bovine rotavirus (BRV) Guangxi isolates, 3 strains of Mycoplasma bovis Guangxi isolates were obtained from Guangxi Veterinary Research Institute, 2 strains of bovine infectious rhinotracheitis virus (IBRV) ), 3 strains of bovine viral diarrhea virus (BVDV), 2 strains of bovine rotavirus (BRV) reference strains were purchased from the China Veterinary Drug Administration, and foot-and-mouth disease (FMDV) inactivated vaccine types A, O and Asia 1 were purchased from Lanzhou Veterinary Research Institute, foot-and-mouth disease inactivated virus type O, A, Asia 1 type, vesicular stomatitis (VSV) inactivated virus New Jersey type (NJ type) and Indian type (IND type), bluetongue disease (BTV) inactivated Live virus (type 4, type 8, type 9, type 15, type 17, type 18) and inactivated v...

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Abstract

The invention provides a visual multiplex fluorescent LAMP detection method which can detect bovine rotavirus (BRV) and enterotoxigenic escherichia coli (ETEC). According to the detection method, twofluorophores are introduced into an LAMP method, a result is directly judged by virtue of colors of a reaction product, for example, red is namely bovine diarrhea caused by the BRV, and green is namely bovine diarrhea caused by the ETEC. The detection method provided by the invention has the advantage that the two viruses can be identified and detected at the same time through once LAMP reaction in one reaction tube. The primer designed by the invention is high in sequence sensitivity, and each reaction can detect 100 copied mixed templates at least; and specificity is good, and a target genecan be efficiently amplified; and clinical detection effect is good. The detection method provided by the invention is convenient and rapid, does not need to use an expensive instrument, is low in cost and can realize field pathogeny detection.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to a visual multiple fluorescence LAMP detection method capable of simultaneously detecting bovine rotavirus (BRV) and enterotoxigenic Escherichia coli (ETEC). Background technique [0002] Calf diarrhea is a common disease in the cattle industry, and its incidence and mortality are the highest within one month of calf birth, causing huge economic losses to the cattle industry. Bovine rotavirus (BRV) and enterotoxigenic E.coli (ETEC) are the main pathogens causing calf diarrhea. Diarrhea caused by bovine rotavirus infection mostly occurs in calves less than 1 month of age, and the severity of the disease varies widely, including asymptomatic infection, mild self-limited diarrhea, and severe diarrhea with severe dehydration. It has been reported that nearly half of neonatal calf diarrhea is associated with rotavirus infection. According to data, the morbidity rate of c...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12Q1/689C12Q1/10C12R1/93C12R1/19
CPCC12Q1/6844C12Q1/689C12Q1/701C12Q2600/16C12Q2531/119C12Q2537/143C12Q2563/107
Inventor 谢芝勋范晴谢志勤黄莉熊文婕黄娇玲张艳芳曾婷婷谢丽基
Owner GUANGXI VETERINARY RES INST
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