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Primer for detecting infectious bronchitis viruses, detection method and kit

A technology for bronchitis and chicken infectivity, applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., can solve common PCR detection sensitivity is not high, unfavorable general laboratory testing services, samples Higher requirements and other issues, to achieve broad market prospects, convenient quality control work, and good specificity

Inactive Publication Date: 2013-07-03
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are following defects in the prior art: at present, the laboratory detection of chicken infectious bronchitis mainly relies on methods such as PCR and fluorescent quantitative PCR, and the detection sensitivity of ordinary PCR is not high, and the requirements for samples are relatively high, and it is easy to cause false positives , the disadvantage of fluorescent quantitative PCR is that the detection relies on relatively high-priced quantitative PCR detectors, which is not conducive to ordinary laboratories to carry out detection services

Method used

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  • Primer for detecting infectious bronchitis viruses, detection method and kit
  • Primer for detecting infectious bronchitis viruses, detection method and kit
  • Primer for detecting infectious bronchitis viruses, detection method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: detection of chicken infectious bronchitis virus RT-LAMP

[0051]All chicken infectious bronchitis viruses used in the present invention are purchased from China Veterinary Drug Control Institute, including M41 strain, H52 strain, Hotle strain and Australia T strain.

[0052] Prepare chicken infectious bronchitis virus RT-LAMP detection reaction solution according to the following composition:

[0053] (1) Reaction solution A: Contains 10× reaction buffer, Bst DNA polymerase, dNTP (1mM each), Betaine solution (5M), MLV, Mg2+, and four pairs of primers. The sequences are:

[0054] N1-F2: 5' ATC AGG GTA CTC GTG ACT CT 3' (SEQ ID NO.1)

[0055] N1-B2: 5' TGC AGG GTA CTC GTG ACT CT 3' (SEQ ID NO.2)

[0056] N1-F3: 5' TGC TGG TAA GGG TGC TGA 3' (SEQ ID NO.3)

[0057] N1-B3: 5' ACTG CGA AAC TTC ACG TGA TG 3' (SEQ ID NO.4)

[0058] N1-FIP: 5' AGG TCC TCC GTC TGA AAA CCG TAT CAG GGT ACT CGT GAC TCT 3' (SEQ ID NO.5)

[0059] N1-BIP: 5' CCT CTG AAT CGT GGC AG...

Embodiment 2

[0075] Embodiment 2: RT-LAMP detects the sensitivity of chicken infectious bronchitis virus

[0076] Extract the total RNA of IBV M41 strain. After measuring the concentration, take 2 μL for 10-fold gradient dilution (10-1~10-10), and use the diluted RNA as a template to conduct 4 groups of experiments: Group A RT-LAMP, Group B RT- SYBR Green I fluorescent dye was added to PCR, C group and D group. The detection sensitivity was compared by four sets of experiments.

[0077] The RT reaction system is: 5x Reaction Buffer 5 μL, 1 μL dNTP, M-MLV Reverse Transcriptase 0.5 μL, RNase inhibitor 0.5 μL, DEPC 3 μL, total system 25 μL.

[0078] The PCR reaction system is: 25 μL of the total system, including 12.5 μL of 2x EX Taq Master Mix, 1 μL of Primer-F, 1 μL of Primer-R, 2 μL of template, and 8.5 μL of ultrapure water. PCR reaction conditions: pre-denaturation at 94°C for 2min, denaturation at 94°C for 30s, annealing temperature at 60°C for 30s, extension at 72°C for 30s, a tota...

Embodiment 3

[0083] Example 3: Rapid RT-LAMP Detection Kit for Chicken Infectious Bronchitis Virus

[0084] A chicken infectious bronchitis virus rapid detection kit comprises the following reagents:

[0085] Fluorescent dye SYBR green I, 10× reaction buffer, Bst DNA polymerase, dNTP (10mM each), Betaine solution (5M), MLV, Mg2+, 4 pairs of primers, the sequence of which is:

[0086] N1-F2: 5' ATC AGG GTA CTC GTG ACT CT 3' (SEQ ID NO.1)

[0087] N1-B2: 5' TGC AGG GTA CTC GTG ACT CT 3' (SEQ ID NO.2)

[0088] N1-F3: 5' TGC TGG TAA GGG TGC TGA 3' (SEQ ID NO.3)

[0089] N1-B3: 5' ACTG CGA AAC TTC ACG TGA TG 3' (SEQ ID NO.4)

[0090] N1-FIP: 5' AGG TCC TCC GTC TGA AAA CCG TAT CAG GGT ACT CGT GAC TCT 3' (SEQ ID NO.5)

[0091] N1-BIP: 5' CCT CTG AAT CGT GGC AGG AGT GTG CTC TAC TAG ATG CCG CTG 3' (SEQ ID NO.6)

[0092] N1-FIC: 5' AGG TCC TCC GTC TGA AAA CCG T 3' (SEQ ID NO.7)

[0093] N1-BIC: 5'CCT CTG AAT CGT GGC AGG AGT G 3' (SEQ ID NO.8).

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Abstract

The invention belongs to the filed of biotechnology and discloses a primer for detecting infectious bronchitis viruses, a detection method and a kit. The primer comprises the following components: a forward direction outer primer 1: N1-F2, a reverse direction outer primer 1: N1-B2, a forward direction outer primer 2: N1-F3, a reverse direction outer primer 2: N1-B3, a forward direction inner primer N1-F1P, a reverse direction inner primer N1-B1P, a forward direction loop primer N1-FIC, and a reverse direction loop primer N1-BIC. The invention further provides the method and the kit for detecting infectious bronchitis viruses. The detecting method and the kit have the characteristics of speediness, sensitiveness, simplicity, convenience, high efficiency, easiness in judgment and the like, and can make up the deficiencies of the existing method for detecting infectious bronchitis viruses.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a primer for detection of chicken infectious bronchitis virus, a detection method and a kit. Background technique [0002] Chicken infectious bronchitis (infectious bronchitis, IB) is an acute, high-contact infectious disease of chickens caused by infectious bronchitis virus (Infectious bronchitis virus, IBV), which can cause infection in chickens of all ages, with respiratory symptoms The main features are kidney disease, egg production and egg quality decline. Mixed infection with opportunistic pathogenic bacteria Mycoplasma gallisepticum (M.G) can lead to increased mortality. It is one of the main diseases affecting the development of poultry industry in all countries in the world. There are many serotypes of the disease, and the unique primer transcription mechanism of its genomic RNA makes the virus highly variable, and the continuous emergence of ne...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 丁铲
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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