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RPA specific primer pair for detecting ALV-A/B/J, crRNA fragment and application thereof

A primer pair and specificity technology, applied in the field of RPA-specific primer pair detection of ALV-A/B/J, can solve the problems of unsuitable field detection, expensive equipment, and high technical requirements

Pending Publication Date: 2021-08-13
SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Purpose of the invention: the present invention aims at the disadvantages of complicated operation, expensive equipment, high technical requirements, long time-consuming and unsuitable for field detection in the above-mentioned existing ALV detection technology and method, and provides a method for diagnosing ALV-A / B / J RPA-specific primer pairs, crRNA fragments, CRISPR-Cas13a detection system, and CRISPR-Cas13a-based detection of ALV-A / B / J related diagnostic products and vaccines, etc., have high specificity and sensitivity, and can Provide a fast, simple and accurate detection method for the diagnosis of ALV-A / B / J infection

Method used

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  • RPA specific primer pair for detecting ALV-A/B/J, crRNA fragment and application thereof
  • RPA specific primer pair for detecting ALV-A/B/J, crRNA fragment and application thereof
  • RPA specific primer pair for detecting ALV-A/B/J, crRNA fragment and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Establishment of the CRISPR-Cas13a system for detecting ALV-A / B / J

[0052] 1. Design of RPA primers and crRNA

[0053] Retrieve the nucleotide sequence of the gp85 gene of ALV-A / B / J from the Genbank database of NCBI, and select a section that is relatively conserved in the gp85 gene of the same subgroup strain and is different from other subgroup ALV strains through sequence alignment and screening. Nucleic acid sequences with large differences are used as target regions. According to the method recommended in the TwistDx instruction manual of the RPA kit seller, several pairs of primers were designed for ALV-A / B / J (Table 1). A 25bp T7 promoter sequence (underlined part of Table 1) was added to the 5' end of the upstream primer, so that the target fragment amplified by RPA could be transcribed as the recognition object RNA of Cas13a. The size of the target fragment obtained after RPA amplification is about 140-190bp, and a 28bp highly conserved crRNA targeti...

Embodiment 2

[0080] Example 2 The specificity and sensitivity of the method for detecting ALV-A / B / J based on CRISPR-Cas13a

[0081] 1. Specificity

[0082] The proviral DNA of other ALV subgroup strains (C, D, E) infecting chickens, the proviral DNA of avian reticuloendotheliosis virus (REV) and the DNA of chicken Marek's virus (MDV) were used as templates, respectively , configure the RPA and Cas13a reaction system according to the method in Example 1 to verify the specificity of the present invention. like image 3 As shown, only ALV-A / B / J has a "T" line that is positive; other nucleic acid tests have only a "C" line that is negative. The results show that the invention has high specificity and no cross-reaction with other chicken tumor viruses.

[0083] 2. Sensitivity

[0084]Use the specific identification primers (H5 / envA, H5 / envB, H5 / H7) of ALV-A / B / J in Table 3 to amplify the corresponding sequences and clone them into the TA vector GEM-T-easy. The recombinant plasmid was used a...

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Abstract

The invention discloses an RPA (recombinase polymerase amplification) specific primer pair, a single-stranded DNA (deoxyribonucleic acid), a crRNA fragment, a CRISPR-Cas13a (clustered regularly interspaced short palindromic repeats) detection system and application thereof in preparation of ALV related diagnostic products and vaccines. RPA amplification can be carried out on target nucleic acid under the condition of constant temperature of 37 DEG C, the requirement for instruments and equipment is low, an amplified target fragment is transcribed into RNA, which is identified by specific crRNA and then is cut by Cas13a, the 'incidental cutting' activity of the Cas13a can cut RNA reporter molecules in the system, the RPA reaction time is about 30 minutes, the SHERLOCK reaction time is about 40 minutes, and the overall detection time is shorter than 2 h; According to the invention, when matched with the application of a lateral flow chromatography test strip, a detection result is visualized, and rapid detection and differential diagnosis of ALVA / B / J can be realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an RPA-specific primer pair for detecting ALV-A / B / J, a crRNA fragment and applications thereof. Background technique [0002] Avian leukemia (AL) is a general term for a variety of neoplastic diseases in poultry caused by avian leukosis virus / sarcoma virus (ALV / RSV) in the family Retroviridae Alpha Oncovirus. Avian leukosis virus (ALV) can cause slow growth, immunosuppression, and decreased egg production in chickens in addition to causing poultry tumors, causing huge losses to the poultry industry. According to the host category of ALV, the characteristics of envelope glycoprotein, virus interference experiment and other molecular biological characteristics, ALV is currently divided into 11 subgroups. Among them, A-D, J and K subgroups are exogenous ALV. C and D subgroups are rare in the wild, E-I subgroup is endogenous ALV, and F-I subgroup mainly infects wild birds. Among the e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/113C12N9/22C12Q1/70C12Q1/6844
CPCC12N15/113C12N9/22C12Q1/702C12Q1/6844C12N2310/20C12Q2531/119C12Q2521/327C12Q2565/625
Inventor 徐晴晴姚永秀维诺·奈尔沈志强
Owner SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY
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