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Nucleic acids and method for synchronous detection and distinguishing of five canine diarrhea viruses and kit

A diarrhea virus, simultaneous detection technology, applied in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc. Viruses, multiple methods detection effect is not ideal and other problems, to achieve the effect of fast reaction, simple operation and high sensitivity

Pending Publication Date: 2018-12-28
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The above-mentioned five kinds of diarrhea viruses in dogs, if traditional detection techniques such as colloidal gold, PCR, etc., have disadvantages in terms of specificity and sensitivity, colloidal gold cannot detect five kinds of viruses at the same time, conventional PCR methods are mainly double and triplet methods, but the actual detection performance of the above-mentioned published multiplex methods is not ideal
At present, the main problem encountered by multiplex conventional PCR or multiplex real-time fluorescent PCR is the decrease in detection sensitivity and specificity caused by multiplex reactions. Resources such as enzymes and cross-reactivity issues
However, at present, there are few reports on whether the simultaneous detection of the five viruses can be realized, mainly because the primers, probes and amplification products used for the detection of these pathogens must be guaranteed to be compatible with each other in order to realize the simultaneous detection of multiple viruses. There can be no cross-reaction between them, otherwise, false positive results are prone to occur, especially the more virus factors are detected, the more difficult it is to design primers and probes

Method used

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  • Nucleic acids and method for synchronous detection and distinguishing of five canine diarrhea viruses and kit
  • Nucleic acids and method for synchronous detection and distinguishing of five canine diarrhea viruses and kit
  • Nucleic acids and method for synchronous detection and distinguishing of five canine diarrhea viruses and kit

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Experimental program
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Effect test

Embodiment 1

[0040] Design and synthesis of embodiment 1 primers and probes

[0041] The genome sequences of standard strains of CAV1, CAV2, CCV, CDV, and CPV were downloaded from the NCBI website, and the conserved regions of the standard strains of each virus were selected by DNAStar software, and primers were designed by Primer5 software, and a total of 5 pairs of primers were designed and optimized. , and carry out CY3 labeling on the 5' end tail sequence of the downstream primer. According to the designed primer interval range, select the highly variable interval of each representative strain to design the probe. In order to improve the influence of the steric hindrance of the probe, 15 T bases within 15 can be added at the 5' end, and the designed probe BLAST was performed to check its specificity. The primers and probes are shown in Table 1 and Table 2.

[0042] Table 1 Specific primer sequences

[0043]

[0044] Table 2 Specific probe sequences

[0045]

Embodiment 2

[0046] Embodiment 2 PCR amplification

[0047] (1) PCR amplification

[0048] CAV1, CAV2, and CCV refer to the gene sequences of each virus registered on the NCBI website and synthesize plasmids by Shanghai Sangong Co., Ltd. CDV and CPV are extracted from clinically positive disease materials and passed plasmid mini-extraction reagents produced by Tiangen Biochemical Technology Co., Ltd. Cassette extraction to prepare plasmids. Using the extracted DNA and cDNA as templates, amplify by PCR. The PCR system is 25 μl: 2×ES TaqMaster Mix 12.5 μl, upstream primer (10 pmol / L) 1.0 μl, downstream primer (10 pmol / L) 1.5 μl, template 1.0 μl, make up the volume to 25 μl with RNase-Free Water. The PCR program was: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min 15 s, a total of 35 cycles; extension at 72°C for 10 min.

[0049] The results of PCR amplification were as figure 1 shown.

[0050] (2) Construction of...

Embodiment 3

[0052] The establishment of the detection method of embodiment 3 gene chip

[0053] (1) Extract the nucleic acid of the sample to be tested and carry out reverse transcription;

[0054] (2) Amplify according to the method described in Example 2

[0055] (3) Chip preparation

[0056] Dilute the oligonucleotide probes synthesized by Shanghai Sangon Bioengineering Co., Ltd. to 40 μmol / L with distilled water, add 5 μL each to the A384 plate, and take 5 μL chip spotting solution for dilution, and use the Personal Arrayer 16 chip spot The sample instrument spots the probes on the aldehyde-based glass substrate (see Table 3), and repeats the spotting of each probe 3 times. The microarray layout includes the spotting quality control probe HEX, the positive quality control probe P , sample solution N for negative quality control probes, and 11 specific probes. After spotting the chip, place the chip at 37°C for more than 12h, then wash it with 0.2% SDS, wash it with clean water, sea...

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Abstract

The invention provides a group of nucleic acids for synchronously detecting and distinguishing five canine diarrhea viruses and also provides a multi-gene-chip high-flux molecular biological detectionmethod for the five canine diarrhea viruses. The nucleic acids include upper and lower primers and hybridization probes of a canine adenovirus type 1, a canine adenovirus type 2, canine coronaviruses, canine distemper viruses and canine parvoviruses of the five canine diarrhea viruses. By adopting the method, multi-gene-chip detection is performed by extracting the nucleic acids of the canine diarrhea viruses in samples to be detected, synchronous and accurate detection and distinguishing of the canine diarrhea viruses in samples to be detected are achieved, and the nucleic acids have the advantages of being good in specificity, high in sensitivity, good in stability, simple and convenient to operate and capable of achieving high-flux quick detection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a nucleic acid and a detection method for synchronous detection and identification of five canine diarrhea viruses, canine adenovirus type 1, canine adenovirus type 2, canine coronavirus, canine distemper virus, and canine parvovirus and test kits. Background technique [0002] Canine adenovirus disease (Canine adeno, CA) is an acute infectious disease of dogs caused by canine adenovirus infection. Virus type 1 (Canine adenovirus type 1, CAV-1) and canine adenovirus type 2 (Canine adenovirus type 2, CAV-2), CAV-1 mainly causes canine infectious hepatitis, and can also infect bears and foxes to cause encephalitis lesions , mainly infecting puppies; CAV-2 mainly causes canine infectious laryngotracheitis, which spreads rapidly and has a rapid course, causing serious harm to dogs. Canine coronavirus disease is an acute contact infectious disease mainly caused by canine cor...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6837C12N15/11
CPCC12Q1/6837C12Q1/701Y02A50/30
Inventor 杨兵周宏专苏霞徐福洲常彦嫣
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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