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Primers and probes for the detection of canine distemper virus and their use

A technology of canine distemper virus and probe, which is applied in the biological field, can solve the problems that there are no detection methods for canine distemper virus, and achieve the effects of saving reaction time, great application potential and strong specificity

Active Publication Date: 2020-04-14
INSPECTION & QUARANTINE TESTING CENT OF HEBEI ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently there is no detection method for canine distemper virus based on nfo probe, LFS and RPA

Method used

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  • Primers and probes for the detection of canine distemper virus and their use
  • Primers and probes for the detection of canine distemper virus and their use
  • Primers and probes for the detection of canine distemper virus and their use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1 primer and probe sequence

[0026] The sequences of primers and probes designed in the present invention are shown in Table 1.

[0027] Table 1 Primers and probes used in experiments

[0028]

[0029]

[0030] Note: FAM: carboxyfluorescein labeling; THF: tetrahydrofuran; C3-spacer: polymerase extension blocker.

Embodiment 2

[0031] The establishment of embodiment 2 LFS RT-RPA method

[0032] The steps of this method are as follows:

[0033] 1. Extract the RNA of the strain to be tested;

[0034] 2. Add the RNA extracted in step 1 and the primers and probes designed in the present invention into the RPA system for amplification, and use LFS to detect the amplification result.

[0035] The specific steps are:

[0036] 1. Extraction of RNA

[0037] According to the instruction of Trizol reagent, the RNA of CDV virus was extracted using Trizol reagent (Invitrogen, Waltham, USA).

[0038] 2. LFS RT-RPA detection

[0039] Using TwistAmp from Twist Company TMnfo kit (TwistDX, Cambridge, UK) to establish the RPA method, the reaction system is 50 μL, of which 10 μmol / L CDV-nfo-F and CDV-nfo-R each 2.1 μL (final concentration 0.42 μmol / L), 10 μmol / L CDV -nfo-P 0.6μL (final concentration 0.12μmol / L), 200U / μL M-MLV reverse transcriptase 1μL (final concentration 4U / μL), 40U / μL RNase inhibitor (TAKARA c...

Embodiment 3

[0040] Embodiment 3 reaction time optimization

[0041] To determine the optimal reaction time of the CDV LFS RT-RPA, a 9.4 × 10 3 Copies in vitro transcribed RNA as a template were held tightly in the hands of three different technicians at room temperature for 5, 10, 15, and 20 min.

[0042] Wherein, the preparation method of CDV in vitro transcription RNA is as follows:

[0043] Using N-F and N-R as primers (N-F: 5′-ATGGCCAGCCTTCTTAAG-3′; N-R: 5′-TTAATTGAGTAGCTCTCTATCA-3′), RT-PCR was performed using CDV virus HB / BD-2017 strain genomic RNA as a template to obtain the CDV N gene , a full length of 1572bp. The reaction system of RT-PCR is 50 μL, including 2×one-step Buffer 25 μL, one-step enzyme mixture (Takara, Dalian, China) 2 μL, N-F (20 μmol / L) 0.5 μL, N-R (20 μmol / L) 0.5 μL, CDV RNA 5 μL , ddH 2 O 17 μL. The RT-PCR reaction conditions were: 50°C, 30min, 1 cycle; 94°C, 2min, 1 cycle; 94°C 30s, 55°C 30s, 72°C 60s, 32 cycles.

[0044] The RT-PCR product was purified...

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Abstract

The invention discloses a lateral flow chromatographic test strip RT-RPA primer and probe combination for detecting canine distemper virus; wherein the sequences of the F primer, R primer, and probe are represented by SEQ ID No.1, SEQ ID No.2, and SEQ ID No.3. The invention also discloses a visual onsite rapid detection method, which uses the primer and probe combination and is used for detectingcanine distemper virus without using any detection equipment. The specificity of the primers and probe is strong, only the detection on canine distemper virus is positive, detection on other viruses is negative, the sensitivity is high, and the detection limit is 9.4*10<1> copies. The method can be effectively applied to the onsite diagnosis of canine distemper, the operation is simple, the reaction time is short, and the method can be used in labs with bad equipment in economic less-developed areas, can be applied to onsite and field diagnosis of canine distemper, and has a good application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer and a probe for detecting canine distemper virus and their application. Background technique [0002] Canine distemper (CD) is a highly contagious disease infecting canines caused by canine distemper virus (CDV), mainly manifested as respiratory symptoms, gastroenteritis, dermatitis, neurological symptoms Wait. The clinical symptoms caused by canine distemper are very extensive and similar to those caused by other epidemic diseases, so it is difficult to effectively diagnose canine distemper from clinical symptoms. Therefore, the effective and rapid diagnosis of canine distemper is of great significance for professionals to take effective measures in time to control the epidemic and prevent the spread of the epidemic. [0003] CDV belongs to the Morbillivirus genus of the Paramyxoviridae family and is a non-segmented non-enveloped negative-strand RNA virus. A series of ca...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2521/507C12Q2522/101C12Q2565/625
Inventor 王建昌王金凤刘立兵石蕊寒
Owner INSPECTION & QUARANTINE TESTING CENT OF HEBEI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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