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CPA primer set, reagent, kit and detection method and application thereof for detecting amdoparvovirus

A technology of Aleutian virus and detection method, which is applied in the field of CPA primer sets for detection of Aleutian virus, can solve the problems of time-consuming, high requirements on experimental conditions, inability to directly detect the virus, limited on-site application, etc., and achieves that the detection method is simple, The amplification results are stable and reliable, and the effect of expanding the scope of application

Pending Publication Date: 2019-04-12
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional virus PCR detection needs to extract nucleic acid, provide precise reaction conditions, and electrophoresis to detect nucleic acid amplification specific bands. It is mainly completed in a specialized laboratory, which is time-consuming and requires high experimental conditions, and its application in the field is limited.
CIEP indirectly determines virus infection by detecting antibodies, and cannot directly detect viruses
ELISA detection and quantification of virus antibodies requires professional precision instruments, the cost of reagents is high, and there are great limitations in field application

Method used

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  • CPA primer set, reagent, kit and detection method and application thereof for detecting amdoparvovirus
  • CPA primer set, reagent, kit and detection method and application thereof for detecting amdoparvovirus
  • CPA primer set, reagent, kit and detection method and application thereof for detecting amdoparvovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Example 1 Aleutian virus CPA primer design and detection

[0116] 1. Primer design

[0117] Referring to the comparison of the AMDV gene sequence (NC_001662), the CPA primer set targeting the VP2 gene of AMDV was designed and screened, including a pair of stripping primers and a pair of single cross-primers, the sequences of which are as follows:

[0118] The nucleotide sequences of the stripped primer pairs include:

[0119] BL-F: 5'-ctgtMacagaaaccaaccaaggta-3' (SEQ ID NO.1);

[0120] BL-R: 5'-ttgaatgttaggRtatctgttgta-3' (SEQ ID NO. 2);

[0121] The nucleotide sequence of single cross primer pair comprises:

[0122] JC-F: 5'-gttggtttggttgctctccaaggactccagctgcgccgttgg-3' (SEQ ID NO. 3);

[0123] JC-R: 5'-actccagctgcgccgttggttggtttggttgctctccaagga-3' (SEQ ID NO. 4).

[0124] 2. CPA amplification reaction

[0125] Taking AMDV genomic DNA as a positive template, the amount of AMDV templates was 5.38×10 1 copies / μL, 5.38×10 2 copies / μL, 5.38×10 3 copies / μL, 5.38×1...

Embodiment 2

[0132] Example 2 CPA primer set specificity test

[0133]1. Select the DNA of canine adenovirus, canine parvovirus, canine distemper virus and mink enteritis virus, as well as the DNA of mink, blue fox and Ussuri raccoon dog tissue as the DNA template, with AMDV as the positive template, ddH 2 O is a negative control, utilizes the amplification system and method described in embodiment 1 to carry out CPA amplification reaction, then carries out agarose gel electrophoresis, the results are shown in figure 2 .

[0134] 2. Result identification

[0135] figure 2 In the agarose electrophoresis diagram, M represents DL2000 Marker, 1 to 4 represent the templates of canine adenovirus, canine parvovirus, canine distemper virus and mink enteritis virus DNA, and 5 to 7 represent the templates of mink, blue fox and crow DNA of Suri raccoon dog tissue, + stands for positive control, - stands for negative control;

[0136] figure 2 The results of agarose electrophoresis showed that...

Embodiment 3

[0137] Embodiment 3 CPA primer set sensitivity test

[0138] 1. The DNA concentration of the AMDV genome extract was tested to be 5.4×10 7 copies / μL, serially diluted 10 times to a dilution concentration of 5.4×10 2 copies / μL, as a template for common PCR.

[0139] 2, CPA reaction system is identical with embodiment 1, common PCR reaction condition is as follows:

[0140] 30 μL reaction system: 1 μL template, 1×PCR buffer, 0.3-0.6 μM stripping primer, Hot StarTaq TM Polymerase 2~3U to 30μL and mix well.

[0141] Reaction program: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, 35 cycles, and finally extension at 72°C for 5 min.

[0142] 3. Result identification

[0143] Take 10 μL of normal PCR products for agarose gel electrophoresis, image 3 In the electropherogram, M stands for DL2000Marker, 10 2 、10 3 、10 4 、10 5 and 10 6 Respectively represent the amount of AMDV template is 5.4×10...

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Abstract

The present invention discloses a CPA primer set, a reagent, a kit and a detection method and an application thereof for detecting amdoparvovirus, and relates to the technical field of virus molecularbiology. The CPA primer set comprises a stripping primer pair and a single cross-primer pair, and nucleotide sequences of the stripping primer pair are SEQ ID NO.1 and SEQ ID NO.2 and the nucleotidesequences of the single cross-primer pair are SEQ ID NO.3 and SEQ ID NO.4. The CPA primer set has good specificity and high sensitivity, and can effectively detect the amdoparvovirus. The present invention also discloses the CPA reagent, the CPA kit and the detection method for detecting the amdoparvovirus. The CPA kit can detect the amdoparvovirus quickly and sensitively, has a detection limit aslow as 1.82x10<2>copies / [mu]L, and can realize rapid visualization and qualitative determination. The detection method is simple, fast, and efficient, does not require complicated equipment, and is suitable for rapid detection on site.

Description

technical field [0001] The invention relates to the technical field of viral molecular biology, in particular to a CPA primer set, reagent, kit, detection method and application for detecting Aleutian virus. Background technique [0002] Aleutian virus (Amdoparvovirus) belongs to parvoviridae (Parvoviridae), parvovirinae (Parvovirinae), Aleutian virus genus. Aleutian virus mainly infects a wide range of hosts such as mustelidae, skunks and red pandas. Aleutian virus is the pathogen of Aleutian disease in mink, ferret, otter and skunk and red panda. Potential pathogens of genus and raccoon dogs. There may be new virus species in the genus Aleutiana, infecting a wider range of hosts. Among them, Aleutian mink disease virus (AMDV) can cause mink Aleutian disease, which is a serious hazard to the mink breeding industry. [0003] At present, the commonly used methods for detecting Aleutian virus mainly include conventional PCR detection, CIEP detection and ELISA detection. Co...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2563/107
Inventor 邵西群韦韬章秀婷
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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