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Dual PCR primer group and kit for detecting novel raccoon dog parvovirus and application of dual PCR primer group and kit

A parvovirus and primer set technology, applied in the field of virus molecular biology, can solve the problems of no identification and rapid detection of nRDPV, etc., and achieve the effect of strong practicability, high sensitivity and high sensitivity

Pending Publication Date: 2022-03-04
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, it is very important to quickly and accurately identify the new raccoon parvovirus (nRDPV), but there is no PCR method for identification and rapid detection of nRDPV in this field

Method used

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  • Dual PCR primer group and kit for detecting novel raccoon dog parvovirus and application of dual PCR primer group and kit
  • Dual PCR primer group and kit for detecting novel raccoon dog parvovirus and application of dual PCR primer group and kit
  • Dual PCR primer group and kit for detecting novel raccoon dog parvovirus and application of dual PCR primer group and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, the establishment of a dual PCR method for detecting a new type of small virus

[0042] Primitive design

[0043]By compete by the VP2 protein gene sequence of the carnivorous virus type 1 (including FPV, MEV, CPV-2A, CPV-2B, CPV-2C, etc.), which has been published on Genbank, and the VP2 protein gene sequence of novel fragrant viral NRDPV. In pair, the difference between NRDPV and other carnivore-based virus type 1 virus VP2 protein gene sequence, by design, screening primers, exploitable reaction conditions, etc., finally get a pair of carnivores for graphic virus 1 general primer 1: SEQ ID NO .1 / seqid No.2 and a pair of NRDPV specific primers 2: SEQ ID NO.3 / SEQ ID NO.4, primer sequences are as follows:

[0044] SEQ ID NO.1: 5'-ctgaagctactattatgagacc-3 ';

[0045] SEQ ID No.2: 5'-ctccttctgatatctTCC-3 ';

[0046] SEQ ID NO.3: 5'-ggctttagatgattcatgc-3 ';

[0047] SEQ ID No.4: 5'-ttatcttgttgaactcctatatatc-3 ';

[0048] A general primer pair 1 amplification ...

Embodiment 2

[0057] Example II, specificity and sensitivity experiment

[0058] Specific experiment

[0059] NRDPV, CPV-2A, CPV-2B, CDV, and CCV nucleic acids were used as a template, and dual PCR reactions were performed by the above optimized conditions, respectively. Agarose gel electrophoresis was carried out on a dual PCR product. The results showed that two fragments were obtained by SEQ ID NO.1 / SEQ IDNO.2, SEQ ID NO.3 / SEQ ID NO.4, i.e. NrDPV specific gene fragment, and only the general gene fragment can only be amplified for CPV-2A and CPV-2b without a specific fragment amplification, and the amplification of the canine disease virus CDV, canine virus CCV is negative (see image 3 . This indicates that dual PCR established based on SEQ ID NO.3 / SEQ ID NO.2 and SEQ ID NO.3 / SEQ ID NO.4 is used for rapid identification of NRDPV and other carnivorous animal original virus 1 type.

[0060] 2. Sensitive experiment

[0061] Using the single PCR method in the first example, NRDPV was used...

Embodiment 3

[0062] Example 3, Clinical Sample Test Experiment

[0063] 50 doubts of 20 貉 貉, Dongping, Linyi and other places were used in the establishment of the double PCR method, and the manure of the viral enteritis was tested and the intestinal sample. Positive, with the vivobial separation culture test results in the compliance rate of 100%, indicating that the dual PCR method established by the present invention is suitable for rapid detection of NRDPV (Table 1). ,

[0064] Table 1 Comparison of dual PCR and virus separation detection

[0065]

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Abstract

The invention provides a dual PCR primer group and a kit for detecting novel raccoon dog parvovirus and application of the dual PCR primer group and the kit. According to the dual PCR primer group and the kit, nRDPV and other carnivorous animal original parvovirus type 1 can be rapidly identified and detected at the same time by adopting dual PCR. If the 261bp product and the 648bp product are amplified at the same time, the detected sample is infected by the novel raccoon dog parvovirus nRDPV; if only 261bp is amplified, the detected sample is infected by the original parvovirus type 1 virus of other carnivorous animals; if the fragments of 261bp and 648bp are not amplified, the detected sample is not infected with the carnivorous animal original parvovirus type 1 virus and the novel raccoon dog parvovirus nRDPV. The PCR detection method for the targeted raccoon dog parvovirus is established for the first time, has the advantages of convenience and rapidness in detection, strong specificity, high sensitivity and the like, and can rapidly and accurately realize nRDPV detection.

Description

Technical field [0001] The present invention relates to a double PCR primer group, a detection kit, and its application thereof, which is a rapid detection of a new type of fragrant virus. Background technique [0002] The farming industry has developed an important industry, and the proportion of animal husbandry in my country is constantly rising, and it is an important part of the modern characteristics of my country. Among the National Livestock Genetic Resources Catalogs released in May 2020, the Agricultural Livestock Genetic Resource Catalog is officially listed as special livestock (non-edible). The infection of Raccoon Dog Parvovirus (RDPV) is an important epidemic disease that is seriously hindering the healthy development of aquaculture, causing great economic losses. [0003] RDPV belongs to the fine virus (Parvoviridae), a small viral subviae (PROURNPARVOVIRUS) carnivore-carnivore 1 (Carnivore Protoparvovirus 1) Member (International Virus Classification Committee (I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 谢之景杜彦霆马欢欢
Owner SHANDONG AGRICULTURAL UNIVERSITY
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