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Triple TaqMan fluorescent quantitative PCR (Polymerase Chain Reaction) kit for simultaneously detecting three circoviruses

A fluorescence quantitative and kit technology, applied in the direction of recombinant DNA technology, resistance to vector-borne diseases, measurement/inspection of microorganisms, etc., can solve problems such as false positives, hazards to operators and the environment, and low sensitivity, and achieve The effect of reducing false negatives, improving the level of virus detection, and supporting timely early warning

Pending Publication Date: 2022-06-07
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Three kinds of circoviruses are now ubiquitous in our country, causing extensive economic losses, but at present, my country distinguishes chicken infectious anemia virus (CAV), circovirus avian origin type 1 (GyG1), and circovirus human origin type 1 (GyH1) mainly Through ordinary PCR technology, as well as molecular biology and immunological detection methods, etc., these methods are harmful to the operator and the environment, or the operation is complicated, or the sensitivity is low or missed detection, false positive and other difficult problems to solve

Method used

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  • Triple TaqMan fluorescent quantitative PCR (Polymerase Chain Reaction) kit for simultaneously detecting three circoviruses
  • Triple TaqMan fluorescent quantitative PCR (Polymerase Chain Reaction) kit for simultaneously detecting three circoviruses
  • Triple TaqMan fluorescent quantitative PCR (Polymerase Chain Reaction) kit for simultaneously detecting three circoviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] The establishment of embodiment 1 CAV, GyG1 and GyH1 triple TaqMan fluorescent quantitative PCR method

[0057] (1) Design of primers and probes

[0058] According to the circovirus sequences registered in GenBank, a set of triple real-time fluorescent quantitative PCR primer-probe sets for three circoviruses were designed with Primerpremier5.0 software. Primers and probes were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., and the sequences are shown in Table 1.

[0059] Table 1 Fluorescent quantitative PCR primer and probe sequences

[0060]

[0061] Get target gene

[0062] Take the DNA of existing strains CAV (Cux-1 strain), GyG1 (HLJ1508 strain) and GyH1 (SDAU-1 strain) as templates, or use the DNA of other existing strains of the same type as templates for ordinary PCR amplification, The amplified product was identified by agarose gel (1.5%) electrophoresis, and purified and recovered using an agarose gel DNA recovery kit (Tiangen).

[0063] Pur...

Embodiment 2 3

[0111] Example 2 Triple TaqMan fluorescent quantitative PCR specificity test

[0112] In order to detect the specificity of the kit of the present invention, the above-mentioned kit of the present invention is used to detect chicken J subgroup leukemia virus (ALV-J, NX0101 strain), avian reticuloendothelial disease virus (REV, SNV strain), Triple circle virus nucleic acid of 3 viruses of Marek's disease virus (MDV, Md5 strain). All three viruses are known strains.

[0113] Specificity test result shows: kit of the present invention only amplifies CAV, GyG1 and GyH1, shows that kit of the present invention can specifically amplify, and not with chicken J subgroup leukemia virus (ALV-J), poultry meshwork There were cross-reactions between 3 viruses including Reptilian Endothelial Proliferation Virus (REV) and Marek's Disease Virus (MDV). Figure 7 , while 3 kinds of viruses such as ALV-J, REV, and MDV did not amplify, had no Ct value, and were judged as negative; it can be see...

Embodiment 3

[0114] Example 3 Triple real-time fluorescent quantitative PCR detection of three kinds of circoviruses

[0115] (1) Sample collection

[0116] A total of 40 cases were collected for this experiment.

[0117] (2) Total DNA extraction

[0118] Soybean-sized livers from each case were mixed and placed in 1.5mL centrifuge tubes, extracted using the tissue genomic DNA extraction kit from Tiangen Company, and operated according to the product instructions to obtain total tissue DNA, which was stored at -20°C for later use.

[0119] (3) Single-plex ordinary PCR detection and single-plex fluorescent quantitative PCR detection

[0120] The single-plex common PCR detection system is shown in Table 4:

[0121] Table 4 Fluorescent quantitative PCR system

[0122]

[0123]

[0124] Single-plex PCR amplification was performed on the three circoviruses respectively. The reaction was carried out in a PCR amplification instrument, and the reaction parameters were: pre-denaturation ...

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Abstract

The invention belongs to the field of virus detection, and particularly provides a triple TaqMan fluorescent quantitative PCR (Polymerase Chain Reaction) kit for simultaneously detecting three circoviruses, which integrates a forward primer, a reverse primer and a specific fluorescent probe of chicken infectious anemia virus (CAV). The kit comprises a forward primer, a reverse primer and a specific fluorescent probe of a circovirus avian type-1 (Gyrovirus galga1, GyG1), and also comprises a reverse primer and a specific fluorescent probe of the circovirus avian type-1 (GyG1). The kit comprises a forward primer, a reverse primer and a specific fluorescent probe of the circovirus human type 1 (Gyrovirus homsa1, GyH1), and further comprises a reverse primer and a specific fluorescent probe of the circovirus human type 1 (Gyrovirus homsa1, GyH1). The kit adopting the composition has the advantages of high sensitivity, high specificity, low pollution and real-time detection, and provides reliable technology and product for early diagnosis, monitoring, early warning, prevention and treatment of clinical circle virus diseases in livestock and poultry farms and animal food safety inspection.

Description

technical field [0001] The invention belongs to the field of virus detection and specifically provides a triple TaqMan fluorescent quantitative PCR kit for simultaneously detecting three circle viruses. Background technique [0002] Gyrovirus is a single-stranded circular DNA virus. The virus genus once belonged to the family Circoviridae (Circoviridae). Since the structure and genetic information of the circle virus are more similar to members of the Anelloviridae (Anelloviridae), so in 2016 In the latest virus taxonomy approved by the International Committee on Taxonomy of Viruses, Gyrovirus was reassigned to the family Lenoviridae. The circovirus genus can be divided into three clades, among which chicken infectious anemia virus (CAV), circovirus avian origin type 1 (GyG1) and circovirus human origin type 1 (GyH1) belong to clade A. [0003] Among them, chicken infectious anemia virus (CAV) is hexagonal, and some virus particles are also spherical, with an average partic...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2600/16C12Q2600/158C12Q2563/107C12Q2531/113C12Q2537/143C12Q2561/113Y02A50/30
Inventor 成子强李佳琛周德方严天行刘圣龙赵钰莹
Owner SHANDONG AGRICULTURAL UNIVERSITY
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