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Real-time fluorescent quantitation PCR reagent kit for detecting porcine circoviruses

A real-time fluorescence quantitative and porcine circovirus technology, which is applied in the detection/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of PCR false positives, missed detection, PCR pollution, etc., and achieve high specificity , The test operation is simple, and the effect of eliminating false positives

Inactive Publication Date: 2020-07-28
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problems of false positives, PCR contamination and missed detection in conventional PCR in the process of detecting porcine circovirus, the present invention provides a new real-time fluorescent quantitative PCR of porcine circovirus 1, 2 and 3 types SYBR Green I, with specificity Good, high sensitivity, low cost

Method used

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  • Real-time fluorescent quantitation PCR reagent kit for detecting porcine circoviruses
  • Real-time fluorescent quantitation PCR reagent kit for detecting porcine circoviruses
  • Real-time fluorescent quantitation PCR reagent kit for detecting porcine circoviruses

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Experimental program
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Effect test

Embodiment 1

[0027] Embodiment 1 Construction of vector

[0028] 1.1 Primer design and synthesis

[0029] According to the ORF1 gene sequences of PCV1, PCV2, and PCV3 in Genbank, a pair of specific primers (Table 1) were designed using Primer 5.0 software, and the primers were synthesized by Shanghai Bioengineering Technology Co., Ltd.

[0030] Table 1 PCV1, PCV2, PCV3 primers

[0031] .

[0032] 1.2 Preparation of standard template

[0033] Refer to the establishment of porcine circovirus type Ⅰ Taq Man fluorescence quantitative PCR detection method, the establishment of porcine circovirus type 2 SYBR Green Ⅰ real-time fluorescent quantitative PCR detection method, the establishment of porcine circovirus type 3 SYBR Green Ⅰ real-time fluorescent quantitative PCR detection method Plasmids T-PCV1, pEASY-Blunt-PCV2, and pEASY-Blunt-PCV3 of type 1, 2, and 3 porcine circoviruses were constructed according to the method in the article, and stored at -80°C for future use.

[0034] The comp...

Embodiment 2

[0040] Embodiment 2 method verification

[0041] 2.1 Specificity test

[0042] Extract the nucleic acid of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine pseudorabies virus (PRV), porcine parvovirus (PPV), PCV1, PCV2, PCV3 infection materials, and reverse transcription of RNA virus Be cDNA, carry out real-time fluorescence quantitative PCR reaction according to the condition in embodiment 1, set negative, positive contrast simultaneously, the result is as follows Figure 4 As shown: only PCV1, PCV2, and PCV3 have fluorescent signals, and others such as PRV, PPV, PRRSV, CSFV, and negative controls have no fluorescent signals, indicating that the method has good specificity.

[0043] 2.2 Sensitivity test

[0044] The recombinant plasmid standard product in embodiment 1 is diluted to 10 times through 10 times gradients 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 、10 9 A total of 9 dilutions were used as template...

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Abstract

The invention provides a real-time fluorescent quantitation PCR reagent kit for detecting porcine circoviruses. The real-time fluorescent quantitation PCR reagent kit comprises an upstream primer, 3 downstream primers and SYBR Green I dye, and can detect PCV1, PCV2 and PCV3 at the same time. The primers and the reagent kit provided by the invention are good in detection specificity and high in sensitivity, and are suitable for accurate and fast detection of 1, 2 and 3 porcine circoviruses in a laboratory or clinical application.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, in particular to a real-time fluorescent quantitative PCR reagent for detecting porcine circovirus, in particular to a real-time fluorescent quantitative PCR of porcine circovirus 1, 2 and 3 types SYBR Green I. Background technique [0002] Porcine circovirus (PCV) is a non-enveloped circular single-stranded DNA virus belonging to the genus Circovirus in the family Circoviridae. It is also the smallest animal virus with autonomous replication ability discovered so far, with a genome of about 2Kb. Before 2016, only two serotypes of porcine circovirus type 1 (porcine circovirus, PCV1) and porcine circovirus type 2 (porcine circovirus, PCV1) were found worldwide, and only PCV2 was considered to be pathogenic. The main pathogen that causes post-weaning multisystmicwasting syndrome (PMWS) in piglets is closely related to porcine dermatitis and npphropathy syndrome (PDNS) and proliferative a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 李俊田瑶时建立彭喆徐绍建李琛吴晓燕王硕刘畅韩红
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI