Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Primer probe group, kit and detection method for PRRSV (porcine reproductive and respiratory syndrome virus) and CSFV (classical swine fever virus) duplex fluorescent quantitative PCR (polymerase chain reaction) detection

A technology of PRRSV-D1-R and PRRSV-D1-F, applied in the field of molecular biology, can solve the problems of low conservation, low detection sensitivity, inaccurate results, etc., and achieve high amplification efficiency and binding rate, fast Effect of differential detection and high detection sensitivity

Pending Publication Date: 2022-05-27
哈尔滨中科基因技术有限公司
View PDF2 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] When RT-PCR is used for simultaneous joint detection of multiple viruses, if there is an interaction between the primers and probe sequence structures of different viruses, it will easily have a negative impact on the detection sensitivity and accuracy, resulting in a decrease in detection sensitivity and inaccurate results.
At the same time, factors such as rapid mutation of the virus itself, multiple genotypes and serotypes will also affect the sensitivity and accuracy of RT-PCR joint detection
The invention patent with the application number CN201110146877.8 "A Combined Detection Method and Kit for Two-color Fluorescent Quantitative PCR of CSFV and PRRSV" can realize simultaneous detection of CSFV and PRRSV in the same sample to be tested. Porcine reproductive and respiratory syndrome virus; however, the gene locus for detection of porcine reproductive and respiratory syndrome is located in the ORF5 gene, which has hypermutation and homologous recombination
The invention patent with the application number CN201510191676.8 "African swine fever virus, swine fever virus and porcine reproductive and respiratory syndrome virus triple fluorescent RT-PCR detection reagent and its method and application", the detection site of PRRSV is NSP2, This site is also not highly conserved, and even in highly pathogenic porcine reproductive and respiratory syndrome, there are still a large number of deletions in the NSP2 gene
If the detected gene locus is mutated or deleted, it may even directly cause a false negative test result, which reduces the specificity of NSP2 mutant strains and misleads the diagnosis of sick pigs

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Primer probe group, kit and detection method for PRRSV (porcine reproductive and respiratory syndrome virus) and CSFV (classical swine fever virus) duplex fluorescent quantitative PCR (polymerase chain reaction) detection
  • Primer probe group, kit and detection method for PRRSV (porcine reproductive and respiratory syndrome virus) and CSFV (classical swine fever virus) duplex fluorescent quantitative PCR (polymerase chain reaction) detection
  • Primer probe group, kit and detection method for PRRSV (porcine reproductive and respiratory syndrome virus) and CSFV (classical swine fever virus) duplex fluorescent quantitative PCR (polymerase chain reaction) detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] The present invention provides a primer probe set for PRRSV and CSVV double fluorescence quantitative PCR detection, comprising a primer probe set for PRRSV and a primer probe set for CSVV.

[0057] Primer probe sets for PRRSV include primers and probes with the following nucleotide sequences:

[0058] Forward primer PRRSV-D1-F: 5′-ACCTGGAAATTCATCACCTC-3′, as shown in SEQ ID No.1;

[0059] Reverse primer PRRSV-D1-R: 5′-CGACAAATGCGTGGTTATCA-3′, as shown in SEQ ID No.2;

[0060] Probe PRRSV-Probe: 5′-FAM-TGCTAGGCCGCAAGTACATTC-BHQ1-3′, as shown in SEQ ID No.3;

[0061] The primer probe set for CSV comprises primers and probes having the following nucleotide sequences:

[0062] Forward primer CSVV-D155-18F: 5′-GGGTGGTCTAAGTCCTGA-3′, as shown in SEQ ID No.4;

[0063] Reverse primer CSVV-D325-18R:5′-CTAATAGTGGGCCTCTGC-3′, as shown in SEQ ID No.5;

[0064] Probe CSFV-Probe: 5′-ROX-CAGTAGTTCGACGTGAGCAGAAG-BHQ2-3′, as shown in SEQ ID No.6.

[0065] The primer probe set for PRRSV and ...

Embodiment 2

[0067] The present embodiment provides a kit for PRRSV and CSVV double fluorescence quantitative PCR detection, comprising a primer probe set for PRRSV in Example 1 and a primer probe set for CSVV: forward primer PRRSV-D1-F, reverse primer PRRSV-D1-R, probe PRRSV-Probe, forward primer CSVV-D155-18F, reverse primer CSFV-D325-18R and probe CSVV-Probe.

[0068] The kit also includes DNA polymerase, reverse transcription mixed enzymes, negative controls, positive controls for PRRSV and CSVV, and PCR buffers containing dNTP.

[0069] PCR buffer containing dNTP contains buffer, magnesium chloride and dNTP, the present example kit uses a commercially available 2X One Step RT-PCR Buffer III.

[0070] The DNA polymerase used in the kit of the present embodiment is a commercially available 5U / μl Of TaKaRa Ex Taq HS.

[0071] Reverse transcription mixed enzyme contains a RNA inhibitor, M-MuLV reverse transcriptase and heat-resistant DNA polymerase, the present embodiment of the kit uses a ...

Embodiment 3

[0081] The present embodiment provides a method for preparing PRRSV positive plasmids and positive standards, the specific steps are as follows:

[0082] Step 1: Obtain the genomic RNA of PRRSV:

[0083] PRRSV is derived from the commercially available vaccine TJM-F92 strain, using the fully automated nucleic acid extractor HERO 32 (Luoyang Aisen) to extract the genomic RNA of PRRSV from the commercially available vaccine (magnetic bead extraction reagent, Luoyang Aisen).

[0084] Step 2: Obtain the target amplification product of PRRSV:

[0085] Take advantage of Takara PrimeScript TMOne Step RT-PCR Kit Ver.2 (RR055) one-step reagent, the genomic RNA of PRRSV as a template, and the amplification primer with the PRRSV ORF6 gene sequence conserved fragment as the amplification target sequence was designed with the following nucleotide sequences:

[0086] PRRSV-SF: 5′-TTTCAGCGGAAAAATGGGGTC-3′, as shown in SEQ ID No.8;

[0087] PRRSV-SR: 5′-TTTCTGCCACCCAACACGAGG-3′, as shown in SEQ I...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a primer probe group, a kit and a detection method for double fluorescent quantitative PCR (polymerase chain reaction) detection of PRRSV (porcine reproductive and respiratory syndrome virus) and CSFV (classical swine fever virus), and belongs to the technical field of molecular biology. In order to further improve the accuracy of the double fluorescent quantitative PCR joint detection of the PRRSV and the CSFV, the invention provides a primer probe group for the double fluorescent quantitative PCR detection of the PRRSV and the CSFV, a primer probe group aiming at the PRRSV and a primer probe group aiming at the CSFV, which are designed by taking an ORF6 gene of the PRRSV and a 5 'UTR non-coding region of the CSFV as amplification target regions, so that the conservative property and the stability of detection gene loci are ensured, and the accuracy of the double fluorescent quantitative PCR joint detection of the PRRSV and the CSFV is improved. The sequence structures of the primer probe group do not interact, the amplification efficiency and binding rate are high, and the detection sensitivity is very high. The detection kit provided by the invention has no cross reaction with similar pathogens, has high specificity and accuracy, and can meet the requirements of large-scale rapid identification and detection.

Description

Technical field [0001] The present invention belongs to the field of molecular biology techniques, in particular to primer probe sets, kits and detection methods for PRRSV and CSVV double fluorescence quantitative PCR detection. Background [0002] Porcine Reproductive and Respiratory Syndrome (PRRS), also known as swine blue ear disease, is mainly caused by the swine reproductive and respiratory syndrome virus PRRSV, which can lead to severe reproductive failure of pregnant sows and respiratory disorders in pigs of all ages, which is a swine infectious disease that seriously affects the economic benefits of breeding. Classical Swine Fever (CSF), mainly caused by the swine fever virus CSV, can lead to high mortality rates and morbidity in pigs, causing devastating economic losses, and is listed as a Class A infectious disease by the OIE of the World Organisation for Animal Health. [0003] Because porcine reproductive and respiratory syndrome and classical swine fever can cause p...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6858C12Q2600/16C12Q2531/113C12Q2563/107C12Q2537/143C12Q2545/113Y02A50/30
Inventor 赵阳魏启超武守霖孙彩云袁丹阳董雪莹
Owner 哈尔滨中科基因技术有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com