Check patentability & draft patents in minutes with Patsnap Eureka AI!

Probe primer combination and kit for identifying PCV2 and PCV3 and application

A primer combination and kit technology, applied in the field of molecular biology, can solve the problems of false positives, low sensitivity, and time-consuming, etc., and achieve the effects of good repeatability and stability, low sensitivity, and time-consuming reduction

Pending Publication Date: 2021-11-30
贵州傲农七环畜牧养殖有限公司 +2
View PDF0 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these conventional methods are time-consuming, have low sensitivity and are prone to false positives
In the PCR detection method, there is only a single detection of PCV2 or PCV3, and real-time fluorescent PCR detection of PCV2 and PCV3 cannot be carried out in one reaction at the same time. Therefore, it is of great significance to establish a fast, sensitive and accurate identification method for PCV2 and PCV3.

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
  • Probe primer combination and kit for identifying PCV2 and PCV3 and application
  • Probe primer combination and kit for identifying PCV2 and PCV3 and application
  • Probe primer combination and kit for identifying PCV2 and PCV3 and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] This embodiment provides a kit for identifying PCV2 and PCV3, which includes a first probe primer set for PCV2, a second probe primer set for PCV3, a positive control, a negative control, and a PCR amplification solution. The first probe primer combination includes the first primer pair shown in SEQ ID NO.1-2 and the first probe shown in SEQ ID NO.3, and the second probe primer combination includes SEQ ID NO.4-5. The second primer pair shown and the second probe shown in SEQ ID NO.6.

[0060] The sequence of the first probe primer combination is as follows:

[0061] Upstream primer (PCV2-F) SEQ ID NO.1:

[0062] 5'-ATGGCGGGAGGAGTAGTT-3';

[0063] Downstream primer (PCV2-R) SEQ ID NO.2:

[0064] 5'-CGCTCTGTGCCCTTTGAA-3';

[0065] The first probe (PCV2-P) SEQ ID NO.3:

[0066] CATAGGGGTCATAGGTGAGGGC.

[0067] The target sequence amplified by the first primer pair is as follows (SEQ ID NO.7):

[0068] ATGGCGGGAGGAGTAGTTTACATAGGGGTCATAGGTGAGG GCTGTGGCCTTTGTTACAAAGTTA...

experiment example 1

[0095] In this experimental example, the condition optimization experiment of dual fluorescent quantitative PCR was carried out.

[0096] The two plasmids extracted in Example 1 were respectively subjected to 10-fold gradient dilution and then the concentration was 10 4 copise / μL, mixed according to the volume ratio of 1:1, and the mixed plasmid solution was used as a template. Set up a total system of 25 μL, and mix the upstream and downstream primers and corresponding probes at different final concentrations of primers and probes. Primers (including the upstream and downstream primers of two pairs of primers) and the corresponding probes (including two probes) were set to 4 ratios (refer to Table 1): 0.5μL: 0.25μL (200nM: 100nM), 0.5μL : 0.5μL (200nM: 200nM), 1μL: 0.5μL (400nM: 200nM), 1μL: 1μL (400nM: 400nM). All primer probes were applied at a concentration of 10 μM. According to the amplification program provided in Example 1 of the present invention, 37°C for 2min, 95...

experiment example 2

[0101] In this experimental example, the kit of Example 1 was used for the fluorescence amplification sensitivity experiment of circovirus type 2.

[0102] The prepared circovirus type 2 standard plasmid was respectively diluted 10 times and used as a template, and the concentration was 10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 Copise / μL for sensitivity determination, using a single-plex qPCR reaction system: DNA template 5 μL, upstream and downstream primers 1 μL (400 nM), probe 0.5 μL (200 nM), PCR amplification solution 12.5 μL, ddH 2 O 6 μL, all primers and probes were applied at a concentration of 10 μM. The amplification program was 37°C for 2min, 95°C for 30s, cycled at 95°C for 10s, and 58°C for 30s (collecting fluorescent signals), a total of 45 cycles, for PCR amplification, and analyzed the standard curve.

[0103] Result reference figure 1 and figure 2 as shown, figure 2 It shows that within the current concentration range of dilution, the amount of temp...

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 discloses a probe primer combination and a kit for identifying PCV2 and PCV3, and an application, and belongs to the technical field of molecular biology. The probe primer combination provided by the invention comprises (a) a first group of probe primer combinations as shown in SEQ ID NO.1-3, and (b) a second group of probe primer combinations as shown in SEQ ID NO.4-6. The kit provided by the invention can simultaneously realize one-time detection of PCV2 infection, PCV3 infection and PCV2 and PCV3 co-infection, thereby solving the problems of long time consumption, low sensitivity and easiness in false positive occurrence in conventional methods, and preventing concentration difference at which samples in a pig farm co-infected with different types of viruses from affecting virus detection rate. The probe primer combination and kit have advantages of high detection sensitivity and high specificity, provide technical support for epidemic surveillance and control of the porcine circovirus, and have good application prospects.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a combination of probes and primers, a kit and an application for identifying PCV2 and PCV3. Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) is a small single-stranded negative-strand, circular non-enveloped DNA virus, which can be divided into three serotypes according to the differences in its genes and antigens: Porcine circovirus Porcine circovirus type 1 (PCV1), porcine circovirus type 2 (PCV2) and the emerging porcine circovirus type 3 (PCV3). PCV1 is considered to be non-pathogenic to pigs; PCV2 can cause diseases such as multisystem wasting syndrome, porcine dermatitis and nephrotic syndrome, and reproductive disorders in weaned piglets, posing a great threat to the pig industry. PCV3 is a novel porcine circovirus associated with porcine dermatitis and nephrotic syndrome, reproductive failure, and multisystem inflammation. ...

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): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 张蓉毛旭明凌勇丁能水龙小敏龙毅吴有林
Owner 贵州傲农七环畜牧养殖有限公司
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

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

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com