Unlock instant, AI-driven research and patent intelligence for your innovation.

Porcine circovirus multiplex real-time fluorescent PCR detection primer pair, probe, and prepared kit

A porcine circovirus and detection kit technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems such as prone to false positives, low sensitivity, time-consuming and labor-intensive, and achieve reliable Good sex, high sensitivity, high sensitivity effect

Active Publication Date: 2022-06-21
LUOYANG PULIKE WANTAI BIOTECH
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these conventional methods are time-consuming, labor-intensive, low-sensitivity, and prone to false positives.
And clinically each genotype of porcine circovirus is often in a mixed infection situation. In the prior art, the PCR detection for porcine circovirus is mainly aimed at the single detection of each type. In addition, the clinical symptoms of PCV2 and PCV3 are similar to each other. Therefore, it is of great significance to establish a rapid, simple, specific, sensitive, and reliable differential detection method.

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
  • Porcine circovirus multiplex real-time fluorescent PCR detection primer pair, probe, and prepared kit
  • Porcine circovirus multiplex real-time fluorescent PCR detection primer pair, probe, and prepared kit
  • Porcine circovirus multiplex real-time fluorescent PCR detection primer pair, probe, and prepared kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0021] As an embodiment of the present invention, the kit further includes an enzyme mixture, the enzyme mixture is composed of Taq DNA polymerase, Tris-HCl, EDTA, DTT and glycerol, Taq DNA polymerase, Tris-HCl, EDTA , DTT and glycerol content were 0.5 U / μl, 20 mM, 0.1 mM, 50% V / V, respectively.

[0022] As an embodiment of the present invention, the kit further includes a PCR reaction solution, the PCR reaction solution consists of the porcine circovirus type 3 real-time fluorescent PCR detection primer pair and probe, reaction buffer, dDTPs and ddH 2 O is mixed in a volume ratio of 6:10:5:19.

[0023] As a preferred embodiment of the present invention, the primer pair concentration is 10 μM, the probe concentration is 5 μM, the reaction buffer is 5×Tris-HCl (pH 8.3), and the dDTPs concentration is 2.5 mM.

[0024] As an embodiment of the present invention, the kit further includes a positive control and a negative control.

[0025] As an embodiment of the present invention...

Embodiment 1

[0049] Example 1 Isolation and identification of porcine circovirus type 3

[0050] 1. Source of disease materials

[0051] In a domestic commercial pig farm, compared with the historical average, the mortality rate of sows increased by 9.4%, the conception rate decreased by 1.2%, and the mummified fetus increased by 8.2%. Clinically, affected sows show signs of anorexia, multifocal papules, spots and superficial dermatitis. Aborted litters contained mummified fetuses of different gestational ages, consistent with symptoms of miscarriage caused by PCV2 infection. Although the overall clinical manifestations and symptoms of abortion observed in sows were consistent with reproductive disorders caused by porcine circovirus type 2, various tissues in all sows, including kidneys, lymph nodes, lungs, skin, and stillbirths, were evaluated by immunocompetent The detection of PCV2, PRRSV, PPV, CSFV and Mycoplasma hyopneumoniae by chemical and quantitative PCR were negative. In order...

Embodiment 2

[0056] Example 2 Screening of specific primers and probes for porcine circovirus multiplex real-time fluorescent PCR method

[0057] 1. Primer and probe design of porcine circovirus

[0058] The specific primers for porcine circovirus PCV1, PCV2 and PCV3 refer to: an oligonucleotide chain with a length of about 20 bases, a highly conserved specific nucleotide fragment of the porcine circovirus ORF2 gene;

[0059] The specific probes for porcine circovirus PCV1, PCV2 and PCV3 refer to: oligonucleotide chains between 13 and 30 bases in length, the 5' end of which is labeled with fluorescent excitation groups such as FAM, HEX or NED The 3' end is labeled with a quenching group that does not emit light by itself.

[0060] The inventors designed multiple real-time fluorescent PCR amplification primers and TaqMan probes for porcine circovirus PCV1, PCV2 and PCV3, the specific sequences of the primer pairs and the probes are shown in the sequence table, and the names and labels corr...

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

PropertyMeasurementUnit
Sensitivityaaaaaaaaaa
Sensitivityaaaaaaaaaa
Sensitivityaaaaaaaaaa
Login to View More

Abstract

The invention relates to a primer pair and a probe for porcine circovirus type 3 real-time fluorescent PCR detection and a kit containing the PCR primer pair and the probe. The PCR primer pair and probe can specifically and highly sensitively detect PCV3 virus DNA samples, and provide a basis for early prevention of pigs infected with porcine circovirus type 3.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to multiple real-time fluorescent PCR detection, in particular to porcine circovirus type 3, type 1, and / or type 2 and multiple real-time fluorescent PCR detection primers, probes and kits. Background technique [0002] Porcine circovirus (PCV) is a single-stranded circular DNA virus with a genome length of about 1.7 kb, which is one of the smallest animal DNA viruses. Two types of PCV have been identified, porcine circovirus type 1 (PCV1) and porcine circovirus type 2 (PCV2). PCV1 was first identified as a contaminant in PK cell cultures in 1974 and was not pathogenic in pigs. PCV2 was first reported in 1998. It can cause porcine circovirus associated diseases (PCVAD) in pigs under clinical conditions, mainly causing multisystem wasting syndrome, pneumonia, porcine dermatitis and nephrotic syndrome in piglets. Reproductive disorders, mainly manifested as respiratory, urinary, intestinal...

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
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12N15/11
CPCC12Q1/686C12Q1/701C12Q2561/113C12Q2563/107C12Q2537/143
Inventor 田克恭李向东孙明张超林
Owner LUOYANG PULIKE WANTAI BIOTECH