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

Universal Cycleave fluorescent PCR detection method for sheep pox virus

A sheeppox virus and virus technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, can solve the problems of diagnosis relying on laboratory testing, asymptomatic hidden infection, etc., and achieve rapid and high Throughput detection speed, easy operation, good specificity

Active Publication Date: 2021-07-30
CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Clinical symptoms such as fever, skin or mucous membrane nodules (or papules), and swollen lymph nodes can preliminarily diagnose diseases caused by sheeppoxviruses, but their diagnosis depends on laboratory tests, especially those with similar symptoms to foot-and-mouth disease, sheep mouth sores, blue When distinguishing common ruminant diseases such as tongue disease, there are also cases of asymptomatic recessive infection

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
  • Universal Cycleave fluorescent PCR detection method for sheep pox virus
  • Universal Cycleave fluorescent PCR detection method for sheep pox virus
  • Universal Cycleave fluorescent PCR detection method for sheep pox virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Screening and Design of Primers and Probes

[0036]Capripoxvirus (CaPV) includes bovine nodular skin disease virus (Lumpy skindisease virus, LSDV), goat pox virus (Goatpox virus, GTPV) and sheep pox virus (Sheeppoxvirus, SPPV). The gene homology between viruses can reach more than 95%, and the homology is relatively high. In order to avoid false negatives when detecting this genus of viruses, first search and download the complete genome sequences of all 27 strains of LSDV from the NCBI database (GenBank accession numbers: MN636843.1, MN636842.1, MN636841.1, MN636840.1, MN636839.1, MN636838.1, MK4418381, MH646674.1, KX764645.1, KX764644.1, KX764643.1, MT992618.1, MT130502.1, AF409137.1, MN995838.1, MN12, MN907.1 KX894508.1, MH893760.2, KY829023.3, KY829023.3, KY702007.1, KX683219.1, MT130502.2, MT643825.1, AF325528.1, AF409138.1), all 12 GTPV genomes have been sequenced (GenBank accession numbers: KX576657.1, AY077836.1, AY077835.1, MN072621.1, MN072620.1, ...

Embodiment 2

[0050] Embodiment 2: the sensitivity of the primer pair of screening, probe

[0051] 1. Preparation of positive plasmid standard

[0052] 1) Preparation of positive plasmid

[0053] The Chinese epidemic strain LSDV / China / XJ / 2019-1 virus was isolated, identified, preserved and provided by the National Center for Alien Animal Disease Research. Use a commercial viral genome extraction kit to extract the viral DNA template according to the instructions, and perform conventional PCR amplification. The specific steps are as follows:

[0054] Reaction system preparation: 25 μL of reaction solution per tube, containing 12.5 μL of 2×Cycleave fluorescent PCR reaction solution, 1 μL of 10 pmol / μL CaPV forward and CaPV reverse, 8.5 μL of double distilled water, and 2 μL of extracted DNA template. Among them, the Platinum Super Green PCR Mix Detection Kit (catalogue number: 00766789) was purchased from Invitrogen. The second is the setting of reaction conditions: pre-denaturation at 95°...

Embodiment 3

[0065] Embodiment 3: repeatability experiment

[0066]Using the primers and probes designed in Example 1 to prepare the reaction system, the working standard 2 to 6 prepared in Example 2 was repeatedly detected by Cycleave fluorescent PCR three times, and the coefficient of variation between batches was calculated; repeated in the same Cycleave fluorescent PCR Detect three times and calculate the intra-assay coefficient of variation. The specific steps are as follows:

[0067] (1) Reaction system preparation: 25 μL of reaction solution per tube, containing 12.5 μL of 2×Cycleave fluorescent PCR reaction solution, 1 μL of 5 pmol / μL CaPV forward and 1 μL of CaPV reverse, 1 μL of 5 pmol / μL CaPV probe, 7.5 μL of double distilled water , 2 μL of the DNA template of the sample to be detected. Among them, Cycleave fluorescent PCR detection kit (catalogue number: CY505A) was purchased from Treasure Bioengineering (Dalian) Co., Ltd. products. (2) Reaction condition setting: pre-denatu...

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 provides a Cycleave fluorescent PCR primer pair and a probe capable of simultaneously detecting sheep pox virus bovine nodular skin diseases, goat pox and sheep pox, and a detection method using the primer pair and the probe. According to the primer pair and the probe group, the sequence of the forward primer is SEQ ID NO: 1; the sequence of the reverse primer is SEQ ID NO: 2; the sequence of the probe is SEQ ID NO: 3, and the 8th site of the probe is an RNA base. The primer, the probe and the method for detecting the capripoxvirus virus through the Cycleave fluorescent PCR have the advantages of being high in detection sensitivity, good in specificity, high in flux and easy and convenient to operate.

Description

technical field [0001] The invention belongs to the technical field of detection of pathogenic microorganisms belonging to the genus of cappypox virus, and specifically relates to a pair of cycleave fluorescent PCR primers and probes capable of simultaneously detecting bovine nodular dermatosis, goat pox and sheep pox of the genus of cappox virus and the application of the primer pair and probe detection methods. Background technique [0002] Bovine nodular skin disease virus (Lumpy skin disease virus, LSDV), goat pox virus (Goatpoxvirus, GTPV) and sheep pox virus (Sheeppox virus, SPPV) are members of Capripoxvirus (CaPV). Viruses are important diseases in domestic ruminants that must be controlled, and have caused huge economic losses to ruminant production and trade in endemic areas worldwide. [0003] Bovine nodular skin disease is an imported exotic animal disease, which was first diagnosed in my country in August 2019 in Ili Kazakh Autonomous Prefecture, Xinjiang. Sin...

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
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2563/107C12Q2525/131
Inventor 南文龙陆游陈义平吴晓东王永杰哈达特木尔巴根巩明霞李林张永强赵永刚屈海龙邹艳丽张雨初王志亮
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT