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Cycleave fluorescent PCR method for detecting bovine lumpy skin disease

A technology for nodular and dermatological diseases, applied in biochemical equipment and methods, resistance to vector-borne diseases, measurement/inspection of microorganisms, etc., to achieve high sensitivity, fast detection speed, and easy operation

Inactive Publication Date: 2021-08-20
CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, there are cases of latent infection without clinical symptoms, all of which need to be confirmed by laboratory testing

Method used

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  • Cycleave fluorescent PCR method for detecting bovine lumpy skin disease
  • Cycleave fluorescent PCR method for detecting bovine lumpy skin disease
  • Cycleave fluorescent PCR method for detecting bovine lumpy skin disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Screening and Design of Primers and Probes

[0032] Bovine nodular skin disease virus (LSDV) belongs to the genus Goatpoxvirus, which also includes Goatpox virus (GTPV) and Sheeppox virus (SPPV). Viruses have high genome homology.

[0033] In order to avoid false positives when detecting LSDV, 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, MN642592.1, MN642592.1, MN07.1 .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, MH381810.1, KC951854.1, MW020570.1, MN072622.1, MN072625.1, MN1723.607.1 1) and all 13 S...

Embodiment 2

[0047] Embodiment 2: the sensitivity experiment of the primer pair of screening, probe

[0048] 1. Preparation of positive plasmid standard

[0049] 1) Preparation of positive plasmid

[0050] The Chinese epidemic strain LSDV / China / XJ / 2019-1 virus was isolated, identified, preserved and provided by the China Center for Animal Health and Epidemiology. 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:

[0051] The reaction solution is 25 μL per tube, containing 12.5 μL of 2×Platinum Super Green PCR Mix reaction solution, 1 μL of 10 pmol / μL LSD forward and LSD 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°C for 5 minute...

Embodiment 3

[0062] Embodiment 3 repeatability experiment

[0063] 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:

[0064] (1) Reaction system preparation: 25 μL of reaction solution per tube, containing 12.5 μL of 2×Cycleave PCR reaction solution, 0.6 μL of 5 pmol / μL LSD forward and 0.6 μL of LSD reverse, 0.8 μL of 5 pmol / μL LSD probe, and 8.5 μL of double distilled water , 2 μL of the DNA template of the sample to be detected. Among them, the Cycleave PCR detection kit (catalogue number: CY505A) was purchased from Bao Biological Engineering (Dalian) Co., Ltd. products. (2) Reaction condition setting: pre-denaturation at ...

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Abstract

The invention provides a Cycleave fluorescent PCR primer pair, a probe and a detection method applying the primer pair and the probe. The provided primer pair has a sequence of a forward primer as shown in SEQ ID NO:1 and a sequence of a reverse primer as shown in SEQ ID NO:2; and the probe has a sequence as shown in SEQ ID NO:3. The provided primers, the probe and the method for detecting bovine lumpy skin disease viruses through the Cycleave fluorescent PCR have advantages of high detection sensitivity, good specificity, high flux and easy and convenient operation.

Description

technical field [0001] The invention belongs to the technical field of detecting bovine disease pathogenic microorganisms, and in particular relates to a pair of Cycleave fluorescent PCR primers and a probe for detecting bovine nodular dermatosis and a detection method using the pair of primers and the probe. Background technique [0002] Lumpy skin disease (LSD) is a bovine nodular skin disease virus (Lumpy skin disease virus, LSDV) caused by poxviridae (Poxviridae), capripoxvirus (Capripoxvirus, CaPV) It is an acute and subacute infectious disease characterized by fever, nodular lesions on the skin and mucous membranes, and enlarged lymph nodes. [0003] Cattle are the specific host of the virus, and the onset can lead to decreased milk production in cows, transient or permanent infertility in bulls, abortion in pregnant cows, leather damage, and secondary bacterial infection leading to death. The disease was prevalent in many countries in Africa, the Middle East, and Eas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/166C12Q2563/107C12Q2545/113Y02A50/30
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
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT