Cycleave fluorescent PCR method for detecting Lumpy Skin Disease (LSD)

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 the effect of simple operation, high sensitivity and fast detection speed

Active Publication Date: 2021-12-03
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 tests

Method used

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  • Cycleave fluorescent PCR method for detecting Lumpy Skin Disease (LSD)
  • Cycleave fluorescent PCR method for detecting Lumpy Skin Disease (LSD)
  • Cycleave fluorescent PCR method for detecting Lumpy Skin Disease (LSD)

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 (Lumpy skin disease virus, LSDV) belongs to the genus Goatpoxvirus, which also includes goatpox virus (GTPV) and sheep poxvirus (Sheeppox virus, SPPV). Studies have shown that three Viruses have high genome homology.

[0033] In order to avoid false positives in the detection of 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, MN07.1K06, MN072 .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, MH...

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. A commercial viral genome extraction kit was used to extract the viral DNA template according to the instructions, and then 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 fo...

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, Cycleave PCR detection kit (catalogue number: CY505A) was purchased from Treasure Bioengineering (Dalian) Co., Ltd. products. (2) Reaction condition setting: pre-denaturation at 95°C fo...

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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. According to the provided primer pair and the probe, the sequence of a forward primer of the primer pair is SEQ ID NO: 1, and the sequence of a reverse primer of the primer pair is SEQ ID NO: 2; and the sequence of the probe is SEQ ID NO: 3. The primer, the probe and the method for detecting the Lumpy Skin Disease (LSD) virus through the Cycleave fluorescent PCR, which are provided by the invention, have the advantages of high detection sensitivity, good specificity, high flux and easiness and convenience for operation.

Description

technical field [0001] The invention belongs to the technical field of detection of 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 reduced 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 once popular in many countries in Africa, the Middle East, 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亮
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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