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
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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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