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Fluorescent quantitative PCR method for detecting infectious spleen and kidney necrosis virus of mandarin fish and corresponding kit

A spleen and kidney necrosis virus, fluorescence quantitative technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of time-consuming, complicated operation, and inability to meet fast, simple and sensitive detection, etc., and achieve high sensitivity High performance, wide application range and stable detection results

Pending Publication Date: 2020-08-04
广东美格基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the detection methods for ISKNV include pathological anatomy, electron microscope observation, PCR detection and nucleic acid probe in situ hybridization. , simple and sensitive detection
Fluorescent quantitative PCR detection method has strong specificity, high sensitivity, and can detect diseases qualitatively or quantitatively. It is currently recognized as the most accurate and rapid disease detection method. At present, no systematic QPCR diagnostic technology for ISKNV has been established for the conserved sequence of ISKNV. , the present invention has found three conserved sequences ATPase, MCP and IRB6 by comparing and analyzing the whole genome sequence of ISKNV, designed highly specific primer probes, and carried out qualitative or quantitative detection of ISKNV, which can be used for the diagnosis and prevention of mandarin fish pathogens. control is important

Method used

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  • Fluorescent quantitative PCR method for detecting infectious spleen and kidney necrosis virus of mandarin fish and corresponding kit
  • Fluorescent quantitative PCR method for detecting infectious spleen and kidney necrosis virus of mandarin fish and corresponding kit
  • Fluorescent quantitative PCR method for detecting infectious spleen and kidney necrosis virus of mandarin fish and corresponding kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1 detection primer amplification standard curve and plasmid sensitivity experiment

[0061] 1. Microbial culture

[0062] The LB medium is used as the medium of the mandarin fish infectious spleen-kidney necrosis virus plasmid, and it grows well in the plate with pH 7.0~7.4. The diameter of the colony on the plate is 2mm, round, smooth and transparent. The vigorously growing microbial samples were selected for subsequent experiments.

[0063] 2. Genomic DNA Extraction

[0064] Genomic DNA was extracted according to the instructions of Qiagen's QIAamp DNA Mini Kit.

[0065] 3. Production of standard plasmids for amplified products

[0066] DNA extracted from mandarin fish infectious spleen-kidney necrosis virus was used to amplify with primers of ATPase, MCP and IRB6 genes respectively. After induction of competent cells, the plasmids were amplified on a large scale. The primer sequences and TaqMan probe sequences of the three genes are shown in SEQ ID N...

Embodiment 2

[0084] Example 2 Plasmid Repeatability Experiment

[0085] 1. Microbial culture

[0086] With embodiment 1.

[0087] 2. Genomic DNA Extraction

[0088] With embodiment 1.

[0089] 3. qPCR detection of 3 target genes

[0090] Carry out qPCR amplification of the sample according to the instructions of TaKaRa Taq PCR Mix, and obtain the Ct value reading corresponding to each pair of primers. The qPCR reaction in this experiment was set at two concentrations of 1×10 6 fg / μL and 1×10 5 fg / μL, 4 biological replicates and 5 technical replicates were set up for each concentration, and the qPCR reaction system and amplification reaction conditions are shown in Table 7.

[0091] Table 7

[0092]

[0093] 4. Result analysis

[0094] In this example, the correlation coefficient and reliability of the amplification curves of the three genes were analyzed, as well as the amplification efficiencies at two concentrations. The measured CT values ​​were all less than 35 cycles, and a...

Embodiment 3

[0102] Embodiment 3 Positive and negative sample detection situation

[0103] 1. Microbial culture

[0104] With embodiment 1.

[0105] 2. Genomic DNA Extraction

[0106] With embodiment 1.

[0107] 3. qPCR detection of 3 target genes

[0108]Carry out qPCR amplification of the sample according to the instructions of TaKaRa Taq PCR Mix, and obtain the Ct value reading corresponding to each pair of primers. Each qPCR reaction was set up with 3 biological repetitions and 3 technical repetitions, and the qPCR reaction system and amplification reaction conditions were the same as in Example 2.

[0109] 4. Result analysis

[0110] as shown in Table 11 and Figure 10 As shown, the qPCR amplification results of three pairs of primers showed that the mandarin fish infectious spleen-kidney necrosis virus showed all positive results of ATPase, MCP and IRB6 genes, while other viruses or bacterial genera showed negative results. Therefore, the detection of ATPase, MCP and IRB6 gene...

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Abstract

The invention discloses a fluorescent quantitative PCR method for detecting infectious spleen and kidney necrosis virus of mandarin fish and a corresponding kit. Specific gene detection is ingeniouslyapplied to distinguish the infectious spleen and kidney necrosis virus of the mandarin fish from strains or viruses of other species, and accurate bacterial genus information is obtained through comprehensive determination. Compared with an existing mainstream detection kit, the kit for detecting the infectious spleen and kidney necrosis virus of the mandarin fish has the advantages of being highin sensitivity, rapid, convenient to use, good in specificity, rigorous and accurate in judgment and the like, and has good application prospects and market value.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and in particular relates to a method for fluorescent quantitative PCR detection of mandarin fish infectious spleen-kidney necrosis virus through a specific gene and a corresponding detection kit. Background technique [0002] Infectious spleen and kidney necrosis virus (ISKNV) belongs to the cytomegalovirus genus in the family Iridoviridae. The mature virus particle is icosahedral with a diameter of 100nm-150nm. The genome is double-stranded DNA. The mature virus Particles generally consist of an envelope, an electron-dense region, and a core. It invades mandarin fish through endocytosis and attaches to spleen tissue cells, resulting in the appearance of a large number of basophilic, homogenized cytoplasmic swollen cells with a diameter of about 15 μm to 20 μm. [0003] Mandarin fish (Siniperca chuatsi) is one of the high-quality freshwater fish species in my country with tender mea...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686
CPCC12Q1/701C12Q1/686C12Q2563/107C12Q2545/114
Inventor 唐伟束文圣杨荣束浩然黄斌蒋华
Owner 广东美格基因科技有限公司
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