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Fluorescent quantitative RT-PCR primer pair, probe, kit and detection method for detecting tilapia lake virus

A RT-PCR, fluorescence quantitative technology, applied in the field of detection, can solve the problem of no sensitivity data, and achieve the effect of high sensitivity and high accuracy

Active Publication Date: 2021-12-07
厦门海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

"A Luohu virus Taq-man probe fluorescent quantitative PCR detection kit and detection method" is based on TiLV-AD-2016, TiLV-4-2011, TiLV-TV1, TiLV-TV2, TiLV-TV3, TiLV-TV4, TiLV-TV5, TiLV-TV6, TiLV-TV7 and their labs isolated from two strains of TiLV in Guangdong and other 11 TiLV S1 gene sequence designed primer probes; no sensitivity data

Method used

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  • Fluorescent quantitative RT-PCR primer pair, probe, kit and detection method for detecting tilapia lake virus
  • Fluorescent quantitative RT-PCR primer pair, probe, kit and detection method for detecting tilapia lake virus
  • Fluorescent quantitative RT-PCR primer pair, probe, kit and detection method for detecting tilapia lake virus

Examples

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Embodiment 1

[0052] Embodiment 1: the screening experiment of primer

[0053] In the design process of the (TiLV RT-qPCR) primer probe sequence of the present invention, reference was made to the complete gene sequence information of 31 fragments 1 of all 30 strains of TiLV, including: strain TH-2013 (GenBank accession number of fragment 1: MN687685), TH-2014 (GenBank accession number of Fragment 1: MN687695), TH-2015 (GenBank accession number of Fragment 1: MN687705), TH-2016-CN (GenBank accession number of Fragment 1: MN687725), TH-2016 -CU (GenBank Accession Number of Fragment 1: MN687715), TH-2017 (GenBank Accession Number of Fragment 1: MN687735), TH-2018-K (GenBank Accession Number of Fragment 1: MN687755), TH-2018-N (Fragment GenBank accession number for 1: MN687745), TH-2019 (GenBank accession number for Fragment 1: MN687765), BD-2017 (GenBank accession number for Fragment 1: MN939372), BD-2017-181 (GenBank accession number for Fragment 1: MT466437), BD-2019-E3 (GenBank Accession ...

Embodiment 2

[0058] The final concentration experiment of embodiment 2 primer, probe

[0059] See Figure 4. A to D in which the final concentrations of TiLV-qP are 0.25, 0.125, 0.0625, 0.03125 μM respectively (corresponding use concentrations are 5, 2.5, 1.25, 0.625 μM respectively). A1 / B1 / C1 / D1~A6 / B6 / C6 / D6 indicate that the final concentrations of TiLV-qF / qR are 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625 μM (the corresponding use concentrations are 10, 5, 2.5, 1.25, 0.625, 0.3125 μM). The probe TiLV-qP with an original concentration of 5 μM, and the primers TiLV-qF and TiLV-qR with an original concentration of 10 μM were respectively subjected to 10-fold serial dilution. First group, Figure 4 In A, 0.5 μL of the probe TiLV-qP with a concentration of 5 μM (corresponding to a final concentration of 0.25 μM) was added to the six fluorescent quantitative PCR tubes, and the concentrations of 10, 5, 2.5, and 1.25 μM were added to tubes A1 to A6. , 0.625, 0.3125 μM primers TiLV-qF and Ti...

Embodiment 3

[0060] Example 3 The annealing temperature and repeatability experiment of RT-qPCR

[0061] See Figure 5 Graph of RT-qPCR annealing temperature and reproducibility results. The annealing temperatures of A to E are 50°C, 55°C, 60°C, 65°C, and 70°C in sequence. The TiLV plasmid with an original concentration of 5 μg / mL was serially diluted 10 times to 5×10 -2 ~5×10 -7 μg / mL. Figure 5 In A, except for the template, the reagents were prepared according to the preferred reaction system, and evenly divided into 6 groups (each group was repeated 3 times) in a total of 18 fluorescent PCR reaction tubes. Add 5×10 -2 ~5×10 -7 ng / μL gradient diluted TiLV plasmid each 1 μL, according to the annealing temperature of 50 ℃ for RT-qPCR; Figure 5 In B, the reaction system is exactly the same, and RT-qPCR is performed according to the annealing temperature of 55°C; Figure 5 In C, the reaction system is exactly the same, and RT-qPCR is performed according to the annealing temperature...

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Abstract

The invention discloses a fluorescent quantitative RT-PCR primer pair, a probe, a kit and a detection method for detecting tilapia lake virus. The sequences of the primer pair are shown as SEQ ID NO: 1 and SEQ ID NO: 2, and the sequence of the probe is shown as SEQ ID NO: 3. The RT-qPCR method adopting the primer probe sequence to be protected is clear in sensitivity data, the sensitivity can reach 2.5 * 10 <-8 > ng / mu L, the specificity is good, and the repeatability is high.

Description

technical field [0001] The invention relates to the detection field, in particular to a fluorescent quantitative RT-PCR primer pair and probe, a kit and a detection method for detecting Luohu virus. Background technique [0002] Tilapia is a major source of dietary protein in many developing countries and an important source of income for many fishers and farmers. Tilapia farming has become an important industry for ensuring global food security and meeting human nutritional needs. [0003] Since 2009, Israel, Ecuador, Egypt, Thailand, Colombia and other countries have successively experienced massive deaths of farmed and wild tilapia. It was not until 2014 that the causative agent was discovered to be a new type of RNA virus - Luohu virus. Luohu virus is a virus that harms tilapia first discovered by American scientists in 2012. It is a new type of RNA virus consisting of 10 unique gene segments with a diameter of 55-75nm. Its taxonomic status has not yet been determined ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2545/113C12Q2563/107Y02A50/30
Inventor 徐淑菲孔凡德朱黄鑫曾韵颖刘启霖林双庆陈信忠方成俊
Owner 厦门海关技术中心
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