Fluorescence RT-PCR primer, probe and kit for detecting Schmallenberg viruses

A technology of RT-PCR and kits, applied in the field of probes and kits, and fluorescent RT-PCR primers, can solve the problems of time-consuming and laborious, difficult to grasp the specific period of viremia, unsuitable for rapid detection, etc., and achieve low cost, good repeatability

Inactive Publication Date: 2015-03-25
中华人民共和国珠海出入境检验检疫局
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Virus isolation is usually the gold standard for diagnosing epidemic diseases. However, since adult sick animals often do not show clinical symptoms, it is difficult to grasp the specific period of viremia, and it is time-consuming and laborious. Therefore, the implementation of virus isolation is subject to certain restrictions and is not suitable for rapid detection. need

Method used

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  • Fluorescence RT-PCR primer, probe and kit for detecting Schmallenberg viruses
  • Fluorescence RT-PCR primer, probe and kit for detecting Schmallenberg viruses
  • Fluorescence RT-PCR primer, probe and kit for detecting Schmallenberg viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Primer Design

[0023] Download multiple nucleocapsid protein gene sequences of Schmallenberg virus (SBV) from Genebank, compare them with DNASTAR 5.07 software, find highly conserved regions, and then select appropriate nucleocapsid protein gene sequences to design two sets Primers and Taqman probes for specific detection of the SBV nucleocapsid protein gene were sent to Dalian Bao Biotechnology Co., Ltd. for synthesis, dissolved in DEPC-treated water, and stored at -80°C in the dark. See Table 1 for primers and probes:

[0024] Table 1 SBV fluorescent RT-PCR primers and probes

[0025]

Embodiment 2

[0026] Embodiment 2 kit composition

[0027] 1. Primers: as shown in Table 1.

[0028] 2. Enzymes: Reverse Transcriptase XL (hereinafter referred to as AMV), TaKaRa Ex TaqTM DNA Polymerase (hereinafter referred to as Ex TaqTM), PrimeScript 1step Enzyme Mix, 2×1Step Buffer are products of Treasure Bioengineering (Dalian) Co., Ltd.

[0029] 3. Fluorescence PCR reagent

[0030] QuantiFast ? RT-PCR for QIAGEN, Germany ? company's product.

[0031] 4. RNA extraction reagent

[0032] Total RNA purification reagent (Trizol) was a product of Invitrogen, USA.

[0033] 5. Schmallenberg virus positive control and negative control

[0034] Positive control: Send the target sequence of the SBV nucleocapsid protein gene to be amplified to Bao Bioengineering (Dalian) Co., Ltd. for whole gene synthesis to prepare the recombinant plasmid pMD-SBV of the target fragment, and place it in LB bacterial culture medium containing Amp Culture was carried out, and the bacterial liquid was har...

Embodiment 3

[0036] Embodiment 3 The establishment of fluorescent RT-PCR reaction system and reaction program

[0037] 1. Viral RNA extraction

[0038] (1) Take n sterilized 1.5 mL Eppendorf tubes, where n is the sum of the tested sample, positive control and negative control (positive control and negative control are marked in the kit), numbered.

[0039] (2) Add 600 μL of lysate to each tube, add 200 μL each of the tested sample, negative control, and positive control, and then add 200 μL of chloroform, shake and mix for 5 s on the mixer (not too strong, so as not to produce an emulsified layer , can also be mixed by hand upside down). Centrifuge at 4°C, 12 000 r / min for 15 min.

[0040] (3) Take the same number of sterilized 1.5 mL Eppendorf tubes as in (1), add 400 μL of isopropanol (pre-cooled at -20 ℃), and mark it. Aspirate the supernatant in each tube of this standard (2) and transfer it to the corresponding tube. The supernatant should be aspirated at least 500μL, and the m...

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Abstract

The invention discloses a fluorescence RT-PCR primer, a probe and a kit for detecting Schmallenberg viruses. The sequences of the primer and the probe are shown as SEQ ID NO. 1-6. The kit comprises the primer, the probe, reverse transcriptase, DNA polymerase, an RNA extraction reagent, a fluorescence PCR reaction reagent, a Schmallenberg virus positive control and a Schmallenberg virus negative control. The Schmallenberg virus fluorescence RT-PCR kit and a detection method provided by the invention are specific, sensitive and quick, have good repeatability and low costs, and the detection method is a favorable method for quickly detecting Schmallenberg viruses.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, in particular to a fluorescent RT-PCR primer, a probe and a kit for detecting Schmallenberg virus. Background technique [0002] Schmallenberg disease (Schmallenberg, SB) is a new viral zoonotic disease found in cattle and sheep in Schmallenberg, North Rhine-Westphalia, Germany in November 2011. The main clinical features of the disease are: abortion and stillbirth of infected ewes, deformity of newborn lambs, decreased survival rate, diarrhea, fever, and decreased milk production of infected cattle, which have caused great harm to animal husbandry. [0003] The current laboratory diagnostic methods for SB mainly include pathogen detection methods such as cell culture isolation of viruses, and serological methods such as enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA) and neutralization test (NT). Virus isolation is usually the gold standard for diagn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701
Inventor 杨素陶旻陈轩黄海超徐海聂沙才华廖秀云罗宝正薄清如
Owner 中华人民共和国珠海出入境检验检疫局
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