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Specific primers and probe for fluorescence RT-PCR detection for bluetongue virus-16

An RT-PCR and bluetongue virus technology, which is applied in the field of specific primer pairs and probes for bluetongue virus type 16 fluorescent RT-PCR detection, can solve problems such as complicated operations, and achieve efficient amplification. , good specificity, and the effect of improving inspection efficiency

Inactive Publication Date: 2015-04-29
YUNNAN ANIMAL SCI & VETERINARY INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The identification of BTV serotypes in conventional clinical samples needs to isolate the virus first, and then determine it by neutralization test. The operation is cumbersome and takes more than one month.

Method used

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  • Specific primers and probe for fluorescence RT-PCR detection for bluetongue virus-16
  • Specific primers and probe for fluorescence RT-PCR detection for bluetongue virus-16

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] (1) Preparation of the template to be tested

[0035] Method 1 Use a commercial RNA extraction kit and follow the instructions.

[0036] Method 2 Extraction method with Trizol nucleic acid extraction reagent

[0037] a. Take 100 ul of the sample and put it into a 1.5 ml centrifuge tube, then add 300 ul of Trizol tissue extract to it, and oscillate fully on the shaker. Then centrifuge at 12000rpm for 15min, and transfer the supernatant to a 1.5ml centrifuge tube;

[0038] b. Add 400ul of pre-cooled isopropanol to the supernatant, shake fully on the shaker, and centrifuge at 12,000 rpm for 10 minutes to obtain RNA precipitation;

[0039] c. Carefully pour off the supernatant, add 600ul 75% ethanol, and wash by hand upside down several times. (Note: do not shake vigorously to prevent RNA fragmentation and difficulty in re-precipitation after RNA precipitation dissolves);

[0040] d. Centrifuge at 12000 rpm for 10 min, slowly discard the supernatant, dry at room tem...

Embodiment 2

[0048] Specificity test of bluetongue virus type 16 real-time fluorescent RT-PCR method:

[0049] In the 25ul reaction system, nucleic acids of bluetongue virus and epidemic hemorrhagic fever virus (EHDV) strains of other serotypes except type 16 were added as templates, and the specific primers and probes of the present invention were used for real-time fluorescent RT -PCR detection, as a result, only bluetongue virus type 16 has a specific amplification curve, while other samples have no amplification curve, confirming that the present invention has good specificity for the primers and probes of the BTV-16 type ( figure 1 ).

Embodiment 3

[0051] Sensitivity test of bluetongue virus type 16 real-time fluorescent RT-PCR method:

[0052] In the 25ul reaction system, after extracting bluetongue virus type 16 nucleic acid to measure the concentration, make eight 10-fold serial dilutions, perform real-time fluorescent RT-PCR detection, and obtain the dilution gradient series amplification curve ( figure 2 ), it was found that the lowest dilution that could be detected was 10 -6 , equivalent to a concentration of 2.18pg / ul RNA.

[0053] Using the present invention for detection is easy and quick to operate, generally can be completed in about 3 hours, and the result can be detected in real time during the detection process, without the need for electrophoresis observation of the amplified product, which avoids the pollution of the amplified product to the experimental environment and the electrophoresis Contamination of the environment by EB at the time of observation.

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Abstract

The invention belongs to the field of virus detection, and relates to primers and a probe sequence for fluorescence RT-PCR detection for bluetongue virus serum-16. A real-time fluorescence RT-PCR detection method for BTV-16 comprises the following steps: adopting a Taqman technology, designing a group of specific primers which are only conservative in BTV-16 VP2 genes and a specific fluorescence labelling probe, and applying a fluorescence PCR instrument to detect a sample. Domestic and overseas BTV-16 viral nucleic acids can be specifically detected by using the fluorescence RT-PCR detection method established by the group of primers and the probe, and other serotypes of BTV nucleic acids cannot be detected. The method has the characteristics of being high in specificity, high in sensitivity, simple to operate, low in time consumption, high in efficiency and the like, and is capable of avoiding possible environmental pollution during a detection process.

Description

technical field [0001] The invention relates to primers and probe sequences for fluorescent RT-PCR detection of bluetongue virus type 16 (BTV-16). Background technique [0002] Bluetongue (Bluetongue, BT) is caused by bluetongue virus (Bluetongue Virus, BTV) of the Reoviridae Circovirus genus. It is transmitted by the bite of blood-sucking insects and infects ruminants to cause non-contact transmission of the disease. The arbovirus disease is characterized by ulcerative inflammatory changes in the oral cavity, nasal cavity and gastrointestinal mucosa. Bluetongue virus mainly causes morbidity and mortality in sheep; cattle and goat animals are often recessively infected, with occasional morbidity and mortality. The "Terrestrial Animal Health Code" revised by the World Organization for Animal Health (OIE) in 2009 listed bluetongue disease as a variety of zoonotic diseases that are legally reported. It is listed as a first-class animal disease in the Species List. [0003] B...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/112C12Q2563/107
Inventor 朱建波杨振兴肖雷李华春
Owner YUNNAN ANIMAL SCI & VETERINARY INST
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