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Nucleic acid combination for detecting pseudorabies virus and application of nucleic acid combination, and kit and method for detecting pseudorabies virus

A technology of pseudorabies virus and kit, applied in the direction of microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of difficult and accurate collection of trigeminal ganglion, missed detection, etc., achieve high sensitivity, avoid The effect of missing detection and improving the detection rate

Pending Publication Date: 2017-05-10
成都海关技术中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in actual testing, the tester does not always take samples personally, and some parts such as the trigeminal ganglion are also difficult to collect accurately. If samples with low virus content such as blood are used for testing, there may be missed detection

Method used

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  • Nucleic acid combination for detecting pseudorabies virus and application of nucleic acid combination, and kit and method for detecting pseudorabies virus
  • Nucleic acid combination for detecting pseudorabies virus and application of nucleic acid combination, and kit and method for detecting pseudorabies virus
  • Nucleic acid combination for detecting pseudorabies virus and application of nucleic acid combination, and kit and method for detecting pseudorabies virus

Examples

Experimental program
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Effect test

Embodiment 1

[0052] The present embodiment provides a nucleic acid combination for detecting pseudorabies virus, which includes a first primer pair and a first probe corresponding to the first primer pair, and a second primer pair and a second probe corresponding to the second primer pair. Needle. The first pair of primers and the first probe are designed according to the conserved sequence of the gB gene of pseudorabies virus, and can be used to detect whether the sample contains pseudorabies virus. The second primer pair and the second probe are designed according to the conserved sequence of the gE gene of pseudorabies virus. By adding the second primer pair and the second probe, it is possible to further distinguish all pseudorabies viruses while detecting whether the sample contains pseudorabies virus. Whether the detected pseudorabies virus is a field strain (gB positive, gE positive) or a gE-deleted commercial vaccine strain (gB positive, gE negative).

[0053] It should be noted t...

Embodiment 2

[0061] This embodiment provides a kit for detecting pseudorabies virus, which includes the nucleic acid combination provided in Embodiment 1. The kit can be used to detect whether a sample contains pseudorabies virus on the ddPCR platform, and at the same time distinguish the detected pathogen, that is, whether the detected pseudorabies virus is a wild strain or a vaccine strain with gE gene deletion, Moreover, the kit provided in this embodiment has good sensitivity and specificity, and can avoid the occurrence of missed detection caused by the small amount of virus contained in the sample.

[0062] Of course, in other embodiments, the kit for detecting pseudorabies virus provided by the present invention may also include one or more of PCR reaction buffer, dNTP, Taq DNA polymerase and the like when performing ddPCR.

Embodiment 3

[0064] This embodiment provides a method for detecting pseudorabies virus, specifically as follows.

[0065] 1. Prepare ddPCR reaction system: 10 μL of 2×ddPCR master mix, 0.9 μL of each primer (900nM) (0.9 μL each of gB-F, gB-R, gE-F, gE-R), and 0.5 μL of each probe (250nM) (0.5 μL each for gB-probe and gE-probe), 2 μL of the DNA template of the sample to be tested, and make up to 20 μL with DEPC-treated water.

[0066] The base sequences of gB-F, gB-R, gE-F, gE-R, gB-probe, and gE-probe are the same as in Example 1.

[0067] 2. Generate microdroplets: microdroplets are formed by wrapping ddPCR reaction system with microdroplet generating oil, the volume ratio of microdroplet generating oil to ddPCR reaction system is 7:2, and the number of microdroplets is 1×10 4 indivual. In this example, the ddPCR reaction system was coated with 75 μL of droplet generating oil on a ddPCR instrument (QX100 microdroplet digital PCR instrument, Bio-Rad, USA) to form microdroplets.

[0068]...

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Abstract

The invention provides a nucleic acid combination for detecting pseudorabies virus and application of the nucleic acid combination, and a kit and a method for detecting the pseudorabies virus, and relates to the technical field of pseudorabies virus detection. The nucleic acid combination for detecting the pseudorabies virus comprises a first primer pair designed according to a conserved sequence of a gB gene of the pseudorabies virus, and a first probe, wherein the first primer pair comprises two primers, and the base sequences of the two primers are respectively as shown in SEQ ID No. 1 and SEQ ID No. 2; the base sequence of the first probe is as shown in SEQ ID No. 3. After the nucleic acid combination is adopted, rabies virus can be rapidly and conveniently detected on a digital polymerase chain reaction (PCR) platform, samples with low rabies virus content can be detected, and missed detection is avoided; the nucleic acid combination has the characteristics of being high in sensitivity, high in specificity, high in detection rate, and the like.

Description

technical field [0001] The invention relates to the technical field of pseudorabies virus detection, in particular to a nucleic acid combination for detecting pseudorabies virus and its application, kit and method. Background technique [0002] At present, pseudorabies virus (Pseudorabies, PRV) belongs to Herpesviridae (Herpesvirdae) a-herpesvirus subfamily (Alpherpesvirinae), is a kind of pregnancy sow abortion, stillbirth, weak fetus, mummified fetus, newborn piglet fever, neurological symptoms , paralysis, exhaustion and death are characterized by infectious diseases, and its mortality rate can reach 100%. Pseudorabies is one of the most serious infectious diseases that harm the pig industry and has caused huge economic losses to the pig industry. The disease exists widely in our country and has caused huge losses. [0003] At present, the detection methods of nucleic acid are mainly traditional PCR, fluorescent quantitative PCR and nucleic acid hybridization technology....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/705C12Q2600/16C12Q2537/143C12Q2563/107C12Q2563/159
Inventor 林华陈世界肖艳杨苗任梅渗安微孙颖杰严玉宝胡娟薛昌华
Owner 成都海关技术中心
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