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Reagent kit, method, primers and probe for quantitative detection of nucleic acid of swine fever virus

A technology for the quantification of swine fever virus and nucleic acid, which is applied in biochemical equipment and methods, microbiological measurement/inspection, and material stimulation analysis. It can solve the problems of high detection cost and hardware requirements, high false positive rate, and poor repeatability. Achieve high specificity, simplify experimental steps, and achieve reliable results

Active Publication Date: 2012-09-19
湖北万德瑞生命科学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This technology has the advantages of high degree of automation, high sensitivity, low molecular weight for detection, and high efficiency, but it also has defects such as high cost, high false positive rate, and poor repeatability.
At present, gene chip technology is far inferior to other detection methods in the detection of influenza, and the detection cost and hardware requirements are relatively high

Method used

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  • Reagent kit, method, primers and probe for quantitative detection of nucleic acid of swine fever virus
  • Reagent kit, method, primers and probe for quantitative detection of nucleic acid of swine fever virus
  • Reagent kit, method, primers and probe for quantitative detection of nucleic acid of swine fever virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] The composition of embodiment 1 kit

[0075] The kit contains: PCR reaction solution, including DEPC-treated water, HotStart Taq DNA polymerase with 5’→3’ exolytic activity, M-MLV reverse transcriptase, dNTP Mix, 10×one-step PCRBuffer, MgCl 2 Solution, CSFV Forward Primer, CSFV Reverse Primer, CSFV LNA Probe; RNA extraction solution is divided into Solution 1, Solution 2, Solution 3, Solution 4 and Solution 5. Solution 1 is Trizol reagent, solution 2 is chloroform, solution 3 is isopropanol, solution 4 is 75% ethanol, solution 5 is DEPC-treated water; negative quality control product is a plasmid DNA fragment that does not contain classical swine fever virus E2 gene; The positive quality control product is a high-concentration swine fever virus genomic DNA fragment; the critical positive quality control product is a low-concentration swine fever virus gene DNA fragment; the working standard is a 127-base-pair nucleus containing the swine fever virus E2 gene The PSG-TS ...

Embodiment 2

[0109] Method for detecting classical swine fever virus nucleic acid on ABI 7300 fluorescent quantitative PCR instrument with kit of the present invention

[0110] (1) Collect samples: collect serum, nasopharyngeal swab, throat swab or disease tissue;

[0111] Serum samples: Collect 5ml of venous blood into 10ml screw-top plastic centrifuge tubes with gaskets (without anticoagulant), centrifuge at 1,000g for 10min, draw serum under aseptic conditions, and divide into several 1ml gaskets with gaskets Put them in screw-top plastic serum tubes (100 μl / tube), and transport them to the laboratory within 48 hours of refrigeration (4-8°C).

[0112] Throat swab and nasopharyngeal swab specimens: first wet the cotton swab with normal saline (do not use specimen transport solution containing penicillin to prevent allergies), nasopharyngeal swab is collected by inserting the cotton swab parallel to the palate into the nostril, and staying After a few seconds, absorb the secretions and w...

Embodiment 3

[0131] The kit of the present invention is used to detect the classical swine fever virus nucleic acid of the clinically confirmed sample according to the method of Example 2. Sample source ×× hospital patient's confirmed sample, the test result of embodiment 3 of the present invention is as follows figure 2 As shown, the test results are shown in the table below:

[0132] serial number

[0133] Clinically confirmed samples 1, 2, 3, 4, and 6 are positive samples, and samples 5 and 7 are negative samples. The test results are consistent, and the accuracy rate is 100%.

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Abstract

The invention discloses a reagent kit, a method, primers and a probe for quantitative detection of nucleic acid of a swine fever virus and belongs to the technical field of biology. The reagent kit comprises PCR (polymerase chain reaction) liquid including DEPC (diethyl pyrocarbonate) treating water, Taq enzyme, M-MLV reverse transcriptase, RNase inhibitor, dNTP Mix, 10X one-step RT-PCR (reverse transcription-polymerase chain reaction) Buffer, MgC12 solution, the forward primer of the swine fever virus, the reverse primer of the swine fever virus and the LNA (locked nucleic acid) probe of the swine fever virus, wherein the forward primer of the swine fever virus refers to: 5'-AACGGYAGTGCTTTCTAYC-3', the reverse primer of the swine fever virus refers to: 5'-GTGGRAAAGGCTTCTCTC-3', and the LNA probe of the swine fever virus refers to: 5'-accActTctGtyCtac-3'. The reagent kit further comprises RNA (ribonucleic acid) extracting solution, negative quality control serum, working standard serum, positive quality control serum and critical positive quality control serum. The real-time fluorescent quantitative PCR technology is used for quantitatively detecting the nucleic acid of the swine fever virus, and accordingly the reagent kit has the advantages of specificity, sensitivity, rapidness and simplicity and convenience to operate.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a kit for quantitative detection of swine fever virus nucleic acid and a detection method thereof. Background technique [0002] Classical Swine Fever Virus (CSFV), belonging to Flaviviridae, Pestivirus genus, has only one serotype, but can be divided into multiple genotypes within the type. Virus particles have an envelope with a diameter of 40-50nm, and the genome is composed of single-stranded negative-strand RNA with only one large open reading frame. For the diagnosis of CSFV, the target regions targeted by nucleic acid are mainly 5'UTR, E2, NS4B and NS5B. The present invention determines that the target gene is E2, and E2 is one of four structural proteins C, Erns, E1 and E2, and is one of the main structural glycoproteins on the capsule. At the same time, studies have also shown that the E2 molecule contains four antigenic domains A, B, C and D, and the A re...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 胡萌王业富周康平邱杨刘建丽
Owner 湖北万德瑞生命科学技术有限公司
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