Fluorescent RT-PCR primers, probe, kit and detection method for detecting bovine torovirus in sample

A RT-PCR and fluorescent probe technology, applied in the field of in vitro nucleic acid detection, can solve the problems of reducing the specificity and sensitivity of ELISA methods, long experimental period, aerosol pollution, etc. The effect of pollution

Inactive Publication Date: 2017-02-15
SHENZHEN BIOEASY BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the ELISA method is a fast and cheap method, because the current bulge virus cannot be cultured in cells, when the excreta or intestinal contents of the diseased animals are used as the antigen source, the experiment produces a high background, which greatly reduces the ELISA. Method specificity and sensitivity
[0004] Sichuan Agricultural University applied for a patent "A RT-PCR Detection Kit for Porcine Ring Aspergillus Virus and Its Detection Method" in 2012, applica...

Method used

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  • Fluorescent RT-PCR primers, probe, kit and detection method for detecting bovine torovirus in sample
  • Fluorescent RT-PCR primers, probe, kit and detection method for detecting bovine torovirus in sample
  • Fluorescent RT-PCR primers, probe, kit and detection method for detecting bovine torovirus in sample

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The linear experiment of embodiment 1 kit

[0059] The target target probe is labeled with a FAM fluorescent group. The linear reference product of the FAM channel of the kit is composed of a plasmid containing the target fragment of bovine bulge virus. The positive plasmid is diluted 10 times with TE buffer to obtain the following gradient solution (S1: 1.0×10 9 copies / ml, S2: 1.0×10 8 copies / ml, S3: 1.0×10 7 copies / ml, S4: 1.0×10 6 copies / ml, S5: 1.0×10 5 copies / ml, S6: 1.0×10 4 copies / ml, S7: 1.0×10 3 copies / ml) as a linear reference product, the experiment shows that the kit is at 1.0×10 9 copies / ml to 1.0×10 3 There is a very good linear relationship between copies / ml, and the linear equation fitted by the kit is: Y=-3.4412x+44.938, correlation coefficient R2=0.99991, where Y represents the Ct value, and x represents the pair of positive reference products value. Experimental result reference figure 1 , standard curve reference figure 2 .

Embodiment 2

[0060] The minimum detection limit experiment of embodiment 2 kit

[0061] The lowest detection limit of the experimental verification kit, using S6 in the linear reference product: 1.0×10 4 copies / ml, S7: 1.0×10 3 copies / ml, S8: 1.0×10 2 Positive plasmids with the same concentration of copies / ml are used as detection limit reference products, and 20 repeated samples are carried out for each concentration. Among them, the detection limit reference products S6 and S7 all show amplification curves, which are positive data, and S8 reference products Only 9 samples amplified S-shaped curves, which did not meet the requirements, so this experiment shows that the detection limit of the kit is 1.0×10 3 copies / ml, that is, 5 copies / reaction, the experimental results refer to image 3 .

Embodiment 3

[0062] The precision experiment of embodiment 3 kits

[0063] This experiment verifies the intra-assay precision of the kit, using S4 in the linear reference product: 1.0×10 6 copies / ml, S6: 1.0×10 4 Two high and low concentrations of positive plasmids, such as copies / ml, were used as precision reference products, and 10 repeated samples were made each. As a result, the coefficient of variation of the two concentration data was less than 5%. The experimental results refer to Figure 4 .

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Abstract

The invention discloses fluorescent RT-PCR primers and a fluorescent probe for detecting bovine torovirus. The primers and the fluorescent probe include a pair of specific forward and reverse primers and a fluorescent probe which are designed in accordance with the conserved sequence of an M gene of the bovine torovirus. The invention also discloses a kit and a detection method for detecting the bovine torovirus. The pair of specific probes and primers are designed are in accordance with the conserved sequence of the gene of the bovine torovirus; it can make a definite diagnosis that a sample is infected with the bovine torovirus when a result of PCR detection is positive; the probe and the primers have the characteristics of being convenient and rapid, high in specificity and high in sensitivity; and the probe and the primers are applicable to the rapid diagnosis of the bovine torovirus in living cattle and cattle meat quarantine as well as to epidemiology studies.

Description

technical field [0001] The invention belongs to the field of in vitro nucleic acid detection, in particular to a fluorescent RT-PCR primer, probe, kit and detection method for detecting Porcine Torovirus (PToV) in a sample, which can be used for the quarantine of cattle, live animals and meat products Rapid diagnosis and epidemiological study of bovine bulge virus. Background technique [0002] Torovirus (Torovirus) is an important member of the Coronaviridae (Coronaviridae) family in the order Nidovirairs, including Equine Torovirus (EToV, originally called Berne virus), Bovine Torovirus (BToV, originally Breda virus), Porcine Torovirus (PToV) and Human Torovirus (HToV). Convex virus has an enveloped virus, which is round, oval, linear or kidney-shaped, and there are also records of rod-shaped and small disc-shaped, with a maximum diameter of 120-140nm. 28,473kb. The virus infects humans and animals, mainly causing digestive and respiratory diseases. It has been more th...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2545/101C12Q2545/113C12Q2561/101
Inventor 李宏朱海黄超杰游腾飞付辉卢和华毕思远汪凤林严义勇
Owner SHENZHEN BIOEASY BIOTECHNOLOGY CO LTD
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