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Nucleic acid, kit and droplet type digital PCR method for detecting infectious bovine rhinotracheitis virus

A rhinotracheitis virus, infectious technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., to achieve the effects of good airtightness, high accuracy, and avoidance of pollution

Inactive Publication Date: 2020-08-21
北京市动物疫病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] For the gB gene of IBRV, the present invention establishes and evaluates a droplet-based digital PCR detection kit to achieve the purpose of accurately detecting IBRV. At present, there is no such kit for detecting IBRV at home and abroad.

Method used

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  • Nucleic acid, kit and droplet type digital PCR method for detecting infectious bovine rhinotracheitis virus
  • Nucleic acid, kit and droplet type digital PCR method for detecting infectious bovine rhinotracheitis virus
  • Nucleic acid, kit and droplet type digital PCR method for detecting infectious bovine rhinotracheitis virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 The establishment of IBRV ddPCR method

[0037] The gB gene sequence of IBRV published by GenBank (if available, accession number: KU198480.1\JN787952.1\KY215944.1\MH751901.1\MG407790.1\KM258883.1\KM258880.1\KM258881.1\JK898220.1 etc.) carried out sequence comparison, and further determined the conserved region of the IBRV gB gene (the 1800-2100th DNA fragment, its sequence is as shown in the sequence of SEQ ID NO.4), and designed for the head, middle and tail of the conserved region Primers and probes were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.; the 5' end of the probe was labeled with a FAM fluorescent group, and the 3' end of the probe was labeled with a BHQ1 quencher group. The designed primers and probe sequences are described in part, and the specific sequences are shown in Table 1.

[0038] Table 1 The sequences of primers and probes for IBRV ddPCR detection

[0039]

[0040] * Indicates the position in the gB gene.

[0041] 2...

Embodiment 2I

[0057] The optimization of embodiment 2 IBRV ddPCR annealing temperature

[0058] The synthetic IBRV gB gene DNA plasmid (pIBRV / ddPCR plasmid) in embodiment 1 is diluted to 1.0 * 10 5 Copies / μL is the template for ddPCR detection. The ddPCR reaction system includes 10 μL of 2×ddPCR Supermix, 2 μL of DNA template, 1.8 μL of forward primer (SEQ ID NO.1) (10 pmol / μL), reverse primer (SEQ ID NO. .2) (10 pmol / μL) 1.8 μL, probe (SEQ ID NO.3) (10 pmol / μL) 0.5 μL, DNase-free water 3.9 μL, the total volume is 20 μL.

[0059] Make 6 duplicate wells, transfer to the droplet generation card, and place it on the droplet generator to generate droplets. The generated micro-droplets were transferred to a 96-well reaction plate and sealed with a PX1 heat sealer at 180°C for 5 s. Place the sealed 96-well reaction plate on a PCR machine for amplification.

[0060] The reaction conditions are: pre-denaturation at 95°C for 10 minutes; denaturation at 94°C for 30 seconds, annealing at 52°C, 54°C...

Embodiment 3I

[0061] The determination of embodiment 3IBRV ddPCR primer concentration and probe concentration

[0062] The synthetic IBRV gB gene DNA plasmid (pIBRV / ddPCR plasmid) in embodiment 1 is diluted to 1.0 * 10 5 Copies / μL is the template for ddPCR amplification. The ddPCR reaction system includes 10 μL of 2×ddPCR Supermix forProbes, 2 μL of DNA template; the final concentration of forward primer (SEQ ID NO.1) and reverse primer (SEQ ID NO.2) 500-900nM, the final concentration of the probe (SEQ ID NO.3) is 100-300nM, the final concentration of the primer is increased by 100nM, and the final concentration of the probe is increased by 50nM for orthogonal test; the rest is made up to 20μL with DNase-free water .

[0063] The reaction parameters were: pre-denaturation at 95°C for 10 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, 40 cycles; 10 min at 98°C; 4°C∞, heating and cooling rate 2°C / s.

[0064] The results show that the final concentration of forward primer and...

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Abstract

The invention provides a nucleic acid for detecting infectious bovine rhinotracheitis virus, a kit and a droplet type digital PCR method. The nucleic acid comprises a forward primer, a reverse primerand a probe, the sequence of the forward primer is shown as SEQ ID NO. 1, the sequence of the reverse primer is shown as SEQ ID NO. 2, and the sequence of the probe is shown as SEQ ID NO. 3. The sensitivity of the droplet type digital PCR detection kit for detecting the infectious bovine rhinotracheitis virus is 1copies / mu L, and only the infectious bovine rhinotracheitis virus can be specificallydetected. When the kit is used for detecting suspected samples of infectious bovine rhinotracheitis viruses, results show that the accuracy of the kit is higher than that of existing qPCR, detectionof trace viruses can be achieved, and absolute quantification can be conducted on nucleic acid without a standard curve. The detection method provided by the invention can realize high-specificity, high-sensitivity and absolute quantitative detection of the infectious bovine rhinotracheitis virus.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a nucleic acid, a kit and a droplet digital PCR method for detecting bovine infectious rhinotracheitis virus. Background technique [0002] Droplet Digital PCR (ddPCR), as the third-generation nucleic acid amplification technology, can process the reaction system into droplets before the amplification reaction to form tens of thousands to hundreds of thousands of liquefied water-in-oil Micro-droplets, nucleic acid molecules are distributed in each micro-droplet. After PCR amplification, the fluorescent signal is detected by the micro-droplet detector. The initial copy number of nucleic acid can be calculated according to the principle of Poisson distribution and the number of positive micro-droplets, which can realize nucleic acid High sensitivity and precise quantification. Droplet digital PCR has been widely used in the fields of gene copy number variation research, tr...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/705C12Q2531/113C12Q2563/159
Inventor 高晓龙程敏姮李月冯小宇梅力王英超韦海涛宋彦军
Owner 北京市动物疫病预防控制中心
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