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Method for detecting infectious bovine rhinotracheitis virus and application thereof

The technology of a rhinotracheitis virus and a detection method is applied in the detection of viral infectious diseases and the detection of bovine infectious rhinotracheitis virus, and can solve the problems of inability to correctly determine the copy number of a target gene, false negatives, and reduced sensitivity, etc. To achieve the effect of short detection time, convenient operation and high sensitivity

Inactive Publication Date: 2010-05-05
新疆出入境检验检疫局检验检疫技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the presence of a large number of complex unknown components in the sample, and the residue of some substances in the sample processing process will affect the PCR amplification, which will reduce the sensitivity of the reaction measurement and even cause false negative results. For fluorescent PCR detection, it is impossible to correctly determine the target gene. copy number
[0004] After searching, there is no report on the fluorescent quantitative PCR detection method of detecting BHV-1 with high detection sensitivity, good specificity, convenient operation, and the ability to indicate and calibrate false negative results and improve accuracy at home and abroad.

Method used

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  • Method for detecting infectious bovine rhinotracheitis virus and application thereof
  • Method for detecting infectious bovine rhinotracheitis virus and application thereof
  • Method for detecting infectious bovine rhinotracheitis virus and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0068] Embodiment one: detection of bovine infectious rhinotracheitis virus

[0069] 1. Design and synthesis of primers and probes:

[0070] According to the BHV-1gB gene sequence, specific primers Ip2, Ir2 and fluorescent probe ITpro of 141 bp were amplified, and primers Ip1 and Ir1 were constructed as templates, in which Ip1 and Ir1 were located outside Ip2 and Ir2, and a fragment with a length of 787 bp was amplified. The probe ITpro sequence was rearranged, primers Ic1 and Ic2 were designed, and the internal standard probe ICpro was constructed by using the principle of base mutation and the artificially added linker sequence.

[0071] 2. Preparation of target DNA template:

[0072] BHV-1 was propagated by cell culture, and after freezing and thawing, the supernatant was taken to extract viral DNA with DNAZo1 extraction solution; the semen was filtered with Sephacryl S-400 gel, and nucleic acid was extracted with DNAZol.

[0073] Viral DNA was used as a template, and Ip1...

Embodiment 2

[0084] Embodiment 2: Obtaining the target template

[0085] BHV-1 was propagated by cell culture, and after freezing and thawing, the supernatant was taken to extract virus DNA with DNAZol extraction solution, and the semen was filtered with Sephacryl S-400 gel, and then nucleic acid was extracted with DNAZol.

[0086] Use viral DNA as a template, use Ip1 and Ir1 as amplification primers for PCR amplification, 20 μL reaction system, in which: Mg 2+ 1.5mmol / L, 0.25mmol / L each dNTP, 1mmol / L each primer, 1.25u Taq DNA polymerase, 5μL template. The amplification conditions were: denaturation at 95°C for 5 minutes; 35 cycles of 96°C for 1 minute, 50°C for 1 minute, and 72°C for 2 minutes; and finally extension at 72°C for 10 minutes. The amplified product was electrophoresed with 1% agarose, purified and recovered by gel, connected to the pMD18-T vector, transformed into DH5α competent cells, and the plasmid was extracted for identification by PCR, enzyme digestion and sequencing....

Embodiment 3

[0091] Embodiment three: the construction of internal standard template

[0092] Using plasmid IT as a template, primers Ip1, Ic1 and Ic2, Ir1 were used as paired primers for PCR amplification, 20 μL reaction system, in which Mg 2+ 1.5mmol / L, 0.25mmol / L each dNTP, 1mmol / L each primer, 1.25u Taq enzyme, 5μl template. The amplification conditions of Ip1 and Ic1 were: 95°C for 5 min; 96°C for 1 min, 56°C for 45 s, 72°C for 45 s, 35 cycles; 72°C for 10 min. The amplification conditions of Ic2 and Ir1 were: 95°C for 5 minutes; 96°C for 1 minute, 62°C for 45s, 72°C for 45s, 35 cycles; 72°C for 10 minutes. Fragments of 227bp and 588bp were amplified respectively, and named Ip1c1 and Ic2r1, see attached Figure 4 .

[0093] Ip1c1 and Ic2r1 were mixed in equal amounts as a template, and PCR amplification was performed with primers Ip1 and Ir1, and the reaction system and amplification conditions were the same as those in Examples 1 and 2. Purify and recover the amplified product an...

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Abstract

The invention discloses a method for detecting infectious bovine rhinotracheitis virus. Internal amplification control (IAC) markers indicating the false negative are added to a polymerase chain reaction (PCR) system; an IAC fragment is built through the rearrangement of the base, the design of the primers and the PCR gene amplification with the bypass method; the added concentration of an internal maker template is 10<2> MuL in each reaction process, the added concentration of the Mg2+ is 2.0 mmol / L, the added concentration of the each dNTP is 0.3 mmol / L, the added concentration of each primer is 0.5 mmol / L, and the added concentration of a probe is 0.2 mmol / L; the primers are amplified at 50 DEG C for 2min in 1 cycle, 95 DEG C for 5min in 1 cycle, 95 DEG C for 15s in 45 cycles and 60 DEG C for 60s in 45 cycles. Therefore, the existence of inhibitory components or the occurrence of false negative indication can be accurately showed. The method is 10-100 times more sensitive than the conventional PCR method and the virus isolation test method and can be widely used for detecting the infectious bovine rhinotracheitis virus.

Description

field of invention [0001] The invention belongs to the field of virus detection, in particular to a detection method of viral infectious diseases, in particular to a detection method and application field of bovine infectious rhinotracheitis virus. Background technique [0002] Infectious bovine rhinotracheitis (IBR) is a viral infectious disease of cattle caused by bovine herpes virus 1, which is referred to as BHV-1. Infected cows often have clinical symptoms such as severe respiratory infection, conjunctivitis, encephalitis, decreased milk production, metritis, enteritis and abortion. The disease is widely distributed in the world, and serological surveys in some provinces in my country also show the existence of the disease. The World Organization for Animal Health (OIE) lists IBR as a B-type animal disease. my country's Animal and Plant Quarantine Law also clearly stipulates that imported and exported cattle must be tested for IBR in order to control the occurrence and...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12R1/93
Inventor 季新成员丽娟黄玲段晓东田延河杨忠于学辉牛国辉
Owner 新疆出入境检验检疫局检验检疫技术中心
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