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Polymerase chain reaction (PCR) detection method universal for viruses

A detection method and virus technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of low repeatability, high quality requirements of virus samples, and not well promoted, and achieve the solution Cumbersome, specific and reproducible effects

Active Publication Date: 2012-08-08
ZHAOQING DAHUANONG BIOLOGIC PHARMA +1
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0009] Studies have proved that the currently used viral general-purpose PCR method can quickly and effectively detect clinically unknown pathogenic infection samples, which is of great significance for production and infectious disease monitoring; but this method has certain limitations, because the relative content of chromosomal DNA from the host Higher, high quality requirements for virus samples, and due to the low repeatability of this method, the application of this technology to directly discover or identify new viruses has not been well promoted

Method used

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  • Polymerase chain reaction (PCR) detection method universal for viruses
  • Polymerase chain reaction (PCR) detection method universal for viruses
  • Polymerase chain reaction (PCR) detection method universal for viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: virus general PCR method specificity test

[0045] 5. Materials

[0046] Virus strains and cells: Specificity test to detect viruses (as shown in the table below) and strains are preserved by the crude drug research and development center of Guangdong Dahuanong Animal Health Products Co., Ltd.

[0047]

[0048] Strain: DH5α strain is preserved by the crude drug research and development center of Guangdong Dahuanong Animal Health Products Co., Ltd.

[0049] Random primer: VVVVVVVVAA, where V is any one of T, G, and C, synthesized by TaKaRa Company; after sequencing, the random primers corresponding to each virus are: H9N1: TTGGGGCGAA;

[0050] PRRSV: GCTGCCTTAA / CGTCTGCTAA; PRV: CTGCTGCTAA;

[0051] IBV: TTGGTGGCAA; EDS: TGTTTCTCAA; NDV: TTTCTCGGAA; PRRSV-PRV: GGCGTGTGAA / CTTGGGGCAA.

[0052] 2. Method steps

[0053] Virus propagation and culture are shown in the following table:

[0054]

[0055] Cell venom sample pretreatment: the above-mentioned...

Embodiment 2

[0068] Embodiment 2: virus universal PCR method susceptibility test

[0069] 5. Materials

[0070] Virus strain, cell: PRV recombinant virus PRV / TK with TK gene deletion of PRV-Ra strain - : Constructed and preserved by the crude drug research and development center of Guangdong Dahuanong Animal Health Products Co., Ltd.; BHK21 cells were purchased from China Veterinary Drug Control Institute.

[0071] Strain: DH5α strain is preserved by the crude drug research and development center of Guangdong Dahuanong Animal Health Products Co., Ltd.

[0072] Random primer: VVVVVVVVAA, where V is any one of T, G, and C, synthesized by TaKaRa Company.

[0073] 2. Method steps

[0074] Virus proliferation: BHK21 cells were cultured according to conventional methods, infected with virus at 5-10 PFU / cell, when the cytopathic pathology (CPE) reached about 90%, the virus was harvested, and the TCID50 of the virus was calculated;

[0075] Cell venom sample pretreatment: take 107.0TCID50 cell...

Embodiment 3

[0087] Example 3: Identification of Unknown Microorganisms

[0088] In a chicken farm, chicken embryos were inoculated with the liver suspension of sick and dead chickens. The whole body of the chicken embryos was severely hemorrhage, and blister-like substances appeared on the allantoic membrane. Various methods such as PCR and serology could not identify unknown microorganisms in the allantoic fluid; this test was carried out to identify the unknown microorganism.

[0089] 2. Materials

[0090] Chicken embryos: purchased from Guangdong Dahuanong

[0091] Random primer: VVVVVVVVAA, where V is any one of T, G, and C, synthesized by TaKaRa Company.

[0092] 3. Method steps

[0093] 1) Microbial proliferation: chicken embryo allantoic cavity inoculation: chicken embryo allantoic fluid containing unknown microorganisms was filtered through a 0.1um filter, 10-day-old SPF chicken embryo allantoic cavity was inoculated, observed for 3-5 days, and chicken embryo allantoic fluid wa...

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Abstract

The invention relates to a virus detection method, in particular to a polymerase chain reaction (PCR) detection method universal for viruses. The method comprises the steps of sample treatment, nucleic acid extraction, amplification, electrophoresis observation, amplification product cloning, genome sequencing analysis and the like. Host genome infection, bacterial infection or exogenous virus infection are avoided by the PCR detection method universal for the viruses, and the method has better specificity and repeatability. The method is used for quickly identifying and diagnosing different viruses, the complexity of selecting and identifying a cause of disease caused by specific PCR one by one is overcome, and the method can be used for quickly detecting a clinically unknown virus infection sample during production and quickly and effectively detecting a new virus or a virus strain with high genetic variation.

Description

technical field [0001] The invention relates to a virus detection method, in particular to a general PCR detection method for viruses. Background technique [0002] In recent years, infectious diseases have become more and more serious, with complex transmission routes and great variability of pathogens, which undoubtedly pose great challenges to clinical testing. Traditional pathogen detection methods, such as diagnosis of pathogens through clinical symptoms, pathological changes, virus isolation, artificial pathogenicity tests, serum separation, electron microscope observation, etc., have a large workload, complex technology, and time-consuming, and are not suitable for the effective monitoring of infectious diseases today. and prevention and control. This requires a fast and flexible method to detect pathogenic nucleic acids to strictly control the occurrence of infectious diseases. [0003] In 1990, American scholars Welsh and Williams established a clinical diagnostic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 谢小雨邵定勇陈瑞爱黄红亮唐兆新任常宝
Owner ZHAOQING DAHUANONG BIOLOGIC PHARMA
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