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Method for detection of viral hemorrhagic sepsis virus based on liquid chip

A technology for viral hemorrhage and sepsis, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection. Short time, no environmental pollution, good specific effect

Inactive Publication Date: 2015-07-29
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cytological diagnostic techniques mainly include the use of cell culture to isolate viruses, histopathological sections, and electron microscope observation, which are cumbersome to operate, have a long detection cycle, and have low sensitivity
Immunological diagnostic techniques mainly include immunofluorescence detection and immune dot hybridization, which have the advantages of strong specificity and high sensitivity, but the steps are quite cumbersome and are not suitable for detection of a large number of samples
Molecular biology diagnostic techniques mainly include polymerase chain reaction (PCR), which is fast and sensitive, but requires agarose gel electrophoresis and staining with ethidium bromide to observe the results. Ethidium bromide is a carcinogen and is harmful to the human body and the environment. Hazardous, cross-contamination problem is more serious

Method used

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  • Method for detection of viral hemorrhagic sepsis virus based on liquid chip
  • Method for detection of viral hemorrhagic sepsis virus based on liquid chip
  • Method for detection of viral hemorrhagic sepsis virus based on liquid chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, the design of specific primer pair and specific probe

[0038] A specific primer pair and a specific probe for amplifying the viral hemorrhagic sepsis virus are designed through a large number of sequence alignments and amplification effect comparisons.

[0039] The specific primer pair is as follows (the target sequence is 191bp):

[0040] F1 (sequence 1 of the sequence listing): 5'-GACGAGGCAAGCAAGGAT-3';

[0041] R1 (SEQ ID NO: 2 of the Sequence Listing): 5'-GTTTGACAGGGCGAGGTT-3'.

[0042] The nucleotide sequence of the specific probe (sequence 3 in the sequence listing) is as follows:

[0043] 5'-AACGGGTACTCGTGATCCTTGC-3'.

Embodiment 2

[0044] Example 2, application of specific primer pairs to aid in the identification of viral hemorrhagic sepsis virus

[0045] Viral hemorrhagic septicemia virus, carp spring viremia virus, infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus and viral neuronecrosis virus were used as the viruses to be tested for the following experiments:

[0046] 1. Use the RNA extraction kit to extract the total RNA of the virus to be tested.

[0047] 2. Using the total RNA obtained in step 1 as a template, using a primer pair composed of F1 and R1, and using an RT-PCR kit, perform RT-PCR amplification on a gradient PCR amplification instrument to obtain RT-PCR amplification products.

[0048] RT-PCR amplification reaction system: In a 0.2mL PCR reaction tube, add 10×RT-PCR buffer 2.5μL, dNTP (2.5mmol / L each) 2.5μL, 10pmol / μL F1 0.5μL, 10pmol / μL R1 0.5μL, Inhibiter 0.5μL, AMV XL 0.5μL, AMV Taq (5U / μL) 0.5μL, 25mmol / L MgCl 2 5.0 μL, total RNA 3.0 μL (about 300ng),...

Embodiment 3

[0052] Example 3, application of primer probe composition to assist in the identification of viral hemorrhagic sepsis virus

[0053] 1. Preparation of primers and probes

[0054] Prepare primers and probes as follows:

[0055] F2: 5'-biotin-GACGAGGCAAGCAAGGAT-3';

[0056] R2: 5'-biotin-GTTTGACAGGGCGAGGTT-3'.

[0057] Probe T1: 5'-NH 2 -AACGGGTACTCGTGATCCTTGC-3'.

[0058] F2 is biotinylated at the 5' end of F1, and R2 is biotinylated at the 5' end of R1. Probe T1 is obtained by amination modification at the 5' end of the single-stranded DNA fragment shown in Sequence 3 of the sequence listing.

[0059] 2. Establishment of liquid phase chip detection system

[0060] 1. Use the RNA extraction kit to extract the total RNA of viral hemorrhagic sepsis virus.

[0061] 2. Using the total RNA obtained in step 1 as a template, using a primer pair composed of F2 and R2, and using an RT-PCR kit, perform RT-PCR amplification on a gradient PCR amplification instrument to obtain RT-PC...

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Abstract

The invention discloses a method for detecting viral haemorrhagic septicaemia virus based on a liquid chip. The method adopts a specific primer pair assisting in identification on the viral haemorrhagic septicaemia virus, and the specific primer pair consists of single strand DNA molecules shown by a sequence 1 in a sequence table and single strand DNA molecules shown by a sequence 2 in the sequence table. The method also protects a primer and probe composition assisting in identification on the viral haemorrhagic septicaemia virus, and the primer and probe composition consists of the specific primer pair and a probe T1; the nucleotide sequence of the probe T1 is shown as a sequence 3 in the sequence table. Specific primers provided by the invention are high in specificity on identification on the viral haemorrhagic septicaemia virus. The primer probe composition provided by the invention is combined with the liquid chip for identifying the viral haemorrhagic septicaemia virus. The method has the advantages of being high in specificity, high in sensitivity, easy and convenient to operate, short in required time and free of environment pollution, avoiding human health threat, and capable of performing high throughput detection.

Description

technical field [0001] The invention relates to a pair of primers for assisting identification of viral hemorrhagic sepsis virus, a primer probe composition and their application, in particular to a method for detecting viral hemorrhagic sepsis virus based on a liquid phase chip. Background technique [0002] Viral hemorrhagic septicemia of salmonids (VHS) is a second-class infectious disease listed in the "List of Class I and Class II Infectious Diseases and Parasitic Diseases of Imported Animals of the People's Republic of China", and is listed in the "Animal Epidemic Prevention of the People's Republic of China". The second-class epidemic diseases stipulated in the Law are important epidemic diseases in the OIE list of the International Office of Epizootics. VHS has plagued European rainbow trout for more than 50 years and is considered the number one killer of economic losses in the European rainbow trout farming industry, and has also been reported in other species. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 岳志芹王群贾鹏尹伟力方绍庆刘宁王颖
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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