Detection kit, primer and probe for simultaneously detecting and identifying classical swine fever, African swine fever and swine vesicular disease

A technology for classical swine fever virus and African swine fever virus, which is applied in the field of inspection and quarantine, can solve the problems of large detection workload, low sensitivity, and difficult to determine results, achieve easy automation, good coverage and versatility, and save detection time. and cost effects

Inactive Publication Date: 2017-07-18
BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the currently established methods are single-target detection for one pathogen. In the actual detection, it needs to be repeated many times to complete the purpose of detecting different pathogens. The detection workload is large and the time is long, which cannot meet the rapid clearance of large-scale animal quarantine. the actual needs of
And it is difficult to realize the differential diagnosis of mixed infections and diseases with similar symptoms in actual samples
While establishing single-target pathogen detection technology, veterinary institutions in various countries have also developed multiplex PCR that can detect multiple viruses at the same time, but the disadvantages of traditional PCR technology, such as low sensitivity and difficult judgment of results, limit its application in multiplex detection

Method used

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  • Detection kit, primer and probe for simultaneously detecting and identifying classical swine fever, African swine fever and swine vesicular disease
  • Detection kit, primer and probe for simultaneously detecting and identifying classical swine fever, African swine fever and swine vesicular disease
  • Detection kit, primer and probe for simultaneously detecting and identifying classical swine fever, African swine fever and swine vesicular disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Composition and use of embodiment 1 test kit

[0039] 1. The composition of the kit

[0040] Table 2 Composition of the kit

[0041]

[0042]

[0043] Among them, 2×Multiplex RT-PCR buffer and Multiplex enzymes were purchased from Path-ID TMMultiplex one-step RT-PCR kit from AB Company.

[0044] The primer mix includes 3 forward primers and 2 reverse primers for CSFV, 2 forward primers and 3 reverse primers for ASFV, and 1 forward primer and 1 reverse primer for SVDV. The primer sequences are shown in Table 1 , the working concentration of the mixed primers is 400nM, and the primers are synthesized by Dalian Bao Biological Company.

[0045] The CSFV probe mixture includes 2 probes for CSFV, 1 probe for ASFV and 1 probe for SVDV. The sequences of the probes are shown in Table 1, and the concentrations are 200nM. The probes were synthesized by Dalian Bao Biological Company.

[0046] The negative control is sterile water without nucleic acid.

[0047] The CSFV po...

Embodiment 2

[0092] Embodiment 2, the sensitivity test of kit

[0093] 1. Materials

[0094] Classical swine fever virus is preserved by our laboratory, and African swine fever virus and porcine vesicular disease virus are recombinant plasmids in vitro.

[0095] 2. Method

[0096] 1) Preparation of positive standard

[0097] Method as described in Example 1.

[0098] 2) Quantitative determination of standard

[0099] Take the prepared in vitro transcribed cRNA and in vitro recombinant DNA and make 200-fold dilutions with RNase-free sterilized water, measure their absorbance values ​​at 260nm and 280nm (OD260 and OD280) with an ultraviolet spectrometer, and calculate the concentration of the sample to be tested and purity. Pure DNA: OD260 / OD280≈1.8 (>1.9, indicating RNA contamination; 2.0, indicating that there may be residual isothiocyanate); pure RNA: 1.72.0 indicates that there may be residual isothiocyanate). Concentration of DNA sample (μg / μL): OD260×dilution factor×50 / 1000; conc...

Embodiment 3

[0115] Embodiment 3, the specificity test of kit

[0116] 1 material

[0117] The viruses used in this experiment are listed in Table 6.

[0118] Table 6 Viruses and nucleic acids used in the specificity test research process

[0119] Virus source Classical swine fever virus (CSFV) The lab saves African swine fever virus (ASFV) USDA Exotic Disease Center Porcine vesicular disease virus recombinant plasmid in vitro Prepared in this laboratory Porcine circovirus type 2 (PCV-2) The lab saves Pseudorabies virus (PRV) The lab saves Porcine parvovirus (PPV) The lab saves transmissible gastroenteritis virus (TGEV) The lab saves

[0120] 2. Method

[0121] 2.1 Use the primers and probes of classical swine fever virus, African swine fever virus and porcine vesicular disease virus to perform fluorescent RT-PCR detection on the other 6 viral nucleic acids in the table to verify the specificity of the primers and probes. ...

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Abstract

The invention discloses a detection kit, a primer and a probe for simultaneously detecting and identifying classical swine fever, African swine fever and a swine vesicular disease. The primer and the probe are shown as a sequence table SEQ ID NO: 1 to SEQ ID NO: 16. The invention further discloses the one-step-method multi-fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit which is rapid, accurate and convenient to use and can be used for simultaneously detecting and identifying the classical swine fever, the African swine fever and the swine vesicular disease. A detection method disclosed by the invention can be used for realizing one-time sampling and one-time analysis and can realize the aim of simultaneously detecting and distinguishing three important viruses, so that the working amount and cost of detection are reduced, the detection of epidemic diseases can be finished within shortest time and the time for preventing and controlling the diseases is earned.

Description

technical field [0001] The invention relates to a one-step multiple fluorescent RT-PCR detection kit, primers and probes capable of simultaneously detecting and differentiating classical swine fever virus (CSFV), African swine fever virus (ASFV) and swine vesicular virus (SVDV). The purpose of achieving one sampling, one analysis, and simultaneously detecting and distinguishing three kinds of viruses belongs to the field of inspection and quarantine. Background technique [0002] Classical swine fever virus (CSFV), African swine fever virus (ASFV) and swine vesicular virus (SVDV) are acute and highly contagious diseases of pigs. The onset characteristics are mainly fever and septic blood type changes. The clinical symptoms are similar and the differential diagnosis is difficult. It is the most serious pathogen that harms the aquaculture industry. Swine fever, African swine fever and porcine vesicular disease are defined by the World Organization for Animal Health (OIE) as p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2600/166C12Q2537/143C12Q2545/113C12Q2563/107
Inventor 史喜菊马贵平柏亚铎刘艳华高峰刘全国李炎鑫高志强李冰玲张玉红郭敏卓刘凌
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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