Reagent and detection method for detecting vesicular stomatitis virus, and applications

A technology for vesicular stomatitis and virus detection, applied in reagents for vesicular stomatitis virus detection, in the field of vesicular stomatitis virus detection, can solve the problem that the detection time needs 70 minutes, there is no vesicular stomatitis virus detection RPA, etc. problems, achieving the effect of low hardware equipment requirements and ensuring production safety

Active Publication Date: 2017-01-25
SHENZHEN CUSTOMS ANIMAL & PLANT INSPECTION & QUARANTINE TECH CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Viral nucleic acid detection is one of the most commonly used methods for pathogen identification. The established VS detection methods include conventional RT-PCR method and real-time fluorescent RT-PCR method, although real-time fluorescent RT-PCR method is faster than conventional RT-PCR method , but the detection time will take at least seventy minutes
At present, there is no research and report on RPA technology for the detection of vesicular stomatitis virus

Method used

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  • Reagent and detection method for detecting vesicular stomatitis virus, and applications
  • Reagent and detection method for detecting vesicular stomatitis virus, and applications
  • Reagent and detection method for detecting vesicular stomatitis virus, and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 1. Materials and methods

[0030] 1. Test nucleic acid

[0031] The templates used in this experiment include VSV-IND nucleic acid, VSV-NJ nucleic acid, and other pathogens: foot-and-mouth disease virus, bluetongue virus, bovine viral diarrhea virus, porcine vesicular disease virus, swine fever virus, porcine reproductive and respiratory syndrome virus , Porcine rotavirus. The nucleic acids and viruses used in this example were provided and preserved by the Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Entry-Exit Inspection and Quarantine Bureau.

[0032] 2. Viral Nucleic Acid Extraction

[0033] In this case, the Thermo virus RNA extraction kit MagMAX was used TM -96 Viral RNA Isolation Kit (AM1836) automatically extracts viral nucleic acid on the automatic magnetic bead extraction and purification system Thermo Scientific KingFisher Flex. The specific operation method is carried out according to the instructions of the nucleic acid extrac...

Embodiment 2

[0062] Using the primers and probes screened out in Example 1, 67 clinical samples were detected, and the VSV-IND nucleic acid and VSV-NJ nucleic acid extracted in Example 1 were respectively used as positive controls; The stomatitis reference (Hole et al., 2010) established a real-time fluorescent RT-PCR method for parallel detection. The 67 clinical samples used in this case were provided and preserved by the Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Entry-Exit Inspection and Quarantine Bureau.

[0063] The test results showed that the results of RPA and real-time fluorescent RT-PCR method were completely consistent, only the positive control had a typical amplification curve, and the other 67 samples had no amplification curve. The results show that the two sets of primers and probes designed in this application have good specificity, and the RPA detection is easy to operate and has a short reaction time, and can quickly and accurately identif...

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Abstract

The invention discloses a reagent and a detection method for detecting the vesicular stomatitis virus, and applications. The reagent for detecting the vesicular stomatitis virus comprises a first primer probe combination set and a second primer probe combination set for detecting the amplification of vesicular stomatitis virus recombinant polymerase; the forward primer, reverse primer and probe of the first primer probe combination set are sequences shown by Seq IP N.1-3; and the forward primer, reverse primer and probe of the second primer probe combination set are sequences shown by Seq IP N.4-6. According to the reagent for detecting the vesicular stomatitis virus, the two sets of primer probe combinations are designed aiming at two subtypes respectively, thus providing a new detection scheme and approach for the detection of the vesicular stomatitis virus. A reagent-based vesicular stomatitis virus RPA detection method is rapid and accurate, is low in requirements on hardware, and applicable to on-site rapid detection, especially suitable for use by inspection and quarantine departments and the like.

Description

technical field [0001] The present application relates to the field of detection of vesicular stomatitis virus, in particular to a reagent for detection of vesicular stomatitis virus and its application, and a detection method of vesicular stomatitis virus based on the reagent. Background technique [0002] Vesicular stomatitis (VS) is an important zoonotic infectious disease caused by vesicular stomatitis virus (VSV), and is listed as a legally notifiable infectious disease by the World Organization for Animal Health (OIE). It is listed as a second-class infectious disease in the list of quarantine and epidemic diseases for imported animals in my country. VSV belongs to Rhabdoviridae and Vesiculovirus in classification. It is a single-stranded negative-sense RNA virus and has two independent serotypes: Indiana type (Indiana, VSV-IND) and New Jersey type (New Jersey, VSV-NJ) . Horses, cattle, and pigs are susceptible, but sheep, goats, and other wild animals can also be inf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2521/507C12Q2522/101
Inventor 林彦星曹琛福花群义曾少灵王潇张彩虹孙洁廖立珊吕建强杨俊兴
Owner SHENZHEN CUSTOMS ANIMAL & PLANT INSPECTION & QUARANTINE TECH CENT
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