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RAA (recombinase aided amplification) constant temperature fluorescent detection primer probe set, kit and method for African swine fever virus CD2V gene

A technology of African swine fever virus, primer probe, applied in the field of molecular biology, to achieve the effect of simple and fast reaction

Active Publication Date: 2020-01-17
南宁众册生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no effective treatment or vaccine for African swine fever, and in the event of an outbreak, the only control strategy is to isolate and remove infected animals

Method used

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  • RAA (recombinase aided amplification) constant temperature fluorescent detection primer probe set, kit and method for African swine fever virus CD2V gene
  • RAA (recombinase aided amplification) constant temperature fluorescent detection primer probe set, kit and method for African swine fever virus CD2V gene
  • RAA (recombinase aided amplification) constant temperature fluorescent detection primer probe set, kit and method for African swine fever virus CD2V gene

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Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1: the design of the RAA constant temperature fluorescence detection primer probe group of African swine fever virus CD2V gene

[0035]This study refers to the African swine fever gene CD2V gene sequence published on GenBank, and uses DNAMAN 6.0 software to compare multiple sequences. A large number of experiments have shown that different primers have a certain influence on the effect and sensitivity of isothermal amplification. Therefore, this study preliminarily designed four sets of primer probes for the African swine fever virus CD2V gene: ASFV-CD2V-1, ASFV-CD2V-2, ASFV-CD2V-3 and ASFV-CD2V-4 (primer sequences are listed in Table 1. shown), these sequences can specifically combine with the corresponding sequences of the African swine fever virus CD2V gene, wherein the size of the target fragment amplified by the above four sets of primers is 107bp, 110bp, 172bp and 197bp respectively, and the positive sample amplification curve is as follows: figure 1 s...

Embodiment 2

[0039] Embodiment 2: RAA constant temperature fluorescent detection kit and detection method of African swine fever virus CD2V gene

[0040] This embodiment provides a kind of RAA thermostatic fluorescence detection kit of ASFV-CD2V, including primer mixture, specific fluorescent probe, A Buffer (20% PEG), B Buffer (280mM MgAc), RAA dry powder reagent, African swine fever Viral standards and ddH 2 O.

[0041] The composition of RAA dry powder reagent is as follows: 1mmol / L dNTP, 90ng / μL SSB protein, 120ng / μL recA recombinase protein (SC-recA / BS-recA) or 30ng / μL Rad51, 30ng / μL Bsu DNA polymerase, 100mmol / L Tricine , 20% PEG, 5mmol / L dithiothreitol, 100ng / μL creatine kinase, Exo exonuclease.

[0042] In the primer mixture described in this embodiment, the base sequence of the forward primer is shown in SEQ ID No.1, the base sequence of the reverse primer is shown in SEQ ID No.2, the forward primer and The molar ratio of the reverse primer is 1:1.

[0043] The specific probe ...

Embodiment 3

[0060] Embodiment 3: Apply the kit of the present invention to carry out actual sample detection

[0061] In order to verify that the primers, probes, and reagents of the present invention can be used normally in actual conditions, the actual conditions are simulated, and the primers, probes, and reagents of the present invention are used to detect actual samples.

[0062] The actual samples are swabs from the mouth and nose of sick pigs and blood samples (serum, plasma and whole blood) of sick pigs, totaling 12. The detection result was consistent with the known PCR detection result, and the coincidence rate was 100%.

[0063] It shows that the primer-probe combination kit of the present invention has the functions of rapid, real-time, sensitive, accurate and convenient detection of African swine fever CD2V gene under body temperature conditions (37°C-39°C) in a fluorescent detection instrument with temperature control function. Advantages of detection.

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Abstract

The invention belongs to the technical field of molecular biology and discloses an RAA (recombinase aided amplification) constant temperature fluorescent detection primer probe set, kit and method forAfrican swine fever virus CD2V gene. A nucleotide sequence of a forward primer of the primer probe set is shown as SEQ ID NO. 1, a nucleotide sequence of a reverse primer is shown as SEQ ID NO. 2, and a nucleotide sequence of a specific fluorescent probe is shown as SEQ ID NO. 3, wherein a fluorescent reporter group is marked in the 5' end direction of the specific fluorescent probe, while a fluorescent quenching group is marked in the 3' end direction of the specific fluorescent probe. The primer probe set and kit provided by the invention help rapidly, conveniently, efficiently and specifically detect whether a sample is infected with an African swine fever wild strain or a CD2V deleted strain under an isothermal condition, do not need complex instruments and professional equipment on site, and are suitable for POCT (point-of-care testing).

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a RAA constant-temperature fluorescence detection primer probe group, a kit and a detection method for African swine fever virus CD2V gene. Background technique [0002] African swine fever (African swine fever, ASF) is an acute, febrile, highly contagious zoonotic disease of domestic pigs and wild boars caused by the African swine fever virus (ASF virus, ASFV). It can be infected, and the morbidity and mortality rate can reach up to 100%. The World Organization for Animal Health lists it as a statutory reportable animal disease, and my country lists it as a first-class animal disease. ASFV has the characteristics of resistance to acid and alkali, cold and heat. Direct contact between healthy pigs and diseased pigs or pollutants is the main transmission route of African swine fever. Pigs can also be transmitted through the bite of vector insects such as ticks...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2521/507C12Q2522/101
Inventor 黄震巨李沛李春燕林晓滕丽琼
Owner 南宁众册生物科技有限公司
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