Cpf1 reagent kit and detection method for quickly detecting nucleic acid of African swine fever virus

An African swine fever virus and detection method technology, applied in the biological field, can solve problems such as expensive and unsuitable for on-site detection in pig farms, and achieve the effects of simple detection method, convenient and quick result interpretation, and high-sensitivity visual detection

Active Publication Date: 2019-12-10
SHANGHAI TECH UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above scheme requires some expensive experimental equipment and is not suitable for on-site testing in pig farms
Recently, a real-time PCR assay improved by a research team based on Universal Probe Library (UPL) probes can be used for molecular diagnosis of ASFV under field conditions (Agüero, Fernández et al.2003), but due to the limitations of battery-powered real-time PCR instruments , the method can only handle a moderate number of samples

Method used

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  • Cpf1 reagent kit and detection method for quickly detecting nucleic acid of African swine fever virus
  • Cpf1 reagent kit and detection method for quickly detecting nucleic acid of African swine fever virus
  • Cpf1 reagent kit and detection method for quickly detecting nucleic acid of African swine fever virus

Examples

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Embodiment example 1

[0048] Implementation Case 1: Rapid and Sensitive Detection of African Swine Fever Virus p72 Gene Fragment

[0049] 1.1 Nucleic acid preparation

[0050] In this case, the p72 gene fragment of African swine fever virus is based on the Genotype II subtype sequence, and the C-terminal 454bp SEQ NO.1 was synthesized by Nanjing GenScript Company and constructed into the pUC57 vector, named pUC57-ASFV-p72.

[0051] Using the RPA amplification primers RPA-F1SEQ NO.2 and RPA-R1SEQ NO.3 in the present invention, referring to the RPA isothermal amplification operation steps, amplified to obtain the sample to be detected. The specific operation is as follows:

[0052] In the case of sensitivity detection, the molecular weight of the pUC57-ASFV-P72 plasmid was calculated, and a 10-fold serial dilution was performed to obtain 2*e10, 2*e9, 2*e8, 2*e7, 2*e6, 2*e5 per microliter , 2*e4, 2*e3, 2*e2, 2*e1 and 2*e0 copy number (copy / μL). 1 μL of gradient dilution samples were subjected to RP...

Embodiment example 2

[0073] Implementation case 2: Rapid genotype identification of African swine fever virus P72 gene fragment

[0074] The rapid detection of African swine fever virus genotype is helpful to understand the source of virus infection and trace the route of virus transmission in detail, and is of great significance to the prevention and control of the virus. In this implementation case, ASFV subtype I, II and III are taken as examples to demonstrate the application of the Cpf1 detection kit of the present invention to type African swine fever virus.

[0075] Based on the fact that Cpf1 performs highly specific recognition and cutting of the target sequence under the guidance of crRNA, a hypothesis is proposed: there are differences in nucleic acid sequences of different genotypes of ASFV, which can lead to the inability of the Cpf1 cutting system to efficiently recognize, manifested as low detection fluorescence values ​​or colloidal gold The test strip has no detection strip. Base...

Embodiment example 3

[0082] Implementation Case 3: Rapid ASFV Detection of Nucleic Acids in Clinical Tissue Samples

[0083] In this implementation case, ASFV rapid detection of nucleic acid in clinical tissue samples was performed. All samples and operations were completed in the national African swine fever regional laboratory, and all operations were carried out in strict accordance with relevant laws and regulations and relevant regulations of the Ministry of Agriculture.

[0084] The clinical DNA samples in this case came from the national African swine fever regional laboratory, including pig tissue samples collected from pig farms infected with African swine fever virus, including 6 tissue organs of lung, spleen, kidney, intestine, heart and liver 12 samples. The total DNA of the tissue was extracted, and qPCR detection and Cpf1 detection were performed respectively. In this example, a rapid nucleic acid release agent purchased from Novizyme was used to obtain pretreated nucleic acid. The...

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Abstract

The invention discloses a Cpf1 reagent kit for quickly detecting nucleic acid of an African swine fever virus. The Cpf1 reagent kit comprises a Cpf1 detection system suitable for quickly detecting theAfrican swine fever virus, and an immune colloidal gold test strip, wherein the Cpf1 detection system comprises specific crRNA protein, specific Cpf1 protein and a single-chain DNA(ssDNA) reporting system in accordance with a p72 gene of the African swine fever virus, the specific crRNA is one or more of crRNAs from ASFV P72 crRNA1 to ASFV P72 crRNA10, and the sequence of the specific crRNA is SEQ NO.4 to SEQ NO.13; and the single-chain DNA(ssDNA) reporting system comprises ssDNA FQreporter for fluorescence detection of a microplate reader and/or ssDNA DB reporter for detecting the immune colloidal gold test strip. According to the Cpf1 reagent kit disclosed by the invention, for the first time, the Cpf1 is used for detecting the African swine fever virus, and has the advantages of beinghigh in sensitivity, high in specificity, short in time consumption, high in flux, independent of large-scale experiment equipment and the like. The advantages enable a detection method based on the immune colloidal gold test strip developed by the invention to be conveniently used in basic laboratories and breeding enterprises to be used for performing detection, identification and diagnosis on basic quick detection of the African swine fever.

Description

technical field [0001] The invention relates to a rapid detection method and kit for African swine fever virus nucleic acid, more specifically, relates to a rapid detection method and kit for African swine fever virus nucleic acid based on Cpf1, and belongs to the field of biotechnology. Background technique [0002] African swine fever (African Swine Fever, ASF) is an acute and severe pig infectious disease caused by African swine fever virus (African Swine FeverVirus, ASFV) infection. %, is the most serious severe infectious disease in the pig industry (Galindo and Alonso 2017). ASF is an animal disease that is required to be reported by the World Organization for Animal Health (OIE). ASFV belongs to the first class of animal pathogenic microorganisms in the list of animal pathogenic microorganisms in my country. [0003] ASFV is a member of the African swine fever virus genus (Asfivirus) of the African swine fever virus family (Asfarviridae), and the virus genome is comp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2521/507C12Q2522/101C12Q2565/625
Inventor 王鑫杰黄行许李广磊
Owner SHANGHAI TECH UNIV
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