Triple fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for African swine fever viruses, swine fever viruses and respiratory syndrome viruses and preparation method and application thereof

An African swine fever virus and RT-PCR technology, applied in the field of triple fluorescent RT-PCR detection reagents and their preparation, can solve problems such as single detection

Inactive Publication Date: 2015-07-01
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Fluorescent PCR and fluorescent RT-PCR methods and public patents for the detection of single African swine fever virus, classical swine fever virus and porcine reproductive and respiratory syndrome virus have been reported at present, and can detect African swine fever virus, classical swine fever virus and porcine reproductive and respiratory syndrome virus simultaneously. The triple fluorescent RT-PCR detection reagent for reproductive and respiratory syndrome virus and its preparation method and application are rarely reported

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  • Triple fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for African swine fever viruses, swine fever viruses and respiratory syndrome viruses and preparation method and application thereof
  • Triple fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for African swine fever viruses, swine fever viruses and respiratory syndrome viruses and preparation method and application thereof
  • Triple fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for African swine fever viruses, swine fever viruses and respiratory syndrome viruses and preparation method and application thereof

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preparation example Construction

[0071] The triple fluorescent RT-PCR detection reagent for African swine fever virus, swine fever virus and porcine reproductive and respiratory syndrome virus of the present invention and its preparation method and application include the following steps.

[0072] The first step is to prepare the positive control material for the African swine fever virus CP530R gene. The process includes:

[0073] (1) In the NCBI website, perform BLAST alignment of the African swine fever virus CP530R gene sequence, and select a conservative sequence for the preparation of the positive control of the African swine fever virus CP530R gene, as shown in SEQ ID NO.4.

[0074] (2) Design and synthesize 4 primers based on the sequence of SEQ ID NO.4, the primer sequences are as follows:

[0075] CP530RF15’-TCTTGCCAAGTCGGTCTCCTTGCTGTCTTTCTTGTCGCCTCAACCATCCCACCGAGTT-3’

[0076] CP530RR15’-AACTCGGTGGGATGGTTGAGGCGACAAGAAAGACAGCAAGGAGACCGACTTGGCAAGA-3’

[0077] CP530RF2: 5’-ACTGGTGTAGTGTCAATAATCCCTATCTTGCCAAGTCGG...

Embodiment 1

[0145] Example 1 Preparation of positive control of African swine fever virus CP530R gene

[0146] Perform BLAST on the African swine fever virus CP530R gene sequence (GenBank accession number: KJ380911.1), and select a conservative sequence suitable for designing fluorescent PCR primers and probes as the reference sequence for preparing the positive control plasmid of the African swine fever virus CP530R gene, such as SEQ ID NO.4 is shown.

[0147] 2. Design and synthesis of amplification primers

[0148] Based on the above sequence, four PCR amplification primers were designed and synthesized. Among them, the CP530RF1 and CP530RR1 sequences are completely complementary and used as templates. CP530RF2 and CP530RR2 partially overlap with the CP530RF1 and CP530RR1 sequences respectively. The synthesized sequences are as follows:

[0149] CP530RF15-TCTTGCCAAGTCGGTCTCCTTGCTGTCTTTCTTGTCGCCTCAACCATCCCACCGAGTT-3

[0150] CP530RR1: 5-AACTCGGTGGGATGGTTGAGGCGACAAGAAAGACAGCAAGGAGACCGACTTGGCAAGA-...

Embodiment 2

[0164] Example 2 Preparation of Swine Fever Virus 5'UTR Gene Positive Control (RNA)

[0165] 1. Selection of reference gene sequence

[0166] Perform BLAST on the swine fever virus 5'UTR gene sequence (GenBank accession number: AY259122), and select a conservative and suitable sequence for designing fluorescent RT-PCR primers and probes as the positive control (RNA) for preparing swine fever virus 5'UTR gene The reference of reference A) is shown in SEQ ID NO.1.

[0167] 2. Design and synthesis of amplification primers

[0168] Based on the above sequence, four PCR amplification primers were designed and synthesized. Among them, CSFV-F1 and CSFV-R1 sequences were completely complementary and used as templates. CSFV-F2 and CSFV-R2 partially overlapped with CSFV-F1 and CSFV-R1 sequences, respectively. The synthesized sequence is as follows:

[0169] CSFV-F2: 5’-CTGGGTGGTCTAAGTCCTGAGTACAGGACAGTCGTCAGTAGTTCGACGTGAGC-3’

[0170] CSFV-F1: 5’-GTCGTCAGTAGTTCGACGTGAGCAGAAGCCCACCTCGAGATGCTATGTGG...

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Abstract

The invention discloses a triple fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for African swine fever viruses, swine fever viruses and respiratory syndrome viruses and a preparation method and application thereof. Three sets of specific primers and Taqman probes as well as positive controls specific to African swine fever virus CP530R genes, swine fever virus 5 minute-UTR genes and swine respiratory syndrome virus NSP2 genes respectively are designed and synthesized, and a rapid, easy and convenient triple fluorescent RT-PCR detection system with high specificity and high sensitivity is established by using the three sets of primers and probes, so that nucleic acids of the African swine fever viruses, the swine fever viruses and the respiratory syndrome viruses can be detected synchronously from a detected sample within 3-4 hours in a rapid, accurate, specific, safe, easy and convenient way. The detection reagent can be applied to synchronous detection of the nucleic acids of trace African swine fever viruses, swine fever viruses and respiratory syndrome viruses in hogs and relevant samples thereof.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a triple fluorescent RT-PCR detection reagent for African swine fever virus, swine fever virus and swine reproductive and respiratory syndrome virus, and a preparation method and application thereof. Background technique [0002] African swine fever (ASF) is an acute, febrile, highly contagious disease of pigs caused by African swine fever virus (ASFV). It is characterized by short course of disease, high fatality rate, clinical symptoms and pathological changes similar to acute swine fever, high fever, skin congestion, abortion, edema and organ hemorrhage. Classical Swine Fever (CSF) is an acute, febrile and highly contact infectious disease of swine caused by swine fever virus (CSFV). It is acute according to the virulence of the epidemic strain and the immune status of pigs. Type, subacute, mild and other clinical symptoms. Porcine Respiratory and Reproductive Syndrome is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143C12Q2561/101
Inventor 董志珍王建华赵祥平王玉玲肖妍赵丹张俊哲柴铭骏陈本龙陈小金王乃福
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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