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Fluorescent quantitative RT-PCR detection kit for feline infectious peritonitis virus

A peritonitis virus, detection kit technology, applied in the direction of viruses, viral peptides, viruses/phages, etc., to achieve the effects of convenient sampling, simple and easy operation procedures, and high detection efficiency

Pending Publication Date: 2022-06-07
昆明海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there is no report on the quantitative detection kit of feline infectious peritonitis virus using Taqman probe real-time fluorescent quantitative RT-PCR technology

Method used

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  • Fluorescent quantitative RT-PCR detection kit for feline infectious peritonitis virus
  • Fluorescent quantitative RT-PCR detection kit for feline infectious peritonitis virus
  • Fluorescent quantitative RT-PCR detection kit for feline infectious peritonitis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Four sets of primers and TaqMan probes are designed according to the full sequence of the N gene of the feline infectious peritonitis virus, as follows:

[0028] F3：5’-AGAAGCCTGAGGAATTGTCTGT-3’(SEQ ID NO:8)

[0029] R3：5’-TCGTAACCTCATCAATCATCTCA-3’(SEQ ID NO:9)

[0030] P3：5’-(FAM)CACAGATGTGTTTGATGACACACAGGT-3’(TAMRA)(SEQ ID NO:10)

[0031] F4：5’-CCAACAACACTTGGCACTCG-3’(SEQ ID NO:11)

[0032] R4：5’-CCAGCTTCTTCAGCAGACCA-3’(SEQ ID NO:12)

[0033] P4：5’-(FAM)CGTTGCCAATGGTAACGCTGCC-3’(TAMRA)(SEQ ID NO:13)

[0034] F5：5’-TTGTCAGAGGCCAGCGTAAG-3’(SEQ ID NO:14)

[0035] R5：5’-TTCCACGAGTGCCAAGTGTT-3’(SEQ ID NO:15)

[0036] P5：5’-(FAM)GGGTTGCAAGGGATGGAGCCA-3’(TAMRA)(SEQ ID NO:16)

[0037]F6：5’-CGGTCTAACTCTCGTGGTCG-3’(SEQ ID NO:17)

[0038] R6：5’-AAGTTCCTTACGCTGGCCTC-3’(SEQ ID NO:18)

[0039] P6：5’-(FAM)ACAGACAGGTGCGTTACCGCA-3’(TAMRA)(SEQ ID NO:19)

[0040] After fluorescence quantitative RT-PCR detection, the above four sets of primers and TaqMan probes detected a large number of cli...

Embodiment 2

[0042] Look for conserved areas of the N gene of feline infectious peritonitis virus; Design synthetic primers and TaqMan probes

[0043] According to the N gene sequence of cat infectious peritonitis virus published by GenBank (AB086903.1, FJ917524.1, A22378.1, FJ917535.1, AB0866881.1, FJ943764.1, FJ917533.1, FJ917532.1, FJ917530.1, FJ917529.1), its conserved region was analyzed, A specific conserved sequence of the N gene of cat infectious peritonitis virus (SEQ IDNO:1) was obtained:

[0044] ATGGCCACACAGGGACAACGCGTCAACTGGGGAGATGAACCTTCCAAAAGACGTGGTCGTTCTAACTCTCGTGGTCGGAAGAATAGTAATATACCT。

[0045] The primer design for this conservative sequence was obtained with two pairs of specific primers and two Taqman probes, the 5' end of the probe fluorescence reporting group is FAM, and the 3' end of the fluorescent quenching group is TAMRA.

[0046] Both the specific detection primer and the Taqman probe are synthesized by Baori Medical Biotechnology Co., Ltd.

[0047] The primers and ...

Embodiment 3

[0055] Quantitative construction and preparation of standard plasmids

[0056] 1. Construction of quantitative standard plasmids

[0057] Depending on the PCR primer designed, genes containing amplified fragments of interest are cloned into plasmid vector pUC57 using recombinant DNA techniques and DNA sequence assays are performed. The recombinant plasmid constructed serves as a quantitative standard plasmid for detecting infectious peritonitis virus in cats, named pUC57-FIPV-N ( Figure 1 )。 Recombinant plasmids are synthesized by Kinko Biotech.

[0058] 2. Preparation of quantitative standard plasmids

[0059] The standard plasmid pUC57-FIPV-N is extracted and purified by alkali cracking, the concentration is determined by a trace nucleic acid protein analyzer, and the copy number is calculated by formula (copy number = 6.02×10 23 × DNA concentration / mass MW, MW = DNA base number ×330), the standard plasmid copy number is calculated to be 3.4x10 11 copies / μL, store at -20 °C.

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Abstract

The invention relates to a fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for a feline infectious peritonitis virus. The kit comprises a specific primer for fluorescent quantitative RT-PCR detection of the feline infectious peritonitis virus and a TaqMan probe, wherein the sequence of the forward primer is SEQ ID NO: 2, and the sequence of the reverse primer is SEQ ID NO: 3; the sequence of the TaqMan probe is as shown in SEQ ID NO: 4. The kit has good sensitivity, specificity and stability, is suitable for rapid diagnosis in the early stage of feline infectious peritonitis virus infection, and has important significance for reducing the transmission of feline infectious peritonitis and improving the detection efficiency of feline infectious peritonitis.

Description

Technical field [0001] The present invention relates to a viral molecular biology detection kit in the field of biotechnology, specifically to a cat infectious peritonitis virus fluorescence quantitative RT-PCR detection kit. Background [0002]Feline Infectious Peritonitis (FIP) is an infectious disease commonly seen in cats caused by feline infectious peritonitis virus (FIPV), with high mortality rate and serious harm to the health of cats. FIPV belongs to the Feline Coronavirus (FCoV), a single-stranded positive-stranded RNA virus with an envelope. Feline infectious peritonitis virus mainly encodes four structural proteins: spike protein, membrane protein, envelope protein and nucleocapsid protein. Studies have shown that the nucleocapsid protein (N protein) of the coronavirus as the most immunogenic protein, its corresponding antibody is the earliest to be produced, so N protein is often used as an indicator of early diagnosis. Cat coronaviruses are divided into gastroenterit...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/50C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C07K14/005C12Q2600/158C12N2770/20022C12Q2531/113C12Q2521/107C12Q2563/107C12Q2545/114C12Q2545/113Y02A50/30
Inventor 韩佃刚董俊叶玲玲艾军杨妮张冲杨云庆李静李瑶瑶罗倩敏董仙兰宿放
Owner 昆明海关技术中心
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