RPA-LFD primer, probe and kit for jointly detecting epidemic hemorrhagic disease virus and Palimer serum group virus

An epidemic hemorrhagic disease and detection primer technology, applied in the field of probes and detection kits, recombinase polymerase-lateral flow chromatography test paper detection primers, can solve the problem of reducing detection efficiency, configuring expensive instruments and equipment, and difficult to provide power supply and other problems, to achieve the effect of strong specificity and good detection sensitivity

Active Publication Date: 2021-08-17
YUNNAN ANIMAL SCI & VETERINARY INST
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Problems solved by technology

[0005] Yang Zhenxing and Li Zhanhong have developed real-time fluorescence quantitative polymerase chain reaction (quantitative real-time polymerase chain reaction, qRT-PCR) and competitive ELISA (competitiveELISA, C-ELISA) methods for the detection of EHDV and PALV respectively. detection, but the above two detection methods have deficiencies
[0006] (1) Inadequacies of qRT-PCR: On the one hand, the detection technology requires expensive equipment (about 300,000 yuan), and on the other hand, well-trained technicians are required to perform experimental operations
In addition, the qRT-PCR detection process needs to go through variable temperature cycles such as denaturation (95°C) and extension (72°C), and it is difficult to provide a stable power supply for field and on-site detection
[0007] (2) Insufficiency of C-ELISA: It mainly detects antibodies in animal blood, and it generally takes 2 to 3 weeks from animal infection with EHDV or PALV to antibody production. Therefore, C-ELISA method cannot be used before a

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  • RPA-LFD primer, probe and kit for jointly detecting epidemic hemorrhagic disease virus and Palimer serum group virus
  • RPA-LFD primer, probe and kit for jointly detecting epidemic hemorrhagic disease virus and Palimer serum group virus
  • RPA-LFD primer, probe and kit for jointly detecting epidemic hemorrhagic disease virus and Palimer serum group virus

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preparation example Construction

[0101] (4) Preparation of positive control template

[0102] According to the whole genome sequences of EHDV and PALV popular in China obtained earlier, two pairs of specific primers were designed: EHDV Seg-1 F: 5'-aaaatgcaatggtcgcaattaccgt-3' (SEQ ID NO.7); EHDV Seg-1 R: 5' -tttttcacccacgcacgtcc-3' (SEQ ID NO.8); PALV Seg-1F: 5'-gtcatattgcttctgcttcaa-3' (SEQ ID NO.9); PALV Seg-1 R: 5'-ccttacccgtgtgctcatcc-3' (SEQ ID NO .10) For the amplification of EHDVSeg-1 DNA fragment and PALV Seg-1 DNA fragment, the lengths of the amplified products are 1174bp and 959bp respectively. The nucleic acid sequences amplified by the PCR reaction are shown in SEQ ID NO.11 and SEQ ID NO.12.

[0103] The specific method is:

[0104] Using Viral DNA / RNA Extraction Kit" Genomic RNA of EHDV and PALV isolated in my country was extracted with Vrial DNA / RNA Kit” (Trans genBiotech), denatured at 94°C for 3 min, and immediately cooled in ice bath, followed by “PrimeScript TM RT Master Mix (Perfect

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Abstract

The invention relates to an RPA-LFD primer, a probe and a kit for jointly detecting an epidemic hemorrhagic disease virus and a Palimer serum group virus, and belongs to the technical field of molecular biological detection of animal viruses. The primer and the nfo probe for jointly detecting EHDV and PALV are designed, the RPA-LFD detection kit capable of jointly detecting the nucleic acids of EHDV and PALV popular in China is constructed on the basis, and the kit has the advantages of specificity, sensitivity, rapidness, high efficiency, isothermal amplification, on-site rapid diagnosis and the like, and is easy to popularize and apply.

Description

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Claims

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Application Information

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Owner YUNNAN ANIMAL SCI & VETERINARY INST
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