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

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

technical field [0001] The invention belongs to the technical field of animal virus molecular biology detection, and in particular relates to a recombinant enzyme polymerase-lateral flow chromatography test paper detection primer, a probe and a detection kit for jointly detecting epidemic hemorrhagic disease virus and Paliam serogroup virus. Background technique [0002] Epizootic haemorrhagic disease virus (EHDV) and palyam serogroup virus (PALV) are both members of the Orbivirus genus of the Reoviridae family and are widely prevalent in Tropical and subtropical regions between 49° north latitude and 35° south latitude. EHDV and PALV are mainly transmitted by blood-sucking bites of female Culicoides spp. to ruminants. EHDV can cause the death of infected white-tailed deer. It was previously believed that only the Ibaraki strain belonging to the EHDV-2 type could cause clinical symptoms similar to bluetongue disease in infected cattle, but in recent years EHDV-1, -6 and -7 ...

AI Technical Summary

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 antibody production. early clinical diagnosis

[0010] Li Zhanhong and others have developed a LAMP visual detection method for EHDV and PALV, but this method also has certain shortcomings

Although both LAMP and RPA are isothermal amplification technologies, and the detection results can also be judged by naked eyes, this technology requires at least two pairs of primers to complete an amplification reaction

Due to the easy formation of primer-dimers between multiple pairs of primers, resulting in non-specific amplification, it is difficult for LAMP to be used for the detection of two or more pathogens, which seriously reduces the detection efficiency

[0011] RPA has been applied in the detection of various pathogens including viruses and bacteria, and has the advantages of strong characteristics, high sensitivity, fast detection speed and isothermal amplification. More importantly, this technology can be used in clinical samples detection, but there is currently no RPA-LFD method for the co-detection of EHDV and PALV

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