Real-time fluorescent RPA kit and test strip RPA kit for rapidly detecting capripoxvirus and application thereof
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A detection kit, a technology for detecting sheep pox virus, which is applied in the field of detection kits for sheep pox virus, test strip RPA kit, and achieves the effects of strong specificity, increased specificity, and simple method
Active Publication Date: 2016-05-11
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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[0010] The present invention is aimed at the GPCR gene of sheeppox virus, establishes and evaluates the RPA (real-timeRPA) detection kit based on fluorescent probe and the LFSRPA detection kit based on test strip to realize the purpose of fast detection sheeppox virus, according to our As far as is known, no such kit has been established at home and abroad for the detection of sheep pox virus
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Embodiment 1
[0054] The establishment of the real-time fluorescent RPA (Real-timeRPA) detection kit and detection method of rapid detection of embodiment 1 sheeppox virus
[0055] 1. Design and preparation of primers and probe sequences
[0056] The inventors of the present invention determined the conserved region of the GPCR gene of sheeppox virus by comparing and analyzing the homologous sequences of GPCR genes from X74443, L39878, EU267274, EU267273, AJ849636, FJ905304, AY560591 and AJ563705 in GenBank. Primers and probes were designed in order to detect as many sheeppox viruses as possible. All primers and probes were synthesized by Sangon Biotech (Shanghai, China). The present invention designed and synthesized three pairs of upstream primers, three pairs of downstream primers and one probe sequence respectively, as shown in Table 1 below.
[0057] Table 1, the sheep pox virus Real-timeRPA primer and probe designed by the present invention
[0058]
[0059]
[0060] 2. Virus...
Embodiment 2
[0090] Example 2 Establishment of the test strip RPA (LFSRPA) detection kit and detection method for rapid detection of sheep pox virus
[0091] 1. Design and preparation of primers and probe sequences
[0092] The inventors of the present invention determined the conserved region of the GPCR gene of the sheeppox virus by comparing and analyzing the homologous sequences of the GPCR genes from X74443, L39878, EU267274, EU267273, AJ849636, FJ905304, AY560591 and AJ563705 in GenBank. Primers and probes were designed in order to detect as many sheeppox viruses as possible. All primers and probes were synthesized by Sangon Biotech (Shanghai, China). The present invention designed and synthesized three pairs of upstream primers, three pairs of downstream primers and one probe sequence respectively, as shown in Table 4 below.
[0093] Table 4, LFSRPA primers and probes designed by the present invention
[0094]
[0095]
[0096] 2. Virus strains, cells and clinical samples ...
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Abstract
The invention discloses a real-time fluorescent RPA kit for rapidly detecting capripoxvirus and application thereof, and further discloses a test strip RPA kit for rapidly detecting the capripoxvirus and application thereof. The two kits comprise primers shown as SEQ ID NO.1 and SEQ ID NO.2 respectively and respective probes, and modified bases and detection platforms of the primers and the probes are different although the primers and the probes aim at a same target sequence. Experiments prove that the sensibility of the real-time fluorescent RPA kit and the sensibility of the test strip RPA kit are both 102 copies per response, and both of the two kits only can specially detect a sheep pox virus and a goat pox virus. A capripoxvirus suspected sample is detected by the two kits separately, and results show that the detection results of the two kits both have the very high conformity with that of qPCR. Therefore, the two kits both can rapidly, efficiently and sensitively detect the sheep pox virus or the goat pox virus, and an effective technological means is supplied to differentiation and diagnosis of the sheep pox virus or the goat pox virus.
Description
technical field [0001] The present invention relates to a kit for detecting goat pox virus and its application, in particular to a real-time fluorescent RPA kit and test strip RPA kit for rapid detection of goat pox virus based on RPA reaction, and also relates to the use of both The invention relates to the rapid detection of sheep pox virus, and the invention belongs to the field of preventive veterinary inspection. Background technique [0002] Thirty years have passed since the birth of PCR technology. From classic PCR, real-time quantitative PCR to the current digital PCR, this technology is constantly changing but has never faded out of our field of vision. However, this technology requires special and expensive thermal cycle instruments and skilled operators, which is time-consuming and labor-intensive, and it is difficult to be used in on-site diagnosis and places with poor experimental conditions. In the laboratories of developing countries, due to the limitation ...
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