On-site rapid detection method and kit for detecting PRRSV
A detection kit and detection method technology, applied in the field of animal husbandry, can solve the problems of long inspection cycle and high requirements for experimental conditions
Active Publication Date: 2018-12-04
SOUTH CHINA UNIV OF TECH
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Problems solved by technology
Although these methods can effectively detect PRRSV virus, there are problems such as long inspection period and high requirements for experimental conditions, and these methods generally need to take pig heart, spleen, kidney, lung, lymph node, blood and other tissues, which are difficult to detect in the absence of experimental facilities Fields such as farms and slaughterhouses can quickly detect viruses in real time, especially not suitable for breeding places
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Embodiment 1
[0048] In the embodiment of the present invention, on-site rapid detection of PRRSV virus based on CRISPR / Cas13a technology, the method is used for non-diagnostic or therapeutic purposes, including the following steps:
[0049] 1) Design target-specific crRNA for two important subtypes of PRRSV virus, namely European type and American type. The sequence of the designed crRNA is shown in SEQ ID NO: 1-6 in the sequence table, and then construct crRNA for in vitro transcription Vector and in vitro transcription and purification.
[0050] 针对欧洲型PRRSV病毒各亚型(NCBI登陆号分别为AY366525,AY588319,DQ489311,DQ864705,EU076704,GQ461593,JF276430,JF276431,JF276432,JF276433,JF276434,JF276435,JX187609,KC492504,KC492505,KC492506,KM196101,LEYPOLYENV) Specific crRNA was designed in the conserved region, and off-target analysis was performed to screen three crRNAs with good specificity and low off-target. The design results are shown in Table 1, that is, as shown in SEQ ID NO: 1-3 in the sequence table.
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Relating to the field of animal husbandry, the invention discloses an on-site rapid detection method and kit for detecting PRRSV. The invention utilizes the specificity of crRNA and the specificity ofRPA primers to effectively guarantee the virus detection accurateness, and within 1-2h at room temperature, whether the virus is infected and the subtype of the infected PRRSV can be determined by test paper, and compared with the traditional PCR based detection method, the precision is improved. According to the invention, the kit equipped with purified freeze-dried LwaCas13a and crRNA can be formed, can achieve quick and convenient detection of PRRSV at pig farms and slaughterhouses, and does not require complex temperature control instruments, like PCR instruments and centrifuges. Only byone constant temperature device, naked-eye observable results can be quickly acquired, and the health status of pig farms can be understood more efficiently, and loss can be avoided. Therefore, the invention provides the fast and convenient PRRS detection method with lower cost.
Description
technical field [0001] The invention relates to the field of animal husbandry, in particular to an on-site rapid detection method and a kit for detecting PRRSV, in particular to using Cas13 / crRNA specific on-site rapid identification and detection of PRRSV virus. Background technique [0002] PRRS is a contagious pig disease caused by porcine PRRS virus (PRRSV). PRRS can cause reproductive disorders in sows and severe respiratory symptoms in piglets, seriously affecting economic benefits. China is an important epidemic area of porcine blue-ear disease. Most of the virus strains isolated in China are highly pathogenic strains, which have brought a lot of losses to China's pig industry. [0003] However, the current detection method for this virus takes a long time to detect and has high requirements for instruments. The more commonly used methods for the detection of the virus mainly include virus isolation, virus antigen antibody detection, detection methods based on rev...
Claims
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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2521/507C12Q2522/101
Inventor 黄黎珍林浩斯钟国瑞李浩健杜红丽白静何昌生谢水林戴仁科
Owner SOUTH CHINA UNIV OF TECH