A test strip for detecting portunus trituberculatus reovirus and its preparation method
A technology for reovirus and portunus trituberculatus, which is applied in the field of immunochemical detection, can solve the problems of unpublished test strips for portunus trituberculatus reovirus, and achieve the effects of rapid detection, simple preparation process and convenient storage
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specific Embodiment 1
[0043] A kind of portunus trituberculatus reovirus detection test strip, such as figure 1 As shown, the test strip consists of a PVC bottom plate 7 and a sample pad 1, a bonding pad 2, a nitrocellulose membrane 3 and a water-absorbing pad 4 that are sequentially overlapped on the PVC bottom plate 7; Orphan virus egg yolk antibody-colloidal gold marker, the detection line 5 coated with Portunus trituberculatus reovirus antigen and the quality control line coated with rabbit anti-chicken egg yolk antibody (IgY) are respectively set on the nitrocellulose membrane 3 6.
[0044] The preparation method of the above-mentioned anti-portunus reovirus yolk antibody-colloidal gold marker is as follows: the anti-portunus trituberculata reovirus yolk antibody is mixed with a colloidal gold solution with a particle diameter of 20-30nm at 7-9μg: 1mL , combined by stirring and shaking for 30-60min under the condition of pH5.4, adding PBST buffer solution containing 10wt% bovine serum albumin...
specific Embodiment 2
[0045] The preparation method of rapid detection portunus trituberculatus reovirus colloidal gold test strip, such as figure 2 As shown, the specific operation steps are as follows:
[0046] (1) Purification of Portunus trituberculatus reovirus
[0047] Take the Portunus crabs that died after being infected with the virus, and dissect them on ice to get their viscera. After weighing, add TNE containing 1mM phenylmethylsulfonyl fluoride (PMSF) that has been ice-bathed in advance at a volume ratio of 1:5-1:20. 2 Homogenize the buffer solution, centrifuge the homogenate at 4°C, 8000g for 20min, and use TNE containing 1mM PMSF for precipitation 2 After resuspending the homogenate in the buffer, centrifuge again at 4°C, 8000g for 20min, combine the supernatant obtained from the two centrifugations, centrifuge the supernatant at 4°C, 6000g for 10min, take the supernatant and centrifuge at 4°C, 38000g for 1.5h , with pre-cooled TM 2 The loose part of the upper layer of the precip...
specific Embodiment 3
[0065] Colloidal gold test strips for the detection of clinical samples, the specific steps are as follows:
[0066] Portunus trituberculatus were purchased from various local supermarkets and farms, and samples of swimming crabs were collected in a certain outbreak of disease and a large number of dead swimming crab farms. Take the tissue samples of Portunus, TNE 2 Add the buffer solution dropwise on the tissue sample, break the tissue, centrifuge at 2000-3000rpm for 5 minutes, take the supernatant, insert the test strip into the sample to be tested, and observe the result after 10 minutes: if only the quality control of the test strip If a red line appears in the area, it is a positive result, that is, the sample contains Portunus reovirus; if the test line and the quality control line of the test strip appear red lines at the same time, it is a negative result, that is, the sample does not contain Portunus reovirus. Each sample was repeated 5 times, and the test results of...
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