Kit and method for detecting feline infectious rhinotracheitis virus
A rhinotracheitis virus and infectious technology, which is applied in the field of bioengineering, can solve the problems of low accuracy and complicated detection method of feline infectious rhinotracheitis virus, and achieve the effect of short time, good reproducibility and high sensitivity
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Embodiment 1
[0032] Embodiment 1 kit composition
[0033] The real-time fluorescent PCR reaction system was 25 μL, including 12.5 μL of 2×TaqMan Master Mix, 0.5 μL (10 μM) of upstream and downstream primers, 1 μL (10 μM) of probe, 5 μL template DNA, and made up to 25 μL with deionized water.
[0034] Upstream primer FHV-1F: 5'-ACGCTACAATTATACTCAAGCCAGAA-3' (SEQ ID NO: 1);
[0035] Downstream primer FHV-1R: 5'-TCAGCTGTTATCTTCGGATCCA-3' (SEQ ID NO: 2).
[0036] The sequences of the probes are as follows:
[0037] FHV-1 Probe: FAM-CGTGCCAAAAATTCCCCAGGCG-TAMRA (SEQ ID NO: 3).
[0038] Real-time fluorescent PCR reaction parameters: 95°C, 30s; 40 cycles, 95°C, 5s; 60°C, 34s. Fluorescent signals were collected at 60°C, detection channel: FAM.
Embodiment 2
[0039] Example 2 The method of using the feline infectious rhinotracheitis virus detection kit
[0040] 1. Processing of samples to be tested
[0041] The samples to be tested generally include cat conjunctival swabs, nasal swabs, oral swabs collected clinically, and virus cell culture fluid. The DNA is extracted by itself, and the positive control does not need to be extracted, and the sample is added directly.
[0042] 2. Detection of fluorescent PCR
[0043] (1) Take 2×TaqMan Master Mix commercial reagents (can also be replaced by fluorescent PCR reagents with equivalent efficiency), upstream and downstream primers and probes according to the number of detection samples n (n=number of samples to be tested + 2) and mix them in a centrifuge In the tube, vibrate on the vortex shaker, pack according to each tube, and cover the tube for later use.
[0044] (2) Now add the negative control solution into an aliquot tube, take the DNA of each sample and add it to the corresponding ...
Embodiment 3
[0054] Embodiment 3 kit specificity test
[0055] Take 5 μL of nucleic acid from 5 control samples including feline calicivirus, feline distemper virus, canine distemper virus, canine parvovirus, and cat kidney cells (F81) as templates for PCR amplification, and set a negative control group at the same time.
[0056] PCR amplification conditions: 95°C, 30s; 40 cycles, 95°C, 5s; 60°C, 34s. Fluorescent signals were collected at 60°C, detection channel: FAM.
[0057] The results of fluorescent PCR showed that only the FHV-1 sample amplified the S-shaped curve, while the feline calicivirus, feline distemper virus, canine distemper virus, canine parvovirus, and cat kidney cells (F81) as the control samples had no such amplification curve appears (see Figure 4 ).
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