Triple fluorescent quantitative PCR kit for simultaneously detecting bovine rotavirus, bovine coronavirus and bovine viral diarrhea virus and application method of triple fluorescent quantitative PCR kit
A technology for bovine viral diarrhea and bovine rotavirus, applied in bovine diarrhea pathogen detection kits, bovine coronavirus and bovine viral diarrhea virus kits, and simultaneous detection of bovine rotavirus, can solve the problem that cannot meet the needs of multiple viruses Requirements for differential diagnosis of sexually transmitted pathogens, complex detection technology, unsuitable batch detection, etc., to achieve stable detection efficiency, high amplification efficiency, and save detection time
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Embodiment 1
[0054] Example 1 Simultaneous detection of bovine rotavirus, bovine coronavirus and bovine viral diarrhea virus triple real-time fluorescent quantitative PCR kit composition and reaction conditions
[0055] A triple fluorescent quantitative PCR kit for simultaneous detection of bovine rotavirus, bovine coronavirus and bovine viral diarrhea virus, including primers and probes, reverse transcriptase, standard positive RNA template, TaqDNA polymerase, and fluorescent quantitative PCR reaction solution;
[0056] (1) The content of each component of the fluorescent quantitative PCR kit - the reaction system is shown in Table 1.
[0057] Table 1
[0058] μL / reaction N responses Volume (μL) PCR reaction solution 12.5 10 125 reverse transcriptase 1 10 10 DNA polymerase (5u / μL) 1.5 10 15 BRV-1 Primers and Probes 3 (including upstream and downstream primers and probes) 10 30 BVDV-2 Primers and Probes 3 (including upstream and downs...
Embodiment 2
[0075] Example 2 Fluorescence Quantitative Kit Repeatability and Stability Detection
[0076]This embodiment is to detect the stability and repeatability of the triple fluorescent quantitative PCR kit for simultaneous detection of bovine rotavirus, bovine coronavirus and bovine viral diarrhea virus in embodiment 1. Using the RNA standard with known template concentration as a template, set up two concentration gradients PI and P2, and set up a negative control at the same time, and conduct intra-assay and inter-assay repeatability experiments. The method is: select three batches of primers and probes in June, September, and November 2019, named 2019-I, 2019-II, and 2019-Ⅲ, and each batch of reagents detects 2 different concentrations of templates 3 times, Seven replicates were set for the same reagent and the same template concentration in the same experiment, and the obtained CT values were analyzed to obtain the intra-assay and inter-assay errors. In principle, only when ...
Embodiment 3
[0088] Example 3 Application of triple real-time fluorescent quantitative PCR kit for simultaneous detection of bovine rotavirus, bovine coronavirus and bovine viral diarrhea virus in the detection of calf diarrhea samples
[0089] In this embodiment, the triple fluorescent quantitative PCR kit for simultaneous detection of bovine rotavirus, bovine coronavirus and bovine viral diarrhea virus in Example 1 is used for the detection of pathogens in cattle farm calf diarrhea samples. The specific testing process and results are as follows:
[0090] (1) Extract and purify sample RNA. RNA was extracted from 100 microliters of diarrhea samples by the magnetic bead method and dissolved in 50 microliters of nuclease-free water.
[0091] (2) Prepare the reaction system according to Table 9.
[0092] Table 9 Fluorescent quantitative PCR reaction system for detection of clinical diarrhea samples
[0093]
[0094] (3) The reaction conditions of the fluorescent quantitative PCR kit ar...
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