Kit for detecting human enteric viruses by real-time fluorescent quantitative PCR
An enterovirus and kit technology, applied in the field of real-time fluorescent PCR detection kits, can solve the problems of time-consuming, unsatisfactory processing of a large number of samples at the same time, laborious and other problems, and achieve the effect of good sensitivity
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Embodiment 1
[0033] Embodiment 1: the development of human enterovirus fluorescent PCR detection reagent
[0034] 1. Design of primers and probes: Through sequence comparison and analysis of the existing enterovirus nucleic acid sequences in the Genbank database and the nucleic acid sequences reported in domestic and foreign published literature, the conserved genes in the 5' non-coding region of enteroviruses The fragment is the amplified target site, select a highly conserved segment with no secondary structure, and use software and manual design of multiple pairs of primers and probes according to the basic principles of primer probe design.
[0035] 2. Selection of samples: According to relevant domestic and foreign literature reports, samples such as feces, serum, throat swabs, cerebrospinal fluid, and herpes fluid can be selected.
[0036] 3. Establishment and optimization of the reaction system
[0037] Sample preparation: 8 enteroviruses identified as positive by virus culture and...
Embodiment 2
[0047] Embodiment 2: human enterovirus fluorescent PCR detection kit and its use
[0048] 1. Prepare a kit including the following components: 1 tube of RNA extraction solution (50ml / tube), 24 tubes of PCR amplification reaction solution (20μl / tube), 1 tube of negative quality control (100μl / tube), 1 tube of positive quality control 1 tube of sample (100 μl / tube), 4 tubes of quantitative reference product (50 μl / tube).
[0049] 2. Specimen collection, transportation and storage
[0050] (1) Specimen collection: The object of specimen collection is clinically diagnosed cases of HFMD at the time of the outbreak and children under 5 years old in communities (villages) near the community (village) without cases or nursery institutions where the cases occurred (as healthy control population).
[0051] Specimens should be shipped frozen with necessary information, such as specimen number, date of onset, and date of specimen collection.
[0052] (2) Specimen storage and delivery: ...
Embodiment 3
[0075] Embodiment 3: Reproducibility experiment of enterovirus fluorescent PCR detection kit
[0076] at 5.0×10 4 The positive reference product of PFU / ml is as the sample to be tested, detects 5.0 * 10 simultaneously with 7 enterovirus fluorescent PCR detection kits of 3 production batches according to the method in embodiment 2 4 Positive reference substance of PFU / ml, the results are attached Figure 5 As shown: the Ct values of different amplification curves are all between 27 and 28, and the calculated coefficient of variation CV=5%, indicating that the kit has good repeatability.
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