Method for simultaneously detecting three groups of rotaviruses A, B and C
A group A rotavirus, virus technology, applied in the simultaneous detection of A, C three groups of rotavirus, B field, can solve the problems of false negative, low detection sensitivity, different amplification efficiency, etc., to achieve strong specificity and sensitivity High, short detection time effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0058] Embodiment 1 Primer probe design and synthesis
[0059] A pair of specific primers, a specific probe with different fluorescent labels and a pair of universal primers were designed respectively for group A rotavirus, B rotavirus and group C rotavirus.
[0060] Table 1 List of detected pathogens
[0061] serial number virus name English abbreviations target gene 1 group A rotavirus RV-A VP6 2 group B rotavirus RV-B VP6 3 Group C rotavirus RV-C VP6
[0062] Multiple gene sequences covering domestic and foreign RV-A, RV-B, and RV-C viruses were downloaded from the NCBI gene bank. The homology comparison was carried out using BioEditor software to determine the conserved regions of the above viral genomes. Primer Premier 5.0 software was used to design highly specific primers and probes in its conserved regions. The sequences of primers and probes were verified by Blast and had good specificity. Label the VIC fluorophore ...
Embodiment 2
[0065] Example 2 Sensitivity Evaluation of Rotavirus Multiplex PCR Detection Method
[0066] 1. Preparation of samples to be tested:
[0067] The construction method of RV-A plasmid: the nucleic acid of group A rotavirus samples is amplified by PCR method using specific primers (i.e. RV-A specific primers in the present invention) to obtain group A rotavirus target gene fragments After purification, it was cloned into the pMD18-T vector by TA, identified by sequencing, and the recombinant vector with the correct sequencing result was transformed into DH5α competent cells, amplified, and RV-A plasmid was obtained.
[0068] The construction method of RV-B plasmid: apply specific primers (i.e. RV-B specific primers in the present invention) and use PCR method to amplify the nucleic acid of group B rotavirus samples to obtain group B rotavirus target gene fragments After purification, it was cloned into the pMD18-T vector by TA, identified by sequencing, and the recombinant vecto...
Embodiment 3
[0089] Example 3 Rotavirus multiplex PCR detection method detection effect evaluation
[0090] 1. Sample processing
[0091] Two cases of stool samples from patients confirmed positive for RV-A, RV-B, and RV-C in clinical testing, and two cases of serum samples from healthy people were selected, and a total of 8 samples were extracted for RNA extraction. Commercial kits can be used to extract RNA, such as using QIAGEN ViralRNA Mini Extraction Kit (CAT: 52904) to extract viral RNA. The extraction steps are well known to those skilled in the art and will not be repeated here.
[0092] 2. PCR amplification reaction
[0093] A total of 8 viral RNA samples extracted above were selected, plus a negative control / positive control reaction, and a total of 10 tubes of PCR reactions were required. Add the viral RNA samples to be tested to reaction tubes 1 to 8, and add 1×10 to tube 9. 6 Copies / ml plasmid positive control substance, add DEPC-ddH2O to No. 10 reaction tube as negative co...
PUM
Property | Measurement | Unit |
---|---|---|
Sensitivity | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com