Kit for synchronously detecting thirteen diarrhea viruses and detection method of kit
A technology for synchronous detection of diarrhea virus, which is applied in the field of detection kits for diarrhea virus, can solve the problems of time-consuming and labor-consuming antibodies, expensive technology costs, and increased costs, achieve good sensitivity and specificity, and avoid low specificity, The effect of saving production cost
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specific Embodiment 1
[0036] The present invention is a kit for simultaneous detection of thirteen kinds of diarrhea viruses, including DEPC water, 5×RT buffer, reverse transcription primer (RT primer mix), RT enzyme (full name reverse transcriptase), solution X, 10× PCR buffer, PCR primers, 25mM magnesium chloride solution, DNA polymerase, positive control (DNA fragments with specific sequences, used for quality control of the entire reaction system), 5×RT buffer, 10×PCR buffer, 25mM magnesium chloride solution, DNA polymerase was ordered from sigma company. The above-mentioned reverse transcription primers include the RT amplification primers of eleven kinds of diarrhea RNA viruses and human RNA internal references in the following table 1, and the above-mentioned PCR primers include the forward and reverse PCRs of the remaining two diarrhea DNA viruses, human DNA internal references, and reaction internal references in the table. The gene sequences of the amplification primers and PCR amplificat...
specific Embodiment 2
[0059] A method for synchronously detecting thirteen diarrhea viruses of the present invention, the detected viruses include human enteric coronavirus, enteric adenovirus 40, 41, enterovirus and its subtype (Coxsackie virus A16 type) , astrovirus, rotavirus A, B, C groups, norovirus, human biechovirus, human bocavirus type 2, sapovirus, small double segmented RNA virus, etc. (see Table 1), and collected patients Samples (fecal specimens, vomit, blood, etc.), nucleic acid extraction, reverse transcription and PCR reactions using patient nucleic acid as a template, and finally separated samples by capillary electrophoresis, the specific steps are as follows:
[0060] 1. Production of kits for the simultaneous detection of thirteen kinds of diarrhea viruses based on the GeXP multiple gene expression genetic analysis system. The components included in the kit are the same as those in Example 1 above;
[0061] 2. Collect samples and extract nucleic acids
[0062] Collect isolated ...
specific Embodiment 3
[0088] Detection Kit Sensitivity and Specificity Analysis
[0089] Sensitivity analysis: After diluting the positive control according to a certain copy number ratio, it is detected by PCR amplification and capillary electrophoresis until no signal is detected. The copy number is the lowest detection line, which is the sensitivity of the kit. Sensitivity up to 40 copies / uL.
[0090] Specificity analysis: Single-plex PCR amplification is detected as a single peak of the target fragment size by capillary electrophoresis.
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