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Method and kit for detecting RV RNA (Rubella Virus Ribose Nucleic Acid)
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A detection kit and rubella virus technology, which is applied in the field of extraction, purification and detection of rubella virus RNA (RV-RNA), achieving high yield, high purity and yield, and good adsorption effect
Active Publication Date: 2015-03-11
SANSURE (SHANGHAI) GENE TECH LTD
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[0005] The purpose of the present invention is to solve the defects of the existing rubella virus fluorescence quantitative PCR detection kit, provide a kind of RV nucleic acid fluorescence PCR detection kit with fast operation, simple and convenient method, high detection sensitivity and wide detection range, which can detect blood plasma, amniotic fluid, etc. Rapid detection of RV-RNA in unknown samples provides a reliable experimental basis for the diagnosis of rubella infection
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Embodiment 1
[0023]This embodiment provides a rubella virus nucleic acid detection kit (a kit for detecting RV RNA), which includes the following components:
[0024] ① RNA extraction solution Ⅰ: composed of sodium lauryl sulfate 0.2-1.0% (mass / volume), triton 1.0-4.0% (volume / volume), guanidine isothiocyanate 0.2-1.0mol / L and 100-400μg / ml of magnetic beads composition;
[0025] ②RNA extraction solution II: including 4-hydroxyethylpiperazineethanesulfonic acid 100-300mmol / L, sodium chloride 100-300mmol / L, solution II pH value is 6.5±0.2;
[0029] ⑥Internal standard (positive internal control): a 100-base-pair artificially synthesized DNA sequence inserted into the recombinant pUC18T vector, that is, a plasmid, with a concentration of 1.00E+04~5.00E+04copies / ml; its base seq...
Embodiment 2
[0043] This embodiment provides the operation steps of the kit described in the above-mentioned embodiment 1 for detecting RV-RNA in unknown samples such as plasma and amniotic fluid:
[0044] 1. Reagent preparation
[0045] 1) Take the corresponding amount of RNA extraction solution Ⅰ (200μl-1ml / person) and internal standard (0.5μl / person) in proportion and mix thoroughly to form RNA extraction solution 1-mix, centrifuge briefly and set aside.
[0046] 2) According to the number of samples to be tested, RV negative control, and RV positive control, take the corresponding amount of PCR reaction solution (38 μl / person) and RV enzyme mixture (2 μl / person) in proportion, and mix well to form a PCR- mix, centrifuged briefly for later use.
[0047] 2. RNA extraction operation
[0048] 1) Lyse the virus: add 200μl~1ml RNA extraction solution 1-mix to each tube, then add 100μl~1ml of the sample to be tested (such as plasma, amniotic fluid), cover the tube cap, shake and mix for 10 ...
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Abstract
The invention provides a method for extracting, purifying and detecting RV RNA (Rubella Virus Ribose Nucleic Acid), and a corresponding kit for detecting the RV RNA. The kit comprises a RNA extracting solution containing magnetic beads, and a PCR (Polymerase Chain Reaction) reaction liquid containing an upstream primer, a downstream primer and a probe, wherein the upstream primer and the downstream primer are used for amplifying target polynucleotides; the probe is used for detecting the target polynucleotides. The kit can be used for detecting the RV RNA and cannot be used for detecting non-RV pathogens, thereby illustrating that the kit has the good specificity. In addition, the RNA is extracted by selecting a method of the magnetic beads which are good in adsorption effect and easy to purify, so that the RNA with a high purity and a high yield can be obtained. Thus, the detection sensitivity, the detection accuracy and the detection stability of the kit are greatly improved, wherein the lower limit of detection of the RNA, namely, the sensitivity of the kit can reach 400copies / ml; the detection range of the kit (the quantitative linear range of the kit) can reach 4.00E+02copies / ml to 4.00E+08copies / ml.
Description
technical field [0001] The invention provides a method for extracting, purifying and detecting rubella virus RNA (RV-RNA), and also provides a corresponding kit for detecting RV RNA. Background technique [0002] Real-time fluorescent PCR technology (FQ-PCR) integrates PCR, molecular hybridization and photochemistry, so that the whole process of PCR amplification and product analysis is carried out under single-tube closed conditions. Compared with other detection technologies, real-time fluorescent PCR technology has the following advantages: (1) Compared with immunological detection, it has higher sensitivity, and theoretically only one gene copy can be detected. (2) Primers are designed according to the unique conserved gene sequences of various pathogens, which can ensure the high specificity of PCR reactions and avoid cross-reactions. (3) The virus can be quantitatively detected, which can reflect the copy number and replication status of the pathogen in the patient. ...
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