A test kit for human staphylococcus detection
A staphylococcus and kit technology, applied in the direction of microorganism-based methods, microorganism measurement/inspection, biochemical equipment and methods, etc., can solve problems such as high cost, delay in disease treatment, lack of specific primers, etc., and meet equipment requirements Effects of low and high specificity
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Embodiment 1
[0016] Embodiment 1: Staphylococcus rpoB gene sequence alignment
[0017] like figure 1 As shown, in the human Staphylococcus rpoB gene sequence, the 5' end of the guide RNA sequence (selected part) of the present invention is the PAM structure of TTTN. The guide RNA sequence does not have a completely consistent sequence in Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus wahlschii, Staphylococcus capitis, and Staphylococcus hemolyticus, and all have several base differences. In addition, there is no PAM structure of TTTN at the 5' end of the homologous sequence in Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus wattleri, Staphylococcus capitum, and Staphylococcus hemolyticus. Therefore, Cas12a protein cannot target To Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus wahlschii, Staphylococcus capitus, Staphylococcus hemolyticus, therefore, only human staphylococcus can show positive reaction during detection, and other stap...
Embodiment 2
[0018] Embodiment 2: The guide RNA specificity determination of the present invention, the steps are as follows:
[0019] (1) Prepare human Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus worbachii, Staphylococcus capitis, Staphylococcus haemolyticus standard strains;
[0020] (2) Take 1ml of the sample to be tested, heat it at 98 degrees Celsius for 5min, and draw 1μl as the test sample;
[0021] (3) Prepare the reaction system, the reaction system is 25 μl, including 1 μl detection sample, 14.75 μl hydrated TwistAmpbasic kit reaction drying ball (TwistDx company), 0.9 μl 10mM RPA-F (sequence is SEQ No.2) and RPA-R ( Sequence is SEQ No.3), 0.375 μl Ribonuclease Inhibitor (Takara Company), 3.5 μl buffer2.1 (NEB Company), 1000nM guide RNA (sequence is SEQ No.1), 250nM Cas12a (NEB Company), 200nM single-stranded DNA Probe (Shanghai Sangong), add water to make up to 25μl;
[0022] (4) React at a constant temperature of 37 degrees Celsius...
Embodiment 3
[0025] Embodiment 3: the detection method based on guide RNA described in the present invention and traditional PCR method specificity and experimental time-consuming comparison
[0026] 1. based on the detection method of guide RNA described in the present invention, the steps are as follows:
[0027] (1) Prepare human Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus worbachii, Staphylococcus capitis, Staphylococcus haemolyticus standard strains;
[0028] (2) Take 1ml of the sample to be tested, heat it at 98 degrees Celsius for 5min, and draw 1μl as the test sample;
[0029] (3) Prepare the reaction system, the reaction system is 25 μl, including 1 μl detection sample, 14.75 μl hydrated TwistAmpbasic kit reaction drying ball (TwistDx company), 0.9 μl 10mM RPA-F (sequence is SEQ No.2) and RPA-R ( Sequence is SEQ No.3), 0.375 μl Ribonuclease Inhibitor (Takara Company), 3.5 μl buffer2.1 (NEB Company), 1000nM guide RNA (sequence is SEQ No...
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