Probe for rapid detection on vibrio cholera sandwiched DNA (Deoxyribonucleic Acid) hybridization, kit and detection method
A technology for vibrio cholerae and detection probes, which is applied in the field of probes, kits and detection fields for rapid detection of vibrio cholerae sandwich DNA hybridization, achieving the effects of high sensitivity, wide application and accurate results
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Embodiment 1
[0033] Example 1, VC Sandwich DNA Hybridization Rapid Detection Kit includes a 96-well plate coated with poly dT, two bottles of concentrated bacterial liquid lysate, one bottle of lysis buffer, one bottle of hybridization solution, and one bottle of probe solution , a bottle of washing solution, a bottle of chromogenic solution, a bottle of stop solution, a bottle of positive control solution and a bottle of negative control solution, wherein:
[0034] The probe solution is composed of the following components: 12.5 μL of 8 μmol / L VC capture probe whose DNA sequence is SEQ ID NO.1; 12.5 μL of 8 μmol / L VC detection probe whose DNA sequence is SEQ ID NO.2; , the volume is 30 μL; the hybridization solution, the volume is 100 μL; the washing solution, which needs to be diluted by 10 times, the volume is 750 μL; the chromogenic solution, the volume is 150 μL; the stop solution, the volume is 100 μL;
Embodiment 2
[0035] Embodiment 2, the design of probe
[0036]In the nucleic acid detection of VC, virulence genes such as ctx, zot, and rtx are often selected as detection objects, but false positive or false negative results are prone to occur in this way. Establishing a detection method with housekeeping genes as the target gene can avoid such problems. The groEL gene encodes the molecular chaperone GroEL, which plays an important role in maintaining cell osmotic pressure. The gene is universal and conserved in nature and The variability among species makes it well suited as a phylogenetic marker.
[0037] According to the groEL gene reference sequence of VC published by GenBank, Clustal W was used to perform multiple alignments to analyze the conserved regions of the sequences. The probe design software Primer 5 was used to design specific probes, respectively marked as: SEQ ID NO1, SEQ ID NO2. The capture probe in the probe group: SEQ ID NO.1; the detection probe: SEQ ID NO.2; the v...
Embodiment 3
[0039] Embodiment 3, the preparation of positive control substance
[0040] The standard positive VC bacteria liquid was cultivated with tryptone soybean broth (TSB) for 48 hours. The concentration of the bacterial solution was determined by the plate colony counting method, and the concentration was controlled at 1.0×10 7 ~1.0×10 8 CFU / mL, a bottle of 5mL.
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