Streptococcus agalactiae loop-mediated isothermal amplification kit and application thereof
A ring-mediated isothermal and lactis streptococcus technology, applied in the field of microbial detection, can solve the problems of being unsuitable for grassroots and on-site detection, high cost, and complicated equipment, and achieve the effects of increasing test costs, easy operation, and avoiding pollution
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Embodiment 1
[0077] The specificity result of embodiment 1 LAMP detection method
[0078] LAMP amplification was performed on 1 strain of Streptococcus agalactiae, 8 strains of negative control bacteria and water control, the results were as follows figure 1 As shown, the Streptococcus agalactiae reaction tube showed a rising curve of turbidity at about 37 minutes, which was a positive result, and the curves of the 8 negative control bacteria reaction tubes and the water control reaction tube showed no amplification, which was a negative result.
Embodiment 2
[0079] Example 2 Sensitivity results of LAMP detection method
[0080] Recombinant plasmid pMD18-T- Sip The starting concentration is 3.55×10 1 ng / μL, LAMP and PCR amplification were performed after 10-fold dilution, the results were as follows figure 2 and image 3 As shown, the results show that the detection limit of the LAMP method is about 3.55×10 -5 ng / μL, while the detection limit of PCR method is 3.55×10 -3 ng / μL.
Embodiment 3
[0081] Example 3 Fluorescence visualization detection results of LAMP detection method
[0082] According to the optimized conditions monitored by the turbidimeter, fluorescent dyes were added to the reactor, and after 60 minutes of reaction at 63°C, observed under ultraviolet light, Figure 4 In order to observe the results, the left tube is the negative control, and the right tube is the reaction with Streptococcus agalactiae as the template, which shows that the established method can be used at the grassroots level. It is only necessary to use the LAMP primer designed by this method in the kit, and after adding the sample, Use an inexpensive water bath to maintain 63°C for 60 minutes, and you can quickly observe the results without opening the cover, avoiding pollution.
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