Application of Loop-mediated Isothermal Amplification to Detect the aph Gene in Staphylococcus
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Abstract:
Since the use of antibiotics in animal breeding, bacterial resistance has become a worldwide concern. Detection of drug-resistance genes is a new method for assessing biosecurity. The aph gene was cloned into the pEASY-T1 vector using PCR-based methods and sequenced. A set of loop-mediated isothermal amplification (LAMP) detection primers was designed against a highly conserved region of the aph gene based on homology with aph loci in GenBank. The reaction conditions were optimized and LAMP detection methods for the aph gene were established. The optimal detection conditions were as follows: 0.8 mol/L betaine and 20 mmol/L Mg2+, completed within 50 min in a 63°C isothermal water bath. Compared to PCR detection, the LAMP method can be completed in a shorter time period with higher specificity. The sensitivity threshold was approximately 20.4 copies for the aph gene and was similar to that of the PCR method. The positive detection rate of the aph gene was 84.2% for 38 Staphylococcus aureus isolates.
