Innovation of qRT-PCR assay for detection of multiple drug resistance gene lsa(E)
Journal Title: Journal of Henan Agricultural University - Year 2023, Vol 57, Issue 4
Abstract
[Objective] To provide a reference for monitoring the prevalence and spread of lsa(E) gene by innovating a real-time quantitative PCR (qRT-PCR) method for the detection of multi-drug resistance gene lsa(E). [Method] Specific primers were designed according to the reference sequence of the lsa(E) gene in GenBank. The recombinant plasmid DNA containing lsa(E) gene was extracted following purification, ligation and transformation and used as the standard substance for detection. The standard curve was drawn and its specificity and repeatability were verified to establish the innovative method of a qRT-PCR assay for multidrug-resistant gene lsa(E). Then, the method was applied to detect the copy numbers and relative abundance of lsa(E) gene in feces, soil, sludge and padding collected from cattle farms. [Result] The results showed that the melting curves of this method were smooth and unimodal, and exhibited a strong specificity; the amplification efficiency was 101.56%; the value of r2 was ≧0.997, indicating a good linearity relationship of the standard curve; the detection range (number of gene copies) of the assay for lsa(E) was wide with 1.71×102 to 1.71×108 copies·μL-1 and its sensitivity was high, and both the variable coefficients of intra and inter batch repeated tests were within 0.45% to 1.66%, with a good repeatability. For environmental samples from cattle farm, the copy number of the lsa(E) gene was from 2.54×104 to 1.52×108 copies·μL-1 and the relative abundance was from 9.39×10-4 to 1.20×10-3 detected by qRT-PCR. However, the conventional PCR method could only detect lsa(E) in environment samples with more than 1×106 copies·μL-1 of the copy number of gene. [Conclusion] This study innovated a qRT-PCR assay for the detection of multidrug-resistant gene lsa(E) with high specificity, sensitivity, and wide range of application. The innovative method could be used in different environment samples (including feces, soil and sludge) collected from cattle farms, and provided a technical and theoretical guidance for monitoring the epidemic and transmission of lsa(E) gene.
Authors and Affiliations
Bo ZHANG, Zheng CHEN, Qiong LI, Weicheng LIU, Congyang DU, Xiangdang DU, Chunyan XU
Keywords
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- EP ID EP769235
- DOI 10.16445/j.cnki.1000-2340.20230515.001
- Views 4
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