Advanced High Resolution Melt Technology for Bacteria Serotyping and Antibiotic Resistance Detection
- Author(s): ZHANG, YANG
- Advisor(s): Fraley, Stephanie
- et al.
Bacteria identification plays an important role in confirming diagnosis of bacterial disease and tracing the source of an outbreak for public health. However, the standard method for bacteria identification which relies on the colony feature of the bacteria cultured from the patient’s specimen is time consuming and lack of accuracy and sensitivity. New technology such as qPCR based sequencing and microarray showed their benefit in this area but are still suffering problems regarding to cost, complexity and accuracy. Therefore, this thesis tries to advance the high resolution melt (HRM) technology, a molecular technique to detect difference in double-stranded DNA, to realize bacteria serotyping. Two methods: intercalating dyes and multiplex PCR were investigated for this purpose. To our excitement, multiplex PCR of V1 to V6 region and V6 region of the gene 16S ribosome RNA of Salmonella followed by HRM can distinguish 5 serotypes of Salmonella under 3 hours, including Newport, Typhimurium, Heidelberg, Enteritidis and Choleraesuis. We further applied this technology to achieve antibiotic resistance detection and bacteria speciation in one test and successfully detected the kanamycin resistance from E. coli. This multiplex PCR combined with HRM will be further combined to digital PCR to build a fast, easy to use, highly specific, highly sensitive and absolute quantitative tool for bacteria diagnosis.